DESC:TECHNICAL FIELD
The present disclosure pertains to the field of molecular oncology/biotechnology. The
present disclosure relates to a method of prognosing and predicting breast cancer
recurrence in patients. More particularly, the present disclosure relates to a method of
prognosing which stratifies early stage breast cancer patients with hormone receptor
positive and Her2 receptor negative invasive ductal/lobular carcinoma of the breast into
low risk or high risk for breast cancer recurrence by employing an IHC based assay which
assesses or measures expression of a combination of 5 biomarkers and by employing a
histopathological analysis which assesses 3 clinical prognostic parameters. The present
disclosure also relates to a combination of 5 biological markers and 3 clinical parameters
employed in the method for prognosis of breast cancer, a kit/test comprising the antibodies
against said markers for said prognosis and an IHC based assay system comprising said
markers.
BACKGROUND OF THE DISCLOSURE
In the field of oncology, the detection, identification and characterization of tumor cells
is an important aspect for diagnosis of cancer and for detection/prognosis of cancer
recurrence. Of the many challenges of medicine, none has had a more controversial
beginning or has experienced more hard-fought progress than the treatment and cure of
cancer and more importantly, prevention of cancer recurrence. Effective treatment for
most patients is needed to reach every organ in the body to pin down the metastatic
disease. More than 70% of cancer patients undergo chemo/radio therapy.
Despite the path breaking progress in oncology therapy from multiple angles, cancer cure
still remains elusive. Advanced solid malignancies remain therapeutic challenges despite
maximal therapy, in part, due to the development of resistance to radiation and
chemotherapy. For eg. Glioblastomas are among the most lethal of cancers with current
therapies offering only palliation. Standard-of-care for glioblastoma consists of surgical
resection, ionizing external beam irradiation, and chemotherapy. Though radiotherapy has
3
been the most effective nonsurgical treatment modality, but yet recurrence is essentially
universal.
At present, about 95% of the patients with hormone receptor positive and Her2 negative
early stage breast cancer get treated with chemotherapy, with majority of early stage
cancer patients getting over-treated with Chemotherapy.
Thus, majority of patients undergoing chemo/radio therapy suffer from un-necessary
severe side effects of the treatment. In addition, many patients also show resistance to the
treatment resulting in treatment failure and cancer recurrence. Existing techniques for
assessing risk of cancer recurrence in the abovementioned hormone receptor positive and
Her2 receptor negative patients are expensive, applicable only for stage I cancer patients
and not predictive in nature.
Thus, in spite of availability of many ways of standardized methods for diagnosis &
prognosis of breast cancer currently, there exists a need to develop prognostic and
predictive tests that can determine risk of cancer recurrence and effectiveness of the
prescribed chemo/radio therapy, with accuracy and at affordable costs, which in-turn will
help devise new drug therapy. Breast cancer recurrence risk in ER+ stage 1 and stage 2
patients being assessed currently by considering clinical parameters and biomarker based
means (Prognostic markers: Age, tumor grade, size, nodes with metastasis (TNM) and
Predictive markers: ER/PR/Her-2-neu) is insufficient, leading to over-treatment of
patients with chemotherapy thereby leading to unnecessary side-effects. Over the past
decade, several molecular tests have been developed to predict risk of recurrence in Breast
Cancer. Treatment of early stage patients in the West today is decided based on the risk
predicted by any of these tests including Oncotype Dx, Mammaprint, PAM50 and
EndoPredict. These tests are primarily based on hormone receptor & proliferative
pathways. These pathways are important, but insufficient to accurately predict risk of
recurrence as Breast cancer is a heterogeneous disease and several pathways regulate the
molecular pathogenesis of the disease. Further, Oncotype Dx, Mammaprint, Endopredict
and PAM50 are used primarily for ER+/PR+/Her2- Stage 1/2 patient who are lymph node
4
negative (LN-) and for limited patients with 1-3 lymph nodes positive. Thus, these tests
are applicable for a highly-limited set of Stage 1 patients which are not common outside
the western world and are largely prognostic with limited chemo-predictivity. Further all
the 4 tests mentioned above are based on gene expression detection in whole FFPE blocks
by microarray or q-PCR, and do not detect protein expression thus unable to prescribe
new targeted drugs apart from also being very expensive and impractical for application
in Asian Patient Cohort, as only ~5% of patients are diagnosed in LN-, Stage 1 cancer.
The instant disclosure therefore provides for a prognostic and predictive method which
addresses all the limitations existing in the prior art for treating cancer and devising new
or personalized drug therapy.
STATEMENT OF THE DISCLOSURE
Accordingly, the present disclosure relates to a method of measuring biomarker
expression, comprising measuring expression of five biomarkers by assaying a biological
sample with a combination of antibodies, wherein the biological sample is obtained from
a subject having early stage ER+/PR+ and Her2- breast cancer or after removal of the
early stage ER+/PR+ and Her2- breast cancer and the five biomarkers are CD44,
ABCC11, N-cadherin, Pan-cadherin and ABCC4; a method of performing
immunohistochemistry (IHC) on a tumor sample obtained from a subject having early
stage ER+/PR+ and Her2- breast cancer or after removal of the early stage ER+/PR+ and
Her2- breast cancer, comprising performing IHC on the tumor sample to optionally detect
hormone receptor expression by detecting whether cells are expressing at least one
receptor selected from the group consisting of estrogen receptor and, progesterone
receptor, and ensuring tumor samples do not express Her2 receptor and performing the
method as above on the tumor sample by IHC to measure the expression of the five
biomarkers CD44, ABCC11, N-cadherin, Pan-cadherin and ABCC4; a method of
prognosing and treating a subject having early stage ER+/PR+ and Her2- breast cancer
or after removal of the early stage ER+/PR+ and Her2- breast cancer, comprising
prognosing whether the subject is at high or low risk for breast-cancer recurrence by
performing, and analyzing the results of, the method as above; and treating the prognosed
5
subject with chemotherapy if prognosed as high risk for breast-cancer recurrence; a
combination of antibodies, comprising antibodies specific for five biomarkers CD44,
ABCC11, N-cadherin, Pan-cadherin and ABCC4; a kit comprising the combination of
antibodies as above and instructions for measuring expression of the five biomarkers
CD44, ABCC11, N-cadherin, Pan-cadherin and ABCC4; an IHC-based assay system,
comprising the combination of antibodies as above; and a method of predicting the
likelihood of recurrence of breast cancer in a subject having early stage ER+/PR+ and
Her2- breast cancer or after removal of the early stage ER+/PR+ and Her2- breast cancer,
comprising: measuring expression level of five biomarkers CD44, ABCC11, N-cadherin,
Pan-cadherin and ABCC4, calculating a relapse score for said subject by measuring the
differential expression levels of each of the biomarkers and their contribution to breast
cancer recurrence along with three clinical parameters, and using said relapse score to
determine the likelihood of breast cancer recurrence.
BRIEF DESCRIPTION OF THE ACCOMPANYING FIGURES
The features of the present disclosure will become fully apparent from the following
description taken in conjunction with the accompanying figures. With the understanding
that the figures depict only several embodiments in accordance with the disclosure and
are not to be considered limiting of its scope, the disclosure will be described further
through use of the accompanying figures:
Figure 1 illustrates current method of prognosis and improvement using CanAssist-
Breast: Overview of factors considered for risk stratification of patients with early stage
Her2 negative breast cancer .
Figure 2 illustrates description of CanAssist-Breast
Figure 3 illustrates Kaplan Meier (KM) curve results of risk stratification of early stage
ER+/PR+ and Her2- breast cancer patients by employing the prognostic method/test of
the instant disclosures. As can be observed from the KM of the instant disclosure, low and
6
high risk patients are separated well and predicted for all cases as there is no intermediate
zone in the KM curve of CanAssist-Breast test.
Figure 4 illustrates a brief work-flow of the method employed in predicting risk of
recurrence in early stage ER+/PR+ and Her2- breast cancer patients, from start to finish.
DETAILED DESCRIPTION OF THE DISCLOSURE
Unless otherwise defined herein, scientific and technical terms used in connection with
the present disclosure shall have the meanings that are commonly understood by those of
ordinary skill in the art. Further, unless otherwise required by context, singular terms shall
include the plural and plural terms shall include the singular as is considered appropriate
to the context and/or application. The various singular/plural permutations may be
expressly set forth herein for the sake of clarity. Generally, nomenclatures used in
connection with, and techniques of biotechnology, molecular oncology, molecular and
cellular biology described herein are those well-known and commonly used in the art.
Certain references and other documents cited herein are expressly incorporated herein by
reference. In case of conflict, the present specification, including definitions, will control.
The materials, methods, figures and examples are illustrative only and not intended to be
limiting.
The present disclosure has utility in the field of oncology for stratifying early stage
ER+/PR+ and Her2- breast cancer patients into high or low risk for breast cancer
recurrence, prior to cancer treatment and/or post-surgery. In an embodiment, the
prognostic and/or predictive test in the instant invention is preferably performed on patient
samples after surgery and before chemotherapy, since the decision to give chemotherapy
is based on test results. The present disclosure thus relates to a prognostic test for detecting
cancer recurrence preferably in early stage ER+/PR+ and Her2- breast cancer as well as
a predictive test which will enable chemotherapy decision and also devising of new drugs
or personalized therapy specifically based on the prognostic outcome of the patient.
7
The present disclosure thus by helping in stratifying the early stage ER+/PR+ and Her2-
breast cancer patients into low or high risk of breast cancer recurrence will therefore
enable to intelligently chalk out a module to treat low and high risk patients, as per the
optimal chemotherapy requirement; thus overcoming under treatment/over treatment of
patients as applicable; and also help in devising new age effective drugs or personalized
therapy for cancer treatment.
Before the method of prognosis, biomarkers, kit comprising antibodies against said
markers and other embodiments of the present disclosure are disclosed and described, it
is to be understood that the terminologies used herein are for the purpose of describing
particular embodiments only and are not intended to be limiting. It must be noted that, as
used in the specification and the appended claims, the singular forms “a,” “an” and “the”
include plural referents unless the context clearly dictates otherwise.
As used herein, “tumor” or “cancer” are terms as well known in the art and used
interchangeably throughout the disclosure. Similarly, the terms “clinical prognostic
factors” “clinical prognostic parameters” or “clinicopathological parameters/clinical
parameters/clinical variables”; “algorithm” or “module” or “equation”; “IHC
scoring/scoring” or “IHC grading/grading”; are one and the same and used
interchangeably throughout the disclosure.
“Status” means a state of gene expression of a set of genetic markers whose expression is
strongly correlated with a particular phenotype. For example, “ER status” means a state
of gene expression of a set of genetic markers whose expression is strongly correlated
with that of ESR1 (estrogen receptor gene), wherein the pattern of these genes' expression
differs detectably between tumors expressing the receptor and tumors not expressing the
receptor.
The terms “ER+” or “ER+ve” or “estrogen receptor positive” are used interchangeably
throughout the disclosure and intend to convey the positive expression of the estrogen
receptor. Similarly, the terms “PR+” or “PR+ve” or “progesterone receptor positive” are
8
used interchangeably throughout the disclosure and intend to convey the positive
expression of the progesterone receptor.
The term “hormone receptor positive” used throughout the disclosure intends to convey
the positive expression of one of or both of estrogen receptor and progesterone receptor.
The terms “Her2-” or “Her2-ve” or “Her2 negative” or “Her-2-neu -ve” or “Her-2-neu
receptor negative” or “Her-2-neu negative” or “negative for Her-2-neu receptor” are used
interchangeably throughout the disclosure and intend to convey the absence of expression
of the human epidermal growth factor receptor 2.
“Marker” or “Bio-marker” means an entire gene/protein, or an EST derived from that
gene, the expression or level of which changes between certain conditions. Where the
expression of the gene correlates with a certain condition, the gene is a marker for that
condition.
The term “High” with regards to risk of breast cancer recurrence means that a patient is
expected to have >9% probability of distant recurrence within five years of initial
treatment of breast cancer.
The term “Low” with regards to risk of breast cancer recurrence means that a patient is
expected to have 9% or lower probability of distant recurrence of breast tumor within five
years of initial treatment of breast cancer.
The term “breast cancer” in the present disclosure refers to hormone receptor positive
invasive ductal carcinoma of the breast or invasive lobular carcinoma of the breast.
Preferably, the carcinoma is hormone receptor positive invasive ductal carcinoma of the
breast.
The term “prognosis” or “prognostic marker” in the present disclosure relates to
predicting disease progression without taking into consideration any reference to
9
therapy/treatment/drug. A very qualitative way of prediction for eg: age, tumor stage,
tumor grade are prognostic markers. This prognosis relates to understanding the longterm
outcome of a disease.
The term “IHC grading” in the present disclosure relates to raw grading values assigned
for each stained IHC slide by the Pathologist based on % staining (0-100%) and Intensity
of staining (0-3).
The term IHC/immunohistochemistry in the present disclosure (devoid of reference to
prior arts) relates to Morphometric immunohistochemistry. The term also intends to
encompass the conventional histopathological techniques associated with the process of
carrying out the IHC. The immunohistochemistry analysis carried out within the present
disclosure encompasses the conventional IHC method, whereby the identification of
biological markers is carried out by visualizing a colored reaction or fluorescence
obtained at completion of the method due to staining of the desired biomarkers present on
the cells cells from the sample.
The terms “CanAssist Breast score” or “CanAssist Breast test score” or “relapse score”
or “prediction score” or “score” in the present disclosure are used interchangeably and
relates to an algorithm generated score computed using the averages of raw grading (for
percentage and/or intensity of staining) for each IHC marker as well as information
regarding tumor size, node status and tumor grade.
The term “tumor grade” in the present disclosure relates to grade, ranging from 1-3
assigned to the tumor based on histopathological features according to the modified
Bloom-Richardson Elston criteria.
The term “tumor stage” in the present disclosure relates to the TNM staging of Breast
Cancer which is based on tumor size (T) Node status (N) and Metastasis in the body (M).
10
The term “predictive” or “predictive marker” in the present disclosure relates to a marker
that not only gives some kind of reference to disease progression but more importantly
will direct a therapy based on the presence of the markers for eg: ER, PR and Her2.
Employing them is prognostic along with a direction as to what a possible therapy could
be. Thus prediction is related to understanding benefit to the patient in response to certain
treatment.
In the present disclosure, the prognostic and predictive test herein is also referred to as
“CanAssist Breast test”.
The present disclosure relates to a method of measuring biomarker expression, comprising
measuring expression of five biomarkers by assaying a biological sample with a
combination of antibodies, wherein the biological sample is obtained from a subject
having early stage ER+/PR+ and Her2- breast cancer or after removal of the breast
cancer; and the five biomarkers are CD44, ABCC11, N-cadherin, Pan-cadherin and
ABCC4. The biomarker expression is measured for prognosis about whether the subject
having cancer is at high or low risk for breast-cancer recurrence at a distant site.
The present disclosure also relates to a method of performing immunohistochemistry
(IHC) on a tumor sample obtained from a subject having early stage ER+/PR+ and Her2-
breast cancer or after removal of the breast cancer, comprising performing IHC on the
tumor sample to detect receptor expression by detecting whether cells are expressing at
least one receptor selected from the group consisting of estrogen receptor and
progesterone receptor and not having Her2 receptor and performing the method as above
on the tumor sample by IHC if receptor expression is detected to measure the expression
of the five biomarkers CD44, ABCC11, N-cadherin, Pan-cadherin and ABCC4.
Alternatively, the IHC is directly performed on samples known to be positive for estrogen
receptor and/or progesterone receptor and negative for Her-2-neu receptor. Thus, the
method of the present disclosure may comprise performing IHC to determine the status
of estrogen receptor, progesterone receptor and Her-2-neu receptor; or may not comprise
11
performing IHC to determine the status of estrogen receptor, progesterone receptor and
Her-2-neu receptor, if such status is already/previously known for a sample.
Thus, preferably, the present disclosure relates to a method of performing
immunohistochemistry (IHC) on a tumor sample obtained from a subject having early
stage ER+/PR+ and Her2- breast cancer or after removal of the breast cancer, comprising
performing IHC on the tumor sample to measure the expression of the five biomarkers
CD44, ABCC11, N-cadherin, Pan-cadherin and ABCC4.
The present disclosure also relates to a method of prognosing and treating a subject having
early stage ER+/PR+ and Her2- breast cancer or after removal of the breast cancer,
comprising prognosing whether the subject is at high or low risk for breast-cancer
recurrence by performing, and analyzing the results of, the method as above; and treating
the prognosed subject with chemotherapy if prognosed with high risk for breast-cancer
recurrence.
The breast cancer referred to in the present disclosure is stage I, II or IIIA hormone
receptor positive, Her2 receptor negative invasive ductal carcinoma, or invasive lobular
carcinoma of the breast.
In embodiments of the present disclosure, the breast-cancer recurrence is prognosed in
patients having early stage ER+/PR+ and Her2- breast cancer or in patients after surgical
removal of the early stage ER+/PR+ and Her2- breast cancer.
The breast cancer recurrence risk in the present disclosure is measured by analyzing the
expression of the five biomarkers along with three clinical parameters only if expression
of at least one of estrogen receptor or progesterone receptor is detected on the tumor cells
which also do not express Her2 receptor. The expression of the five biomarkers is
thereafter measured on basis of percentage of cells (0-100) stained and staining intensity
of cells (0-3).
12
The CanAssist-Breast test further comprises assessing three clinical parameters viz node
status, tumor grade, and tumor size, wherein the node status includes tumor node positive
and tumor node negative, the tumor grade includes Grades 1, 2 and 3 according to the
modified Bloom-Richardson-Elston criteria, and the tumor size includes T1, T2, or T3
according to the TNM classification of breast cancer.
The tumor sample obtained from the subject is collected, fixed, sectioned,and IHC is
performed by adding primary antibodies and secondary antibodies conjugated with a
detectable label and detecting a color or fluorescence from the detectable label.
The present disclosure also relates to a combination of antibodies specific for the five
biomarkers CD44, ABCC11, N-cadherin, Pan-cadherin and ABCC4.
The present disclosure also relates to a kit comprising the combination of antibodies and
instructions for measuring expression of the five biomarkers CD44, ABCC11, Ncadherin,
Pan-cadherin and ABCC4.
In embodiments of the present disclosure, the kit also comprises at least one member from
IHC reagents such as xylene, isopropanol, ethanol, buffer solutions, protein blocking
agents, primary antibodies, secondary antibodies labeled with enzymes such as
horseradish peroxidase (HRP) or alkaline phosphatase (AP) or with fluorescent tags such
as fluorescein isothiocyanate (FITC) or phycoerythrin (PE) or with molecule
biotin/streptavidin, and substrates such as diamino benzydene (DAB) or p-nitrophenyl
phosphate (PNPP).
The present disclosure also relates to an IHC-based assay system, comprising the
combination of antibodies specific for CD44, ABCC11, N-cadherin, Pan-cadherin, and
ABCC4.
In embodiments of the present disclosure, the IHC-based assay system further comprises
at least one member from IHC reagents such as xylene, isopropanol, ethanol, buffer
13
solutions, protein blocking agents, primary antibodies, secondary antibodies labeled with
enzymes such as horseradish peroxidase (HRP) or alkaline phosphatase (AP) or with
fluorescent tags such as fluorescein isothiocyanate (FITC) or phycoerythrin (PE) or with
molecule biotin/streptavidin, and substrates such as diamino benzydene (DAB) or pnitrophenyl
phosphate (PNPP).
The present disclosure also relates to a method of predicting the likelihood of recurrence
of breast cancer in a subject having early stage ER+/PR+ and Her2- breast cancer or after
removal of the breast cancer, comprising: measuring expression level of five biomarkers
CD44, ABCC11, N-cadherin, Pan-cadherin and ABCC4; calculating a relapse score for
said subject by measuring the differential expression levels of each of the biomarkers and
their contribution to breast cancer recurrence along with three clinical parameters; and
using said relapse score to determine the likelihood of breast cancer recurrence.
In embodiments of the present disclosure, the method further comprises assessing clinical
parameters node status, tumor grade, and tumor size, wherein the node status includes
tumor node positive and tumor node negative, the tumor grade includes Grades 1, 2 and
3, and the tumor size includes T1, T2, or T3.
In further embodiments of the present disclosure, the breast cancer is stage I, II or IIIA
hormone receptor positive, Her-2-neu receptor negative invasive ductal carcinoma, or
invasive lobular carcinoma of the breast. Accordingly, the term ‘early stage’ used to
describe the stage of breast cancer of the present disclosure refers to breast cancer at stage
I, II or IIIA.
The method of the present disclosure thus relates to a cost-effective, accurate, robust,
prognostic and predictive, ISO-certified, Morphometric IHC test, which is useful for
ER+/PR+ and Her2- breast cancer patients across Stages I, II and IIIA of cancer.
The method of the present disclosure is a ‘New’ biomarker/ ‘New’ biomarker combination
leading to ‘broadly distributed prognostic and predictive’ test which can assess ‘risk of
14
recurrence’ and can offer ‘targeted’ treatments to pre or post-menopausal patients, ER+ve
and/or PR+ve, Her2 –ve, and Lymph node –ve or +ve patients in stage I, stage II or stage
IIIA cancer.
The present disclosure therefore relates to a method of prognosing early stage ER+/PR+
and Her2- breast cancer patients for risk of breast cancer recurrence. The risk of
recurrence is considered high if the probability of recurrence is greater than about 9% and
low if it is less than or equal to about 9%.
In a non-limiting embodiment, the method of prognosis involves employing a
morphometric IHC based assay to predict the risk of breast cancer recurrence.
In a preferred embodiment, the breast cancer patients are patients, with I, II or IIIA stage
hormone receptor positive, Her2 negative invasive ductal or lobular carcinoma of the
breast.
In another preferred embodiment, hormone receptor positive includes ER+ (Estrogen
receptor positive) and/or PR + (Progesterone receptor positive).
In preferred embodiments of the present disclosure, the IHC based assay quantifies
expression of a 5 biomarker combination in the early stage ER+/PR+ and Her2- breast
cancer patient sample. Said biomarker expression is considered along with 3 specific
clinical prognostic factors/clinicopathological parameters by employing histopathological
analysis, to classify patients into high risk or low risk of recurrence based on a CanAssist
Breast score which is devised by a statistical algorithm (Figure 2).
In preferred embodiments of the present disclosure, the clinical prognostic factors are
selected from a group comprising, but not limiting to tumor size, node status and tumor
grade, which are conventionally known and well understood by a person skilled in the art.
15
In an exemplary embodiment, the node status includes tumor node positive and tumor
node negative including N0 and N1, and tumor grade includes Grades 1, 2 and 3 according
to the Bloom-Richardson-Elston criteria and tumor size as T1, T2, or T3 according to the
TNM classification of Breast Cancer.
In further preferred embodiments of the present disclosure, the CanAssist Breast score or
relapse score is an estimate of the patient’s risk of recurrence that is based on combined
expression of the marker combination along with the clinical factors, which is computed
based on a machine-learning statistical algorithm or module. The algorithm or composite
module of the present disclosure relates to a machine learning based risk classifier which
stratifies patients as low or high risk of recurrence within 5 years from diagnosis. The
relapse score is computed on a scale of 1-100 and scores of 15.5 and below are classified
as low risk, and above 15.5 are classified as high risk.
The CanAssist-Breast algorithm/algorithm of the present disclosure is
constructed/developed in a series of steps which are defined as follows:
1) Formulation of the problem,
2) Pre-processing of raw data,
3) Classifier selection and assessment, and
4) Selection of the final 5 biomarkers and 3 clinical parameters.
In embodiments of the present disclosure, the algorithm development includes:
Formulation of the problem-
The problem of predicting breast cancer recurrence within five years (prognostic problem)
is formulated as a binary classification problem with two outcomes, Recurrence and No
Recurrence. The goal is to develop a classifier which computes a relapse score (also called
CanAssist-Breast score) (a number between 0 and 100) which corresponds to five-year
probability of recurrence for the given patient. State-of-the-art machine learning
methodology is used to solve this problem.
Pre-processing of raw data:
16
The raw training set data consists of about 298 samples which are represented by 57 IHC
measurements or features (where each feature corresponds to a marker, and its staining
pattern which is evaluated based on location, intensity and % of staining) and clinical
variables/parameters/clinical prognostic factors (grade, age and TNM status). The dataset
contains about 68 recurred samples (breast cancer recurrence within five years) and about
230 non-recurred samples (breast cancer recurrence-free within five years). The raw data
is pre-processed as follows:
- TNM codes are converted into three discrete variables (size, number of nodes, metastasis
status)
- the discrete variables (TNM and grade) are one-hot encoded
- all variables are centered-scaled to zero mean and unit standard deviation
Subsequently, classifier assessment proceeds as per the well-defined statistical learning
paradigm involving cross-validation for the assessment of statistical predictions.
A representative set of the complete training set is provided herewith by way of table no.
3 (cumulative of tables 3A, 3B, 3C and 3D), wherein IHC measurements are provided for
160 data samples.
Classifier Selection and Assessment:
The classifier selection considers two types of parameters: internal ("weights") and
external ("hyperparameters"). The external hyperparameters that are selected include cost,
number of variables used in the classifier, and decision threshold. The internal parameters
are selected using core classifier algorithms such as Support Vector Machine, Random
Forest and Deep Learning. External hyperparameter selection is more complex, and
includes an extensive grid search over a plausible set of values of the hyperparameters.
For each fixed hyperparameter n-tuple, the performance of the classifier is computed
using repeated cross-validation.
The following classifiers are evaluated: 1) Support Vector Machine (SVM) with linear
and Radial Basis Function (RBF) kernel 2) Random Forest 3) ElasticNet [ESL] 4) multilayer
perceptron 5) normal mixture modeling.
17
Model selection is performed using Receiver Operator Graph (ROG) whereby each
classifier is represented by its sensitivity and specificity. The principal model selection
criterion is maximum sensitivity among classifiers with greater than 90% specificity. The
ROG displays a potentially large family of classifiers which cover the full grid of external
parameters, and the statistics are computed using cross-validation. The RBF SVM proved
superior by the selected criteria (sensitivity and specificity) and is therefore selected for
model assessment. This problem is also evaluated as a survival problem, whereby the goal
is to estimate actual probability of survival at various time points for a given patient. A
regularized Cox model is used in this approach. However, as this approach proved inferior
to the performance achieved by the classifiers, it is not carried forward.
Selection of the final 5 biomarkers:
The training set is evaluated for a total of 41 biomarkers (listed in table 1 below) most
commonly associated in the literature with the type of breast cancer provided by the
present disclosure. However, no meaningful relevance was observed for 24 biomarkers
with respect to their ability in being prognostic in nature. The data for the remaining 17
markers was analyzed more closely for the training set, and based on the interplay of
markers, and their relevance in correlation with the prognosis of breast cancer, five
distinct IHC markers are chosen to be part of the algorithm along with 3 clinical
parameters/clinical prognostic factors. To select the markers, the markers are ranked by
the absolute value of Pearson correlation coefficient between the marker value and the
outcome (Recurrence/No Recurrence). This method is chosen, as no other marker
selection algorithm matches or surpasses its performance. Each IHC marker corresponds
to multiple IHC features (numeric values), since the protein levels are evaluated in the
cell nucleus, cytoplasm and/or membrane. The marker selection process is integrated
inside the cross-validation loop to avoid selection bias. The top 5 markers chosen from
this list are as follows: CD44, Pan-Cadherin, N-Cadherin, ABCC11 and ABCC4.
18
Marker
Code Biomarkers
A CD44
B CD24
C ABCG2
D ALDH1A1
E ESA
F ABCC4
G CD133
H NANOG
I Oct3/4
J SOX2
K APC
L P-CADHERIN
M HIF2A
N THY1
O p-B-CATENIN
P FOXA1
Q CD15
R ABCC11
S1 NESTIN
T Ki67
U N-CADHERIN
V E-CADHERIN
W Pan-CADHERIN
X Klf4
Y Alpha v beta 3
Z Klk6
ZA Total b-cat
ZB MAGE-A9
ZC MAGE-A11
ZD CxCR4
ZE IFITM1
ZF Nrf2
ZF p-Nrf2
ZG CD147
19
ZH XBP1
ZI Notch 1
ZJ DLL-3
ZK GATA3
ZL HSP 70
ZM Integrin beta 6
ZN MDR1
Table 1
Accurate risk stratification using the method of the present disclosure enables a physician
to design an optimal therapeutic strategy for patients, i.e., patients who are stratified as
low risk can be spared of chemotherapy and patients who are stratified as high risk to
require adjuvant chemotherapy. The method of the present disclosure when compared to
prior art prognostic tests is capable of separating high and low risk patients well, without
having any intermediate risk category of patients, thus being accurate and capable of
predicting all patients into either high/low risk of recurrence (Figure 3).
The markers employed in the IHC based assay of the present disclosure function in
molecular pathways other than hormone receptor regulation and proliferation. Said
molecular pathways are involved in initiation and progression of breast cancer including
apoptosis, self-renewal, angiogenesis, hypoxia, and drug resistance etc. Therefore,
antigens towards these markers in the instant disclosure are from said molecular pathways
and which are localized to different parts of a cell, not limiting to cell membrane.
However, these markers have not been used in the art till date for breast cancer disease
prognosis and recurrence prediction.
The IHC based assay of the present disclosure is developed and validated on both tumor
node negative and tumor node positive patients from tumor stage ranging from Stage I to
Stage IIIA and hence is a broad spectrum assay. The IHC based assay of the present
disclosure is developed and validated on Hormone receptor positive (ER+ and/or PR+) and
Her2 -ve samples. In other words, the IHC based assay of the present disclosure is
20
applicable for patient samples where at least one of or both of ER and PR are +ve; and
Her2 is -ve.
The method of identifying unique biomarker combination and employing the same for
possible prognosis of breast cancer recurrence has been devised through rigorous
experimentation and research and carefully studying the data sets over a period of more
than 5 years, which involved biomarker selection and analysis in training samples set by
employing IHC assay for biomarker combination shortlisting; testing for presence of
shortlisted biomarker combination and clinicopathological parameters; followed by
clinical validation of test-samples for predicting breast cancer recurrence.
The training sample set of the present disclosure relates to patient tumor samples of 298
patients, which satisfied the inclusion criteria of the present disclosure and includes
samples of women having invasive ductal or lobular carcinoma of the breast; tumor stage
I, II and III, and Age of the patient being preferably below 74 years; Positive for
expression of hormone receptor ER and/or PR; Negative for expression of Her2 status;
having minimum of 5 years follow-up and known clinical outcome data. This training
sample set in the present disclosure is analyzed by IHC for ~41 gene-biomarker
expression, most commonly associated with breast cancer in the literature. Based on the
relevance of the markers in prognosis of breast cancer vis-à-vis the patient outcome, 17
markers were shortlisted for final screening and analysis. Each biomarker is graded
quantitatively and this data along with three clinical parameters/clinical prognostic factors
viz tumor size, tumor grade and node status is analyzed by the Statistician using Support
Vector Machine based model. 5 biomarkers are shortlisted for their consistent prognostic
ability and an algorithm is developed using staining intensity (0-3) and percentage of
staining (0-100) of said 5 biomarkers in conjunction with three clinical prognostic
parameters. The algorithm developed gives a certain score ranging from 1-100 to each
patient’s tumor sample, and if the score is under the cutoff point then the patient has a low
probability of distant recurrence in the first 5 years and if the score is higher than the cutoff
point, then the patient has a higher probability of distant recurrence.
21
The predictive ability of the 5 biomarker plus 3 clinical parameter combination arrived at
from the training sample is further validated with a validation sample set. The validation
sample set comprises patient tumor samples of around 700 patients, which again satisfy
the inclusion criteria as aforementioned except that tumor stage was restricted until Stage
3A. The validation sample of the present disclosure is analyzed by IHC for the shortlisted
5 biomarker combination and information on percentage staining and biomarker staining
intensity in conjunction with 3 clinical prognostic parameters is assessed with the
statistical algorithm, for risk stratification. Results from the said analysis are compared
with the known outcomes for each sample (from the patient history provided for each of
the patients in validation set) to determine the accuracy of the 5 biomarker plus 3 clinical
parameter combination. A representative set of the complete validation set is provided
herewith by way of table no. 4, wherein IHC measurements are provided for 299 data
samples.
In some embodiments of the present disclosure, the biomarker selection and analysis by
IHC assay involves shortlisting about 17 proteins belonging to different molecular
pathways involved in initiation and progression of breast cancer including apoptosis, selfrenewal,
angiogenesis, hypoxia and drug-resistance. Analyzing expression of these 17
genes in patients samples (Training set for 298 patient samples: having minimum 5 year
follow-up data and known clinical outcome) is done by employing IHC, wherein the IHC
assay involves analysis by observing the patient sample slides and scoring/grading of the
samples based on percentage tumor stained and staining intensity for all the about 17
genes and further analyzing the scoring/grading data using known statistical tools and
shortlisting most significant biomarker combination of 5 genes of the present disclosure.
The 5 biomarkers analyzed by the IHC based assay of the present disclosure are CD44,
ABCC11, N-cadherin, Pan-cadherin and ABCC4.
In a preferred embodiment, the selection/inclusion criteria for the training patient set
samples involve selection of samples which are positive for hormone receptor status ER
and/or PR; negative for Her2 status; and positive or negative for tumor node status; and
tumor stage ranging from stage I, II, or IIIA.
22
In exemplary embodiments of the present disclosure, the statistical tools involve,
multivariate analysis, Cox regression, Lasso and SVM models.
The present disclosure thus relates to a prognostic test which analyses the expression of a
combination of five markers CD44, ABCC11, N-cadherin, Pan-cadherin and ABCC4 for
detecting cancer recurrence in early stage ER+/PR+ and Her2- breast cancer as well as a
predictive test which will enable devising of new drugs or personalized therapy
specifically based on the prognostic outcome of the patient.
The present disclosure thus provides a tool for analyzing expression of 5 markers, i.e.,
CD44, ABCC11, N-cadherin, Pan-cadherin and ABCC4 and three clinical parameters
thereby helping in stratifying the early stage ER+/PR+ and Her2- breast cancer patients
into low or high risk of breast cancer recurrence.
In a non-limiting embodiment, the risk of recurrence is high if the CanAssist Breast score
is higher than designated cut off point or low if the CanAssist Breast score is less than the
designated cut off point.
In a preferred embodiments of the present disclosure, the testing of validation samples is
done by employing the composite module or algorithm which takes into consideration
and computes the expression of 5 most relevant biomarker combination data (which is
based on the IHC analysis data of the training-set) in combination with the 3
clinicopathological parameters/clinical prognostic factors, for risk stratification of
samples. The clinical validation of samples for breast cancer prognosis involves
employing about 700 test samples of breast cancer in a retrospective study with the same
inclusion criteria as for the training set and analyzing the test samples by the composite
algorithm/module of the instant disclosure, which enables predicting the risk of distant
recurrence in patients with early stage ER+/PR+ and Her2- stage breast cancer and
thereby validating by comparing the same with actual outcomes of the respective patient
history data.
23
In preferred embodiments of the present disclosure, manual grading is carried out before
inputting the data in the statistical algorithm or module and the grading comprises of
assessing the percentage of staining along with the intensity of staining of each of the
biomarkers. The statistical algorithm or module employed in the present disclosure
considers the grading values of % of staining and intensity of staining and clinical
prognostic parameters of a sample and arrives at a predictive score.
In preferred embodiments of the present disclosure, the method of prognosis of the instant
disclosure provides significant prognosis of breast cancer risk recurrence in terms of
providing NPV (negative predictive value) of about 95%, when compared with the patient
history data available.
In an exemplary embodiment, the validation study module of the instant disclosure is
depicted in the flow-chart as below:
Conducting model retrospective validation study on additional 700 breast
cancer patients (Stages I-IIIA).
IHC is performed on the tumor blocks for selected 5markers.
‘CanAssist Breast relapse score’ is calculated based on the IHC results and 3 clinical
parameters using the composite algorithm/Breast model equation.
Predicted ‘risk of recurrence’/results are compared with actual outcomes confirming the
prediction.
The present disclosure, further relates to a method of identifying expression of biological
marker combination on cells in a biological sample being or suspected of being a early
stage tumor/ cancer, said method comprising acts of:
24
a) collecting, fixing, sectioning and treating the biological sample (which
fulfills all the inclusion criteria as provided by the instant invention) with
IHC reagents, followed by antigen retrieval using predetermined buffer
solution and heat and adding primary antibody against ER/PR and/or Her-
2-neu receptor (in case the ER/PR/Her-2-neu status is not previously
known) followed by antibodies against biological marker combination of
CD44, ABCC11, N-cadherin, Pan-cadherin and ABCC4; and
b) adding secondary antibody conjugated with an enzyme and reagents for
obtaining a colored reaction (i.e: substrate specific to the enzyme) or
fluorescence for identifying positive expression of the hormonal receptors
ER and/or PR; and biological marker combination.
Preferably, the present disclosure relates to a method of identifying expression of
biological marker combination on cells in a biological sample, having one or both of ER
and PR positive, and Her-2-neu negative, being or suspected of being a tumor/ cancer,
said method comprising acts of:
a) collecting, fixing, sectioning and treating the biological sample (which
fulfills all the inclusion criteria as provided by the instant invention) with
IHC reagents, followed by antigen retrieval using predetermined buffer
solution and heat and adding antibodies against biological marker
combination of CD44, ABCC11, N-cadherin, Pan-cadherin and ABCC4;
and
b) adding secondary antibody conjugated with an enzyme and reagents for
obtaining a colored reaction (i.e: substrate specific to the enzyme) or
fluorescence for identifying expression of the biological marker
combination.
The biological samples are first checked for presence of ER and/or PR receptors and for
absence of Her-2-neu receptors by IHC assay and only if the samples match the inclusion
criteria of the instant invention and presence of ER+ and/or PR+ receptor status is
confirmed (along with absence of Her-2-neu), said samples are considered for biomarker
25
combination expression using IHC assay. The samples are not tested for the 5 biomarker
combination of the instant invention, if the hormone receptor status is found negative for
both ER and PR expression. Alternatively, if the status of ER/PR/Her-2-neu receptors on
the samples are already/previously known, the samples can directly be tested for the 5
biomarker combination of the present disclosure.
In another non-limiting embodiment of the present disclosure, the collecting, fixing,
sectioning and treating is carried out under predetermined conditions by conventional
immunohistochemistry technique. All possible ways and means to carry out
immunohistochemistry known to a person skilled in the art is envisaged by the method
and assay of the present disclosure.
In another non-limiting embodiment of the present disclosure, the samples are subjected
to H&E (Haemotoxylin and Eosin) staining to measure the tumor content before the IHC
assay of the present disclosure is performed.
In exemplary embodiments of the present disclosure, the process of identifying the
biomarker in patient samples is carried out by collecting the tumor sample from the
patients having inclusion criteria mentioned above. The sample is processed using
formalin and other organic solvents and then embedded in paraffin to make a block; and
the block is sectioned on a slide. The slide is then passed through a series of organic
solvents to rehydrate the tumor. Thereafter, antigen retrieval is carried out under
controlled experimental conditions, using specific buffer solutions and heat by Multiple
Epitope Retrieval System (MERS) or water bath at 90°C. This is followed by cooling the
section on the slide to room temperature (RT), followed by adding a blocking agent. This
is followed by adding of primary antibody against the antigen of interest; which is in turn
followed by addition of secondary antibody conjugated with an enzyme. Finally,
appropriate reagents are added for obtaining a coloured reaction. The staining of the
section is observed for identification of percentage of staining and intensity of staining,
optionally along with location of staining for biomarker expression in patient
tumor/cancer samples. Thus, IHC reagents employed in the present disclosure include at
26
least one of xylene, isopropanol, ethanol, buffer solutions, protein blocking agents,
primary antibodies, secondary antibodies labeled with enzymes such as horseradish
peroxidase (HRP) or alkaline phosphatase (AP) or with fluorescent tags such as
fluorescein isothiocyanate (FITC) or phycoerythrin (PE) or with molecule
biotin/streptavidin, and substrates such as diamino benzydene (DAB) or p-nitrophenyl
phosphate (PNPP).
Once the biological sample was identified for the ER/PR and/or Her-2-neu and found
positive for ER and/or PR and negative for Her-2-neu by employing IHC assay, such
samples were identified for 5 biomarker combination expression and scored for
percentage of tumors stained (0-100) and staining intensity (0-3) again by using IHC
assay. Alternatively, if the biological sample is already known to be ER+ and/or PR+ and
Her-2-neu -ve, IHC assay is directly performed to identify expression of 5 biomarker
combination of the present disclosure. Manual grading is carried out before inputting the
data in the statistical algorithm or module and the grading comprises computing the
percentage of staining along with the intensity of staining of each of the biomarkers. This
IHC based data was analyzed using various statistical tools and information obtained
therefrom was fed into a statistical algorithm along with 3 clinicopathological parameters
data, for breast cancer risk stratification.
The present disclosure further relates to a method of prognosis of a subject having cancer
or suspected of having early stage ER+/PR+ and Her2- breast cancer for cancer
recurrence, said method comprising acts of:
a) collecting biological sample from the subject having positive expression
of at least one receptor selected from a group comprising estrogen receptor
and progesterone receptor and absence of Her-2-neu receptor in cells of
the sample;
b) carrying out IHC analysis on the cells of step (a) with antibody against
biological marker combination of CD44, ABCC11, N-cadherin, Pancadherin
and ABCC4;
27
c) identifying expression of the biological marker combination along with
clinical prognostic parameters; and
d) inputting this information in a statistical algorithm or module for
predicting the prognosis of the subject (Figure 4).
In embodiments of the present disclosure, the immunohistochemistry analysis is carried
out by conventional method and wherein the identification of markers is carried out by
visualizing a colored reaction or fluorescence obtained at completion of the method due
to staining of the cells from the sample.
In further embodiments of the present disclosure, parameters employed for analyzing
expression of biomarkers are selected from a group comprising percentage of staining,
intensity of staining and optionally location of staining or any combination thereof; and
wherein the location of the staining is selected from a group comprising cell membrane,
cytoplasm and nucleus or any combination thereof.
In further embodiments of the present disclosure, manual grading is carried out before
inputting the data in a statistical algorithm or module and the grading comprises
computing the percentage of staining along with the intensity of staining of each of the
biomarkers. The statistical algorithm or module employed in the present disclosure
considers the grading scores and clinical prognostic parameters of a sample and arrives at
a predictive score, in order to predict the prognosis as being high risk or low risk for breast
cancer recurrence. The statistical algorithm or module computes the prediction score by
taking into account the expression data of the 5 biomarker combination and clinical
prognostic parameters (which is devised from the retrospective study on training set and
respective patient history data).
Thus, the present disclosure provides an assay or a tool for prognosis of breast cancer
recurrence by studying and analyzing expression of a specific combination of biomarkers
along with clinical prognostic parameters. The assay is performed by carrying out an
immunohistochemistry based method which helps in studying and analyzing said
28
expression of biomarkers. Initially, as an optional step, the assay or method provides for
identification of estrogen receptor and/or progesterone receptor, and proceeds to the next
step only if one or both of the receptors are present. However, if the information on status
of estrogen receptor and/or progesterone receptor is already known for a sample/patient,
this step is not performed. In the next step, the assay provides for identification of
expression of biomarkers CD44, ABCC11, N-cadherin, Pan-cadherin and ABCC4;
preferably 5 biomarker combination of CD44, ABCC11, N-cadherin, Pan-cadherin and
ABCC4, followed by grading the expression on the basis of parameters including
percentage of staining and intensity of staining. The data from said grading along with
status of clinical prognostic parameters is inputted into a proprietary statistical algorithm
or module for obtaining a prediction score. The proprietary statistical algorithm or module
is built on the basis of data obtained by retrospective study of 298 patient samples
(training set) and comprises of expression data pertaining to each of the 5 biomarkers and
clinical prognostic parameters. Since the retrospective study takes into consideration the
patient history of all the samples, the expression data is calculated by the algorithm or
module accordingly and a relapse score is computed. The relapse score is computed on a
scale of 1-100 and scores of 15.5 and below are classified as low risk, and above 15.5 are
classified as high risk. Lastly, the prediction score stratifies the respective patient into low
risk or high risk category, based on the prediction of the prognosis of the subject. The
patients are classified as high risk if the patient is expected to have >9% probability of
distant recurrence within five years of initial treatment of breast cancer. Similarly, the
patients are classified as low risk if the patient is expected to have 9% or lower probability
of distant recurrence of breast tumor within five years of initial treatment of breast cancer.
The cutoff of 9% risk of recurrence for 'low risk' patients is line with population
recurrence rate of 10% and is lower than what similar prognostic tests offer for their low
risk categories. As it can be understood lower the cutoff for the low risk category, higher
would be usefulness of the same.
The present disclosure also relates to marker(s) employed in the method of prognosis of
the instant disclosure, which are CD44, ABCC11, N-cadherin, Pan-cadherin and ABCC4,.
29
In preferred embodiments of the present disclosure, the marker acts as a bio-marker for
prognosis of cancer recurrence in patients having breast cancer or suspected of having
breast cancer.
In preferred embodiments of the present disclosure, the marker(s) of the instant disclosure
are employed in combination for breast cancer prognosis.
The present disclosure also relates to method of using the markers of the present
disclosure for prognosis of breast cancer in samples of patients having breast cancer or
suspected of having breast cancer.
The present disclosure also relates to a kit for prognosis of a subject having early stage
ER+/PR+ and Her2- breast cancer or suspected of having early stage ER+/PR+ and Her2-
breast cancer, said kit comprising antibody against biological markers CD44, ABCC11,
N-cadherin, Pan-cadherin and ABCC4, optionally along with at least one member from
IHC reagents such as xylene, isopropanol, ethanol, buffer solutions, protein blocking
agents, primary antibodies, secondary antibodies labeled with enzymes such as
horseradish peroxidase (HRP) or alkaline phosphatase (AP) or with fluorescent tags such
as fluorescein isothiocyanate (FITC) or phycoerythrin (PE) or with molecule
biotin/streptavidin, and substrates such as diamino benzydene (DAB) or p-nitrophenyl
phosphate (PNPP), for performing immunohistochemistry and instruction manual.
A more complete understanding can be obtained by reference to the following specific
examples, which are provided for purposes of illustration only and are not intended to
limit the scope of the disclosure.
EXAMPLES
Biomarkers & antibodies
The biomarkers and antibodies against said biomarker being used in the instant disclosure
are selected from one or more of the following:
30
CD44; ABCC11; N-cadherin; Pan-cadherin; ABCC4. These antibodies are purchased
from reputed Antibody manufacturers including Thermo Scientific, Novus, Abnova
Spring, and AbCam.
The organic solvents, reagents (buffer, detergent etc), secondary antibody and enzymes
(substrate specific to the enzyme), mentioned in the detailed protocol below are only for
the purposes of illustration and should not be construed to be limiting in nature. The
instant disclosure envisages and encompasses all possible combinations and alternatives
of such solvents, reagents/substrates, secondary antibody and enzymes known and
commonly used by a person skilled in the art.
Furthermore, the abbreviations referred in the instant disclosure have the following
meaning (table no. 2):
Column Name Description
Outcome 1- Good Outcome/ No recurrence
0- Bad Outcome/Recurrence
Sample No. Unique patient code
TNM
T- Tumor size (0-n)
N- Node status (0-n)
M- Metastases
(present - 1, absent - 0, unknown - x)
Stage Stage of disease based on TNM at the time of diagnosis
Age Age of patient at the time of diagnosis
ER Percentage expression of Estrogen Receptor in Nucleus of tumor cells
ER-I Intensity of marker Estrogen Receptor expression in Nucleus of tumor cells
PR Percentage expression of Progesterone Receptor in Nucleus of tumor cells
PR-I Intensity of marker Progesterone Receptor expression in Nucleus of tumor
cells
31
Her-2
Her-2-neu Status
0-Negative
1-Positive
A%-M Percentage expression of marker A on Membrane of tumor cells
AI-M Intensity of marker A expression on Membrane of tumor cells
C%-C Percentage expression of marker C in Cytoplasm of tumor cells
CI-C Intensity of marker C expression in Cytoplasm of tumor cells
C%-N Percentage expression of marker C in Nucleus of tumor cells
CI-N Intensity of marker C expression Nucleus of tumor cells
F%-M Percentage expression of marker F on Membrane of tumor cells
FI-M Intensity of marker F expression on Membrane of tumor cells
F%-C Percentage expression of marker F in Cytoplasm of tumor cells
FI-C Intensity of marker F expression in Cytoplasm of tumor cells
K%-M Percentage expression of marker K on Membrane of tumor cells
KI-M Intensity of marker K expression on Membrane of tumor cells
K%-C Percentage expression of marker K in Cytoplasm of tumor cells
KI-C Intensity of marker K expression in Cytoplasm of tumor cells
M%-C Percentage expression of marker M in Cytoplasm of tumor cells
MI-C Intensity of marker M expression in Cytoplasm of tumor cells
N%-C Percentage expression of marker N in Cytoplasm of tumor cells
NI-C Intensity of marker N expression in Cytoplasm of tumor cells
O%-M Percentage expression of marker O on Membrane of tumor cells
32
OI-M Intensity of marker O expression on Membrane of tumor cells
P%-N Percentage expression of marker P in Nucleus of tumor cells
PI-N Intensity of marker P expression Nucleus of tumor cells
R%-M Percentage expression of marker R on Membrane of tumor cells
RI-M Intensity of marker R expression on Membrane of tumor cells
R%-C Percentage expression of marker R in Cytoplasm of tumor cells
RI-C Intensity of marker R expression in Cytoplasm of tumor cells
U%-M Percentage expression of marker U on Membrane of tumor cells
UI-M Intensity of marker U expression on Membrane of tumor cells
U%-C Percentage expression of marker U in Cytoplasm of tumor cells
UI-C Intensity of marker U expression in Cytoplasm of tumor cells
V%-M Percentage expression of marker V on Membrane of tumor cells
VI-M Intensity of marker V expression on Membrane of tumor cells
V%-C Percentage expression of marker V in Cytoplasm of tumor cells
VI-C Intensity of marker V expression in Cytoplasm of tumor cells
W%-M Percentage expression of marker W on Membrane of tumor cells
WI-M Intensity of marker W expression on Membrane of tumor cells
W%-C Percentage expression of marker W in Cytoplasm of tumor cells
WI-C Intensity of marker W expression in Cytoplasm of tumor cells
ZA1%-M Percentage expression of marker ZA1 on Membrane of tumor cells
ZA1I-M Intensity of marker ZA1 expression on Membrane of tumor cells
33
ZA1%-C Percentage expression of marker ZA1 in Cytoplasm of tumor cells
ZA1I-C Intensity of marker ZA1 expression in Cytoplasm of tumor cells
ZB%-C Percentage expression of marker ZB in Cytoplasm of tumor cells
ZBI-C Intensity of marker ZB expression in Cytoplasm of tumor cells
ZC%-M Percentage expression of marker ZC on Membrane of tumor cells
ZCI-M Intensity of marker ZC expression on Membrane of tumor cells
ZC%-C Percentage expression of marker ZC in Cytoplasm of tumor cells
ZCI-C Intensity of marker ZC expression in Cytoplasm of tumor cells
ZD%-C Percentage expression of marker ZD in Cytoplasm of tumor cells
ZDI-C Intensity of marker ZD expression in Cytoplasm of tumor cells
ZF2%-N Percentage expression of marker ZF2 in Nucleus of tumor cells
ZF2I-N Intensity of marker ZF2 expression Nucleus of tumor cells
Date of Diagnosis Date of diagnosis of breast cancer
Date of Last follow
up Date of latest visit of patient at the hospital
Date of Death Date of Death of patient due to disease
Current Status
Current Status
0 - Dead
1 - Alive
Status Unknown Current status is unknown
DFS (in months) DFS: Disease Free Survival (in months)
TTP 1st Recurrence
(in months) TTP: Time to Tumor Progression 1st Recurrence (in months)
Date of 1st
Recurrence Date of diagnosis of 1st recurrence
Location of 1st
Recurrence Site of 1st recurrence Eg: Lung, Liver, brain
34
TTP 2nd Recurrence
(in months) TTP: Time to Tumor Progression 2nd Recurrence (in months)
Date of 2nd
Recurrence Date of diagnosis of 2nd recurrence
Location of 2nd
Recurrence Site of 2nd recurrence Eg: Lung, Liver, brain
TTP 3rd Recurrence
(in months) TTP: Time to Tumor Progression 3rd Recurrence (in months)
Date of 3rd
Recurrence Date of diagnosis of 3rd recurrence
Location of 3rd
Recurrence Site of 3rd recurrence Eg: Lung, Liver, brain
TTP 4th Recurrence
(in months) TTP: Time to Tumor Progression 4th Recurrence (in months)
Date of 4th
Recurrence Date of diagnosis of 4th recurrence
Location of 4th
Recurrence Site of 4th recurrence Eg: Lung, Liver, brain
Table 2
Examples 1: Identification and development of 5 biomarker combination of the
present disclosure
Sample selection and H&E Staining
All studies are performed with approval of the Institutional Review Board and Ethics
Committees of the Hospitals participating in the study. Informed consent is waived
according to Indian Council of Medical Research (ICMR) guidelines since the study is
retrospective, observational, non-interventional and anonymized. For the present study,
women with Stage I, II and III Invasive Ductal Carcinoma (IDC) of the Breast, Aged <74,
ER+ve and/or PR+ve, Her2-ve, with minimum 5-year follow up and known clinical
outcome are considered. Majority of the patients are in Stage II. All patient samples are
stripped of personal identifiers, information is collected on age and calendar year of
diagnosis, surgery, tumor (size, grade, histologic type, and ER status), nodal status,
radiation treatment, hormonal therapy or chemotherapy, and clinical follow-up, including
local, loco-regional, or distant recurrences, second primary malignancies, and death or
35
date of last visit. Primary breast tumor surgical samples less than 15 years old (from either
Modified Radical Mastectomy or Breast Conserving Surgery) are used.
H&E staining was carried out to ensure that samples mandatorily have a major
component of IDC, and have at least 30% tumor content. Samples with extensive necrosis,
crush artifacts, poorly processed tissue, only DCIS component and singly scattered cells
are excluded.
ER/PR staining is carried out for all samples, and only those samples that are ER+ and/or
PR+ are included.
IHC assay in the prognostic test of the instant disclosure and Development of the
CanAssist Breast algorithm
A total of 298 tumor samples having the inclusion criteria mentioned above are considered
for the IHC assay. The technique used for the identification of markers in a patient sample
in the present disclosure involves Immunohistochemistry (IHC).
The process of identifying the biomarker in patient samples is carried out using the
technique as below:
Tumor sample is collected from the patients identified above. The sample is processed
using formalin and other organic solvents and then embeded in paraffin to make a block
?
The block is sectioned on a slide
?
The slide is passed through a series of organic solvents to rehydrate the tumor
?
Antigen retrieval is carried out under controlled experimental conditions, using specific
buffer solution and heat, using Multiple Epitope Retrieval System (MERS) or Water
Bath at 90°C.
?
36
This is followed by cooling the section on the slide to room temperature (RT)
?
Followed by adding blocking agent
?
Followed by adding of primary antibody against the antigen of interest
?
Followed by adding of secondary antibody conjugated with an enzyme
?
Followed by adding of reagents for obtaining a coloured reaction
?
Observing staining for grading of % of staining and intensity of staining, optionally
along with location of staining for biomarker expression in patient tumor/cancer
samples.
The above process is carried out and then repeated for identifying expression of each of
the biomarkers in each sample. Initially, a total of 41 biomarkers most commonly
associated in the literature with breast cancer are considered for screening each sample.
However, no meaningful relevance was observed for 24 biomarkers with respect to their
ability of being prognostic in nature. The data from the remaining 17 markers was
analyzed in detail and used for the training set for determining critical biomarkers to be
used for the Statistical algorithm development.
The stained slides (slides containing tumor section from training set samples stained with
antibodies against each of the 17 markers) are thereafter analyzed manually by a
Pathologist for scoring/grading Each sample is scored for percentage of tumors stained
(0-100) and the staining intensity (0-3) for each of the markers. A representative set of the
complete training set is provided herewith by way of table no. 3 (cumulative of tables 3A,
3B, 3C and 3D) wherein IHC measurements for each of the 17 markers are provided for
160 data samples, along with consideration of clinical prognostic parameters (tumor size,
node status and tumor grade).
37
To select the most relevant prognostic markers, the markers are ranked by the absolute
value of Pearson correlation coefficient between the marker value and the outcome
(Recurrence/No Recurrence from the corresponding patient history). This method is
chosen, as no other marker selection algorithm matches or surpasses its performance.
Each IHC marker corresponds to multiple IHC features (numeric values), since the protein
and intensity levels are evaluated in the cell nucleus, cytoplasm and/or membrane. The
marker selection process is integrated inside the cross-validation loop to avoid selection
bias. Based on comparison of scoring against the patient history available for each of the
samples, a set of 5 most relevant biomarkers is selected for its ability of being prognostic
in nature. These markers are CD44, ABCC11, N-cadherin, Pan-cadherin and ABCC4.
38
Sl.
No.
Outcome TNM Grade Stage Age ER ERI
PR PRI
Her-
2
A%-
M
AIM
C%-
C
CIC
C%-
N
CIN
1 1 T1N0M0 2 1 43 80 2 60 3 0 22.5 2 52.5 1.5 52.5 1.75
2 1 T1N0M0 3 1 38 95 3 60 3 1 20 2 27.5 1.25 47.5 2
3 1 T1N0M0 2 1 53 90 3 60 2 0 12.5 0.5 27.5 1 12.5 0.5
4 1 T1N0M0 2 1 57 80 3 90 3 0 55 2 42.5 1.25 50 2
5 1 T1N0M0 3 1 47 70 3 25 3 0 12.5 0.5 47.5 1.5 12.5 0.5
6 1 T1N0M0 3 1 56 0 0 35 1 1 50 2 12.5 0.25 22.5 1
7 1 T1N0M0 2 1 43 95 3 80 3 1 57.5 2 22.5 0.75 50 2
8 1 T1N0M0 2 1 56 50 2 50 3 0 47.5 2 25 1 32.5 1.75
9 1 T1N0M0 2 1 73 80 2 90 3 0 25 1.25 35 1.25 35 1.5
10 1 T1N0M0 2 1 54 80 3 90 3 0 57.5 1.5 12.5 0.25 57.5 1.5
11 1 T1N0M0 1 1 53 80 2 90 3 0 50 2 37.5 0.75 22.5 1
12 1 T1cN0M0 2 1 49 90 3 90 3 0 52.5 1.5 12.5 0.25 47.5 1.5
13 1 T1cN0M0 1 1 41 83 3 80 3 0 47.5 1.75 12.5 0.25 25 1.25
14 1 T1N0M0 3 1 59 70 3 0 0 0 27.5 1.25 45 0.75 30 1.25
15 1 T1N0M0 2 1 51 80 2 50 2 0 12.5 0.5 52.5 1.75 12.5 0.5
16 1 T1N0M0 2 1 60 35 1.5 0 0 0 27.5 1.5 45 1.25 12.5 0.5
17 1 T1N0M0 3 1 57 75 2 50 2 0 12.5 0.5 37.5 1.5 12.5 0.5
18 1 T1N0M0 2 1 33 40 2 5 2 0 17.5 0.75 50 1.25 12.5 0.5
19 1 T1N0M0 1 1 47 75 2.5 90 3 0 22.5 1.5 52.5 1.5 12.5 0.5
20 1 T1N0M0 2 1 65 80 2 0 0 1 52.5 1.5 52.5 1.5 12.5 0.5
21 1 T1N0M0 2 1 37 20 1.5 70 2.5 0 17.5 1.5 25 1.25 12.5 0.5
22 1 T1N0M0 2 1 37 80 2 25 2 0 17.5 1.5 45 1.5 12.5 0.5
23 1 T1N0M0 2 1 35 75 2 70 2 0 27.5 1.5 50 1.25 12.5 0.5
24 1 T1N0M0 2 1 47 90 3 90 3 0 50 1.5 52.5 1.5 12.5 0.5
25 1 T2N0M0 2 2A 58 60 3 30 3 0 27.5 2 12.5 0.25 42.5 1.75
26 1 T2N0M0 2 2A 26 75 3 100 3 0 22.5 1.75 12.5 0.25 50 2
27 1 T2N0M0 3 2A 54 95 3 95 3 1 32.5 1.75 52.5 1.5 12.5 0.5
28 1 T2N0M0 3 2A 61 75 3 0 0 1 22.5 1 47.5 1.5 12.5 0.5
29 1 T2N0M0 3 2A 54 90 3 20 3 0 30 1.75 47.5 1 55 1.5
30 1 T2N0M0 2 2A 48 75 2 90 3 1 30 1.5 57.5 1.5 12.5 0.5
31 1 T2N0M0 2 2A 54 100 3 100 3 0 12.5 0.5 57.5 1 57.5 2
32 1 T2N0M0 2 2A 23 65 3 10 3 1 12.5 0.5 52.5 1 37.5 1.5
33 1 T2N0M0 3 2A 62 70 3 0 0 0 17.5 1.25 47.5 1.25 57.5 2
34 1 T2N0M0 3 2A 46 30 2 0 0 0 32.5 2 22.5 1 17.5 2
35 1 T2N0M0 3 2A 64 80 3 10 3 1 17.5 2 47.5 1.25 42.5 1.5
36 1 T2N0M0 2 2A 63 90 3 90 3 1 12.5 0.5 60 1.25 42.5 1.5
39
37 1 T2N0M0 3 2A 49 5 3 0 0 1 20 1.25 37.5 1.25 42.5 1.5
38 1 T2N0M0 3 2A 54 90 3 70 3 0 12.5 0.5 32.5 1.25 22.5 1
39 1 T2N0M0 2 2A 66 10 1.5 40 2 0 30 2 27.5 1 27.5 1.5
40 1 T2N0M0 2 2A 45 40 2 90 3 0 17.5 1.25 47.5 1.25 17.5 1.5
41 1 T2N0M0 3 2A 80 80 3 75 3 1 57.5 2 37.5 0.75 57.5 2
42 1 T2N0M0 3 2A 53 75 3 75 3 1 50 2 22.5 0.75 47.5 1.75
43 1 T2N0M0 2 2A 43 10 2 50 2 0 17.5 1.25 37.5 1.25 12.5 0.5
44 1 T2N0M0 2 2A 66 75 2 20 2 0 32.5 1.25 47.5 1.25 22.5 1.25
45 1 T2N0M0 1 2A 77 90 3 30 2.5 0 32.5 1.5 12.5 0.25 27.5 1.25
46 1 T2N0M0 1 2A 48 60 2.5 70 3 0 50 1.5 12.5 0.25 12.5 0.5
47 1 T2N0M0 2 2A 50 60 3 25 2 0 12.5 0.5 12.5 0.25 12.5 0.5
48 1 T2N0M0 2 2A 53 30 2 50 2 0 47.5 1.5 27.5 1 27.5 0.75
49 1 T2N0M0 2 2A 53 80 2.5 80 3 1 42.5 2 37.5 0.75 37.5 1
50 1 T2N0M0 2 2A 65 90 3 90 3 0 42.5 1 42.5 1.25 32.5 1.5
51 1 T2N0M0 2 2A 52 80 3 35 2 0 25 1 52.5 1.5 27.5 1
52 1 T2N0M0 2 2A 38 70 2 0 0 0 12.5 0.5 17.5 0.75 32.5 1.5
53 1 T2N0M0 2 2A 63 70 2.5 50 2 0 22.5 1.25 32.5 0.75 47.5 1.5
54 1 T2N0M0 1 2A 56 80 2.5 50 2 1 30 1.25 52.5 1.5 12.5 0.5
55 1 T2N0M0 1 2A 62 90 3 65 2.5 1 20 1.5 52.5 1.5 12.5 0.5
56 1 T2N0M0 3 2A 41 30 2 40 2.5 0 47.5 2 32.5 1 27.5 1.5
57 1 T2N0M0 2 2A 42 30 2 30 2 1 22.5 2 52.5 1.5 12.5 0.5
58 1 T2N0M0 3 2A 55 10 2 50 2 1 22.5 1.5 52.5 1.5 12.5 0.5
59 1 T2N0M0 3 2A 45 60 2 40 2 0 57.5 2 52.5 1.5 12.5 0.5
60 1 T2N0M0 2 2A 65 70 2 0 0 0 22.5 1 47.5 1.25 12.5 0.5
61 1 T2N0M0 3 2A 49 60 3 30 2 0 20 1.25 42.5 1.25 12.5 0.5
62 1 T2N0M0 2 2A 67 70 2.5 30 2.5 0 42.5 1.25 52.5 1.5 17.5 1.5
63 1 T2N0M0 3 2A 56 20 3 20 3 0 42.5 1.5 12.5 0.25 12.5 0.5
64 1 T2N0M0 2 2A 57 0 0 25 1.5 0 25 1.5 42.5 1.5 20 1.25
65 1 T2N0M0 2 2A 49 20 1.5 35 1.5 0 27.5 1.5 52.5 1.5 17.5 1.5
66 1 T2N0M0 2 2A 54 60 2 10 2 0 12.5 0.5 52.5 1.5 12.5 0.5
67 1 T2N0M0 2 2A 57 10 2 0 0 1 22.5 1 52.5 1 12.5 0.5
68 1 T2N0M0 2 2A 43 40 2.5 40 3 0 12.5 0.5 47.5 1.5 12.5 0.5
69 1 T2N0M0 2 2A 41 60 2.5 20 2.5 0 20 1.5 47.5 1.5 12.5 0.5
70 1 T2N0M0 2 2A 40 60 2.5 70 2.5 0 37.5 1.5 47.5 1 12.5 0.5
71 1 T2N0M0 3 2A 50 0 0 20 2 1 17.5 1.25 42.5 1.25 12.5 0.5
72 1 T2N0M0 3 2A 43 90 3 60 3 0 20 1 12.5 0.25 42.5 1.5
73 1 T2N0M0 2 2A 42 80 3 50 3 0 22.5 2 42.5 1.25 12.5 0.5
74 1 T2NxM0 3 2A 46 0 0 25 2.5 0 37.5 1.5 12.5 0.25 27.5 1.25
75 1 T2N0M0 2 2A 38 70 2 45 2 1 27.5 1.5 45 1.25 12.5 0.5
40
76 1 T2N0M0 2 2A 69 60 1.5 10 2 1 30 1.5 42.5 1.25 27.5 1.5
77 1 T2N0M0 2 2A 38 75 2 0 0 0 47.5 1.75 22.5 1 12.5 0.5
78 1 T2N0M0 2 2A 64 90 2.5 80 2 0 45 1.5 42.5 1.25 12.5 0.5
79 1 T2N0M0 3 2A 54 65 2.5 45 2 0 12.5 0.5 52.5 1.5 12.5 0.5
80 1 T2N0M0 3 2A 73 0 0 30 2 0 50 2 52.5 1.5 12.5 0.5
81 1 T2N0M0 3 2A 35 80 2.5 45 2.5 0 27.5 1 50 1.25 12.5 0.5
82 1 T2N0M0 3 2A 60 35 2 5 2 1 12.5 0.5 47.5 1 12.5 0.5
83 1 T2N0M0 2 2A 52 80 2 55 2 1 22.5 1.5 47.5 1.5 12.5 0.5
84 1 T2N0M0 2 2A 49 25 2 0 0 1 47.5 1.5 17.5 1.25 12.5 0.5
85 1 T2N0M0 2 2A 53 90 2.5 40 2 0 22.5 1.5 52.5 1.5 12.5 0.5
86 1 T2N0M0 2 2A 59 65 2.5 80 2.5 0 12.5 0.5 52.5 1.5 12.5 0.5
87 1 T2N0M0 2 2A 51 90 2.5 70 2 0 35 1.5 25 1 12.5 0.5
88 1 T2N0M0 3 2A 53 70 2.5 35 2 0 15 1.5 40 1.25 12.5 0.5
89 1 T2N0M0 3 2A 60 0 0 35 2 1 40 1.5 35 1.5 12.5 0.5
90 1 T1N1M0 2 2A 52 0 0 15 2 1 32.5 1.75 12.5 0.25 42.5 2
91 1 T1N1M0 2 2A 45 35 2 0 0 0 32.5 1.5 22.5 0.75 42.5 1.5
92 1 T1N1M0 2 2A 33 40 2 90 2.5 1 12.5 0.5 52.5 1.5 37.5 1.25
93 1 T1N1M0 3 2A 51 60 2.5 40 2 1 12.5 0.5 52.5 1.5 12.5 0.5
94 1 T1N1M0 3 2A 52 80 3 30 3 0 55 2 17.5 0.75 12.5 0.5
95 1 T1N1M0 3 2A 53 65 3 70 3 0 55 2 17.5 0.75 47.5 1.5
96 1 T1N1M0 2 2A 51 60 2 50 2 0 42.5 1.5 52.5 1.25 12.5 0.5
97 1 T1N1M0 2 2A 37 70 3 70 3 0 42.5 1.5 37.5 1 12.5 0.5
98 1 T1N1M0 1 2A 41 70 2.5 90 3 0 30 1.5 52.5 1.5 12.5 0.5
99 1 T1N1M0 3 2A 37 0 0 20 2 0 50 1.75 52.5 1.5 17.5 1.5
100 1 T1N1M0 3 2A 50 50 2 25 2.5 0 12.5 0.5 47.5 1.25 12.5 0.5
101 1 T1N1M0 2 2A 55 75 2 65 2 0 32.5 1.75 47.5 1.25 12.5 0.5
102 1 T2N1M0 3 2A 51 70 2.5 30 2.5 1 17.5 1.5 37.5 1.25 12.5 0.5
103 1 T2N1M0 2 2A 62 80 2.5 0 0 0 12.5 0.5 47.5 1.5 12.5 0.5
104 1 T2N1M0 2 2A 45 80 2.5 90 3 0 12.5 0.5 47.5 1 12.5 0.5
105 1 T2N1M0 2 2A 54 30 1.5 0 0 0 17.5 1.5 50 1.25 12.5 0.5
106 1 T2N1M0 1 2A 60 90 2.5 75 2.5 0 30 1.5 52.5 1.25 12.5 0.5
107 1 T2N1M0 1 2A 35 80 2.5 70 2 0 22.5 1.5 47.5 1.5 12.5 0.5
108 1 T2N1M0 2 2A 56 90 3 5 2.5 0 30 1.5 52.5 1.25 12.5 0.5
109 1 T2N0M0 2 2A 69 60 1.5 90 2.5 0 42.5 1.5 50 1.5 12.5 0.5
110 1 T2N0M0 2 2A 52 65 2 80 2.5 0 12.5 0.5 42.5 0.75 12.5 0.5
111 1 T2N0M0 2 2A 42 85 3 90 2.5 0 37.5 1.5 47.5 1.5 50 1.5
112 1 T2N0M0 3 2A 50 60 2 0 0 1 15 1.5 52.5 1.5 12.5 0.5
113 1 T2N0M0 3 2A 40 50 2 50 2 1 15 1.5 52.5 1.25 12.5 0.5
114 1 T2N0M0 3 2A 42 80 3 70 3 0 20 1.5 52.5 1.5 12.5 0.5
41
115 1 T2N1M0 2 2B 58 90 2.5 30 2 1 30 1.5 52.5 1.5 12.5 0.5
116 1 T2N1M0 2 2B 55 80 3 0 0 0 17.5 2 12.5 0.25 42.5 2
117 1 T2N1M0 3 2B 36 70 1.5 90 3 0 22.5 1 52.5 1.75 12.5 0.5
118 1 T2N1M0 3 2B 40 60 3 20 3 0 22.5 2 12.5 0.25 35 2
119 1 T2N1M0 3 2B 40 0 0 20 1 0 12.5 0.5 37.5 1 32.5 1.25
120 1 T2N1M0 2 2B 44 100 3 100 3 0 30 2 57.5 1.5 32.5 1.5
121 1 T2N1M0 3 2B 51 50 3 90 3 1 12.5 0.5 37.5 1 52.5 2
122 1 T2N1M0 3 2B 61 100 3 25 3 0 30 2 22.5 1.25 17.5 1
123 1 T2N1M0 3 2B 41 100 3 100 3 1 22.5 2 30 1 42.5 1.5
124 1 T2N1M0 2 2B 50 20 2 0 0 1 25 1.25 52.5 1.25 22.5 1
125 1 T2N1M0 2 2B 64 80 3 80 3 0 22.5 1.5 47.5 1.5 12.5 0.5
126 1 T2N1M0 2 2B 55 80 3 80 3 0 12.5 0.5 32.5 1 37.5 1.5
127 1 T2N1M0 2 2B 62 30 1 0 0 0 20 1.25 12.5 0.25 12.5 1.5
128 1 T2N1M0 3 2B 35 70 3 90 3 0 55 1.75 20 1.25 60 2
129 1 T2N1M0 3 2B 46 85 3 90 3 0 52.5 2 52.5 1 32.5 1.5
130 1 T2N1M0 2 2B 62 50 3 50 3 0 57.5 2 32.5 1.25 17.5 1
131 1 T2N1M0 2 2B 67 10 1.5 15 2 0 22.5 1.25 12.5 0.25 12.5 0.5
132 1 T2N1M0 2 2B 47 80 3 60 2 0 12.5 0.5 47.5 1.25 27.5 1
133 1 T2N1M0 2 2B 61 50 2.5 0 0 0 32.5 1 52.5 1.5 12.5 0.5
134 1 T2N1M0 3 2B 61 50 3 70 2 1 57.5 2 52.5 1.5 12.5 0.5
135 1 T2N1M0 2 2B 54 65 3 20 2 0 25 1.75 50 1.5 12.5 0.5
136 1 T2N1M0 1 2B 52 60 2.5 60 2 0 45 1.5 47.5 1.25 12.5 0.5
137 1 T2N1M0 3 2B 53 30 2 20 2 0 12.5 0.5 42.5 1 20 1.5
138 1 T2N1M0 3 2B 37 75 2.5 35 2.5 0 50 1.75 50 1.5 12.5 0.5
139 1 T2N1M0 2 2B 30 50 2 60 2 0 12.5 0.5 52.5 1.25 12.5 0.5
140 1 T2N1M0 3 2B 48 35 1.5 10 2 0 52.5 1.75 52.5 1.5 12.5 0.5
141 1 T2N1M0 2 2B 42 90 3 90 3 0 15 1.5 47.5 1.25 42.5 1.25
142 1 T2N1M0 2 2B 53 65 2.5 0 0 0 12.5 0.5 42.5 1 12.5 0.5
143 1 T2N1M0 2 2B 36 40 2 15 2 0 27.5 1.75 52.5 1.5 12.5 0.5
144 1 T2N1M0 2 2B 48 80 2 90 2.5 0 12.5 0.5 42.5 1 12.5 0.5
145 1 T2N1M0 2 2B 45 20 2 20 2.5 0 30 1.5 45 1.25 12.5 0.5
146 1 T2N1M0 1 2B 66 65 2 10 2 0 15 1.5 45 1 12.5 0.5
147 1 T2N1M0 2 2B 61 90 3 70 2 0 17.5 1.25 42.5 1.25 20 1
148 1 T2N1M0 1 2B 53 75 3 70 3 1 47.5 1.5 32.5 1 42.5 1.5
149 1 T2N1M0 2 2B 57 80 2.5 30 1.5 0 20 1 47.5 1 52.5 1.5
150 1 T2N1M0 3 2B 37 60 3 80 3 1 55 2 37.5 0.75 35 1
151 0 T1N0M0 3 1 61 100 3 100 3 0 57.5 2 52.5 1 57.5 2
152 0 T2N0M0 3 2A 54 100 3 100 3 0 60 2 42.5 1 12.5 0.5
153 0 T2N0M0 3 2A 51 0 0 20 1.5 1 55 2 47.5 1.25 37.5 1.5
42
154 0 T2N0M0 2 2A 41 40 2 100 3 0 22.5 1.25 60 1 35 1.25
155 0 T2N0M0 3 2A 27 0 0 25 1.5 1 57.5 2 12.5 0.25 55 1.5
156 0 T2N0M0 3 2A 33 0 0 30 3 1 57.5 2 57.5 1.75 60 2
157 0 T2N0M0 2 2A 82 35 2 0 0 1 52.5 2 57.5 1.75 37.5 1.5
158 0 T2N0M0 2 2A 47 80 2.5 60 2.5 0 20 1.5 52.5 1.5 12.5 0.5
159 0 T2N0M0 3 2A 51 100 3 10 1 1 52.5 2 12.5 0.25 42.5 1.25
160 0 T2N0M0 3 2A 49 80 2 100 3 0 47.5 2 32.5 1 25 1.25
Table 3A
43
Sl.
No
Outcome Sample No. F%-M FIM
F%-
C
FIC
K%-
M
KIM
K%-
C
KIC
M%-
C
MIC
N%-
C
NIC
O%-
M
OIM
P%-
N
PIN
R%-
M
RIM
R%-
C
RIC
1 1 MMBVSSSMMKPR/AAJH/0467/06 22.85714286 1.25 22.5 1.25 22.5 1.75 57.5 2 60 2 45 1.75 12.5 0.5 22.5 1 24 1.25 25 1.5
2 1 MMBVSSSMMKPR/ABMH/4734/07 25.71428571 1.25 30 0.5 30 1.5 57.5 1.75 52.5 1.5 37.5 1.25 17.5 1 45 2 20 0.5 27.5 1.5
3 1 MMBVSSSMMKPR/HLRG/1219/03 18.57142857 1.25 12.5 1 12.5 0.5 17.5 1 42.5 1.5 47.5 1.25 32.5 1.25 27.5 1.5 26 1 37.5 1.25
4 1 MMBVSSSMMKPR/ACRG/2834/03 32.85714286 1 57.5 0.5 32.5 1.5 55 2 27.5 1.25 52.5 2 12.5 0.5 20 1.5 20 0.5 20 1.25
5 1 MMBVSSSMMKPR/AVRG/1397/04 24.28571429 1 12.5 0.5 25 1.5 55 2 47.5 1.75 50 1.75 12.5 0.5 27.5 1.5 20 0.5 22.5 1
6 1 MMBVSSSMMKPR/AEMH/6521/06 27.14285714 1.75 12.5 0.5 17.5 1 47.5 2 47.5 1.5 22.5 1.5 12.5 0.5 12.5 0.5 20 0.5 17.5 1
7 1 MMBVSSSMMKPR/AFJH/0478/06 15.71428571 1 12.5 1 17.5 1.25 52.5 2 42.5 1.5 47.5 1.75 12.5 0.5 32.5 1.5 40 1.25 32.5 1
8 1 MMBVSSSMMKPR/HTMX/0675/08 12.85714286 0.5 22.5 1.25 12.5 0.5 55 2 52.5 1.25 57.5 1.5 12.5 0.5 35 1.5 30 1 37.5 1.25
9 1 MMBVSSSMMKPR/IRRG/5724/08 30 1.25 32.5 1.25 32.5 1.5 47.5 1.75 30 1 37.5 1.25 37.5 1.25 27.5 1 48 1.5 32.5 1.25
10 1 MMBVSSSMMKPR/JMRG/1480/08 18.57142857 1.25 20 1.5 35 1.25 47.5 2 32.5 1.25 42.5 1.25 22.5 1 45 1.25 24 1 20 1
11 1 MMBVSSSMMKPR/HCGA1/222/10 12.85714286 0.5 50 2 17.5 1 37.5 1 52.5 1.5 52.5 1.75 12.5 0.5 22.5 1.5 20 0.5 12.5 1.25
12 1 MMBVSSSMMKPR/KIDC/0164/06 25.71428571 1.25 42.5 1.5 32.5 1.25 42.5 1.25 30 1.25 42.5 1.75 27.5 1.5 50 2 28 1 30 0.5
13 1 MMBVSSSMMKPR/KJDC/0167/06 15.71428571 1.25 45 1.5 25 1.5 47.5 2 27.5 1.25 47.5 1.75 12.5 0.5 42.5 1.5 28 1 47.5 1
14 1 MMBVSSSMMKPR/KNDC/0176/05 27.14285714 1.5 50 0.5 27.5 1.5 47.5 1.5 42.5 1.25 42.5 1.5 27.5 1.5 47.5 1.75 30 1 12.5 1.5
15 1 MMBVSSSMMKPR/MH11/3187/09 12.85714286 0.5 12.5 1.5 12.5 0.5 42.5 1.5 37.5 1.5 52.5 1.75 60 2 57.5 2 20 0.5 52.5 0.5
16 1 MMBVSSSMMKPR/HCGA4/5535/06 12.85714286 0.5 52.5 1.75 17.5 1.25 47.5 1.5 47.5 1.5 52.5 1.75 12.5 0.5 12.5 0.5 20 0.5 50 1.5
17 1 MMBVSSSMMKPR/SP5/6544/08 12.85714286 0.5 50 1.75 17.5 1 37.5 1.25 47.5 1 52.5 1.75 22.5 1.25 22.5 1.5 24 1.25 47.5 1.5
18 1 MMBVSSSMMKPR/SP6/5298/06 34.28571429 1.5 50 1.5 40 1.25 47.5 1.5 45 1.25 47.5 1.75 37.5 1.25 12.5 0.5 46 1.5 52.5 1.5
19 1 MMBVSSSMMKPR/SP6/6116/06 12.85714286 0.5 52.5 1.75 20 0.75 47.5 1.25 47.5 1.25 52.5 1.75 12.5 0.5 40 1.25 36 1.25 52.5 1.75
20 1 MMBVSSSMMKPR/SP8/6543/06 34.28571429 1.5 52.5 1.75 50 1.25 52.5 1.75 52.5 1.5 52.5 1.75 50 1.25 12.5 0.5 52 1.25 52.5 1.5
21 1 MMBVSSSMMKPR/SP9/1761/97 15.71428571 1 52.5 1.75 20 1.25 47.5 1.5 50 1.5 52.5 1.75 20 1.25 12.5 0.5 20 0.5 52.5 1.75
22 1 MMBVSSSMMKPR/SP9/2006/02 20 1 52.5 1.75 27.5 1.25 52.5 1.75 50 1.75 50 1.75 22.5 1.25 12.5 0.5 28 1.25 52.5 1.75
23 1 MMBVSSSMMKPR/SP10/4338/05 15.71428571 1.25 52.5 0.5 27.5 1.25 52.5 1.5 50 1.5 52.5 1.75 20 1 12.5 0.5 24 1.25 47.5 1.5
24 1 MMBVSSSMMKPR/SP13/0242/10 12.85714286 0.5 12.5 0.5 42.5 1.25 50 1.5 47.5 1.5 52.5 1.75 30 1 42.5 1.5 30 1 17.5 1.75
25 1 MMBVSSSMMKPR/ADRG/1440/02 18.57142857 1 12.5 0.5 32.5 1.75 37.5 2 52.5 2 27.5 1 22.5 1 32.5 1.5 26 1 27.5 1
44
26 1 MMBVSSSMMKPR/AGMH/2268/04 21.42857143 1.25 12.5 1.5 12.5 0.5 47.5 1.5 60 2 20 1.25 20 1.25 50 2 20 0.5 47.5 1.25
27 1 MMBVSSSMMKPR/AHMH/2180/03 18.57142857 1.5 22.5 0.5 17.5 1.25 57.5 2 52.5 1.75 55 1.5 12.5 0.5 27.5 1.5 26 1.25 47.5 1.5
28 1 MMBVSSSMMKPR/AJMH/1975/03 38.57142857 1.75 12.5 0.5 32.5 1.5 52.5 1.75 47.5 1.5 12.5 0.5 47.5 1.25 57.5 2 32 1.25 52.5 1.5
29 1 MMBVSSSMMKPR/AKMH/1186/03 12.85714286 0.5 12.5 1.25 12.5 0.5 55 2 57.5 1.5 60 1.5 12.5 0.5 45 1.75 44 1.25 12.5 1.5
30 1 MMBVSSSMMKPR/ALMH/4227/05 22.85714286 1.25 42.5 0.5 12.5 0.5 55 1.5 47.5 1.75 52.5 1.5 22.5 1.25 17.5 1.25 36 1.25 47.5 0.5
31 1 MMBVSSSMMKPR/AMMH/2138/05 12.85714286 0.5 12.5 0.5 52.5 1.5 52.5 1.5 50 1.25 42.5 1.75 32.5 1.75 12.5 0.5 20 0.5 42.5 1.5
32 1 MMBVSSSMMKPR/ANMH/6528/05 12.85714286 0.5 12.5 1.5 12.5 0.5 37.5 1.25 47.5 1.5 37.5 1.25 12.5 0.5 37.5 1.75 24 1.5 22.5 1.5
33 1 MMBVSSSMMKPR/AOMH/1621/05 24.28571429 1 52.5 1.5 12.5 0.5 57.5 2 47.5 1.5 25 1 12.5 0.5 17.5 1.25 24 1.25 27.5 1
34 1 MMBVSSSMMKPR/APMH/6408/05 12.85714286 0.5 20 0.5 12.5 0.5 47.5 2 57.5 1 12.5 0.5 45 1.75 12.5 0.5 20 0.5 17.5 1.5
35 1 MMBVSSSMMKPR/AQMH/1408/06 22.85714286 1.25 12.5 1 35 1.5 37.5 1.5 22.5 1.5 30 1 25 1.25 47.5 1.5 36 1 12.5 1.25
36 1 MMBVSSSMMKPR/ARMH/4622/06 12.85714286 0.5 52.5 1 32.5 1.5 55 1.5 47.5 1.25 47.5 1.75 45 1.5 50 1.75 32 1 25 0.5
37 1 MMBVSSSMMKPR/ASRG/5174/03 20 1 32.5 1 12.5 0.5 37.5 1.25 42.5 1.25 47.5 1.5 52.5 1.5 12.5 0.5 40 1.5 27.5 1
38 1 MMBVSSSMMKPR/ATMH/4074/01 17.14285714 1 17.5 1 17.5 1 47.5 1.75 52.5 1.75 30 1.25 12.5 0.5 20 1.25 20 0.5 22.5 1.5
39 1 MMBVSSSMMKPR/AYRG/3670/02 12.85714286 0.5 30 1 12.5 0.5 60 2 50 2 32.5 1.5 17.5 1.25 12.5 0.5 20 0.5 45 1.25
40 1 MMBVSSSMMKPR/AZRG/1472/02 32.85714286 1 47.5 1 22.5 1.25 50 1.75 47.5 1.75 47.5 2 12.5 0.5 12.5 0.5 36 1 42.5 1.5
41 1 MMBVSSSMMKPR/BAJH/0102/07 28.57142857 1 37.5 0.5 12.5 0.5 27.5 1 47.5 1.5 37.5 1.5 52.5 1.25 42.5 2 38 1 42.5 1.5
42 1 MMBVSSSMMKPR/HSMH/4789/07 17.14285714 1 12.5 0.5 22.5 1.25 50 2 32.5 1.5 37.5 1.5 12.5 0.5 47.5 2 20 0.5 17.5 1.25
43 1 MMBVSSSMMKPR/HKRG/3754/03 12.85714286 0.5 12.5 1.75 12.5 0.5 45 1.5 57.5 1.5 47.5 1.25 22.5 1.25 22.5 1.25 24 1 52.5 1
44 1 MMBVSSSMMKPR/IMRG/5141/08 12.85714286 0.5 42.5 1.5 20 1 60 2 47.5 1.25 57.5 2 17.5 1 27.5 1.25 32 1.5 57.5 2
45 1 MMBVSSSMMKPR/IQRG/5751/08 37.14285714 2 42.5 1.5 12.5 0.5 27.5 1.25 57.5 1.25 42.5 1.25 27.5 1 50 1.5 20 0.5 32.5 2
46 1 MMBVSSSMMKPR/JNRG/6963/08 18.57142857 1 42.5 1.5 25 1.25 50 2 45 1.25 57.5 2 30 0.75 42.5 1.25 32 1.25 47.5 1.25
47 1 MMBVSSSMMKPR/JORG/6520/08 17.14285714 1.5 47.5 1.5 27.5 1.25 52.5 2 40 1.75 55 2 12.5 0.5 42.5 1.25 32 1.25 52.5 1.5
48 1 MMBVSSSMMKPR/IXRG/1912/08 17.14285714 1 47.5 2 37.5 1.25 25 1.5 47.5 1.5 47.5 1.75 12.5 0.5 27.5 1 30 1.5 37.5 1.75
49 1 MMBVSSSMMKPR/IHRG/1461/07 18.57142857 1 52.5 1.25 32.5 1.25 47.5 2 60 1.5 45 1 12.5 0.5 42.5 1 40 1.5 47.5 1.75
50 1 MMBVSSSMMKPR/INRG/6074/08 15.71428571 1 27.5 1.75 12.5 0.5 55 2 52.5 1.5 47.5 1.5 12.5 0.5 25 1.25 26 1.5 27.5 1.5
51 1 MMBVSSSMMKPR/JPRG/7119/08 18.57142857 1 47.5 1.5 35 1.25 52.5 2 45 1.5 50 1.75 45 1.25 37.5 1.25 28 1.25 37.5 1.5
52 1 MMBVSSSMMKPR/IARG/1209/01 12.85714286 0.5 22.5 1.5 37.5 1.25 42.5 2 47.5 2 20 1 12.5 0.5 12.5 0.5 20 0.5 37.5 1.5
45
53 1 MMBVSSSMMKPR/JQRG/6998/08 18.57142857 1 32.5 1.5 22.5 1.25 52.5 2 42.5 1.25 57.5 2 27.5 0.75 55 1.5 32 1 30 1.25
54 1 MMBVSSSMMKPR/HCGA1/5042/08 18.57142857 1.25 47.5 1.75 22.5 1 52.5 1.75 52.5 1.5 52.5 1.5 20 1.25 17.5 1.5 20 0.5 42.5 1
55 1 MMBVSSSMMKPR/RG20/7272/08 18.57142857 1 47.5 0.5 42.5 1.25 52.5 1.75 52.5 1.75 52.5 1.75 37.5 1 57.5 1.25 30 1 42.5 1.5
56 1 MMBVSSSMMKPR/MH10/826/06 12.85714286 0.5 12.5 1.25 12.5 0.5 50 1.5 42.5 1.75 42.5 1.5 50 2 47.5 2 20 0.5 17.5 1.5
57 1 MMBVSSSMMKPR/MH12/6907/09 15.71428571 1.25 47.5 1.25 50 1.25 52.5 1.5 52.5 1.5 52.5 1.75 40 1.5 45 2 28 1.25 42.5 1
58 1 MMBVSSSMMKPR/MH13/5276/09 21.42857143 1.25 22.5 1.5 47.5 1.25 52.5 1.5 52.5 1.5 52.5 2 22.5 1 45 2 20 0.5 52.5 1
59 1 MMBVSSSMMKPR/MH13/2852/02 20 1.5 52.5 1.75 22.5 1.25 52.5 1.5 45 1.75 42.5 1.5 12.5 0.5 52.5 2 24 1 52.5 1.5
60 1 MMBVSSSMMKPR/RG24/2672/09 17.14285714 1 55 1.5 22.5 1.25 55 1.75 47.5 1.25 52.5 1.5 12.5 0.5 12.5 0.5 28 1 47.5 1.75
61 1 MMBVSSSMMKPR/RG24/786/09 27.14285714 1.25 50 1.5 27.5 1.5 55 1.75 50 1.5 52.5 1.5 22.5 1 12.5 0.5 40 1.25 52.5 1.5
62 1 MMBVSSSMMKPR/RG24/3773/09 17.14285714 1 50 1.5 40 1.75 47.5 1.5 40 1.75 37.5 1.5 32.5 1.25 15 1.5 24 1 45 1.75
63 1 MMBVSSSMMKPR/RG24/3821/09 18.57142857 1 52.5 1.75 25 1.25 55 1.75 32.5 1.25 45 1.5 12.5 0.5 12.5 0.5 24 1 47.5 1.25
64 1 MMBVSSSMMKPR/RG25/1072/09 17.14285714 1 52.5 1.75 17.5 1.5 55 1.75 22.5 1.25 50 1.75 42.5 1.25 12.5 0.5 20 0.5 52.5 1.75
65 1 MMBVSSSMMKPR/HCGA5/7545/07 30 1.25 52.5 1.75 52.5 1.25 52.5 1.75 42.5 1.25 52.5 1.75 22.5 1 20 1.25 48 1.25 52.5 1.5
66 1 MMBVSSSMMKPR/SP2/2851/03 17.14285714 1 47.5 1.75 22.5 1 55 1.75 52.5 1.5 50 1.75 12.5 0.5 12.5 0.5 24 1.5 50 1.75
67 1 MMBVSSSMMKPR/SP2/06011/08 15.71428571 1.5 55 1.75 17.5 1.25 55 1.75 52.5 1.5 52.5 1.5 12.5 0.5 12.5 0.5 26 1.25 50 1.5
68 1 MMBVSSSMMKPR/SP2/5595E/04 32.85714286 1.5 50 1.75 47.5 1.25 47.5 1.25 47.5 1.5 42.5 1.5 32.5 1.25 12.5 0.5 40 1.5 50 1.75
69 1 MMBVSSSMMKPR/SP3/12267/07 31.42857143 1.75 52.5 1.5 50 1.75 50 1.5 47.5 1.25 50 1.75 47.5 1.75 12.5 0.5 44 1.5 50 1.75
70 1 MMBVSSSMMKPR/SP3/09935/08 34.28571429 1.5 50 1.75 52.5 1.75 52.5 1.75 45 1 50 1.75 47.5 1.5 12.5 0.5 50 1.5 50 1.75
71 1 MMBVSSSMMKPR/SP4/04418/08 18.57142857 1.5 52.5 1.75 32.5 1.5 52.5 1.75 32.5 1.5 50 1.75 17.5 1 12.5 0.5 30 1.5 12.5 1.5
72 1 MMBVSSSMMKPR/KFDC/0149/07 24.28571429 1.25 50 1.25 27.5 1.25 47.5 1.5 32.5 1.5 37.5 1.25 47.5 1.5 50 1.5 36 1 27.5 0.5
73 1 MMBVSSSMMKPR/BDRG/5248/04 12.85714286 0.5 37.5 1.5 25 1.25 52.5 2 32.5 1.5 47.5 1.5 25 1.5 12.5 0.5 20 0.5 12.5 1.25
74 1 MMBVSSSMMKPR/KODC/0178/05 17.14285714 1 37.5 1.75 27.5 1.25 47.5 1.5 17.5 1.5 12.5 0.5 12.5 0.5 12.5 0.5 20 0.5 52.5 0.5
75 1 MMBVSSSMMKPR/SP5/2503/08 30 1.75 55 1.75 47.5 1.75 50 1.75 37.5 1.5 42.5 1.5 27.5 1 17.5 1.25 38 1.5 47.5 1.75
76 1 MMBVSSSMMKPR/SP5/2049/09 34.28571429 1.75 50 1.75 52.5 1.75 52.5 1.75 45 1.25 50 1.75 30 1.25 12.5 0.5 40 1.5 45 1.75
77 1 MMBVSSSMMKPR/SP5/2933/09 35.71428571 1.75 52.5 1.75 55 1.75 30 1.5 45 1.5 47.5 1.25 50 1.5 12.5 0.5 48 1.5 50 1.75
78 1 MMBVSSSMMKPR/SP5/5414/09 28.57142857 1.5 50 1 25 1.5 40 0.75 52.5 1.5 45 1.5 50 1.5 22.5 1.25 30 1 50 1.5
79 1 MMBVSSSMMKPR/SP5/6317/09 12.85714286 0.5 42.5 1.75 12.5 0.5 30 1 52.5 1.5 52.5 1.75 17.5 1 25 1.25 28 1.25 52.5 1.5
46
80 1 MMBVSSSMMKPR/SP5/5630/07 35.71428571 1.5 52.5 1.75 55 1.75 55 1.75 50 1.75 50 1.75 47.5 1.25 27.5 1.75 50 1.5 52.5 1.75
81 1 MMBVSSSMMKPR/SP6/4628/02 27.14285714 1.5 52.5 1.75 32.5 1.25 42.5 1.75 52.5 1.75 52.5 1.75 42.5 1.25 12.5 0.5 42 1.25 52.5 1.5
82 1 MMBVSSSMMKPR/SP6/215/05 12.85714286 0.5 52.5 1.25 17.5 1 52.5 1.75 52.5 1.5 50 1.75 47.5 1.25 12.5 0.5 20 0.5 52.5 1.75
83 1 MMBVSSSMMKPR/SP6/4283/08 27.14285714 1 47.5 1.5 52.5 1.5 52.5 1.5 52.5 1.5 52.5 1.75 42.5 1.25 40 1.25 44 1.5 50 1.75
84 1 MMBVSSSMMKPR/SP6/3004/09 32.85714286 1.5 47.5 1.75 47.5 1.5 47.5 1.5 52.5 1.75 37.5 1.25 42.5 1.25 12.5 0.5 44 1.5 52.5 1.75
85 1 MMBVSSSMMKPR/SP7/6675/07 32.85714286 1.25 52.5 1.75 47.5 1.25 52.5 1.5 52.5 1.75 52.5 1.5 47.5 1.25 17.5 1.25 48 1.25 52.5 1.5
86 1 MMBVSSSMMKPR/SP7/4924/06 15.71428571 1.25 52.5 1.75 17.5 1 50 1.5 47.5 1.5 52.5 1.75 17.5 1 40 1.5 20 0.5 50 1.75
87 1 MMBVSSSMMKPR/SP8/7537/09 17.14285714 1.25 50 1.75 27.5 1.25 52.5 1.75 37.5 1.25 37.5 1.75 17.5 1.25 22.5 1.5 28 1 47.5 1.75
88 1 MMBVSSSMMKPR/SP8/3347/09 28.57142857 1.25 52.5 1.5 42.5 1.25 50 1.5 52.5 1.5 50 1.75 40 1.25 27.5 1.5 42 1.5 52.5 1.5
89 1 MMBVSSSMMKPR/SP8/7252/08 20 1.5 50 0.5 47.5 1.25 52.5 1.75 52.5 1.5 52.5 1.75 42.5 1.25 17.5 1.25 28 1.25 17.5 1.75
90 1 MMBVSSSMMKPR/AURG/1691/04 34.28571429 2 12.5 0.5 25 1.25 37.5 1.25 12.5 0.5 42.5 1.75 32.5 1.25 32.5 1.5 24 1 12.5 1.25
91 1 MMBVSSSMMKPR/AWRG/3418/04 27.14285714 1 12.5 1.5 12.5 0.5 20 2 32.5 1 27.5 1.5 12.5 0.5 12.5 0.5 34 1.25 27.5 0.5
92 1 MMBVSSSMMKPR/HCGA1/6737/09 12.85714286 0.5 47.5 1.5 42.5 1 47.5 1 47.5 1.5 37.5 1.75 12.5 0.5 22.5 1.5 20 0.5 52.5 1.25
93 1 MMBVSSSMMKPR/HCGA3/5860/09 12.85714286 0.5 47.5 1 17.5 1.25 52.5 1.5 45 1.5 52.5 1.75 12.5 0.5 42.5 1.25 24 1.25 12.5 1.5
94 1 MMBVSSSMMKPR/BBMH/6814/07 24.28571429 1 37.5 0.5 27.5 1.75 50 2 42.5 2 37.5 1.5 30 1.5 37.5 2 26 1.25 22.5 0.5
95 1 MMBVSSSMMKPR/BEMH/3166/01 27.14285714 1.25 12.5 1.75 45 1.5 35 1.5 17.5 1 12.5 0.5 12.5 0.5 17.5 1 20 0.5 47.5 1.25
96 1 MMBVSSSMMKPR/RG25/2941/09 27.14285714 1 47.5 1.75 42.5 1.5 42.5 1.75 47.5 1.5 47.5 1.25 42.5 1.5 12.5 0.5 40 1 52.5 1.5
97 1 MMBVSSSMMKPR/SP1/1287/98 27.14285714 1.25 52.5 1.75 37.5 1.25 50 1.5 42.5 1.5 47.5 1.25 12.5 0.5 12.5 0.5 40 1.25 55 1.5
98 1 MMBVSSSMMKPR/SP2/00396/06 21.42857143 1.5 55 1.75 25 1.25 52.5 1.5 47.5 1.25 52.5 1.75 22.5 1 20 1.5 30 1.5 52.5 1.75
99 1 MMBVSSSMMKPR/SP2/5090/03 12.85714286 0.5 55 1.75 32.5 1.5 50 1.5 52.5 1.75 52.5 1.75 22.5 1 12.5 0.5 26 1.25 55 1.75
100 1 MMBVSSSMMKPR/SP3/00076/06 17.14285714 1.5 52.5 1.75 37.5 1.5 52.5 1.75 52.5 1.5 47.5 1.5 20 1.5 12.5 0.5 32 1.5 42.5 1.75
101 1 MMBVSSSMMKPR/SP5/5658/09 17.14285714 1.5 55 1.75 12.5 0.5 47.5 1.5 47.5 1.5 45 1.25 32.5 1.5 12.5 0.5 20 0.5 52.5 1.75
102 1 MMBVSSSMMKPR/SP9/1431/07 18.57142857 1.25 52.5 1.75 37.5 1 52.5 1.75 47.5 1.5 52.5 1.75 22.5 1.25 12.5 0.5 24 1.25 52.5 1.75
103 1 MMBVSSSMMKPR/SP9/00521/07 15.71428571 1 52.5 1.75 32.5 1.25 42.5 1.25 47.5 1.5 52.5 1.75 20 1.25 32.5 1.25 24 1 47.5 1.75
104 1 MMBVSSSMMKPR/SP9/919/99 18.57142857 1 52.5 1.25 25 1 52.5 1.5 52.5 1.5 50 1.5 12.5 0.5 15 1.5 32 1.25 52.5 1.75
105 1 MMBVSSSMMKPR/SP10/1693/03 15.71428571 1.5 52.5 1.5 22.5 1 47.5 1.5 42.5 1.5 52.5 1.75 40 1.5 40 1.5 20 0.5 50 1.25
106 1 MMBVSSSMMKPR/SP11/2802/01 32.85714286 1.25 47.5 1.5 45 1.25 50 1.5 52.5 1.5 52.5 1.75 22.5 1.5 52.5 1.5 48 1.25 50 1.5
47
107 1 MMBVSSSMMKPR/SP11/1002/05 24.28571429 1.25 47.5 1.25 37.5 1.25 42.5 1.5 52.5 1.75 47.5 1.5 42.5 1.25 42.5 1.5 36 1.25 47.5 1.5
108 1 MMBVSSSMMKPR/SP11/4945/09 18.57142857 1 47.5 1.5 45 1.25 50 1.75 50 1.5 52.5 1.75 37.5 1.25 42.5 1.5 40 1.25 50 1.75
109 1 MMBVSSSMMKPR/RG32/47/10 17.14285714 1 47.5 1.5 37.5 1.25 52.5 1.75 50 1.75 47.5 1.5 42.5 1.25 17.5 1.25 40 1.25 40 1.5
110 1 MMBVSSSMMKPR/RG32/7449/09 31.42857143 1.5 47.5 0.75 50 1.5 42.5 1.25 42.5 1.5 42.5 1.5 52.5 1.25 12.5 0.5 50 1.5 50 1.5
111 1 MMBVSSSMMKPR/SP12/0353/09 12.85714286 0.5 47.5 0.75 50 1.25 42.5 1.25 50 1.25 15 1.5 37.5 1.5 55 1.5 32 1.25 50 1.5
112 1 MMBVSSSMMKPR/SP12/2027/09 12.85714286 0.5 47.5 1.25 37.5 1.25 50 1.5 50 1.5 52.5 1.75 35 1.25 20 1.25 24 1 52.5 1.25
113 1 MMBVSSSMMKPR/SP12/1631/09 15.71428571 1.5 47.5 1.5 42.5 1.5 42.5 1.5 42.5 1.25 52.5 1.75 27.5 1.5 52.5 1.5 24 1 50 1.75
114 1 MMBVSSSMMKPR/RG34/1091/10 24.28571429 1.25 40 1.25 40 1.25 52.5 1.75 52.5 1.75 52.5 1.75 40 1.25 27.5 1.25 36 1.25 52.5 1.75
115 1 MMBVSSSMMKPR/SP13/6097/08 20 1.25 47.5 0.5 42.5 1.25 47.5 1.75 50 1 52.5 1.75 30 1.25 15 1.5 30 1.25 47.5 1.5
116 1 MMBVSSSMMKPR/BFMH/2540/04 12.85714286 0.5 12.5 1.5 35 1.5 57.5 1.5 57.5 1.5 25 1.25 12.5 0.5 20 1.25 26 1.25 47.5 1.5
117 1 MMBVSSSMMKPR/BGMH/3558/03 30 1.25 32.5 0.5 32.5 1.25 52.5 1.75 55 1.75 47.5 1.5 12.5 0.5 57.5 2 30 1.25 25 1.25
118 1 MMBVSSSMMKPR/BIMH/4596/05 17.14285714 1.25 12.5 1.5 50 1.5 27.5 1.25 37.5 1.25 20 1 30 1.25 12.5 0.5 24 1 37.5 1.25
119 1 MMBVSSSMMKPR/BJMH/1942/07 30 1.5 27.5 1.25 27.5 1.25 52.5 1.25 52.5 2 37.5 1.5 32.5 2 12.5 0.5 20 0.5 37.5 1.25
120 1 MMBVSSSMMKPR/BKMH/3435/06 17.14285714 1.25 32.5 1 32.5 1.5 52.5 1.5 12.5 0.5 47.5 1.5 12.5 0.5 22.5 2 20 0.5 20 1.25
121 1 MMBVSSSMMKPR/BLJH/2584/05 24.28571429 1.25 22.5 1.5 37.5 1.75 47.5 2 17.5 1 12.5 0.5 27.5 1.5 22.5 1 20 0.5 25 1
122 1 MMBVSSSMMKPR/BMMH/3591/08 12.85714286 0.5 52.5 1.25 25 1.25 52.5 1.75 32.5 1.5 30 1 50 2 47.5 2 20 0.5 42.5 1.5
123 1 MMBVSSSMMKPR/BNMH/3242/06 18.57142857 1.25 52.5 0.5 12.5 0.5 27.5 1.25 22.5 1.5 47.5 1.5 12.5 0.5 47.5 1.75 20 0.5 27.5 1.25
124 1 MMBVSSSMMKPR/BORG/0945/03 32.85714286 1 12.5 1.5 22.5 1.25 22.5 1 50 1.25 47.5 2 47.5 1.25 20 1.25 30 1 22.5 1
125 1 MMBVSSSMMKPR/BPRG/3398/03 18.57142857 1 40 0.5 12.5 0.5 47.5 2 52.5 1.25 52.5 1.5 20 1.25 12.5 0.5 20 0.5 27.5 1
126 1 MMBVSSSMMKPR/BQRG/4390/03 17.14285714 1 12.5 1.75 12.5 0.5 50 1.5 42.5 1.25 12.5 0.5 32.5 1.25 17.5 1.25 44 1.25 22.5 1.25
127 1 MMBVSSSMMKPR/BTRG/3606/03 35.71428571 2 32.5 0.5 25 1.25 52.5 2 40 1.5 32.5 1.25 37.5 1.25 12.5 0.5 28 1.25 12.5 1
128 1 MMBVSSSMMKPR/BUMH/6186/04 12.85714286 0.5 12.5 0.5 12.5 0.5 40 1.5 12.5 0.5 42.5 1.5 20 1.25 50 1.75 28 1 12.5 0.5
129 1 MMBVSSSMMKPR/BVMH/6080/07 12.85714286 0.5 12.5 1.5 40 1.5 55 1.25 47.5 1.5 42.5 1 30 1.25 17.5 1.25 20 0.5 42.5 0.5
130 1 MMBVSSSMMKPR/DMMX/3990/07 22.85714286 1.25 40 1.75 42.5 1.5 47.5 1.75 42.5 1.5 47.5 1.75 52.5 2 50 1.5 26 1.25 47.5 1.25
131 1 MMBVSSSMMKPR/ SP1/2238/01 15.71428571 1 52.5 1.5 25 1.25 52.5 1.75 42.5 1.25 45 1.25 42.5 1 12.5 0.5 30 1 42.5 1.5
132 1 MMBVSSSMMKPR/RG15/1464/08 12.85714286 0.5 47.5 1.75 22.5 1 52.5 1.5 52.5 1.75 52.5 1.75 12.5 0.5 47.5 1.5 20 0.5 52.5 1.5
133 1 MMBVSSSMMKPR/RG22/1931/09 17.14285714 1 52.5 1.5 22.5 1 52.5 1.5 47.5 1.5 52.5 1.75 12.5 0.5 12.5 0.5 20 0.5 37.5 1.75
48
134 1 MMBVSSSMMKPR/MH14/6324/08 27.14285714 1.25 47.5 1.5 52.5 1.5 42.5 1.25 52.5 1.5 52.5 1.75 17.5 1.25 50 2 38 1.5 52.5 1.75
135 1 MMBVSSSMMKPR/ RG24/805/09 21.42857143 1 47.5 1.5 27.5 1.25 47.5 1.5 47.5 1.5 50 1.75 17.5 1 12.5 0.5 28 1 45 1.5
136 1 MMBVSSSMMKPR/HCGA4/6374/08 21.42857143 1 47.5 1.5 47.5 1.25 52.5 1.5 52.5 1.5 52.5 1.5 25 1 12.5 0.5 44 1.25 50 1.5
137 1 MMBVSSSMMKPR/SP1/3187/99 15.71428571 1 47.5 1.75 17.5 1 50 1.5 47.5 1.5 42.5 1.5 37.5 1 12.5 0.5 24 1 50 1.5
138 1 MMBVSSSMMKPR/SP1/1543/00 27.14285714 1.25 47.5 1.75 47.5 1.5 50 1.75 47.5 1.25 27.5 1.5 12.5 0.5 12.5 0.5 44 1.5 52.5 1.5
139 1 MMBVSSSMMKPR/SP2/10817/07 17.14285714 1 55 1.5 37.5 1.25 52.5 1.75 50 1.25 52.5 1.75 17.5 1 17.5 1.25 36 1.5 55 1.5
140 1 MMBVSSSMMKPR/SP2/3424/03 24.28571429 1.5 45 1.75 47.5 1.5 47.5 1.25 52.5 1.5 50 1.5 12.5 0.5 12.5 0.5 34 1.25 47.5 1.75
141 1 MMBVSSSMMKPR/SP2/489/02 15.71428571 1.25 47.5 1.75 27.5 1.25 42.5 1.25 42.5 1 50 1.5 12.5 0.5 35 1.25 26 1.25 42.5 1.75
142 1 MMBVSSSMMKPR/SP3/5454/05 27.14285714 1.5 55 1.5 52.5 1.5 52.5 1.5 47.5 1.5 47.5 1.5 45 1.5 12.5 0.5 38 1.5 55 1.75
143 1 MMBVSSSMMKPR/SP3/00070/06 15.71428571 1.25 52.5 1.75 17.5 1.25 52.5 1.75 52.5 1.75 52.5 1.75 12.5 0.5 12.5 0.5 24 1.5 50 2
144 1 MMBVSSSMMKPR/SP4/11639/06 18.57142857 1.25 50 1.75 42.5 1.75 52.5 1.5 47.5 1.25 47.5 1 12.5 0.5 12.5 0.5 28 1.25 47.5 1.5
145 1 MMBVSSSMMKPR/SP4/12823/07 12.85714286 0.5 47.5 1.75 17.5 1.25 47.5 1.5 50 1.5 52.5 1.75 12.5 0.5 12.5 0.5 24 1.25 55 1.5
146 1 MMBVSSSMMKPR/SP4/14511/07 24.28571429 1.5 52.5 0.5 35 1.5 47.5 1.75 50 1.5 50 1.5 17.5 1 12.5 0.5 30 1.5 12.5 1.75
147 1 MMBVSSSMMKPR/IIRG/4358/08 12.85714286 0.5 12.5 1.5 32.5 1.5 47.5 1.75 42.5 1.75 37.5 1.5 42.5 1.75 12.5 0.5 26 1 42.5 0.5
148 1 MMBVSSSMMKPR/IVRG/1658/08 25.71428571 1.5 47.5 1.25 12.5 0.5 37.5 1.5 25 1 30 1 12.5 0.5 22.5 1 34 1.25 47.5 1.5
149 1 MMBVSSSMMKPR/JRRG/6716/08 15.71428571 1 47.5 1.25 27.5 1.25 50 2 50 1.5 50 1.75 12.5 0.5 52.5 1.25 30 1 20 1.25
150 1 MMBVSSSMMKPR/JSMH/7129/07 12.85714286 0.5 37.5 1.75 40 2 47.5 2 32.5 1.25 37.5 1.5 37.5 1.5 45 2 20 0.5 50 1.25
151
0 MMBVSSSMMKPR/CPMH/1187/05 22.85714
1.25 47.5 0.5 45 1.5 57.5 2 42.5 1.75 12.5 0.5 45 1.75 47.5 2 48 1.25 17.5 1.5
152 0 MMBVSSSMMKPR/CSMH/3038/04 35.71429 1.25 12.5 0.5 32.5 1.25 52.5 2 50 1.75 47.5 1.75 52.5 1.5 52.5 1.5 20 0.5 27.5 1
153 0 MMBVSSSMMKPR/CTMH/2852/04 12.85714 0.5 12.5 1.5 37.5 1.75 55 1.75 52.5 1.75 52.5 1.75 27.5 1.25 12.5 0.5 52 1.25 50 1.25
154 0 MMBVSSSMMKPR/CUMH/1591/03 17.14286 1.25 55 1.75 12.5 0.5 52.5 2 32.5 2 52.5 1.75 20 1.25 57.5 2 30 1 17.5 1
155 0 MMBVSSSMMKPR/CWMH/3473/06 20 1.25 47.5 1.5 12.5 0.5 12.5 0.5 12.5 0.5 27.5 1 45 1.75 12.5 0.5 20 0.5 27.5 1
156 0 MMBVSSSMMKPR/CXJH/3121/05 35.71429 1.25 47.5 1.5 27.5 1.75 32.5 2 47.5 1.75 50 2 12.5 0.5 12.5 0.5 34 1.25 42.5 1
49
Table 3B
Sl.
No Outcome Sample No.
U%-
M
UIM
U%-C
UIC
V%-
M
VIM
V%-
C
VIC
W%-
M
WIM
W%-
C
WIC
ZA1%-
M
ZA1IM
ZA1%-
C
ZA1IC
ZB%-
C
1 1 MMBVSSSMMKPR/AAJH/0467/06 42.5 1.5 56.36363636 2 60 2 10 0.25 52.5 2 50 2 47.5 1.75 37.5 1.5 37.5
2 1 MMBVSSSMMKPR/ABMH/4734/07 42.5 1.75 49.09090909 2 60 2 10 0.25 60 2 7.5 0.5 47.5 1.5 42.5 1.5 37.5
3 1 MMBVSSSMMKPR/HLRG/1219/03 27.5 1.25 38.18181818 2 15 0.5 20 1.25 47.5 1.25 47.5 1.75 50 1.75 37.5 1.25 42.5
4 1 MMBVSSSMMKPR/ACRG/2834/03 27.5 1.5 52.72727273 1.75 55 1.5 10 0.25 52.5 1.75 42.5 1.5 17.5 1 42.5 1.5 57.5
5 1 MMBVSSSMMKPR/AVRG/1397/04 22.5 1.5 54.54545455 2 22.5 1.25 25 1.25 27.5 1.25 50 1.75 22.5 1 42.5 1.25 52.5
6 1 MMBVSSSMMKPR/AEMH/6521/06 12.5 0.5 47.27272727 1.5 30 1.25 10 0.25 22.5 1.25 27.5 1.75 32.5 1.75 12.5 0.5 37.5
7 1 MMBVSSSMMKPR/AFJH/0478/06 32.5 1.25 50.90909091 1.75 55 2 10 0.25 52.5 1.5 12.5 1.5 12.5 0.5 12.5 0.5 47.5
8 1 MMBVSSSMMKPR/HTMX/0675/08 17.5 1 34.54545455 1.5 40 1.5 40 1.25 25 1.25 37.5 1.75 47.5 1.75 37.5 1.5 47.5
9 1 MMBVSSSMMKPR/IRRG/5724/08 47.5 1.5 41.81818182 1.75 50 1.5 45 1.25 45 1 37.5 1.75 47.5 1.5 37.5 1 52.5
10 1 MMBVSSSMMKPR/JMRG/1480/08 22.5 1 49.09090909 1.75 60 1.75 47.5 1 52.5 2 42.5 2 22.5 1.25 50 1.75 42.5
11 1 MMBVSSSMMKPR/HCGA1/222/10 17.5 1.25 49.09090909 1.25 50 1.5 40 1.25 47.5 1.25 22.5 1.25 42.5 1.75 12.5 0.5 47.5
12 1 MMBVSSSMMKPR/KIDC/0164/06 32.5 1 52.72727273 1.75 55 2 10 0.25 45 1.5 32.5 1.5 47.5 2 12.5 0.5 47.5
13 1 MMBVSSSMMKPR/KJDC/0167/06 22.5 1.25 49.09090909 1.25 55 2 35 1.25 52.5 2 37.5 2 45 1.75 12.5 0.5 50
14 1 MMBVSSSMMKPR/KNDC/0176/05 32.5 1 49.09090909 1.25 45 2 35 1.75 47.5 1.5 42.5 1.75 55 2 12.5 0.5 47.5
15 1 MMBVSSSMMKPR/MH11/3187/09 42.5 1.5 50.90909091 1.5 50 1.25 40 1 42.5 1.5 42.5 1.75 47.5 1.5 12.5 0.5 47.5
157 0 MMBVSSSMMKPR/CYJH/0878/06 18.57143 1.25 52.5 1.5 32.5 1.75 55 2 12.5 0.5 37.5 1.25 12.5 0.5 45 2 24 1.75 52.5 1.5
158 0 MMBVSSSMMKPR/RG26/5002/09 34.28571 1.25 52.5 1.25 47.5 1.25 52.5 1.75 42.5 1.25 50 1.75 20 1.25 27.5 1.25 28 1.25 37.5 1.75
159 0 MMBVSSSMMKPR/DAMH/7095/09 34.28571 1.25 35 1.5 52.5 1.5 22.5 1.5 12.5 0.5 12.5 0.5 37.5 1.5 57.5 2 44 1.25 22.5 1.25
160 0 MMBVSSSMMKPR/DBMH/9616/08 24.28571 1.5 52.5 1 30 1.5 35 1.5 25 1.5 22.5 1 57.5 1.75 42.5 1.25 24 1.25 22.5 1
50
16 1 MMBVSSSMMKPR/HCGA4/5535/06 12.5 0.5 49.09090909 1.5 15 0.5 10 0.25 47.5 1.5 47.5 1.5 17.5 1.5 17.5 1 52.5
17 1 MMBVSSSMMKPR/SP5/6544/08 12.5 0.5 49.09090909 1.5 15 0.5 10 0.25 25 1.5 45 1.5 12.5 0.5 47.5 1.25 47.5
18 1 MMBVSSSMMKPR/SP6/5298/06 37.5 1.25 52.72727273 1.75 55 1.75 50 1.5 50 1.5 45 1.75 50 1.5 50 1.5 45
19 1 MMBVSSSMMKPR/SP6/6116/06 27.5 1 49.09090909 1.5 52.5 1.5 50 1.5 50 1.5 47.5 1.75 50 1.75 50 1.75 52.5
20 1 MMBVSSSMMKPR/SP8/6543/06 52.5 1.5 52.72727273 1.75 55 1.5 45 1.5 52.5 1.75 47.5 1.75 52.5 1.75 52.5 1.75 52.5
21 1 MMBVSSSMMKPR/SP9/1761/97 37.5 1.5 52.72727273 1.75 32.5 1.5 45 1.5 32.5 1 47.5 1.75 37.5 1.5 40 1.25 52.5
22 1 MMBVSSSMMKPR/SP9/2006/02 27.5 1.25 49.09090909 1.75 42.5 1.75 45 1.5 47.5 1.75 45 1.75 50 1.75 42.5 1 47.5
23 1 MMBVSSSMMKPR/SP10/4338/05 12.5 0.5 49.09090909 1.25 47.5 1.75 47.5 1.5 52.5 1.5 47.5 1.75 52.5 1.5 47.5 1.75 47.5
24 1 MMBVSSSMMKPR/SP13/0242/10 47.5 1.25 52.72727273 1.75 55 1.5 50 1.25 50 1.5 42.5 1.25 52.5 1.5 47.5 1.5 47.5
25 1 MMBVSSSMMKPR/ADRG/1440/02 32.5 1.5 49.09090909 1.75 40 1.5 10 0.25 27.5 1 7.5 0.5 42.5 1 12.5 0.5 42.5
26 1 MMBVSSSMMKPR/AGMH/2268/04 12.5 0.5 50.90909091 2 55 2 10 0.25 50 2 7.5 0.5 42.5 1.75 32.5 1.5 52.5
27 1 MMBVSSSMMKPR/AHMH/2180/03 32.5 1.25 49.09090909 1.75 55 2 55 1.75 57.5 2 27.5 1.25 52.5 1.75 20 1.25 50
28 1 MMBVSSSMMKPR/AJMH/1975/03 37.5 1.25 52.72727273 1.25 60 2 10 0.25 55 2 7.5 0.5 57.5 2 12.5 0.5 37.5
29 1 MMBVSSSMMKPR/AKMH/1186/03 12.5 0.5 56.36363636 1.5 60 2 37.5 1.5 60 2 7.5 0.5 27.5 1 52.5 1.75 57.5
30 1 MMBVSSSMMKPR/ALMH/4227/05 20 1.25 38.18181818 1.25 37.5 1.5 10 0.25 45 1.5 7.5 0.5 52.5 2 12.5 0.5 42.5
31 1 MMBVSSSMMKPR/AMMH/2138/05 22.5 1.5 27.27272727 1.25 60 2 10 0.25 57.5 2 7.5 0.5 47.5 1.75 12.5 0.5 37.5
32 1 MMBVSSSMMKPR/ANMH/6528/05 22.5 1.5 52.72727273 2 60 2 55 1.75 52.5 2 22.5 2 12.5 0.5 37.5 1.5 42.5
33 1 MMBVSSSMMKPR/AOMH/1621/05 37.5 2 34.54545455 1.5 55 2 55 1.5 52.5 1.75 42.5 2 27.5 1.5 27.5 1 30
34 1 MMBVSSSMMKPR/APMH/6408/05 42.5 2 52.72727273 2 57.5 2 10 0.25 55 2 47.5 1.75 42.5 1.75 32.5 1.75 37.5
35 1 MMBVSSSMMKPR/AQMH/1408/06 47.5 2 52.72727273 2 60 2 10 0.25 57.5 2 22.5 1.25 50 1.75 12.5 0.5 25
36 1 MMBVSSSMMKPR/ARMH/4622/06 27.5 1.25 49.09090909 1.5 15 0.5 20 1.25 42.5 1.5 32.5 1 60 2 12.5 0.5 55
37 1 MMBVSSSMMKPR/ASRG/5174/03 20 1.75 41.81818182 1.5 35 1.25 50 1.25 27.5 1.25 17.5 1.25 32.5 1.25 37.5 1.5 47.5
38 1 MMBVSSSMMKPR/ATMH/4074/01 30 1.25 49.09090909 2 15 0.5 40 1.75 37.5 1.5 47.5 1.75 42.5 1.5 12.5 0.5 27.5
39 1 MMBVSSSMMKPR/AYRG/3670/02 12.5 0.5 52.72727273 1.5 15 0.5 45 1.5 12.5 0.5 42.5 1.75 17.5 1.25 42.5 1.5 52.5
40 1 MMBVSSSMMKPR/AZRG/1472/02 20 1.25 50.90909091 1.5 22.5 1.25 25 1.25 47.5 1.5 42.5 1.75 12.5 0.5 47.5 1.25 52.5
41 1 MMBVSSSMMKPR/BAJH/0102/07 40 1.75 50.90909091 2 45 1.5 20 1.25 47.5 1.25 42.5 1.25 47.5 1.75 37.5 2 42.5
42 1 MMBVSSSMMKPR/HSMH/4789/07 27.5 1 49.09090909 1.5 15 0.5 52.5 1.75 22.5 1 37.5 1.5 47.5 2 12.5 0.5 32.5
51
43 1 MMBVSSSMMKPR/HKRG/3754/03 12.5 0.5 54.54545455 1.5 15 0.5 42.5 1.25 12.5 0.5 37.5 1.5 27.5 1.25 42.5 1.25 47.5
44 1 MMBVSSSMMKPR/IMRG/5141/08 12.5 0.5 47.27272727 2 15 0.5 10 0.25 22.5 1.5 37.5 1.5 27.5 1 52.5 1.75 47.5
45 1 MMBVSSSMMKPR/IQRG/5751/08 12.5 0.5 52.72727273 2 15 0.5 10 0.25 32.5 1.5 47.5 1.75 37.5 1.5 42.5 1.25 27.5
46 1 MMBVSSSMMKPR/JNRG/6963/08 32.5 1.25 49.09090909 1.75 25 1.25 45 1.5 32.5 1 27.5 1.25 42.5 1.5 37.5 1.75 47.5
47 1 MMBVSSSMMKPR/JORG/6520/08 27.5 1.25 54.54545455 2 52.5 1.75 52.5 1.25 52.5 2 42.5 1.5 47.5 1.75 42.5 1.75 42.5
48 1 MMBVSSSMMKPR/IXRG/1912/08 12.5 0.5 58.18181818 2 45 1.5 37.5 1.25 27.5 1.25 37.5 1.75 27.5 1.5 42.5 1.5 57.5
49 1 MMBVSSSMMKPR/IHRG/1461/07 37.5 1 52.72727273 2 55 2 20 1.25 47.5 2 32.5 1.75 57.5 2 12.5 0.5 47.5
50 1 MMBVSSSMMKPR/INRG/6074/08 12.5 0.5 50.90909091 2 15 0.5 10 0.25 42.5 1.5 22.5 1.25 12.5 0.5 52.5 2 55
51 1 MMBVSSSMMKPR/JPRG/7119/08 12.5 0.5 34.54545455 1 55 1.5 10 0.25 52.5 1.75 42.5 2 52.5 1.75 42.5 1.75 27.5
52 1 MMBVSSSMMKPR/IARG/1209/01 37.5 1 38.18181818 1.5 45 1.5 20 1.5 42.5 1.75 7.5 0.5 35 1.25 37.5 1.25 27.5
53 1 MMBVSSSMMKPR/JQRG/6998/08 25 1 54.54545455 1.75 57.5 1.75 22.5 1.25 55 2 42.5 2 52.5 1.75 47.5 1.5 37.5
54 1 MMBVSSSMMKPR/HCGA1/5042/08 17.5 1 52.72727273 1 40 1.5 27.5 0.75 52.5 1.75 22.5 1.5 22.5 1 37.5 1 42.5
55 1 MMBVSSSMMKPR/RG20/7272/08 37.5 1.25 52.72727273 1.75 50 1.5 45 1.25 47.5 1.5 42.5 1.5 27.5 1 47.5 1.5 52.5
56 1 MMBVSSSMMKPR/MH10/826/06 12.5 0.5 32.72727273 1.5 50 1.75 45 1.5 47.5 1.5 35 1.25 45 1.5 17.5 1.25 27.5
57 1 MMBVSSSMMKPR/MH12/6907/09 37.5 1.25 52.72727273 1.5 45 1.5 45 1.5 45 1.25 32.5 1 42.5 1.5 37.5 1.25 52.5
58 1 MMBVSSSMMKPR/MH13/5276/09 47.5 1.5 52.72727273 1.5 40 1.5 50 1.5 37.5 1.5 32.5 1.5 12.5 0.5 37.5 1.25 47.5
59 1 MMBVSSSMMKPR/MH13/2852/02 32.5 1.5 52.72727273 1.5 57.5 1.75 52.5 1.25 55 1.75 50 1.5 50 1.5 37.5 1.25 42.5
60 1 MMBVSSSMMKPR/RG24/2672/09 17.5 1 52.72727273 1.75 20 1 50 1.25 17.5 1 37.5 1.25 12.5 0.5 37.5 0.75 52.5
61 1 MMBVSSSMMKPR/RG24/786/09 32.5 1.25 49.09090909 1.5 40 1.25 35 1 47.5 1.5 42.5 1.5 42.5 1.25 22.5 1 42.5
62 1 MMBVSSSMMKPR/RG24/3773/09 32.5 1.5 52.72727273 1.5 55 1.5 50 1.25 50 1.75 45 1.5 47.5 1.5 47.5 1.5 47.5
63 1 MMBVSSSMMKPR/RG24/3821/09 20 1.25 52.72727273 1.75 25 1.5 47.5 1.25 42.5 1.5 37.5 1.5 37.5 1.5 25 1 32.5
64 1 MMBVSSSMMKPR/RG25/1072/09 27.5 1 49.09090909 1.25 15 0.5 20 0.75 27.5 1 37.5 1.25 12.5 0.5 25 1 52.5
65 1 MMBVSSSMMKPR/HCGA5/7545/07 47.5 1.25 50.90909091 1.5 50 1.5 40 1 52.5 1.5 47.5 1.5 52.5 1.5 47.5 1.25 52.5
66 1 MMBVSSSMMKPR/SP2/2851/03 17.5 1.25 49.09090909 1.5 30 1.75 52.5 1.5 55 1.75 50 1.75 22.5 1 50 1.5 47.5
67 1 MMBVSSSMMKPR/SP2/06011/08 17.5 1 52.72727273 1.75 27.5 1.5 52.5 1.5 27.5 1.5 50 1.75 25 1.25 50 1.5 52.5
68 1 MMBVSSSMMKPR/SP2/5595E/04 42.5 1.5 52.72727273 1.75 52.5 1.75 47.5 1.5 50 1.5 42.5 1.5 50 1.5 50 1.75 47.5
69 1 MMBVSSSMMKPR/SP3/12267/07 42.5 1.5 50.90909091 1.75 45 1.5 42.5 1.25 50 1.75 42.5 1.5 52.5 1.75 37.5 1.5 52.5
52
70 1 MMBVSSSMMKPR/SP3/09935/08 45 1.5 50.90909091 1.75 57.5 1.75 35 1.25 55 1.75 42.5 1.5 52.5 1.75 47.5 1.5 52.5
71 1 MMBVSSSMMKPR/SP4/04418/08 25 1.5 52.72727273 1.75 27.5 1.25 47.5 1.5 32.5 1.25 50 2 37.5 1.5 42.5 1.5 52.5
72 1 MMBVSSSMMKPR/KFDC/0149/07 32.5 1.25 41.81818182 1.5 47.5 1.75 10 0.25 45 1.5 27.5 1.5 50 2 12.5 0.5 27.5
73 1 MMBVSSSMMKPR/BDRG/5248/04 12.5 0.5 50.90909091 1.25 15 0.5 40 1.25 22.5 1 42.5 1.75 37.5 1.75 47.5 2 40
74 1 MMBVSSSMMKPR/KODC/0178/05 22.5 1 41.81818182 1.5 25 1.5 25 1.25 12.5 0.5 20 1.5 37.5 2 12.5 0.5 22.5
75 1 MMBVSSSMMKPR/SP5/2503/08 17.5 1 52.72727273 1.5 55 1.75 45 1.25 55 1.75 50 1.75 55 1.75 55 1.75 52.5
76 1 MMBVSSSMMKPR/SP5/2049/09 52.5 1.5 54.54545455 1.75 55 1.75 50 1.5 55 1.75 50 1.75 55 1.75 50 1.5 45
77 1 MMBVSSSMMKPR/SP5/2933/09 52.5 1.5 50.90909091 1.5 50 1.5 25 1.25 52.5 1.75 45 1.5 55 1.75 42.5 1.5 52.5
78 1 MMBVSSSMMKPR/SP5/5414/09 12.5 0.5 50.90909091 1.5 52.5 1.5 45 1.25 50 1.75 42.5 1.5 52.5 1.75 47.5 1.75 47.5
79 1 MMBVSSSMMKPR/SP5/6317/09 12.5 0.5 50.90909091 1.5 25 1.5 50 1.5 40 1.5 45 1.75 52.5 1.75 47.5 1.75 50
80 1 MMBVSSSMMKPR/SP5/5630/07 52.5 1.5 52.72727273 1.75 55 1.75 50 1.5 55 1.75 50 1.75 55 1.75 27.5 1.5 52.5
81 1 MMBVSSSMMKPR/SP6/4628/02 40 1.25 50.90909091 1.5 50 1.75 47.5 1.5 50 1.5 45 1.5 40 1.5 50 1.5 52.5
82 1 MMBVSSSMMKPR/SP6/215/05 12.5 0.5 52.72727273 1.75 22.5 1.5 50 1.5 27.5 1.5 47.5 1.75 25 1.5 50 1.5 52.5
83 1 MMBVSSSMMKPR/SP6/4283/08 22.5 1 52.72727273 1.25 45 1.5 50 1.5 50 1.5 45 1.5 52.5 1.75 52.5 1.5 52.5
84 1 MMBVSSSMMKPR/SP6/3004/09 47.5 1.5 50.90909091 1.5 55 1.75 50 1.5 50 1.75 40 1.5 50 1.75 50 1.75 32.5
85 1 MMBVSSSMMKPR/SP7/6675/07 47.5 1.25 52.72727273 1.75 55 1.5 47.5 1.25 52.5 1.5 45 1.75 52.5 1.5 50 1.5 52.5
86 1 MMBVSSSMMKPR/SP7/4924/06 22.5 1.25 50.90909091 1.5 47.5 1.5 50 1.5 47.5 1.5 47.5 1.75 25 1.5 47.5 1.25 47.5
87 1 MMBVSSSMMKPR/SP8/7537/09 12.5 0.5 49.09090909 1.25 32.5 1.5 35 1 35 1.5 37.5 1.5 20 1.5 47.5 1.5 47.5
88 1 MMBVSSSMMKPR/SP8/3347/09 45 1.5 50.90909091 1.5 52.5 1.5 47.5 1.25 47.5 1.75 42.5 1.5 47.5 1.5 50 1.5 45
89 1 MMBVSSSMMKPR/SP8/7252/08 35 1.5 52.72727273 1.75 55 1.5 50 1.5 42.5 1.5 45 1.75 50 1.5 50 1.5 50
90 1 MMBVSSSMMKPR/AURG/1691/04 22.5 1 43.63636364 1.5 40 1.25 22.5 1.25 32.5 1 15 1.75 42.5 1.75 37.5 1.5 42.5
91 1 MMBVSSSMMKPR/AWRG/3418/04 27.5 1.5 49.09090909 1.5 25 1.25 10 0.25 27.5 1.5 7.5 0.5 27.5 1.25 47.5 1.75 47.5
92 1 MMBVSSSMMKPR/HCGA1/6737/09 12.5 0.5 52.72727273 1.75 35 1.5 40 1 50 1.75 17.5 1 42.5 1.5 25 1 52.5
93 1 MMBVSSSMMKPR/HCGA3/5860/09 22.5 1.25 52.72727273 1.5 55 1.75 50 1.25 42.5 1.5 32.5 1.25 47.5 1.5 42.5 1.5 52.5
94 1 MMBVSSSMMKPR/BBMH/6814/07 17.5 1 49.09090909 1.75 35 1.5 40 1.5 22.5 1 20 1 52.5 1 12.5 0.5 52.5
95 1 MMBVSSSMMKPR/BEMH/3166/01 25 1.5 49.09090909 1.5 50 1.25 20 1.25 47.5 1.5 7.5 0.5 57.5 2 12.5 0.5 17.5
96 1 MMBVSSSMMKPR/RG25/2941/09 37.5 1.25 52.72727273 1.5 20 1.25 10 0.25 52.5 1.5 47.5 1.5 12.5 0.5 47.5 1.5 52.5
53
97 1 MMBVSSSMMKPR/SP1/1287/98 27.5 1.25 52.72727273 1.75 47.5 1.5 45 1.25 50 1.5 45 1.75 52.5 1.5 52.5 1.75 17.5
98 1 MMBVSSSMMKPR/SP2/00396/06 42.5 1.5 52.72727273 2 57.5 1.75 52.5 1.5 52.5 1.75 47.5 1.75 57.5 1.75 42.5 1.25 52.5
99 1 MMBVSSSMMKPR/SP2/5090/03 22.5 1 52.72727273 1.5 45 1.75 52.5 1.5 22.5 1.5 47.5 1.75 42.5 1.75 42.5 1.5 52.5
100 1 MMBVSSSMMKPR/SP3/00076/06 42.5 1.25 52.72727273 1.75 55 1.5 45 1.25 55 1.75 45 1.5 42.5 1.5 52.5 1.75 55
101 1 MMBVSSSMMKPR/SP5/5658/09 12.5 0.5 54.54545455 1.75 40 1.5 50 1.5 42.5 1.5 50 1.75 45 1.5 42.5 1.5 52.5
102 1 MMBVSSSMMKPR/SP9/1431/07 32.5 1.25 49.09090909 1.5 27.5 1.5 50 1.5 35 1.5 45 1.75 37.5 1.5 37.5 1 47.5
103 1 MMBVSSSMMKPR/SP9/00521/07 37.5 1.25 49.09090909 1.75 50 1.75 45 1.5 50 1.75 45 1.75 52.5 1.75 25 1.5 52.5
104 1 MMBVSSSMMKPR/SP9/919/99 42.5 1.25 52.72727273 1.75 15 0.5 10 0.25 17.5 1.5 47.5 1.75 17.5 1.25 52.5 1.5 52.5
105 1 MMBVSSSMMKPR/SP10/1693/03 47.5 1.25 49.09090909 1.25 55 1.75 50 1.5 52.5 1.75 47.5 1.75 52.5 1.75 47.5 1.25 45
106 1 MMBVSSSMMKPR/SP11/2802/01 52.5 1.5 50.90909091 1.75 52.5 1.5 42.5 1.25 52.5 1.5 45 1.5 52.5 1.5 40 1.5 52.5
107 1 MMBVSSSMMKPR/SP11/1002/05 47.5 1.25 45.45454545 1.5 45 1.5 37.5 1.25 52.5 1.5 42.5 1.5 45 1.5 42.5 1.5 50
108 1 MMBVSSSMMKPR/SP11/4945/09 42.5 1.25 49.09090909 1.5 50 1.5 45 1 47.5 1.5 42.5 1.5 45 1.5 42.5 1.5 37.5
109 1 MMBVSSSMMKPR/RG32/47/10 42.5 1.25 50.90909091 1.5 52.5 1.75 42.5 1.25 37.5 1 45 1.5 50 1.5 45 1.25 47.5
110 1 MMBVSSSMMKPR/RG32/7449/09 50 1.5 40 1.5 52.5 1.75 22.5 1 40 1.5 35 1.5 52.5 1.5 45 1.25 37.5
111 1 MMBVSSSMMKPR/SP12/0353/09 50 1.25 52.72727273 1.5 55 1.5 50 1.25 52.5 1.5 32.5 1.5 57.5 1.75 20 1.5 12.5
112 1 MMBVSSSMMKPR/SP12/2027/09 47.5 1.25 52.72727273 1.75 45 1.5 45 1.25 50 1.5 45 1.75 42.5 1.5 42.5 1.25 47.5
113 1 MMBVSSSMMKPR/SP12/1631/09 47.5 1.5 52.72727273 1.75 60 1.75 55 1.5 52.5 1.75 47.5 1.75 57.5 1.75 37.5 1.5 37.5
114 1 MMBVSSSMMKPR/RG34/1091/10 42.5 1.25 52.72727273 1.75 40 1.25 40 1.25 57.5 1.5 52.5 1.75 47.5 1.5 47.5 1.5 50
115 1 MMBVSSSMMKPR/SP13/6097/08 42.5 1.25 52.72727273 1.5 55 1.5 50 1.25 52.5 1.5 45 1.75 52.5 1.5 47.5 1.5 50
116 1 MMBVSSSMMKPR/BFMH/2540/04 17.5 1.25 32.72727273 1.25 27.5 2 10 0.25 37.5 1.5 7.5 0.5 55 2 42.5 1.25 27.5
117 1 MMBVSSSMMKPR/BGMH/3558/03 12.5 0.5 56.36363636 1.25 62.5 2 27.5 1.25 60 2 20 1.5 42.5 1.75 27.5 1.25 47.5
118 1 MMBVSSSMMKPR/BIMH/4596/05 37.5 1.5 34.54545455 1.5 57.5 2 10 0.25 42.5 2 7.5 0.5 47.5 1.75 37.5 1.75 32.5
119 1 MMBVSSSMMKPR/BJMH/1942/07 20 1.5 41.81818182 1.25 50 2 10 0.25 52.5 2 7.5 0.5 52.5 2 12.5 0.5 42.5
120 1 MMBVSSSMMKPR/BKMH/3435/06 47.5 2 56.36363636 2 40 1.5 50 1.75 57.5 2 47.5 2 32.5 1 12.5 0.5 42.5
121 1 MMBVSSSMMKPR/BLJH/2584/05 37.5 1.5 56.36363636 2 40 1.5 35 1.75 52.5 1.75 42.5 1.75 12.5 0.5 37.5 1.5 42.5
122 1 MMBVSSSMMKPR/BMMH/3591/08 37.5 1.75 49.09090909 2 60 2 10 0.25 60 2 20 1.75 50 1.75 12.5 0.5 40
123 1 MMBVSSSMMKPR/BNMH/3242/06 42.5 1.75 50.90909091 1.5 60 2 25 1.75 60 2 15 1.5 52.5 1.75 37.5 1.5 42.5
54
124 1 MMBVSSSMMKPR/BORG/0945/03 30 1.25 50.90909091 2 45 1 10 0.25 47.5 1.75 42.5 1.75 47.5 1.25 52.5 1.75 52.5
125 1 MMBVSSSMMKPR/BPRG/3398/03 37.5 1.25 52.72727273 2 20 1 10 0.25 25 1.5 42.5 1.25 25 1 47.5 1.75 47.5
126 1 MMBVSSSMMKPR/BQRG/4390/03 42.5 1.5 49.09090909 1.5 30 1.25 10 0.25 37.5 1.5 37.5 1.75 42.5 1.5 27.5 1.25 50
127 1 MMBVSSSMMKPR/BTRG/3606/03 35 1.5 52.72727273 2 30 1 25 0.75 40 1.5 22.5 1.5 32.5 1.25 42.5 1.75 57.5
128 1 MMBVSSSMMKPR/BUMH/6186/04 42.5 1.25 41.81818182 1.5 15 0.5 10 0.25 32.5 1.5 27.5 1.75 42.5 1.25 25 1.25 25
129 1 MMBVSSSMMKPR/BVMH/6080/07 25 1 49.09090909 1.25 40 1 40 1.5 32.5 1.5 47.5 1.75 57.5 2 12.5 0.5 52.5
130 1 MMBVSSSMMKPR/DMMX/3990/07 20 1.25 52.72727273 1.5 60 2 20 1.75 60 2 15 1.75 52.5 2 12.5 0.5 47.5
131 1 MMBVSSSMMKPR/ SP1/2238/01 27.5 1.25 52.72727273 1.5 15 0.5 10 0.25 20 1.25 37.5 1.5 25 1.25 50 1.5 47.5
132 1 MMBVSSSMMKPR/RG15/1464/08 25 1.25 49.09090909 1.25 50 1.5 45 1.25 47.5 1.75 17.5 1 47.5 1.75 27.5 1.75 47.5
133 1 MMBVSSSMMKPR/RG22/1931/09 22.5 1 52.72727273 1.5 27.5 1.25 45 1.5 27.5 1.25 42.5 1.25 17.5 1 42.5 1.5 42.5
134 1 MMBVSSSMMKPR/MH14/6324/08 37.5 1 45.45454545 1.25 60 2 25 1.25 47.5 2 7.5 0.5 12.5 0.5 12.5 0.5 47.5
135 1 MMBVSSSMMKPR/ RG24/805/09 20 1 52.72727273 1.75 35 1 50 1.5 52.5 1.5 47.5 1.75 32.5 1 25 1.25 52.5
136 1 MMBVSSSMMKPR/HCGA4/6374/08 52.5 1.5 52.72727273 1.5 55 1.5 50 1.25 50 1.5 47.5 1.5 37.5 1.25 32.5 1 52.5
137 1 MMBVSSSMMKPR/SP1/3187/99 20 1 49.09090909 1.5 55 1.75 50 1.5 32.5 1.25 47.5 1.75 20 1 45 1.5 37.5
138 1 MMBVSSSMMKPR/SP1/1543/00 32.5 1.25 52.72727273 1.5 45 1 25 0.75 47.5 1.5 45 1.75 52.5 1.75 52.5 1.75 52.5
139 1 MMBVSSSMMKPR/SP2/10817/07 30 1 45.45454545 1.5 57.5 1.75 52.5 1.5 52.5 1.75 47.5 1.75 55 1.75 50 1.5 47.5
140 1 MMBVSSSMMKPR/SP2/3424/03 22.5 1.25 52.72727273 1.5 30 1.5 45 1.5 50 1.5 47.5 1.75 42.5 1.5 55 1.75 42.5
141 1 MMBVSSSMMKPR/SP2/489/02 12.5 0.5 47.27272727 1.25 50 1.5 42.5 1 42.5 1.5 42.5 1.25 30 1.5 32.5 1.5 37.5
142 1 MMBVSSSMMKPR/SP3/5454/05 50 1.75 50.90909091 1.75 52.5 1.5 40 1.25 47.5 1.5 37.5 1.5 50 1.75 50 1.5 52.5
143 1 MMBVSSSMMKPR/SP3/00070/06 25 1.5 50.90909091 1.75 20 1.5 42.5 1 47.5 1.75 42.5 1.5 47.5 1.5 50 1.75 52.5
144 1 MMBVSSSMMKPR/SP4/11639/06 45 1.25 52.72727273 1.5 37.5 1.5 42.5 1.5 50 1.75 45 1.5 45 1.75 47.5 1.25 50
145 1 MMBVSSSMMKPR/SP4/12823/07 17.5 1.25 52.72727273 1.5 25 1.5 35 1.25 27.5 1.25 42.5 1.75 25 1.5 42.5 1.25 50
146 1 MMBVSSSMMKPR/SP4/14511/07 30 1.5 50.90909091 1.5 35 1.25 50 1.5 37.5 1.75 47.5 1.5 42.5 1.75 45 1.75 37.5
147 1 MMBVSSSMMKPR/IIRG/4358/08 42.5 1.25 49.09090909 1.5 35 1.25 40 1.25 47.5 1.5 37.5 1.5 47.5 2 52.5 1.5 47.5
148 1 MMBVSSSMMKPR/IVRG/1658/08 42.5 1.25 50.90909091 1.5 15 0.5 15 1.25 52.5 1.75 27.5 1.25 60 2 12.5 0.5 52.5
149 1 MMBVSSSMMKPR/JRRG/6716/08 22.5 1 52.72727273 1.75 22.5 1.25 47.5 1.75 42.5 1.5 32.5 1.5 17.5 1.5 52.5 1.75 47.5
150 1 MMBVSSSMMKPR/JSMH/7129/07 25 1.25 50.90909091 1.5 47.5 1.75 37.5 1.5 52.5 1.75 32.5 1.5 52.5 2 45 1.5 37.5
55
151 0 MMBVSSSMMKPR/CPMH/1187/05 27.5
1.25 23.63636 0.5
55
1.5 45 1.5 57.5 2 7.5 0.5 52.5 2 12.5 0.5 37.5
152 0
MMBVSSSMMKPR/CSMH/3038/04 17.5 1.5 52.72727 1.5 32.5 1.5 45 1.5 50 1.5 7.5 0.5 52.5 1.75 47.5 1 42.5
153 0
MMBVSSSMMKPR/CTMH/2852/04 30 1.5 56.36364 1.75 42.5 1.75 45 1.5 60 2 20 1.75 52.5 1.75 12.5 0.5 22.5
154 0
MMBVSSSMMKPR/CUMH/1591/03 12.5 0.5 45.45455 1.5 47.5 1.75 47.5 1.5 52.5 1.5 7.5 0.5 12.5 0.5 22.5 1 37.5
155 0
MMBVSSSMMKPR/CWMH/3473/06 17.5 1.25 23.63636 0.5 55 1.5 50 1.25 52.5 2 7.5 0.5 52.5 2 32.5 2 42.5
156 0
MMBVSSSMMKPR/CXJH/3121/05 45 1.25 36.36364 1.25 40 1.5 10 0.25 60 2 7.5 0.5 52.5 1.5 12.5 0.5 42.5
157 0
MMBVSSSMMKPR/CYJH/0878/06 12.5 0.5 56.36364 2 55 2 10 0.25 32.5 1.75 7.5 0.5 12.5 0.5 12.5 0.5 32.5
158 0
MMBVSSSMMKPR/RG26/5002/09 52.5 1.25 52.72727 1.75 55 2 55 1.75 52.5 1.75 47.5 1.5 52.5 1.75 52.5 1.5 52.5
159 0
MMBVSSSMMKPR/DAMH/7095/09 50 1.5 47.27273 1.25 60 2 10 0.25 60 2 15 1.5 47.5 2 37.5 1.75 22.5
160 0
MMBVSSSMMKPR/DBMH/9616/08 32.5 1.25 50.90909 2 60 2 37.5 1.5 50 1.75 32.5 1.5 57.5 2 57.5 1.25 47.5
Table 3C
Sl.
No. Outcome Sample No. ZC%-M ZCI-M ZC%-C ZCI-C ZD%-C ZDI-C ZF2%-N ZF2I-N
1 1 MMBVSSSMMKPR/AAJH/0467/06 33.33333333 0.666666667 76.36363636 2.5 83.91608392 3.333333333 74.59016393 2.666666667
2 1 MMBVSSSMMKPR/ABMH/4734/07 33.33333333 0.666666667 87.27272727 3.333333333 78.67132867 2.666666667 85.24590164 4
3 1 MMBVSSSMMKPR/HLRG/1219/03 33.33333333 0.666666667 32.72727273 2.5 83.91608392 2.666666667 63.93442623 4
4 1 MMBVSSSMMKPR/ACRG/2834/03 40 1.166666667 65.45454545 2.5 41.95804196 2 101.2295082 4
5 1 MMBVSSSMMKPR/AVRG/1397/04 33.33333333 0.666666667 87.27272727 3.333333333 83.91608392 4 26.63934426 2.666666667
56
6 1 MMBVSSSMMKPR/AEMH/6521/06 50 1.333333333 0 0.833333333 73.42657343 2.666666667 74.59016393 4
7 1 MMBVSSSMMKPR/AFJH/0478/06 33.33333333 0.666666667 76.36363636 2.5 94.40559441 4 0 0
8 1 MMBVSSSMMKPR/HTMX/0675/08 33.33333333 0.666666667 76.36363636 2.5 57.69230769 2.666666667 85.24590164 2.666666667
9 1 MMBVSSSMMKPR/IRRG/5724/08 46.66666667 1 87.27272727 3.333333333 52.44755245 3.333333333 10.6557377 2.666666667
10 1 MMBVSSSMMKPR/JMRG/1480/08 45 1.166666667 98.18181818 3.333333333 73.42657343 3.333333333 63.93442623 2.666666667
11 1 MMBVSSSMMKPR/HCGA1/222/10 33.33333333 0.666666667 76.36363636 2.5 62.93706294 2 10.6557377 2
12 1 MMBVSSSMMKPR/KIDC/0164/06 43.33333333 1.166666667 76.36363636 2.5 52.44755245 2 26.63934426 3.333333333
13 1 MMBVSSSMMKPR/KJDC/0167/06 40 1 76.36363636 2.916666667 73.42657343 2 10.6557377 2
14 1 MMBVSSSMMKPR/KNDC/0176/05 50 1.166666667 76.36363636 2.916666667 52.44755245 2.666666667 10.6557377 2.666666667
15 1 MMBVSSSMMKPR/MH11/3187/09 41.66666667 1.333333333 87.27272727 3.333333333 73.42657343 2 21.31147541 2
16 1 MMBVSSSMMKPR/HCGA4/5535/06 33.33333333 0.666666667 87.27272727 2.916666667 83.91608392 3.333333333 0 0
17 1 MMBVSSSMMKPR/SP5/6544/08 33.33333333 0.666666667 76.36363636 2.5 73.42657343 2.666666667 26.63934426 2.666666667
18 1 MMBVSSSMMKPR/SP6/5298/06 53.33333333 1 76.36363636 2.083333333 73.42657343 3.333333333 21.31147541 2
19 1 MMBVSSSMMKPR/SP6/6116/06 40 1 76.36363636 2.5 83.91608392 3.333333333 79.91803279 3.333333333
20 1 MMBVSSSMMKPR/SP8/6543/06 56.66666667 1.333333333 87.27272727 2.5 83.91608392 2.666666667 21.31147541 2
21 1 MMBVSSSMMKPR/SP9/1761/97 36.66666667 1 87.27272727 2.916666667 78.67132867 3.333333333 0 0
22 1 MMBVSSSMMKPR/SP9/2006/02 38.33333333 1.166666667 81.81818182 2.916666667 83.91608392 3.333333333 10.6557377 2.666666667
23 1 MMBVSSSMMKPR/SP10/4338/05 38.33333333 1.166666667 81.81818182 2.916666667 83.91608392 3.333333333 5.327868852 2.666666667
24 1 MMBVSSSMMKPR/SP13/0242/10 45 1.166666667 87.27272727 2.916666667 83.91608392 2.666666667 63.93442623 4
25 1 MMBVSSSMMKPR/ADRG/1440/02 40 1.166666667 54.54545455 2.5 52.44755245 2.666666667 95.90163934 3.333333333
26 1 MMBVSSSMMKPR/AGMH/2268/04 33.33333333 0.666666667 65.45454545 2.916666667 20.97902098 1.333333333 10.6557377 2.666666667
27 1 MMBVSSSMMKPR/AHMH/2180/03 33.33333333 0.666666667 43.63636364 3.333333333 83.91608392 2.666666667 31.96721311 3.333333333
28 1 MMBVSSSMMKPR/AJMH/1975/03 33.33333333 0.666666667 87.27272727 3.333333333 94.40559441 4 21.31147541 2.666666667
29 1 MMBVSSSMMKPR/AKMH/1186/03 33.33333333 0.666666667 87.27272727 3.333333333 94.40559441 4 15.98360656 2.666666667
30 1 MMBVSSSMMKPR/ALMH/4227/05 45 1.166666667 65.45454545 3.333333333 62.93706294 1.333333333 0 0
31 1 MMBVSSSMMKPR/AMMH/2138/05 41.66666667 1 87.27272727 2.916666667 73.42657343 4 74.59016393 2.666666667
32 1 MMBVSSSMMKPR/ANMH/6528/05 33.33333333 0.666666667 87.27272727 3.333333333 83.91608392 4 31.96721311 2.666666667
57
33 1 MMBVSSSMMKPR/AOMH/1621/05 33.33333333 0.666666667 54.54545455 2.916666667 73.42657343 3.333333333 0 0
34 1 MMBVSSSMMKPR/APMH/6408/05 33.33333333 0.666666667 87.27272727 3.333333333 73.42657343 3.333333333 15.98360656 2.666666667
35 1 MMBVSSSMMKPR/AQMH/1408/06 33.33333333 0.666666667 10.90909091 1.666666667 83.91608392 3.333333333 74.59016393 2.666666667
36 1 MMBVSSSMMKPR/ARMH/4622/06 41.66666667 1 76.36363636 2.916666667 83.91608392 4 79.91803279 2.666666667
37 1 MMBVSSSMMKPR/ASRG/5174/03 36.66666667 1 65.45454545 2.5 78.67132867 4 26.63934426 2.666666667
38 1 MMBVSSSMMKPR/ATMH/4074/01 33.33333333 0.666666667 65.45454545 3.333333333 62.93706294 2.666666667 85.24590164 4
39 1 MMBVSSSMMKPR/AYRG/3670/02 33.33333333 0.666666667 98.18181818 3.333333333 73.42657343 2.666666667 0 0
40 1 MMBVSSSMMKPR/AZRG/1472/02 48.33333333 1.166666667 87.27272727 2.916666667 73.42657343 3.333333333 0 0
41 1 MMBVSSSMMKPR/BAJH/0102/07 56.66666667 1.5 87.27272727 2.916666667 83.91608392 4 79.91803279 3.333333333
42 1 MMBVSSSMMKPR/HSMH/4789/07 33.33333333 0.666666667 65.45454545 2.083333333 73.42657343 4 95.90163934 3.333333333
43 1 MMBVSSSMMKPR/HKRG/3754/03 36.66666667 1 87.27272727 2.5 68.18181818 2.666666667 15.98360656 1.333333333
44 1 MMBVSSSMMKPR/IMRG/5141/08 33.33333333 0.666666667 98.18181818 3.333333333 104.8951049 4 31.96721311 2.666666667
45 1 MMBVSSSMMKPR/IQRG/5751/08 33.33333333 0.666666667 76.36363636 2.5 83.91608392 2.666666667 74.59016393 4
46 1 MMBVSSSMMKPR/JNRG/6963/08 45 1.166666667 98.18181818 3.333333333 94.40559441 1.333333333 53.27868852 2
47 1 MMBVSSSMMKPR/JORG/6520/08 46.66666667 1.5 87.27272727 3.333333333 94.40559441 4 31.96721311 2.666666667
48 1 MMBVSSSMMKPR/IXRG/1912/08 43.33333333 1.166666667 76.36363636 3.333333333 94.40559441 4 0 0
49 1 MMBVSSSMMKPR/IHRG/1461/07 33.33333333 0.666666667 87.27272727 2.916666667 83.91608392 4 10.6557377 1.333333333
50 1 MMBVSSSMMKPR/INRG/6074/08 33.33333333 0.666666667 65.45454545 2.916666667 73.42657343 4 21.31147541 1.333333333
51 1 MMBVSSSMMKPR/JPRG/7119/08 36.66666667 1 76.36363636 2.5 83.91608392 3.333333333 31.96721311 1.333333333
52 1 MMBVSSSMMKPR/IARG/1209/01 33.33333333 0.666666667 76.36363636 2.5 78.67132867 3.333333333 95.90163934 3.333333333
53 1 MMBVSSSMMKPR/JQRG/6998/08 46.66666667 1 92.72727273 3.333333333 78.67132867 2.666666667 58.60655738 3.333333333
54 1 MMBVSSSMMKPR/HCGA1/5042/08 36.66666667 1 87.27272727 2.5 83.91608392 3.333333333 10.6557377 2
55 1 MMBVSSSMMKPR/RG20/7272/08 38.33333333 1.166666667 87.27272727 2.5 83.91608392 2.666666667 0 0
56 1 MMBVSSSMMKPR/MH10/826/06 36.66666667 1 87.27272727 2.916666667 41.95804196 2 69.26229508 3.333333333
57 1 MMBVSSSMMKPR/MH12/6907/09 40 1.166666667 87.27272727 2.916666667 73.42657343 3.333333333 15.98360656 2
58 1 MMBVSSSMMKPR/MH13/5276/09 33.33333333 0.666666667 87.27272727 2.916666667 83.91608392 2.666666667 0 0
59 1 MMBVSSSMMKPR/MH13/2852/02 38.33333333 1.166666667 87.27272727 2.083333333 83.91608392 2 95.90163934 3.333333333
58
60 1 MMBVSSSMMKPR/RG24/2672/09 43.33333333 1 76.36363636 1.666666667 68.18181818 2.666666667 0 0
61 1 MMBVSSSMMKPR/RG24/786/09 53.33333333 1.166666667 65.45454545 2.083333333 62.93706294 2.666666667 0 0
62 1 MMBVSSSMMKPR/RG24/3773/09 33.33333333 0.666666667 76.36363636 2.5 83.91608392 2 79.91803279 3.333333333
63 1 MMBVSSSMMKPR/RG24/3821/09 43.33333333 1 76.36363636 1.666666667 52.44755245 2 10.6557377 3.333333333
64 1 MMBVSSSMMKPR/RG25/1072/09 38.33333333 1.166666667 65.45454545 2.083333333 83.91608392 2.666666667 0 0
65 1 MMBVSSSMMKPR/HCGA5/7545/07 55 1 87.27272727 2.916666667 83.91608392 3.333333333 74.59016393 3.333333333
66 1 MMBVSSSMMKPR/SP2/2851/03 36.66666667 1.166666667 92.72727273 2.916666667 83.91608392 3.333333333 0 0
67 1 MMBVSSSMMKPR/SP2/06011/08 36.66666667 1.166666667 92.72727273 2.916666667 83.91608392 3.333333333 0 0
68 1 MMBVSSSMMKPR/SP2/5595E/04 33.33333333 0.666666667 81.81818182 2.5 83.91608392 3.333333333 31.96721311 3.333333333
69 1 MMBVSSSMMKPR/SP3/12267/07 50 1.333333333 81.81818182 2.916666667 78.67132867 2.666666667 53.27868852 2.666666667
70 1 MMBVSSSMMKPR/SP3/09935/08 58.33333333 1.333333333 87.27272727 2.916666667 73.42657343 2.666666667 0 0
71 1 MMBVSSSMMKPR/SP4/04418/08 33.33333333 0.666666667 65.45454545 2.5 89.16083916 3.333333333 0 0
72 1 MMBVSSSMMKPR/KFDC/0149/07 50 1.166666667 65.45454545 2.916666667 52.44755245 2 42.62295082 2.666666667
73 1 MMBVSSSMMKPR/BDRG/5248/04 33.33333333 0.666666667 70.90909091 2.916666667 78.67132867 3.333333333 0 0
74 1 MMBVSSSMMKPR/KODC/0178/05 33.33333333 0.666666667 76.36363636 2.5 41.95804196 2.666666667 74.59016393 2.666666667
75 1 MMBVSSSMMKPR/SP5/2503/08 43.33333333 1.166666667 87.27272727 2.5 83.91608392 2 0 0
76 1 MMBVSSSMMKPR/SP5/2049/09 53.33333333 1.166666667 76.36363636 2.5 73.42657343 2.666666667 0 0
77 1 MMBVSSSMMKPR/SP5/2933/09 50 1 32.72727273 2.083333333 52.44755245 2 21.31147541 2.666666667
78 1 MMBVSSSMMKPR/SP5/5414/09 43.33333333 1 76.36363636 2.083333333 73.42657343 2.666666667 42.62295082 2.666666667
79 1 MMBVSSSMMKPR/SP5/6317/09 33.33333333 0.666666667 81.81818182 2.083333333 83.91608392 3.333333333 69.26229508 2.666666667
80 1 MMBVSSSMMKPR/SP5/5630/07 43.33333333 1.166666667 87.27272727 2.5 73.42657343 2.666666667 90.57377049 4
81 1 MMBVSSSMMKPR/SP6/4628/02 45 1.166666667 81.81818182 2.5 83.91608392 3.333333333 37.29508197 2.666666667
82 1 MMBVSSSMMKPR/SP6/215/05 41.66666667 1 87.27272727 2.5 83.91608392 3.333333333 0 0
83 1 MMBVSSSMMKPR/SP6/4283/08 53.33333333 1 76.36363636 2.083333333 83.91608392 3.333333333 85.24590164 3.333333333
84 1 MMBVSSSMMKPR/SP6/3004/09 50 1.166666667 65.45454545 2.5 68.18181818 3.333333333 26.63934426 1.333333333
85 1 MMBVSSSMMKPR/SP7/6675/07 55 1.166666667 87.27272727 2.5 83.91608392 2.666666667 31.96721311 2
86 1 MMBVSSSMMKPR/SP7/4924/06 33.33333333 0.666666667 87.27272727 2.916666667 83.91608392 3.333333333 0 0
59
87 1 MMBVSSSMMKPR/SP8/7537/09 38.33333333 1.166666667 65.45454545 2.5 68.18181818 2.666666667 31.96721311 3.333333333
88 1 MMBVSSSMMKPR/SP8/3347/09 53.33333333 1.333333333 87.27272727 2.916666667 73.42657343 3.333333333 95.90163934 3.333333333
89 1 MMBVSSSMMKPR/SP8/7252/08 43.33333333 1.166666667 81.81818182 2.916666667 78.67132867 2.666666667 63.93442623 2.666666667
90 1 MMBVSSSMMKPR/AURG/1691/04 43.33333333 1 76.36363636 2.916666667 73.42657343 3.333333333 53.27868852 2.666666667
91 1 MMBVSSSMMKPR/AWRG/3418/04 36.66666667 1.166666667 87.27272727 2.916666667 73.42657343 2.666666667 0 0
92 1 MMBVSSSMMKPR/HCGA1/6737/09 46.66666667 1.166666667 87.27272727 2.5 83.91608392 2 0 0
93 1 MMBVSSSMMKPR/HCGA3/5860/09 36.66666667 1.166666667 92.72727273 2.5 73.42657343 3.333333333 63.93442623 4
94 1 MMBVSSSMMKPR/BBMH/6814/07 33.33333333 0.666666667 43.63636364 1.666666667 73.42657343 2 21.31147541 3.333333333
95 1 MMBVSSSMMKPR/BEMH/3166/01 53.33333333 1 76.36363636 2.083333333 15.73426573 2 85.24590164 4
96 1 MMBVSSSMMKPR/RG25/2941/09 53.33333333 1.166666667 76.36363636 2.5 83.91608392 3.333333333 95.90163934 4
97 1 MMBVSSSMMKPR/SP1/1287/98 45 1.333333333 87.27272727 2.916666667 68.18181818 3.333333333 53.27868852 3.333333333
98 1 MMBVSSSMMKPR/SP2/00396/06 36.66666667 1.333333333 92.72727273 2.916666667 83.91608392 3.333333333 42.62295082 4
99 1 MMBVSSSMMKPR/SP2/5090/03 36.66666667 1.166666667 76.36363636 2.5 83.91608392 3.333333333 53.27868852 2.666666667
100 1 MMBVSSSMMKPR/SP3/00076/06 45 1.333333333 87.27272727 2.916666667 83.91608392 3.333333333 0 0
101 1 MMBVSSSMMKPR/SP5/5658/09 46.66666667 1.166666667 87.27272727 2.916666667 68.18181818 2.666666667 15.98360656 2.666666667
102 1 MMBVSSSMMKPR/SP9/1431/07 40 1.166666667 76.36363636 2.5 83.91608392 3.333333333 0 0
103 1 MMBVSSSMMKPR/SP9/00521/07 40 1 76.36363636 2.5 83.91608392 3.333333333 42.62295082 2.666666667
104 1 MMBVSSSMMKPR/SP9/919/99 41.66666667 1.166666667 87.27272727 2.916666667 83.91608392 3.333333333 85.24590164 2.666666667
105 1 MMBVSSSMMKPR/SP10/1693/03 36.66666667 1 87.27272727 2.5 83.91608392 2.666666667 85.24590164 3.333333333
106 1 MMBVSSSMMKPR/SP11/2802/01 56.66666667 1.166666667 87.27272727 2.5 83.91608392 3.333333333 95.90163934 3.333333333
107 1 MMBVSSSMMKPR/SP11/1002/05 46.66666667 1.166666667 76.36363636 2.5 73.42657343 3.333333333 95.90163934 3.333333333
108 1 MMBVSSSMMKPR/SP11/4945/09 51.66666667 1.166666667 87.27272727 2.916666667 83.91608392 3.333333333 74.59016393 2
109 1 MMBVSSSMMKPR/RG32/47/10 46.66666667 1 87.27272727 2.5 68.18181818 2.666666667 0 0
110 1 MMBVSSSMMKPR/RG32/7449/09 56.66666667 1.333333333 70.90909091 2.916666667 62.93706294 2 5.327868852 4
111 1 MMBVSSSMMKPR/SP12/0353/09 53.33333333 1.166666667 76.36363636 2.916666667 10.48951049 1.333333333 79.91803279 2.666666667
112 1 MMBVSSSMMKPR/SP12/2027/09 41.66666667 1.166666667 87.27272727 2.916666667 83.91608392 2.666666667 0 0
113 1 MMBVSSSMMKPR/SP12/1631/09 40 1.166666667 87.27272727 2.916666667 78.67132867 1.333333333 95.90163934 2.666666667
60
114 1 MMBVSSSMMKPR/RG34/1091/10 43.33333333 1.166666667 87.27272727 2.916666667 83.91608392 3.333333333 63.93442623 4
115 1 MMBVSSSMMKPR/SP13/6097/08 41.66666667 1 87.27272727 2.916666667 83.91608392 3.333333333 74.59016393 2.666666667
116 1 MMBVSSSMMKPR/BFMH/2540/04 33.33333333 0.666666667 87.27272727 2.083333333 62.93706294 2.666666667 85.24590164 2.666666667
117 1 MMBVSSSMMKPR/BGMH/3558/03 33.33333333 0.666666667 87.27272727 3.333333333 31.46853147 2 85.24590164 2.666666667
118 1 MMBVSSSMMKPR/BIMH/4596/05 50 1.166666667 43.63636364 2.916666667 52.44755245 2.666666667 37.29508197 2
119 1 MMBVSSSMMKPR/BJMH/1942/07 33.33333333 0.666666667 54.54545455 2.5 73.42657343 3.333333333 63.93442623 3.333333333
120 1 MMBVSSSMMKPR/BKMH/3435/06 43.33333333 1.166666667 87.27272727 3.333333333 89.16083916 4 42.62295082 2.666666667
121 1 MMBVSSSMMKPR/BLJH/2584/05 33.33333333 0.666666667 65.45454545 2.5 83.91608392 4 0 0
122 1 MMBVSSSMMKPR/BMMH/3591/08 33.33333333 0.666666667 81.81818182 3.333333333 62.93706294 2.666666667 95.90163934 3.333333333
123 1 MMBVSSSMMKPR/BNMH/3242/06 33.33333333 0.666666667 87.27272727 2.5 83.91608392 4 31.96721311 2
124 1 MMBVSSSMMKPR/BORG/0945/03 50 1.166666667 87.27272727 2.916666667 83.91608392 3.333333333 26.63934426 1.333333333
125 1 MMBVSSSMMKPR/BPRG/3398/03 33.33333333 0.666666667 65.45454545 2.916666667 78.67132867 3.333333333 10.6557377 2.666666667
126 1 MMBVSSSMMKPR/BQRG/4390/03 33.33333333 0.666666667 103.6363636 3.333333333 73.42657343 2.666666667 37.29508197 2
127 1 MMBVSSSMMKPR/BTRG/3606/03 36.66666667 1.166666667 65.45454545 2.5 73.42657343 2 10.6557377 1.333333333
128 1 MMBVSSSMMKPR/BUMH/6186/04 41.66666667 1.166666667 87.27272727 2.916666667 0 0 0 0
129 1 MMBVSSSMMKPR/BVMH/6080/07 33.33333333 0.666666667 81.81818182 2.5 52.44755245 2 53.27868852 2.666666667
130 1 MMBVSSSMMKPR/DMMX/3990/07 38.33333333 1 87.27272727 2.916666667 62.93706294 2.666666667 31.96721311 2.666666667
131 1 MMBVSSSMMKPR/ SP1/2238/01 36.66666667 1.166666667 87.27272727 2.916666667 41.95804196 2.666666667 0 0
132 1 MMBVSSSMMKPR/RG15/1464/08 33.33333333 0.666666667 87.27272727 2.5 83.91608392 2.666666667 42.62295082 1.333333333
133 1 MMBVSSSMMKPR/RG22/1931/09 38.33333333 1 87.27272727 2.5 83.91608392 2.666666667 0 0
134 1 MMBVSSSMMKPR/MH14/6324/08 33.33333333 0.666666667 87.27272727 2.916666667 73.42657343 2.666666667 85.24590164 2
135 1 MMBVSSSMMKPR/ RG24/805/09 56.66666667 1.166666667 76.36363636 2.5 73.42657343 2.666666667 0 0
136 1 MMBVSSSMMKPR/HCGA4/6374/08 41.66666667 1 87.27272727 2.5 83.91608392 3.333333333 0 0
137 1 MMBVSSSMMKPR/SP1/3187/99 36.66666667 1 87.27272727 2.916666667 73.42657343 2.666666667 79.91803279 4
138 1 MMBVSSSMMKPR/SP1/1543/00 46.66666667 1.166666667 87.27272727 2.5 62.93706294 3.333333333 42.62295082 2.666666667
139 1 MMBVSSSMMKPR/SP2/10817/07 45 1.166666667 87.27272727 2.916666667 83.91608392 3.333333333 63.93442623 2.666666667
140 1 MMBVSSSMMKPR/SP2/3424/03 43.33333333 1.166666667 81.81818182 2.5 83.91608392 3.333333333 0 0
61
141 1 MMBVSSSMMKPR/SP2/489/02 33.33333333 0.666666667 54.54545455 2.083333333 83.91608392 3.333333333 85.24590164 3.333333333
142 1 MMBVSSSMMKPR/SP3/5454/05 46.66666667 1.333333333 76.36363636 2.5 78.67132867 3.333333333 0 0
143 1 MMBVSSSMMKPR/SP3/00070/06 33.33333333 0.666666667 87.27272727 2.5 83.91608392 3.333333333 0 0
144 1 MMBVSSSMMKPR/SP4/11639/06 45 1.166666667 81.81818182 2.083333333 78.67132867 3.333333333 21.31147541 2
145 1 MMBVSSSMMKPR/SP4/12823/07 36.66666667 1.166666667 81.81818182 2.916666667 83.91608392 3.333333333 15.98360656 2
146 1 MMBVSSSMMKPR/SP4/14511/07 36.66666667 1.166666667 87.27272727 2.916666667 89.16083916 3.333333333 0 0
147 1 MMBVSSSMMKPR/IIRG/4358/08 38.33333333 1.166666667 65.45454545 2.916666667 73.42657343 4 0 0
148 1 MMBVSSSMMKPR/IVRG/1658/08 33.33333333 0.666666667 0 0.833333333 89.16083916 4 85.24590164 4
149 1 MMBVSSSMMKPR/JRRG/6716/08 46.66666667 1 87.27272727 3.333333333 78.67132867 3.333333333 0 0
150 1 MMBVSSSMMKPR/JSMH/7129/07 46.66666667 1 76.36363636 2.5 83.91608392 3.333333333 0 0
151 0 MMBVSSSMMKPR/CPMH/1187/05 53.33333333 1.166666667 87.27272727 2.916666667 78.67132867 2.666666667 95.90163934 4
152 0 MMBVSSSMMKPR/CSMH/3038/04 33.33333333 0.666666667 76.36363636 2.916666667 36.71328671 2 74.59016393 4
153 0 MMBVSSSMMKPR/CTMH/2852/04 36.66666667 1.166666667 87.27272727 2.916666667 68.18181818 4 95.90163934 3.333333333
154 0 MMBVSSSMMKPR/CUMH/1591/03 40 1 43.63636364 2.5 83.91608392 4 0 0
155 0 MMBVSSSMMKPR/CWMH/3473/06 38.33333333 1 54.54545455 2.083333333 36.71328671 2.666666667 26.63934426 2.666666667
156 0 MMBVSSSMMKPR/CXJH/3121/05 36.66666667 1 87.27272727 2.916666667 73.42657343 2.666666667 95.90163934 3.333333333
157 0 MMBVSSSMMKPR/CYJH/0878/06 33.33333333 0.666666667 65.45454545 2.5 73.42657343 3.333333333 0 0
158 0 MMBVSSSMMKPR/RG26/5002/09 40 1.333333333 87.27272727 2.916666667 73.42657343 3.333333333 42.62295082 2.666666667
159 0 MMBVSSSMMKPR/DAMH/7095/09 46.66666667 1 43.63636364 1.666666667 10.48951049 1.333333333 63.93442623 4
160 0 MMBVSSSMMKPR/DBMH/9616/08 33.33333333 0.666666667 87.27272727 2.5 73.42657343 2.666666667 74.59016393 2.666666667
Table 3D
62
Once the combination of 5 biomarkers is selected, based on the data obtained by
retrospective study of 298 patient samples (training set), scores of these 5 biomarkers
are coupled with the three clinical parameters (tumor size, grade and node status) to
construct/develop the CanAssist-Breast algorithm/algorithm of the present disclosure
which provides a CanAssist Breast relapse score which helps in stratifying patients as
low risk or high risk of recurrence within 5 years from diagnosis. The relapse score is
computed on a scale of 1-100 and scores of 15.5 and below are classified as low risk,
and above 15.5 are classified as high risk. The risk of recurrence is considered high if
the probability of recurrence is greater than about 9% and low if it is less than or equal
to about 9%.
Example 2: Validation studies of 5 biomarker and 3 clinical parameter combination
of the present disclosure and correlation of CanAssist Breast relapse score with
recurrence in early stage ER+/PR+ and Her2- breast cancer patients
The prognostic test developed in example 1 is validated on a validation sample set of
700 early stage ER+/PR+ and Her2- breast cancer patients. A representative set of the
complete validation set is provided herewith by way of table no. 4, wherein IHC
measurements are provided for 299 data samples.
63
Sl.
No
Age Grade TNM A%-M W%-C U%-C R%-M F%-M WI-C Outcome DFS TTP Chemo Details
CanAssist
Breast
Score
1 53 1 T1N0M0 37.5 22.5 38.2 48 32.86 1.3 Recurrence 35 Chemo Naive 45.6
2
66 2 T1N0M0 57.5 45 50.9 48 34.29 1.8 Recurrence 60 Chemo Naive
15.6
3
72 2 T2N0M0 50 47.5 52.7 44 35.71 1.5 Recurrence 44 Chemo Naive
19.4
4
42 2 T2N0M0 52.5 47.5 52.7 42 32.86 1.8 Recurrence 52 Chemo Naive
11.5
5
43 3 T1N0M0 52.5 42.5 50.9 40 27.14 1.5 Recurrence 55 Chemo Naive
20.8
6
69 3 T2N0M0 15 40 38.2 34 21.43 1.5 Recurrence 43 Chemo Naive
13.9
7
40 3 T2N0M0 42.5 37.5 49.1 44 27.14 1.8 Recurrence 46 Chemo Naive
24.1
8
52 2 T2N1M0 17.5 42.5 49.1 44 30.00 1.3 Recurrence 53 Chemo Naive
23.8
9
44 2 T2N0M0 52.5 48.75 51.8 31 20.71 1.5 No Recurrence 76 Chemo Naive
8.3
10
62 1 T2N0M0 52.5 50 52.7 38 20.71 1.8 No Recurrence 80 Chemo Naive
8.3
11
62 2 T1N0M0 28.75 43.75 49.1 35 22.14 1.6 No Recurrence 65 Chemo Naive
2.5
12
61 1 T1N0M0 27.5 43.75 49.1 50 32.86 1.6 No Recurrence 64 Chemo Naive
8.1
13
41 2 T2N0M0 15 45 52.7 29 12.86 1.6 No Recurrence 68 Chemo Naive
9
14
59 2 T1N0M0 50 46.25 50.9 29 18.57 1.8 No Recurrence 61 Chemo Naive
5.5
64
15
59 3 T2N2M0 12.5 38.75 50.9 25 15.00 1.4 No Recurrence 74 Chemo Naive
7.1
16
71 3 T2N1M0 22.5 42.5 52.7 30 17.86 1.4 No Recurrence 75 Chemo Naive
7.5
17
59 2 T1N0M0 32.5 45 50.9 36 19.29 1.8 No Recurrence 74 Chemo Naive
4
18
65 3 T2N0M0 12.5 43.75 50.0 24 14.29 1.8 No Recurrence 88 Chemo Naive
6.7
19
57 2 T2N0M0 35 46.25 51.8 45 25.00 1.5 No Recurrence 137 Chemo Naive
9.8
20
67 2 T1N0M0 36.25 42.5 50.0 41 15.71 1.4 No Recurrence 69 Chemo Naive
11.5
21
43 2 T1N0M0 22.5 32.5 51.8 27 18.57 1.1 No Recurrence 66 Chemo Naive
4
22
74 1 T2N0M0 21.25 40 47.3 28 15.71 1.5 No Recurrence 65 Chemo Naive
5.6
23
59 2 T1N0M0 43.75 47.5 52.7 37 24.29 1.8 No Recurrence 78 Chemo Naive
3.7
24
42 2 T1N0M0 17.5 30 40.0 23 12.86 1.5 No Recurrence 71 Chemo Naive
3.4
25
72 3 T2N0M0 37.5 40 47.3 26 14.29 1.5 No Recurrence 103 Chemo Naive
5.4
26
56 2 T2N0M0 12.5 46.25 52.7 28 19.29 1.6 No Recurrence 60 Chemo Naive
5.9
27
72 2 T1N0M0 42.5 46.25 51.8 40 27.14 1.8 No Recurrence 60 Chemo Naive
3.7
28
51 2 T2N0M0 50 47.5 53.6 22 12.86 1.8 No Recurrence 60 Chemo Naive
13.1
65
29
57 3 T2N0M0 35 47.5 52.7 37 22.86 1.5 No Recurrence 85 Chemo Naive
10.3
30
72 2 T2N1M0 52.5 45 52.7 40 25.71 1.6 No Recurrence 93 Chemo Naive
15
31
50 2 T2N0M0 22.5 40 51.8 46 17.86 1.5 No Recurrence 109 Chemo Naive
12.4
32
75 2 T3N1M0 18.75 42.5 52.7 24 15.71 1.3 No Recurrence 130 Chemo Naive
10.9
33
73 1 T1N0M0 20 41.25 49.1 25 12.86 1.5 No Recurrence 79 Chemo Naive
12.7
34
64 3 T2N0M0 12.5 45 51.8 25 14.29 1.9 No Recurrence 68 Chemo Naive
9.1
35
75 2 T2N2M0 25 48.75 53.6 24 15.71 1.8 No Recurrence 86 Chemo Naive
12.4
36
66 1 T1N0M0 18.75 41.25 51.8 28 20.71 1.6 No Recurrence 69 Chemo Naive
7.7
37
64 2 T2N0M0 37.5 50 52.7 26 16.43 1.6 No Recurrence 86 Chemo Naive
6.8
38
57 1 T1N1M0 32.5 43.75 49.1 24 16.43 1.4 No Recurrence 77 Chemo Naive
10.5
39
73 2 T2N1M0 13.75 45 53.6 36 20.71 1.8 No Recurrence 83 Chemo Naive
8.4
40
67 3 T1N0M0 27.5 42.5 53.6 28 15.00 1.6 No Recurrence 60 Chemo Naive
8.4
41
69 2 T2N0M0 46.25 46.25 52.7 26 15.71 1.5 No Recurrence 63 Chemo Naive
7.8
42
57 2 T1N0M0 12.5 42.5 52.7 20 12.86 1.6 No Recurrence 63 Chemo Naive
15
66
43
70 2 T1N0M0 40 46.25 52.7 22 15.00 1.5 No Recurrence 63 Chemo Naive
9
44
63 3 T1N0M0 50 42.5 54.5 31 17.14 1.5 No Recurrence 69 Chemo Naive
13
45
51 3 T2N0M0 52.5 37.5 50.9 20 14.29 1.4 No Recurrence 71 Chemo Naive
11.1
46
71 2 T2N0M0 45 46.25 47.3 20 14.29 1.4 No Recurrence 76 Chemo Naive
6.7
47
60 3 T2N2M0 50 37.5 50.9 20 12.86 1.4 No Recurrence 60 Chemo Naive
9.8
48
72 1 T2N0M0 25 45 53.6 31 21.43 1.9 No Recurrence 67 Chemo Naive
6.9
49
56 1 T1N0M0 51.25 46.25 52.7 38 21.43 1.6 No Recurrence 67 Chemo Naive
7.4
50
71 3 T1N0M0 22.5 48.75 53.6 29 17.14 1.6 No Recurrence 61 Chemo Naive
9.8
51
70 2 T2N0M0 51.25 45 52.7 37 23.57 1.5 No Recurrence 65 Chemo Naive
10.1
52
74 2 T2N1M0 45 48.75 53.6 42 32.86 1.6 No Recurrence 72 Chemo Naive
16.1
53
69 2 T2N0M0 37.5 46.25 53.6 24 21.43 1.5 No Recurrence 76 Chemo Naive
5.2
54
71 3 T2N0M0 40 35 50.9 32 20.00 1.4 No Recurrence 65 Chemo Naive
11.7
55
53 3 T2N0M0 42.5 46.25 51.8 33 21.43 1.5 No Recurrence 70 Chemo Naive
10
56
70 2 T2N0M0 37.5 47.5 50.0 28 22.86 1.8 No Recurrence 64 Chemo Naive
3
67
57
57 2 T2N0M0 48.75 42.5 54.5 32 21.43 1.4 No Recurrence 64 Chemo Naive
9.9
58
68 3 T2N0M0 43.75 42.5 50.0 25 15.00 1.5 No Recurrence 84 Chemo Naive
7.4
59
50 2 T1N0M0 12.5 42.5 51.8 33 18.57 1.4 No Recurrence 81 Chemo Naive
7.6
60
50 2 T1N0M0 40 50 54.5 33 25.00 1.8 No Recurrence 133 Chemo Naive
4.1
61
53 2 T1N0M0 17.5 37.5 49.1 44 26.43 1.4 No Recurrence 86 Chemo Naive
10.1
62
65 1 T1N0M0 28.75 47.5 53.6 39 19.29 1.6 No Recurrence 62 Chemo Naive
8.7
63
55 2 T1N0M0 37.5 43.75 51.8 28 17.14 1.6 No Recurrence 66 Chemo Naive
4.8
64
61 1 T2N0M0 31.25 46.25 51.8 30 17.14 1.4 No Recurrence 66 Chemo Naive
7.6
65
48 3 T2N0M0 25 45 52.7 22 12.86 1.1 No Recurrence 79 Chemo Naive
7.7
66
53 2 T1N0M0 36.25 45 50.0 22 17.14 1.6 No Recurrence 82 Chemo Naive
5.3
67
40 3 T2N0M0 15 36.25 54.5 22 16.43 1.5 No Recurrence 84 Chemo Naive
4.4
68
66 2 T1N0M0 47.5 40 50.9 42 27.14 1.3 No Recurrence 135 Chemo Naive
17.4
69
48 3 T2N0M0 21.25 48.75 54.5 30 17.14 1.8 No Recurrence 81 Chemo Naive
7.8
70
46 2 T2N0M0 40 48.75 51.8 29 15.71 1.8 No Recurrence 89 Chemo Naive
6.4
68
71
59 3 T2N0M0 16.25 47.5 52.7 29 18.57 1.6 No Recurrence 89 Chemo Naive
6.2
72
60 3 T2N0M0 15 48.75 53.6 30 25.71 1.6 No Recurrence 84 Chemo Naive
6.8
73
68 1 T1N0M0 12.5 43.75 43.6 20 20.71 1.5 No Recurrence 98 Chemo Naive
10.8
74
59 3 T2N1M0 18.75 45 52.7 39 29.29 1.6 No Recurrence 86 Chemo Naive
16
75
62 3 T1N0M0 15 48.75 53.6 31 15.71 1.8 No Recurrence 78 Chemo Naive
13
76
58 2 T2N0M0 16.25 47.5 53.6 29 21.43 1.5 No Recurrence 91 Chemo Naive
5.2
77
62 3 T1N0M0 26.25 47.5 51.8 27 20.00 1.6 No Recurrence 60 Chemo Naive
6.4
78
55 3 T1N0M0 12.5 46.25 52.7 23 14.29 1.8 No Recurrence 92 Chemo Naive
13.9
79
50 2 T2N0M0 18.75 46.25 54.5 23 19.29 1.6 No Recurrence 78 Chemo Naive
7.1
80
56 3 T1N0M0 41.25 43.75 51.8 35 28.57 1.4 No Recurrence 61 Chemo Naive
14.2
81
54 3 T1N0M0 42.5 46.25 52.7 28 14.29 1.5 No Recurrence 91 Chemo Naive
10.6
82
57 3 T2N0M0 28.75 47.5 52.7 22 12.86 1.5 No Recurrence 99 Chemo Naive
7.9
83
61 2 T1N0M0 21.25 36.25 51.8 29 16.43 1.3 No Recurrence 60 Chemo Naive
5.2
84
60 3 T1N1M0 20 40 50.9 28 20.00 1.4 No Recurrence 87 Chemo Naive
8.3
69
85
65 3 T2N0M0 30 47.5 53.6 30 20.00 1.8 No Recurrence 67 Chemo Naive
5.8
86
43 2 T1N0M0 48.75 47.5 52.7 32 20.00 1.6 No Recurrence 60 Chemo Naive
5.9
87
65 2 T2N0M0 37.5 37.5 41.8 29 16.43 1.3 No Recurrence 61 Chemo Naive
6.2
88
67 3 T1N0M0 51.25 47.5 52.7 35 24.29 1.5 No Recurrence 62 Chemo Naive
13.2
89
62 2 T2N0M0 26.25 42.5 52.7 31 23.57 1.6 No Recurrence 60 Chemo Naive
2.3
90
52 1 T3N0M0 16.25 43.75 52.7 30 19.29 1.5 No Recurrence 63 Chemo Naive
16.1
91
57 2 T2N0M0 22.5 47.5 40.0 28 22.86 1.6 No Recurrence 64 Chemo Naive
5.7
92
63 2 T1N0M0 41.25 41.25 48.2 30 18.57 1.6 No Recurrence 81 Chemo Naive
3
93
49 2 T1N0M0 12.5 25 47.3 24 14.29 1.0 No Recurrence 68 Chemo Naive
1.9
94
68 2 T1N0M0 43.75 36.25 53.6 35 22.14 1.3 No Recurrence 72 Chemo Naive
10.6
95
56 1 T1N0M0 17.5 47.5 52.7 27 15.71 1.6 No Recurrence 63 Chemo Naive
16.5
96
63 2 T2N0M0 22.5 41.25 52.7 36 26.43 1.5 No Recurrence 60 Chemo Naive
4.1
97
70 2 T1N0M0 40 35 52.7 35 24.29 1.3 No Recurrence 76 Chemo Naive
9.2
98
68 1 T2N0M0 55 42.5 54.5 26 23.57 1.5 No Recurrence 105 Chemo Naive
11.2
70
99
59 1 T1N0M0 50 41.25 49.1 35 18.57 1.4 No Recurrence 99 Chemo Naive
8.9
100
40 3 T1N0M0 38.75 45 49.1 36 23.57 1.6 No Recurrence 70 Chemo Naive
8.2
101
56 1 T2N0M0 12.5 47.5 52.7 30 21.43 1.8 No Recurrence 68 Chemo Naive
9.9
102
62 2 T2N1M0 26.25 40 54.5 33 20.00 1.5 No Recurrence 69 Chemo Naive
6.3
103
61 3 T2N0M0 43.75 45 50.9 33 23.57 1.6 No Recurrence 92 Chemo Naive
9.7
104
68 2 T2N1M0 47.5 45 50.9 33 24.29 1.4 No Recurrence 94 Chemo Naive
11.8
105
53 2 T1N0M0 27.5 36.25 52.7 31 20.00 1.4 No Recurrence 91 Chemo Naive
3.9
106
45 3 T1N0M0 16.25 45 52.7 31 20.71 1.4 No Recurrence 88 Chemo Naive
8.9
107
50 2 T2N1M0 30 42.5 53.6 25 16.43 1.4 No Recurrence 84 Chemo Naive
5.2
108
42 3 T2N0M0 15 48.75 52.7 24 16.43 1.6 No Recurrence 61 Chemo Naive
7.8
109
53 3 T2N0M0 13.75 47.5 53.6 36 20.00 1.5 No Recurrence 94 Chemo Naive
9.2
110
55 2 T1N0M0 45 42.5 50.0 41 29.29 1.6 No Recurrence 98 Chemo Naive
6.9
111
48 3 T2N0M0 12.5 48.75 54.5 26 15.00 1.5 No Recurrence 74 Chemo Naive
10
112
31 3 T1N0M0 40 43.75 53.6 31 19.29 1.3 No Recurrence 73 Chemo Naive
12.3
71
113
53 3 T1N0M0 12.5 42.5 45.5 30 20.71 1.5 No Recurrence 67 Chemo Naive
6.3
114
65 2 T1N1M0 12.5 40 44.5 37 21.43 1.3 No Recurrence 64 Chemo Naive
14.1
115
40 3 T1N0M0 31.25 40 51.8 35 24.29 1.3 No Recurrence 72 Chemo Naive
12.5
116
69 3 T2N0M0 17.5 42.5 47.3 36 15.00 1.5 No Recurrence 71 Chemo Naive
8.9
117
74 3 T2N0M0 36.25 35 47.3 30 17.86 1.3 No Recurrence 72 Chemo Naive
9.4
118
62 2 T2N0M0 32.5 40 52.7 32 21.43 1.3 No Recurrence 85 Chemo Naive
5.7
119
62 2 T1N0M0 12.5 42.5 50.9 28 16.43 1.6 No Recurrence 95 Chemo Naive
7.8
120
55 2 T1N0M0 42.5 47.5 52.7 36 21.43 1.5 No Recurrence 66 Chemo Naive
6
121
52 1 T2N0M0 40 40 51.8 42 17.14 1.4 No Recurrence 60 Chemo Naive
12
122
56 3 T2N1M0 13.75 46.25 52.7 30 22.86 1.5 No Recurrence 110 Chemo Naive
8.5
123
63 2 T2N0M0 28.75 47.5 52.7 34 20.00 1.5 No Recurrence 78 Chemo Naive
4.5
124
58 1 T1N0M0 35 47.5 47.3 26 17.14 1.6 No Recurrence 82 Chemo Naive
8.4
125
59 2 T1N0M0 43.75 43.75 45.5 32 22.14 1.5 No Recurrence 110 Chemo Naive
4.3
126
44 2 T1N0M0 16.25 37.5 50.9 26 15.00 1.3 No Recurrence 67 Chemo Naive
5.7
72
127
54 2 T2N0M0 25 42.5 49.1 35 23.57 1.5 No Recurrence 65 Chemo Naive
3
128
58 1 T1N0M0 28.75 45 47.3 30 16.43 1.6 No Recurrence 60 Chemo Naive
7.7
129
61 2 T1N0M0 57.5 42.5 52.7 22 24.29 1.5 No Recurrence 61 Chemo Naive
9.4
130
66 1 T1N0M0 50 47.5 49.1 30 22.86 1.6 No Recurrence 71 Chemo Naive
5.8
131
63 2 T1N0M0 12.5 42.5 46.4 24 12.86 1.5 No Recurrence 66 Chemo Naive
8.7
132
52 2 T1N0M0 12.5 43.75 52.7 20 12.86 1.3 No Recurrence 71 Chemo Naive
13.9
133
65 2 T1N0M0 50 47.5 54.5 32 17.14 1.5 No Recurrence 62 Chemo Naive
9.9
134
49 1 T1N0M0 40 30 50.0 20 12.86 1.3 No Recurrence 63 Chemo Naive
9
135
72 2 T2N0M0 50 42.5 52.7 34 14.29 1.8 No Recurrence 63 Chemo Naive
9.9
136
54 2 T2N1M0 18.75 46.25 50.9 32 17.14 1.4 No Recurrence 63 Chemo Naive
7.4
137
68 1 T1N0M0 51.25 38.75 50.9 26 20.00 1.1 No Recurrence 62 Chemo Naive
11
138
64 2 T1N0M0 45 45 54.5 20 15.00 1.5 No Recurrence 65 Chemo Naive
11.6
139
47 2 T1N0M0 36.25 41.25 48.2 22 18.57 1.5 No Recurrence 63 Chemo Naive
2.7
140
57 3 T1N0M0 25 42.5 45.5 20 12.86 1.5 No Recurrence 67 Chemo Naive
6
73
141
62 2 T1N0M0 13.75 35 54.5 34 18.57 1.4 No Recurrence 68 Chemo Naive
7.8
142
72 3 T1N0M0 27.5 33.75 43.6 28 15.71 1.3 No Recurrence 68 Chemo Naive
6
143
67 3 T1N0M0 12.5 46.25 54.5 31 25.00 1.6 No Recurrence 61 Chemo Naive
9.3
144
50 3 T2N0M0 52.5 43.75 49.1 29 14.29 1.4 No Recurrence 70 Chemo Naive
14.2
145
26 2 T2N0M0 50 45 50.9 35 22.86 1.5 No Recurrence 72 Chemo Naive
8.4
146
59 3 T1N0M0 50 32.5 49.1 31 19.29 1.3 No Recurrence 68 Chemo Naive
16.9
147
69 2 T1N0M0 52.5 37.5 50.0 42 31.43 1.8 No Recurrence 62 Chemo Naive
11.5
148
72 2 T1N0M0 48.75 43.75 53.6 32 14.29 1.4 No Recurrence 65 Chemo Naive
11.8
149
66 2 T2N0M0 37.5 32.5 47.3 30 25.00 1.3 No Recurrence 60 Chemo Naive
7.2
150
62 2 T1N0M0 33.75 45 52.7 20 17.14 1.5 No Recurrence 60 Chemo Naive
7.7
151
43 2 T1N0M0 30 42.5 51.8 31 25.00 1.4 No Recurrence 60 Chemo Naive
3.6
152
61 2 T1N0M0 48.75 43.75 51.8 24 15.71 1.4 No Recurrence 68 Chemo Naive
8.4
153
65 1 T1N0M0 27.5 33.75 52.7 22 12.86 1.3 No Recurrence 62 Chemo Naive
11.5
154
60 3 T2N2M0 37.5 47.5 52.7 28 28.57 1.8 Recurrence 12 Chemo Treated
16.7
74
155
41 3 T3N0M0 12.5 45 50.9 42 22.86 1.3 Recurrence 37 Chemo Treated
30.1
156
47 2 T2N2M0 50 37.5 45.5 50 35.71 1.3 Recurrence 45 Chemo Treated
100
157
62 2 T2N2M0 15 42.5 49.1 40 27.14 1.3 Recurrence 54 Chemo Treated
27.2
158
50 3 T2N2M0 15 42.5 47.3 40 31.43 1.5 Recurrence 40 Chemo Treated
39.4
159
50 2 T2N1M0 12.5 42.5 49.1 44 12.86 1.5 Recurrence 50 Chemo Treated
19.8
160
48 2 T2N1M0 12.5 45 52.7 40 12.86 1.8 Recurrence 26 Chemo Treated
15.8
161
45 2 T2N0M0 52.5 37.5 52.7 50 32.86 1.5 Recurrence 18 Chemo Treated
35.5
162
57 1 T1N1M0 12.5 47.5 50.9 40 21.43 1.5 Recurrence 21 Chemo Treated
16.2
163
57 2 T2N2M0 12.5 42.5 52.7 40 21.43 1.5 Recurrence 27 Chemo Treated
19.4
164
36 2 T2N1M0 30 45 50.9 46 21.43 1.5 Recurrence 45 Chemo Treated
16.8
165
52 3 T2N1M0 35 42.5 52.7 42 27.14 1.5 Recurrence 35 Chemo Treated
25.4
166
57 2 T1N2M0 45 45 52.7 30 15.71 1.3 Recurrence 49 Chemo Treated
18.9
167
46 2 T3N3M0 42.5 47.5 52.7 48 30.00 1.8 Recurrence 20 Chemo Treated
60.8
168
48 2 T1N2M0 12.5 47.5 52.7 40 17.14 1.8 Recurrence 9 Chemo Treated
27.2
75
169
55 2 T2N1M0 16.25 47.5 53.6 32 17.14 1.8 No Recurrence 84 Chemo Treated
9.2
170
29 3 T2N0M0 43.75 45 50.0 32 25.71 1.5 No Recurrence 87 Chemo Treated
11.8
171
47 3 T2N0M0 41.25 43.75 50.0 42 26.43 1.6 No Recurrence 96 Chemo Treated
17.7
172
51 3 T2N0M0 15 47.5 53.6 31 19.29 1.6 No Recurrence 87 Chemo Treated
6.9
173
56 2 T1N0M0 46.25 40 50.9 28 12.86 1.3 No Recurrence 87 Chemo Treated
9.8
174
49 2 T2N0M0 32.5 42.5 52.7 40 22.86 1.5 No Recurrence 89 Chemo Treated
6.6
175
49 2 T2N0M0 30 50 54.5 24 34.29 1.9 No Recurrence 83 Chemo Treated
9.5
176
48 2 T2N2M0 28.75 45 54.5 28 15.00 1.6 No Recurrence 83 Chemo Treated
10.7
177
74 2 T2N0M0 48.75 48.75 52.7 28 20.71 1.9 No Recurrence 60 Chemo Treated
6.5
178
65 3 T2N1M0 15 43.75 50.0 36 24.29 1.4 No Recurrence 60 Chemo Treated
13.9
179
55 2 T2N1M0 12.5 33.75 50.0 41 22.86 1.3 No Recurrence 64 Chemo Treated
15.6
180
61 2 T2N1M0 12.5 43.75 49.1 28 16.43 1.5 No Recurrence 68 Chemo Treated
5.9
181
47 1 T1N0M0 26.25 45 53.6 22 18.57 1.6 No Recurrence 61 Chemo Treated
13.3
182
37 2 T1N0M0 33.75 35 47.3 27 17.86 1.4 No Recurrence 68 Chemo Treated
1.8
76
183
52 2 T1N0M0 35 45 50.9 37 25.00 1.6 No Recurrence 60 Chemo Treated
3
184
47 2 T1N0M0 42.5 46.25 52.7 29 19.29 1.5 No Recurrence 60 Chemo Treated
5.7
185
68 3 T2N0M0 55 42.5 50.9 24 18.57 1.5 No Recurrence 66 Chemo Treated
12.4
186
37 2 T2N0M0 20 42.5 52.7 20 24.29 1.5 No Recurrence 64 Chemo Treated
4.2
187
48 3 T2N0M0 23.75 46.25 52.7 27 17.14 1.5 No Recurrence 61 Chemo Treated
5.6
188
50 1 T2N0M0 22.5 40 37.3 24 20.00 1.5 No Recurrence 121 Chemo Treated
7.8
189
60 3 T2N0M0 37.5 32.5 50.9 33 24.29 1.8 No Recurrence 60 Chemo Treated
9.7
190
65 2 T2N0M0 13.75 17.5 41.8 26 17.14 1.0 No Recurrence 63 Chemo Treated
0
191
53 2 T2N0M0 40 52.5 52.7 20 17.86 1.6 No Recurrence 69 Chemo Treated
9.6
192
62 2 T2N0M0 22.5 45 50.9 26 18.57 1.5 No Recurrence 62 Chemo Treated
3.1
193
61 2 T2N0M0 16.25 41.25 46.4 20 12.86 1.4 No Recurrence 74 Chemo Treated
3.6
194
68 3 T2N0M0 22.5 43.75 50.9 34 20.00 1.5 No Recurrence 60 Chemo Treated
6.3
195
47 2 T1N1M0 31.25 48.75 52.7 28 17.86 1.8 No Recurrence 94 Chemo Treated
9.4
196
60 3 T2N0M0 15 47.5 50.9 24 15.71 1.6 No Recurrence 75 Chemo Treated
6.6
77
197
49 2 T2N0M0 22.5 47.5 52.7 24 16.43 1.6 No Recurrence 72 Chemo Treated
6.7
198
42 2 T2N0M0 25 45 50.9 26 12.86 1.6 No Recurrence 60 Chemo Treated
6.4
199
39 2 T2N1M0 20 45 51.8 27 16.43 1.5 No Recurrence 71 Chemo Treated
5.6
200
61 2 T2N1M0 12.5 46.25 52.7 29 19.29 1.5 No Recurrence 60 Chemo Treated
7.4
201
48 3 T2N1M0 12.5 47.5 52.7 27 18.57 1.9 No Recurrence 64 Chemo Treated
9.2
202
49 2 T2N1M0 12.5 37.5 52.7 28 14.29 1.4 No Recurrence 71 Chemo Treated
5.8
203
56 3 T2N1M0 12.5 32.5 41.8 26 24.29 1.0 No Recurrence 60 Chemo Treated
9.7
204
59 2 T2N1M0 42.5 37.5 45.5 30 18.57 1.3 No Recurrence 67 Chemo Treated
9.1
205
61 2 T2N1M0 42.5 42.5 52.7 34 15.71 1.5 No Recurrence 82 Chemo Treated
9.4
206
47 2 T2N1M0 12.5 42.5 52.7 40 24.29 1.5 No Recurrence 93 Chemo Treated
11.1
207
45 2 T2N1M0 22.5 45 52.7 20 12.86 1.6 No Recurrence 79 Chemo Treated
8.8
208
61 3 T2N1M0 12.5 42.5 51.8 36 17.86 1.4 No Recurrence 71 Chemo Treated
12.7
209
67 3 T2N1M0 12.5 45 54.5 24 17.14 1.8 No Recurrence 118 Chemo Treated
8.6
210
57 2 T2N1M0 17.5 47.5 41.8 22 15.71 1.5 No Recurrence 72 Chemo Treated
7.4
78
211
56 2 T2N1M0 42.5 22.5 38.2 20 17.14 1.5 No Recurrence 61 Chemo Treated
7.1
212
76 3 T2N2M0 18.75 47.5 47.3 22 18.57 1.8 No Recurrence 78 Chemo Treated
10.6
213
40 2 T3N1M0 25 37.5 50.9 32 22.86 1.5 No Recurrence 127 Chemo Treated
15.3
214
70 2 T2N2M0 12.5 45 50.9 30 16.43 1.8 No Recurrence 127 Chemo Treated
13.3
215
47 3 T2N2M0 23.75 42.5 41.8 22 17.14 1.5 No Recurrence 64 Chemo Treated
9.6
216
48 3 T2N2M0 20 45 50.9 26 12.86 1.6 No Recurrence 85 Chemo Treated
10.1
217
58 3 T2N2M0 12.5 45 53.6 24 16.43 1.6 No Recurrence 62 Chemo Treated
10.7
218
55 3 T2N0M0 31.25 47.5 49.1 24 12.86 1.6 No Recurrence 61 Chemo Treated
6.5
219
40 3 T2N0M0 38.75 42.5 50.0 25 17.14 1.5 No Recurrence 60 Chemo Treated
5.5
220
34 2 T2N0M0 12.5 46.25 51.8 24 12.86 1.8 No Recurrence 60 Chemo Treated
11.5
221
57 3 T2N0M0 15 45 50.0 22 27.14 1.5 No Recurrence 60 Chemo Treated
5.5
222
73 2 T2N0M0 20 46.25 50.9 34 17.86 1.6 No Recurrence 64 Chemo Treated
4.5
223
53 2 T2N0M0 37.5 45 50.0 27 17.86 1.5 No Recurrence 64 Chemo Treated
3.6
224
55 3 T2N0M0 21.25 47.5 53.6 20 12.86 1.8 No Recurrence 62 Chemo Treated
10.8
79
225
60 3 T2N0M0 25 43.75 53.6 20 17.86 1.8 No Recurrence 65 Chemo Treated
6.4
226
63 3 T2N0M0 18.75 47.5 52.7 23 14.29 1.8 No Recurrence 60 Chemo Treated
8.6
227
58 2 T2N0M0 38.75 43.75 50.0 39 24.29 1.5 No Recurrence 61 Chemo Treated
7
228
55 2 T1N0M0 21.25 43.75 53.6 23 14.29 1.4 No Recurrence 62 Chemo Treated
10.4
229
60 3 T3N0M0 12.5 41.25 53.6 34 17.14 1.5 No Recurrence 60 Chemo Treated
16.6
230
66 3 T2N0M0 16.25 50 53.6 26 15.71 1.6 No Recurrence 61 Chemo Treated
8.9
231
41 3 T2N0M0 17.5 47.5 50.9 28 17.14 1.6 No Recurrence 60 Chemo Treated
5.7
232
43 3 T2N0M0 12.5 47.5 52.7 26 15.71 1.6 No Recurrence 60 Chemo Treated
7.9
233
47 2 T1N0M0 46.25 27.5 49.1 20 20.00 1.1 No Recurrence 83 Chemo Treated
5.5
234
62 2 T1N0M0 12.5 40 49.1 28 27.86 1.5 No Recurrence 81 Chemo Treated
3
235
57 2 T2N0M0 55 48.75 51.8 43 31.43 1.6 No Recurrence 63 Chemo Treated
14.2
236
52 3 T2N0M0 47.5 47.5 53.6 25 22.14 1.6 No Recurrence 86 Chemo Treated
9.6
237
85 2 T2N0M0 12.5 48.75 54.5 30 16.43 1.8 No Recurrence 60 Chemo Treated
10.2
238
64 3 T2N0M0 41.25 43.75 51.8 31 20.00 1.8 No Recurrence 77 Chemo Treated
7
80
239
67 2 T3N0M0 20 37.5 52.7 26 18.57 1.3 No Recurrence 84 Chemo Treated
11.2
240
61 2 T1N0M0 15 43.75 51.8 36 18.57 1.6 No Recurrence 64 Chemo Treated
7.2
241
55 2 T2N0M0 40 47.5 52.7 36 22.86 1.6 No Recurrence 84 Chemo Treated
4.6
242
66 3 T2N0M0 35 42.5 51.8 35 25.00 1.5 No Recurrence 84 Chemo Treated
10.2
243
39 3 T2N0M0 12.5 30 50.9 24 16.43 1.1 No Recurrence 63 Chemo Treated
1.7
244
53 2 T2N0M0 46.25 40 48.2 36 14.29 1.4 No Recurrence 62 Chemo Treated
11.7
245
54 2 T2N0M0 23.75 46.25 52.7 24 21.43 1.3 No Recurrence 61 Chemo Treated
4.8
246
58 2 T1N0M0 36.25 48.75 53.6 32 27.14 1.8 No Recurrence 72 Chemo Treated
3.2
247
40 3 T1N0M0 36.25 46.25 50.9 39 28.57 1.9 No Recurrence 86 Chemo Treated
7.8
248
67 2 T1N0M0 27.5 47.5 52.7 28 15.71 1.8 No Recurrence 99 Chemo Treated
8.5
249
61 3 T2N0M0 16.25 41.25 51.8 35 23.57 1.4 No Recurrence 61 Chemo Treated
8.1
250 45 1 T3N3M0 22.5 42.5 52.72727273 26 21.42857143 1.5 Recurrence 54 Chemo Treated 24
251 69 2 T4N1M0 20 47.5 52.72727273 44 24.28571429 1.5 Recurrence 42 Chemo Treated 57.5
252 61 3 T4N1M0 47.5 45 52.72727273 36 21.42857143 1.5 Recurrence 21 Chemo Treated 70.6
81
253 55 3 T2N2M0 22.5 47.5 52.72727273 46 27.14285714 1.5 Recurrence 59 Chemo Treated 38.9
254 44 2 T3N1M0 45 47.5 52.72727273 44 31.42857143 1.5 Recurrence 12 Chemo Treated 42.3
255 64 2 T1N0M0 14.16666667 45 47.27272727 36 18.57142857 1.166666667 No Recurrence 60 Chemo naïve 9.6
256 63 2 T2N2M0 22.5 43.75 51.81818182 30 20 1.625 No Recurrence 75 Chemo Treated 9.3
257 48 1 T1N1M0 30 42.5 47.27272727 33 28.57142857 1.625 No Recurrence 60 Chemo Treated 6.7
258 50 2 T2N1M0 32.5 45 50 36 18.57142857 1.5 No Recurrence 83 Chemo Treated 7.7
259 41 2 T2N2M0 16.25 45 50.90909091 26 16.42857143 1.375 No Recurrence 62 Chemo Treated 9.5
260 60 2 T2N0M0 16.25 45 51.81818182 32 18.57142857 1.5 No Recurrence 64 Chemo Treated 4.5
261 49 3 T2N0M0 20 27.5 52.72727273 32 21.42857143 1.375 No Recurrence 65 Chemo Treated 7
262 50 2 T2N0M0 52.5 46.25 52.72727273 37 22.14285714 1.5 No Recurrence 73 Chemo Treated 10.4
263 49 2 T2N0M0 22.5 47.5 54.54545455 27 15.71428571 1.75 No Recurrence 62 Chemo Treated 8.4
264 50 3 T2N1M0 27.5 47.5 51.81818182 32 21.42857143 1.625 No Recurrence 69 Chemo Treated 7.9
265 55 2 T2N2M0 16.25 45 49.09090909 28 14.28571429 1.625 No Recurrence 61 Chemo Treated 11.5
266 57 2 T2N0M0 37.5 40 53.63636364 32 12.85714286 1.375 No Recurrence 67 Chemo Treated 8.9
82
267 41 2 T2N0M0 45 47.5 52.72727273 45 20 1.5 No Recurrence 84 Chemo Treated 13.2
268 48 1 T2N0M0 15 43.75 54.54545455 35 16.42857143 1.25 No Recurrence 75 Chemo Treated 12.5
269 63 3 T2N0M0 22.5 46.25 54.54545455 30 15 1.625 No Recurrence 76 Chemo Treated 8
270 65 2 T2N0M0 32.5 46.25 54.54545455 38 23.57142857 1.625 No Recurrence 72 Chemo Treated 4.8
271 55 2 T2N0M0 27.5 46.25 52.72727273 34 22.85714286 1.5 No Recurrence 62 Chemo Treated 3.7
272 61 2 T2N0M0 41.25 45 52.72727273 33 25.71428571 1.375 No Recurrence 60 Chemo Treated 7
273 67 2 T2N0M0 45 47.5 52.72727273 34 27.14285714 1.625 No Recurrence 61 Chemo Treated 5.6
274 74 2 T2N1M0 20 46.25 51.81818182 35 30 1.625 No Recurrence 62 Chemo Treated 8.1
275 66 2 T1N0M0 15 47.5 54.54545455 35 28.57142857 1.625 No Recurrence 61 Chemo Treated 6.3
276 61 2 T2N0M0 36.25 50 52.72727273 35 25.71428571 1.875 No Recurrence 60 Chemo Treated 3
277 35 2 T2N0M0 40 42.5 50.90909091 40 25.71428571 1.5 No Recurrence 63 Chemo Treated 8.4
278 57 2 T2N1M0 15 46.25 51.81818182 38 23.57142857 1.5 No Recurrence 66 Chemo Treated 9.5
279 34 3 T2N0M0 20 46.25 52.72727273 39 27.14285714 1.625 No Recurrence 62 Chemo Treated 9.1
280 41 2 T2N0M0 50 47.5 49.09090909 39 25.71428571 1.5 No Recurrence 73 Chemo Treated 10.8
83
281 55 2 T2N0M0 43.75 46.25 51.81818182 40 27.14285714 1.625 No Recurrence 68 Chemo Treated 7.1
282 55 2 T1N0M0 43.75 46.25 51.81818182 40 27.14285714 1.625 No Recurrence 60 Chemo Treated 8.5
283 63 2 T2N1M0 27.5 42.5 52.72727273 36 21.42857143 1.5 No Recurrence 60 Chemo Treated 7.2
284 49 2 T2N0M0 17.5 47.5 52.72727273 40 15.71428571 1.75 No Recurrence 82 Chemo Treated 9.1
285 65 2 T2N1M0 25 45 52.72727273 40 25.71428571 1.5 No Recurrence 67 Chemo Treated 10.4
286 40 2 T2N0M0 33.33333334 45 53.93939394 37.33333333 24.28571429 1.333333334 No Recurrence 94 Chemo Treated 7.7
287 43 1 T1N0M0 15.83333333 43.33333334 53.33333333 30 18.09523809 1.25 No Recurrence 101 Chemo Treated 12.6
288 44 3 T2N2M0 12.5 46.25 51.81818182 24 14.28571429 1.625 No Recurrence 69 Chemo Treated 11.3
289 59 2 T1N1M0 15 46.25 52.72727273 28 16.42857143 1.625 No Recurrence 62 Chemo Treated 13.2
290 73 2 T2N1M0 35 45 52.72727273 34 18.57142857 1.625 No Recurrence 68 Chemo Treated 6.5
291 61 2 T2N1M0 26.25 47.5 52.72727273 31 34.28571429 1.625 No Recurrence 91 Chemo Treated 10.4
292 37 2 T2N1M0 40 47.5 52.72727273 33 21.42857143 1.75 No Recurrence 62 Chemo Treated 5.9
293 55 2 T2N1M0 12.5 40 49.09090909 30 19.28571429 1.5 No Recurrence 69 Chemo Treated 4.4
294 37 2 T2N0M0 41.25 37.5 52.72727273 26 15.71428571 1.5 No Recurrence 91 Chemo Treated 5.2
84
Table 4
295 67 2 T2N0M0 32.5 47.5 52.72727273 36 22.85714286 1.5 No Recurrence 61 Chemo Treated 4.6
296 38 2 T2N0M0 25 42.5 52.72727273 30 23.57142857 1.625 No Recurrence 85 Chemo Treated 2.3
297 49 2 T1N0M0 31.25 42.5 50.90909091 31 22.85714286 1.625 No Recurrence 92 Chemo Treated 2
298 65 2 T2N2M0 23.75 36.25 47.27272727 25 17.85714286 1.375 No Recurrence 66 Chemo Treated 4.5
299 40 2 T2N0M0 12.5 32.5 50 30 35.71428571 1.375 No Recurrence 67 Chemo Treated 8.5
85
The samples employed for validation studies are subjected to exact same inclusion criteria
as provided in example 1 except that Stages used were I, II and IIIA (thus excluding IIIB
and IIIC), and each sample is scored for percentage of tumors stained (0-100) and staining
intensity (0-3) for the 5 markers - CD44, ABCC11, N-cadherin, Pan-cadherin and
ABCC4. The protocol followed for IHC measurements remain identical to that provided
in example 1. The results from grading along with three clinical parameters are fed into
the proprietary statistical CanAssist Breast algorithm for obtaining a prediction score
(CanAssist Breast relapse score) for stratifying patients into high risk or low risk of breast
cancer recurrence
The prediction score for each sample is compared with the respective patient’s medical
history with respect to relapse of the disease within 5 years to understand the accuracy of
the present method. The results showed that the method of the present disclosure provides
a 95% NPV which signifies high predictive capability when compared with the respective
patient’s medical history data available.
Example 3: Synergistic interplay of markers within the 5 marker combination of the
present disclosure
The 5 biomarker combination along with the three clinical parameters of the tumor of the
present disclosure is a synergistic combination of markers, which play an important role
in prognosis of early stage ER+/PR+ and Her2- breast cancer. Though the individual
expression of some of the markers in two samples may be identical, the recurrence profile
need not necessarily be the same. It is the interplay of these markers which when assessed
by way of the CanAssist Breast algorithm, provides the accurate results for a sample. The
validation sample set provided in example 2 above provides good data points to
understand the significance of the combination of markers, vis-à-vis individual markers.
As can be seen from table no. 5 provided below (derived from validation sample data of
table no. 4), even when one (scenarios 1, 3, 8 and 9), two (scenarios 4, 5, 6 and 7) or three
markers (scenarios 2 and 10) between two different samples have similar IHC staining
grades, the overall outcome is not necessarily the same. Therefore, no single marker can
86
determine prognosis, and it is the specific combination of 5 biomarkers that is critical for
accurate prognostication.
Scenario Sl. No. Age
A%-M W%-C U%-C R%-M F%-M WI-C
CanAssist
Score Outcome
1
8 52 17.5 42.5 49.1 44 30.00 1.3 23.8 Recurred
24 42
17.5 30 40.0 23 12.86 1.5 3.4
Not
recurred
2
7 40 42.5 37.5 49.1 44 27.14 1.8 24.1 Recurred
27 72
42.5 46.25 51.8 40 27.14 1.8 3.7
Not
recurred
3
5 43 52.5 42.5 50.9 40 27.14 1.5 20.8 Recurred
14 59
50 46.25 50.9 29 18.57 1.8 5.5
Not
recurred
4
5 43 52.5 42.5 50.9 40 27.14 1.5 20.8 Recurred
27 72
42.5 46.25 51.8 40 27.14 1.8 3.7
Not
recurred
5
3 72 50 47.5 52.7 44 35.71 1.5 19.4 Recurred
28 51
50 47.5 53.6 22 12.86 1.8 13.1
Not
recurred
6 155 41 12.5 45 50.9 42 22.86 1.3 30.1 Recurred
15 59
12.5 38.75 50.9 25 15.00 1.4 7.1
Not
recurred
7
156 47 50 37.5 45.5 50 35.71 1.3 100 Recurred
47 60
50 37.5 50.9 20 12.86 1.4 9.8
Not
recurred
8
159 50 12.5 42.5 49.1 44 12.86 1.5 19.8 Recurred
47 60
50 37.5 50.9 20 12.86 1.4 9.8
Not
recurred
9
154 60 37.5 47.5 52.7 28 28.57 1.8 16.7 Recurred
22 74
21.25 40 47.3 28 15.71 1.5 5.6
Not
recurred
2 66 57.5 45 50.9 48 34.29 1.8 15.6 Recurred
87
10 17 59
32.5 45 50.9 36 19.29 1.8 4
Not
recurred
Table 5
Example 4: Prognostic test of the instant disclosure v/s prior known means of
prognostication
Risk stratification of patients into high/low risk of breast cancer recurrence in the prior
art is carried out by analyzing patient samples for presence or absence of hormone
receptor status and clinical Stage of the disease. Eg: If a Her2-, early stage breast cancer
patient’s sample shows any of the combination of receptor expression including ER+/PR+
or ER+/PR- or ER-/PR+ then the said patient is considered at low risk for recurrence
compared to a patient not expressing both ER and PR receptors but such a patient is still
prescribed to have chemotherapy treatment. However, chemotherapy is not beneficial in
majority (~80%) of such ER+ and/ PR+, Her2- early stage breast cancer patients in terms
of preventing recurrence and often leads to reduction in quality of life (Figure 1).
On the contrary, the prognostic test of the instant disclosure is performed on these Her2-,
early stage breast cancer patients with ER+ and /or PR+ disease, which includes assessing
expression of a 5 marker combination comprising (CD44, ABCC11, N-cadherin, Pancadherin
and ABCC4). The prognostic test of the instant disclosure does not proceed if
the hormone receptor status is found negative for both the markers ER or PR. The
prognostic test of the instant disclosure is performed by carrying out an IHC based assay
for expression of said 5 biomarker combination, followed by grading the expression of
the biomarkers on the basis of parameters including percentage of staining and intensity
of staining. The data from said grading along with status of clinical prognostic parameters
is inputted into a proprietary statistical algorithm or module for obtaining a prediction
score. This score helps in deciphering if the patient falls under the high risk category or
low risk category. Accordingly, a low risk patient is advised to take optimum therapy as
per the clinician’s decision (Figure 1) thus leading to improved quality of life.
88
Therefore, if a patient sample was analyzed by the prior known method, said patient would
be classified only based on hormone receptor status, and most often advised to take
aggressive chemotherapy which may not be beneficial and will reduce quality of life.
However, on the contrary as the instant prognostic test would take into consideration the
5 biomarker combination expression which are specific to tumor biology involved in
recurrence and compute a relapse score which is assessed based on biomarker data and
clinical prognostic factors, said information could render the same patient sample into low
risk and be advised treatment accordingly. Thus, the doctors employing the prognostic
test of the instant disclosure can very accurately assess the patient samples and stratify
them effectively into high risk or low risk for cancer recurrence and devise a suitable and
optimum treatment module.
Advantages of the method and the biomarker combination of the present disclosure:
1. The method of the present disclosure uses biomarkers from pathways other
than hormone regulation and proliferation pathways thereby enhancing its
predictive potential.
2. Importantly, the prognostic test is developed and validated on Node negative
and Node positive patients from tumor stages I-IIIA and this makes it a broad
based test with wider applicability and enhanced utility worldwide. Other
prognostic tests available in the art have mostly been developed in Stage I
node negative patients and have reduced applicability in node positive
patients.
3. The method of the present disclosure is not only prognostic, but also
chemopredictive (i.e, can decide benefit of chemotherapy to patients), unlike
other methods in the art, some of which are only prognostic. Hence, the
prognostic test of the instant disclosure can help reduce unwanted
chemotherapy and prescribe additional targeted therapy via a central lab
facility.
4. The method of the present disclosure uses biomarker combination hitherto
unknown to have prognostic application in Breast Cancer. Further, these
89
marker combination(s) are also selected for their potential to be targeted for
developing new drugs to treat Breast Cancer in the future. No earlier known
method has considered this factor in their test development protocol. This is
possible because the biomarkers employed by the present disclosure are
membrane associated and hence can be targeted to make new drugs.
5. All other prognostic methods developed so far are expensive and not widely
used in geographies outside the Western world. However, the method of the
present disclosure is the first test to be affordable in India and worldwide,
thereby enhancing its utility and reach.
6. The prognostic and predictive test of the present disclosure can be a
companion test for new drugs to come in the market.
7. The method of prognosis or the prognostic test of the instant disclosure is
robust and a simple IHC based test with high NPV when compared to any
other prognostic tests available in the prior art for breast cancer detection and
prognosis.
8. The test of the present disclosure is developed on patients of Stage I, II and
IIIA. The reason IIIA is included is there are a group of these patients also
shown to do well without chemotherapy. On the contrary, the methods of the
prior art have been developed on patient samples from stage I breast cancer
but however, validated on Stage II breast cancer and node positive patients.
This means that since these models are not built on that stage II data set, they
are not as robust and applicable on the Stage II cancers as training and
validation should always preferably be done on cohorts with identical
inclusion criteria.
Although the disclosure and exemplification has been provided by way of illustrations
and examples for the purpose of clarity and understanding, it is apparent to a person
skilled in the art that various changes and modifications can be practiced without
departing from the spirit or scope of the disclosure. Accordingly, the foregoing
descriptions and examples should not be construed as limiting the scope of the present
disclosure.
90
The description of the embodiments of the present disclosure reveals the general nature
of the embodiments that are readily suitable for modification and/or adaptation for various
applications by applying the current knowledge. Such specific embodiments of the
disclosure, without departing from the generic concept, and, therefore, such adaptations
and modifications should and are intended to be comprehended and considered within the
meaning and range of equivalents of the disclosed embodiments.
It is also to be understood that the phrases or terms employed herein are for the purpose
of description and not intended to be of any limitation. Throughout the present disclosure,
the word “comprise”, or variations such as “comprises” or “comprising” wherever used,
are to be understood to imply the inclusion of a stated element, integer or step, or group
of elements, integers or steps, but not the exclusion of any other element, integer or step,
or group of elements, integers or steps.
Where a numerical limit or range is stated herein, the endpoints are included. Also, values
and sub-ranges within a numerical limit or range are specifically included as if explicitly
written out.
With respect to the use of any plural and/or singular terms in the present disclosure, those
of skill in the art can translate from the plural to the singular and/or from the singular to
the plural as is considered appropriate to the context and/or application. The various
singular/plural permutations may be expressly set forth herein for the sake of clarity.
Any discussion of documents, acts, materials, devices, articles and the like that has been
included in this specification is solely for the purpose of providing a context for the
present disclosure. It is not to be taken as an admission that any or all of these matters
form a part of the prior art base or are common general knowledge in the field relevant to
the present disclosure, as it existed anywhere before the priority date of this application.
The contents of all references, patents, and published patent applications cited throughout
this application are incorporated herein by reference for all purposes. ,CLAIMS:We Claim:
1. A method of measuring biomarker expression, comprising measuring expression
of five biomarkers by assaying a biological sample with a combination of
antibodies, wherein:
the biological sample is obtained from a subject having ER+/PR+ and Her2-
breast cancer or after removal of the breast cancer; and
the five biomarkers are CD44, ABCC11, N-cadherin, Pan-cadherin and
ABCC4.
2. A method of performing immunohistochemistry (IHC) on a tumor sample
obtained from a subject having breast cancer or after removal of the breast cancer,
comprising:
performing IHC on the tumor sample to optionally detect receptor expression
by detecting whether cells are expressing at least one receptor selected from
the group consisting of estrogen receptor and progesterone receptor and not
expressing Her2; and
performing the method as claimed in claim 1 on the tumor sample by IHC to
measure the expression of the five biomarkers CD44, ABCC11, N-cadherin,
Pan-cadherin and ABCC4.
3. A method of prognosing and treating a subject having ER+/PR+ and Her2- breast
cancer or after removal of the early stage ER+/PR+ and Her2- breast cancer,
comprising:
prognosing whether the subject is at high or low risk for breast-cancer
recurrence by performing, and analyzing the results of the method as claimed
in claim 2; and
treating the prognosed subject with chemotherapy if prognosed with high risk
for breast-cancer recurrence.
4. The method as claimed in any of the preceding claims, wherein the breast cancer
is stage I, II or IIIA hormone receptor positive, Her-2-neu receptor negative
invasive ductal carcinoma, or invasive lobular carcinoma of the breast.
5. The method as claimed in claim 3, wherein the breast-cancer recurrence is
prognosed after surgical removal of the breast cancer.
92
6. The method as claimed in claim 2, wherein the expression of the five biomarkers
is measured if expression of at least one of estrogen receptor or progesterone
receptor is detected; and wherein the sample is negative for Her-2-neu receptor.
7. The method as claimed in claim 2, wherein the IHC is morphometric IHC; and
wherein expression of the five biomarkers is measured on basis of percentage of
cells stained and staining intensity of cells.
8. The method as claimed in claim 2, further comprising assessing clinical
parameters node status, tumor grade, and tumor size, wherein the node status
includes tumor node positive and tumor node negative, the tumor grade includes
Grades 1, 2 and 3, and the tumor size includes T1, T2, or T3.
9. The method as claimed in claim 2, wherein the tumor sample obtained from the
subject is collected, fixed and sectioned prior to the IHC; and wherein the IHC is
performed by adding primary antibodies and secondary antibodies conjugated
with a detectable label or enzyme or molecule, and detecting a color or
fluorescence from the detectable label.
10. A combination of antibodies, comprising antibodies specific for five biomarkers
CD44, ABCC11, N-cadherin, Pan-cadherin and ABCC4.
11. A kit comprising: the combination of antibodies as claimed in claim 10; and
instructions for measuring expression of the five biomarkers CD44, ABCC11, Ncadherin,
Pan-cadherin and ABCC4.
12. The kit as claimed in claim 11, further comprising at least one member of IHC
reagents selected from xylene, isopropanol, ethanol, buffer solutions, protein
blocking agents, primary antibodies, secondary antibodies labeled with enzymes
such as horseradish peroxidase (HRP) or, alkaline phosphatase (AP) or with
fluorescent tags such as fluorescein isothiocyanate (FITC) or, phycoerythrin (PE)
or with molecule biotin or streptavidin, and substrates such as diamino benzydene
(DAB) or p-nitrophenyl phosphate (PNPP).
13. An IHC-based assay system, comprising the combination of antibodies as claimed
in claim 10.
14. The IHC-based assay system as claimed in claim 13, further comprising at least
one member of IHC reagents selected from xylene, isopropanol, ethanol, buffer
solutions, protein blocking agents, primary antibodies, secondary antibodies
labeled with enzymes such as horseradish peroxidase (HRP) or, alkaline
phosphatase (AP) or with fluorescent tags such as fluorescein isothiocyanate
93
(FITC) or, phycoerythrin (PE) or with molecule biotin/streptavidin, and substrates
such as diamino benzydene (DAB) or p-nitrophenyl phosphate (PNPP).
15. A method of predicting the likelihood of recurrence of breast cancer in a subject
having ER+/PR+ and Her2- breast cancer or after removal of the breast cancer,
comprising: measuring expression level of five biomarkers CD44, ABCC11, Ncadherin,
Pan-cadherin and ABCC4;
calculating a relapse score for said subject by measuring the differential
expression levels of each of the biomarkers and their contribution to breast cancer
recurrence; and
using said relapse score to determine the likelihood of breast cancer recurrence.
16. The method as claimed in claim 15, further comprising assessing clinical
parameters node status, tumor grade, and tumor size, wherein the node status
includes tumor node positive and tumor node negative, the tumor grade includes
Grades 1, 2 and 3, and the tumor size includes T1, T2, or T3.
17. The method as claimed in claim 15, wherein the breast cancer is stage I, II or IIIA
hormone receptor positive, Her-2-neu receptor negative invasive ductal
carcinoma, or invasive lobular carcinoma of the breast.