Field of the Invention
The present invention relates to a method of predicting chemo prediction for solid tumor
patients (except for neurological cancers). More particularly, the present invention
relates to a method of measuring chemo prediction assay which is done on a coated
layer of matrigel. This matrigel is an artificial basement membrane. Furthermore, this
invention also relates to a method of measuring chemo prediction assay on which a
layer of mesothelial cells is coated from the same patient. Moreover, this invention also
relates to a method of measuring chemo prediction assay which not only predicts first
line chemo prediction but also second line or recurrent Cancer Chemo Prediction Assay.
In this process, this assay therefore is a replica of the clinical situation in an ex vivo
situation giving rise than to a more authenticated replication of the behavior of cells in
vivo.
Background of the Invention and Related Prior Art
Cancer treatments have included chemotherapy (i.e., drugs), radiation therapy,
immunotoxins (i.e., antibodies specific for a particular tumor cell attached to a toxin), and
biological modifiers. As some treatments work better than others for different kinds of
tumors and in different patients (e.g., some chemotherapy agents work well than others
for different tumors), it is desirable to have an idea of the particular treatment having the
best likelihood of being effective before administering to a specific patient. Cancer
screening tests are of two basic types: invasive and non-invasive. Taking bladder cancer
as an example, the primary invasive test used is the endoscopic examination, which is
usually repeated every three months for patients with histories of non-invasive
carcinoma of the bladder, and may also be performed periodically on members of high-
risk groups such as dye and paint factory workers.
Among non-invasive diagnostic tests, urinary cytology is the most reliable currently in
use. The accuracy of urinary cytologies is usually between 60% and 80%, but can be
considerably less accurate in the detection of low-grade, low-stage disease.
Furthermore, the use of intravesical antineoplastic drugs increases the number of false
positive results for this test.
The measurement of other diagonistic markers in the urine, e.g., LDH, CPK, CEA,
tryptophan, and polyamines, has not yet proved reliable enough for routine use.
It is known that tumor growth is associated with angiogenesis (the growth of new blood
vessels), and it is believed that the mechanism for such tumor-induced angiogenesis is
the secretion by tumors of one or more angiogenesis-promoting factors. Tumor-induced
angiogenesis probably occurs as a result of first the migration of, followed by the
proliferation of capillary endothelial cells.
The patent document US6355427 describes the lifetime probability of a woman
developing breast cancer can now be determined based on an allelic variation found in
the 3'UTR of the prohibitin gene. The probability is dependent on the sequence of the
3'UTR at position 729, i.e., whether there is a thymine (T) or a cytosine (C) or both at this
position. Polymorphism at position 729 is also disclosed as a susceptibility indicator for
hereditary breast cancer in men. Determining the sequence at the position 729 can be
done by any number of standard techniques. Preferably, the sequence is determined by
amplifying this region by PCR and subjecting it to an RFLP analysis.
The other document US6440676 states the identification of characteristic, nucleic acid
signals is a useful and important discovery which allows for compositions, assays, kits
and reagents suitable for the characterization of various brain cancers. Provided herein
are reagents and methods for ascertaining the propensity of a cell for malignant
phenotype said cell being isolated or in a biological sample, said method comprises
assaying a cell or biological sample to be tested for a signal indicating the transcription
of a nucleic acid transcript. In a preferred embodiment, the nucleic acids are
substantially identical to the sequences of Table I, SEQ ID NOS. 1-9, and known as
CINN 1, CIN 2, OP2C2-6, OP7C3-1, OP9 A4-2, OP11 C1-3, OP11 G2-10, FAS OP13
C1-D, FAS OP17 C1-D, dek, laminin .alpha.-chain gene, .alpha.-NAC gene, ribosomal
protein L35a, and ribosomal protein L7a. Also provided are methods for monitoring
cancer progression or the effectiveness of a treatment regimen, and methods for
identifying compounds that affect expression of genes involved in cancer.
According to the Merck Manual of Diagnosis and Therapy, 16th ed., Merck & Co., 1992,
cancer can develop in any tissue of any organ at any age. Most cancers detected at an
early stage are potentially curable; thus, physicians need a heightened awareness of
predisposing inherited and environmental factors. The ability to screen patients for
genetic predisposition for cancer can greatly assist in the monitoring of high-risk patients
for early signs of cancer, and thus allowing for early intervention.
The other document Professional Guide to Disease, 3rd ed., Springhouse Corp., 1989
says the malignant brain tumors (for example glioma, meningiomas, and schwannomas)
are common, with an incidence of 4.5 per 100,000. The most common tumor types in
adults are gliomas and meningiomas. The most common tumors in children are
astrocytomas, medulloblastomas, ependymomas, and brain stem gliomas. In children,
brain tumors are one of the most common causes of death from cancer.
The patent document US7094538 describes a method for determining risk of cancer
other than breast cancer comprising the steps of: a) determining the base identity of a
portion of genomic DNA from a patient cell sample, said genomic DNA comprising a
prohibitin gene comprising a 3' untranslated region, said portion corresponding to
position 729 as defined in SEQ ID NO:1 of said prohibitin gene in said untranslated
region; and b) correlating said base identity at position 729 as defined in SEQ ID NO:1 of
said genomic DNA with germline polymorphisms at position 729 indicative of a risk for
said cancer.
The document US7618790 states the field of cancerology. More particularly, the method
for the diagnosis of breast cancer in a human patient by determining the presence of
nerve growth factor (NGF) in a biological sample derived from this patient, it being
possible for said method to be used both in early diagnosis, screening, therapeutic-
follow up and prognosis, and in the diagnosis of relapse in the case of breast cancer. In
addition, due to the ability of breast cancer cells to produce NGF, the present invention
also relates to therapy. In women, breast cancer is the primary cause of mortality due to
cancer in industrialized countries. It is estimated that the minimum size of a tumor that
can be detected by mammography is 1 cm. Breast cancers develop slowly. However,
this small tumor has an evolutive past of 8 years, on average, at the time of diagnosis.
The etiology of breast cancer is not well defined. Familial predispositions have been
demonstrated. Age is the most important risk factor. Thus, the risk increases by 0.5%
per year of age in countries in the west. Other risk factors are known, such as the
number of pregnancies and the age of the first pregnancy, breast-feeding, the age at
puberty and at the menopause, estrogenic treatments after the menopause has
occurred, stress and nutrition.
Gorelik, E., et al. states the new Short-Term Assay for in Vivo Evaluation of the Effects
of Anticancer Drugs on Human Tumor Cell Lines. Cancer Research, Vol. 47, (1987)
describes injecting microcapsules filled with human tumor cells into nude mice and
administering drugs to evaluate their effectiveness in vivo.
Ovarian cancer remains the most lethal gynecologic malignancy. Despite the
introduction of taxanes in the 1980s, the recurrence rates and overall survival for ovarian
cancer have remained poor, with less than one third of advanced stage cases surviving
beyond five years. Information regarding the physiology and biology of these tumors and
in vivo responses to standard chemotherapeutics is limited by current clinical
management guidelines, consisting of surgical cytoreduction followed by chemotherapy.
Ovarian cancer patients initially respond to surgery and chemotherapy, but the disease
often recurs between six months and two years. Recurrent disease frequently becomes
unresponsive to chemotherapy and eventually leads to death.
The other document 1193/KOLNP/2003 states the methods for identification of
cancerous cells by detection of expression levels of TTK, as well as diagnostic,
prognostic and therapeutic methods that take advantage of the differential expression of
these genes in mammalian cancer. Such methods can be used in determining the ability
of a subject to respond to a particular therapy, e.g., as the basis of rational therapy. In
addition, the invention provides assays for identifying pharmaceuticals that modulate
activity of these genes in cancers in which these genes are involved, as well as methods
of inhibiting tumor growth by inhibiting activity of TTK.
According to the document 44/MUMNP/2009 discloses a diagnostic assay for
determining presence of lung cancer in a patient depends, in part, on ascertaining the
presence of an antibody associated with lung cancer using random polypeptides. The
assay predicted lung cancer prior to evidence of radiographically detectable cancer
tissue.
The patent document 4400/DELNP/2009 states a methods for identifying cancer patients
eligible to receive Bcl-2 family inhibitor therapy and for monitoring patient response to
Bcl-2 family inhibitor therapy comprise assessment of the expression levels of the
biomarker combinations set out in tables in a patient tissue sample. The methods of the
invention allow more effective identification of patients to receive Bcl-2 family inhibitor
therapy and of determination of patient response to the therapy.
Cancer researchers have long been interested in discovering ways to predict the
response of different individuals' tumors to different chemotherapeutic agents. However,
tumors generally include cancer cells from numerous different tumor cell lines which live
together in equilibrium. Different cell lines may be more resistant to chemotherapy than
others. Thus, because certain individuals may have tumors with drug-resistant cell lines
intermingled with "generic" tumor cell lines, medical personnel cannot simply assume
that administration of a drug which is ordinarily effective against a certain type of tumor
in most individuals will be effective in a particular individual suffering from that type of
tumor. It has long been recognized that because of the heterogeneous nature of tumors
and the likelihood of adaptive mutation due to the inherent instability of the tumor
genome, a blanket or "off-the-shelf phamaceutical agent for curing cancer, even a
particular type of cancer, is unlikely. Several studies have expressly noted that blanket
treatment of a group of patients with a specific anti-cancer drug or drugs often works
very well for a subpopulation of the patients, but then either works minimally or not at all
for the remainder of the patients because these patients suffer from drug-resistant
tumors. According to Lacombe et al. (1994) and Smit et al. (1992) doxorubicin is
commonly administered for lung carcinoma due to its high effectiveness in certain cases,
but for certain patients, treatment is virtually ineffective. In short, generic treatment
methods frequently do not work with specific individuals because of varying tumor
phenotypes.
In response to this problem, some researchers have proposed the use of in vitro
pharmacosensitivity assays to substitute for the lack of in vivo dam for individual cancer
cases: by testing an individual's tumor response to a chemotherapeutic agent in vitro,
the individual's tumor response to that agent in vivo can supposedly be predicted. Most
pharmacosensitivity assays currently in use are at least partially based on the pioneering
soft agar tumor culture assay of Salmon et al. (1977). Other exemplary
pharmacosensitivity assays are the MTT (3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl
tetrazolium bromide) test and the dye exclusion assay test. Schadendorf et al. (1993)
states that the prior art contains several in vitro studies of tumor response according to
these assays and correlates in vitro and in vivo results.
Unfortunately, the prior art pharmacosensitivity assays suffer from several drawbacks
which have prevented their widespread use.
First, as noted by the prior art, some assays are difficult, expensive, and time-consuming
to perform. The last of these disadvantages is possibly the greatest hurdle to widespread
use of pharmacosensitivity assays since medical personnel frequently cannot afford to
expend extended amounts of time performing a pharmacosensitivity assay because of
the threat of disease progression. Owing to this disadvantage, the remainders of this
disclosure are addressed to a "short-term" assay, that is, a pharmacosensitivity assay
that takes less than approximately one week to complete between the time of detection
of the disease and the completion of the assay.
Second, as noted by many of the aforementioned studies, many of the prior art
pharmacosensitivity assays are not applicable to all types of tumors, e.g., liquid tumors
or very hard tumors. As a result, a specific prior art assay cannot be used for all patients,
and different patients require different tests tailored to their specific types of tumors. This
results in an increase in the overall cost of testing for all patients, and tends to have the
effect of reserving assay-based treatment for the wealthy few who can afford such
individualized care.
Third, the prior art pharmacosensitivity assays simply do not present an accurate
reflection of in vivo drug application. Many of the assays suffer from low correlation
between in vitro and in vivo results for particular (or all) tumor types and cell lines.
Due to the disadvantages listed above, the use of short-term pharmacosensitivity assays
has been discouraged for use in cancer patient treatment, especially for the primary
treatment of newly diagnosed patients; patients for whom it is believed that effective
treatment exists; and for patients with a drug-sensitive tumor who fail the first trial of
chemotherapy. It is believed in the art that short-term phamacosensitivity assays may
provide a benefit insofar as they can deter patient exposure to the toxicity of drugs that
are unlikely to be effective, but otherwise the assays cannot recommend a patient
treatment regimen superior to one devised by an experienced medical practitioner who
exercises sound judgment.
None of the currently available first line chemo prediction assays (ChemoFx by Precision
Therapeutics, USA, MICK assay by Diatech Oncology, USA and Extreme Drug
resistance assay (EDR) by Exiqon, USA creates an *ex vivo* condition as completely as
is described in this assay (use of basement membrane matrix, use of normal cells
obtained from the patient where the tumor grows). The assays of all these companies
grow tumor cells in vitro in the laboratory on un-coated plates. EDR assay involves
growing tumor cells like our assay but they culture the cells in soft agar is not a clinical
representation of the scenario.
Summary of the Invention
The present invention relates to a method of predicting chemo therapy for first line and
recurrent patients. More particularly, the present invention relates to a method of
measuring chemo prediction assay which is done on a coated layer of matrigel. This
matrigel is an artificial basement membrane. Furthermore, this invention also relates to a
method of measuring chemo prediction assay on which a layer of mesothelial cells is
coated from the same patient. Moreover, this invention also relates to a method of
measuring chemo prediction assay which not only predicts first line chemo prediction but
also second line or recurrent Cancer Chemo Prediction Assay. In this process, this
assay therefore is a replica of the clinical situation in an ex wVo situation giving rise than
to a more authenticated replication of the behavior of cells in vivo or in vitro. Currently
there are no available methods to test for best therapy towards recurrent cancer patients
(second line chemotherapy) other than this assay.
Detailed description of the Invention
Accordingly, the main object of the present invention is to provide a method of chemo
prediction assay, which obviates the drawbacks in the present invention.
Another object of the present invention is to provide a method of measuring chemo
prediction assay and this method take advantage of the differential response of the
chemo drugs to the tumor cells for first line and recurrent patients. Such methods can be
useful in determining the ability of a subject to respond to a particular therapy.
Still other object of the present invention to provide to a method of measuring chemo
prediction assay which is done on a coated layer of matrigel which is an artificial
basement membrane.
Still another object of the present invention is to provide a method of measuring chemo
prediction assay on which a layer of mesothelial cells is coated from the same patient.
Yet another object of the invention is to a method of measuring chemo prediction assay
which not only predicts first line chemo prediction but also second line or Recurrent
Cancer Chemo Prediction Assay. In this process, this assay therefore is a replica of the
clinical situation in an ex vivo situation giving rise than to a more authenticated
replication of the behavior of cells in vivo or in vitro.
Yet another object of the invention is to a method of measuring chemo prediction assay
which is done on a biological sample obtained as a tumor from surgery or from FNAC
or FNAB.
The steps of the assay are explained as follows:
Step 1: Procurement of tissue: The tissue is procured during surgery from the
operating room under sterile conditions. Briefly, the performing surgeon removes a piece
of tumor and transfers it into a sterile 50 ml tube containing 10 ml of sterile RPMI1640
medium {without Fetal Bovine Serum or FBS). The surgeon then uses a cervical brush
to collect normal peritoneal cells from the organ of choice of the surgeon and the brush
is transferred to a 15 ml sterile tube containing 5 ml of RPMI1640 (without FBS). The 2
tubes are then transferred to the laboratory under room temperature conditions in a
sterile box. This method ensures that the sample can be stable even 3 days post
surgery.
Step 2: Laboratory method: The normal cells in the brush are harvested under sterile
conditions by 10 ml RPMI1640 (with 10% FBS). The cells are counted and 100000 cell
suspension are added to matrigel (Becton Dickinson) coated 96 well plate and incubated
in a C02 incubator with 5% C02 and 37C for 2 hours.
Step 3. The tumor is transferred under sterile conditions on a 60 mm dish and a pure
tumor piece (without surrounding tissues) with a size of 2 - 4mm is surgically excised. 50
ml of RPMI160 (with 10%FBS) is injected 50 times in the tumor by a 26 gauge needle
and 10 ml syringe. The effusion of cells is collected in a 50 ml tube. The cells are
washed twice with PBS (phosphate buffered saline) and re-suspended in 10 ml
RPMI160 (with 10%FBS). The cells are counted and at the end of 2 hours of incubation
of normal cells, 100000 tumor cells are added on top of the normal cells. Prior to this
step, microscopic observation is done to ensure that the normal cells have adhered and
formed a monolayer on the plates. The plates are kept overnight in 5% C02 and 37C in
a C02 incubator.
Step 4. 18 hours after cell addition, RPMI1640 medium is removed and fresh medium is
added.
Step 5: In the 96 well plate, row A1 - A12 is used as Control (without drug) and in rows B
to H, seven different chemo drugs are added as per the following protocol in triplicates.
The concentration of the drugs will depend on the type of cancer. The four doses
selected will be in the area under curve for toxicity assays in cell lines of the particular
cancer.
B1-B3 = Drug 1 {dose 1), B4-B6 = Drug 1 (dose 2), B7-B9 = Drug 1 (dose 3), B10-B12 =
Drug 1 (dose 4).
C1-C3 = Drug 2 (dose 1), C4-C6 = Drug 2 (dose 2), C7-C9 = Drug 2 (dose 3), C10-C12
= Drug 2 (dose 4).
D1-D3 = Drug 3 (dose 1), D4-D6 = Drug 3 (dose 2), D7-D9 = Drug 3 (dose 3), D10-D12
= Drug 3 (dose 4).
E1-E3 = Drug 4 (dose 1), E4-E6 = Drug 4 (dose 2), E7-E9 = Drug 4 (dose 3), E10-E12 =
Drug 4 (dose 4).
F1-F3 = Drug 5 (dose 1), F4-F6 = Drug 5 (dose 2), C7-C9 = Drug 5 (dose 3), F10-F12 =
Drug 5 (dose 4).
G1-G3 = Drug 6 (dose 1), G4-G6 = Drug 6 (dose 2), G7-G9 = Drug 6 (dose 3), G10-
G12 = Drug 6 (dose 4).
H1-H3 = Drug 7 (dose 1), H4-H6 = Drug 7 (dose 2), H7-H9 = Drug 7 (dose 3), H10-H12
= Drug 7 (dose 4).
The chemo drugs are chosen according the physicians' requirements for a particular
type of cancer.
Commonest examples of anti cancer drugs to be tested are:
1. Paclitaxel
2. Carboplatin
3. Etoposide
4. Topotecan
5. Doxorubicin
6. Cyclophosphamide
7. Methotrexate
8. 5 Fluoro-uracil
9. Vincristine
10. Gemcitabine
11. Epirubicine
Step 6: After drug addition, the plates are incubated in 5% C02 and 37°C in a C02
incubator. The next day another round of drugs are applied according to the same
protocol.
Step 7. The next day (that is 48 hours after 1st drug treatment) the medium from the
wells with the same drug are collected in a 15 ml tube.
Example:
Medium from B1 to B12 are collected in the same tube. The plates are washed twice
with PBS and the washings are collected in the same tube. These are the floating cells
that have responded to chemo drugs and have either died or floated up in the medium.
Step 8. The plates are then fixed for 15 mins in 100% methanol and stained with Cell
stain solution (Chemicon) for 5 minutes. The stain is then washed away. The best
functional drug for 1st line chemo is predicted by calculating according to the formula
given below.
Formula of calculation for first line chemoprediction: Number of normal cells added -
Number of tumor cells added: Number of remaining normal cells - Number of
remaining tumor cells.
This takes into account the toxicity of the chemo drug to the normal cells as well as the
tumor cells. This is done by automated microscopy followed by image analysis with
Imagepro software. This is immediately reported to the clinician.
Step 9: The tube of cells treated with the best functional chemo drug is washed with
PBS and the viable cells are counted in a hemocytometer by trypin blue dye exclusion
method. The cells are resuspended in 10ml RPMI1640 (with 10% FBS) and equal
number of cells are added on 96 well nanoculture microhoneycomb plate (SCIVAX) and
incubated in 5%C02 and 37C in a C02 incubator.
Step 10: 5-10 days after incubation of the chemo-drug challenged cells, colony of cells
start appearing. These cells are allowed to grow to about 50% confluence.
Step 11: These cells are then again subjected to chemo-drugs and the experiment is
carried on as described above. The best drug prediction is done by the formula given
below:
Formula for calculation of second line chemo-prediction: Number of tumor cells
remaining in the plate - Number of tumor cells added.
A lesser numerical value predicts greater efficiency of chemo drug to the patient who
has already undergone first line chemotherapy and where the disease has recurred. This
prediction is immediately reported to the clinician.
The whole procedure takes anywhere from 2 weeks to 3 weeks to complete depending
on cell growth post first line chemotherapy. The clinician is already ready with laboratory
information about the next course of medicine for the particular patient where the cancer
is going to recur.
An in vitro assay for identifying a candidate agent that reduces growth of a cancerous
cell which comprises: a) calculation for first line chemo-prediction; b) calculation for
second line chemo-prediction; in which a lesser numerical value predicts greater
efficiency of chemo drug to the patient who has already undergone first line
chemotherapy and where the disease has recurred.
The novelty of the present invention lies in the assay on a coated layer of matrigel which
is an artificial basement membrane, on which we coat a layer of mesothelial cells from
the same patient. This creates an ex vivo situation that mimics the actual clinical
situation which the other assays do not use. Also the other assays only test for first line
chemo prediction, while our assay not only predicts first line chemo prediction but also
second line or Recurrent Cancer Chemo Prediction Assay.
However, this should not be construed to limit the scope of the present invention.
The procedure is well-suited for everyday performance in a laboratory. Because the
procedure may be used for different types of tumors, and because it may be run for
numerous different patients continuously or simultaneously, it is cost-effective for use
with practically all cancer patients. The cost of the procedure can be further reduced if
one or more of the steps of the procedure are automated.
Apart from the treatment of cancer, the procedure is expected to have application in the
treatment of neo-adjuvant therapy, wherein treatment is applied to an individual with
evidence of disease due to a high likelihood of the presence of microscopic metastases.
Although the preferred embodiments of the present invention have been described
above, it should be understood that the present invention is not limited thereto and that
other modifications will be apparent to those skilled in the art without departing from the
spirit of the invention.
The scope of the present invention, therefore, should be determined solely by the
appended claims.
I/We claim:
1. A method of measuring chemo prediction assay which comprises the steps of:
(a) preparing a cancer cell suspension from a cancer specimen obtained from a
human cancer patient;
(b) preparing a control sample from the normal cells of the body;
(c) preparing several drug samples from several putative cancer cell growth-
inhibiting drugs and the cancer cell suspension;
(d) seven different chemo drugs are added as per the following protocol in triplicates;
(e) incubating the control sample and drug samples;
(f) staining the control samples and drug sample with a Cell stain solution
Chemicon;
(g) determining the cancer cell viability in the control sample and the drug samples
by use of automated microscopy followed by image analysis and
characterized in that cells are then again subjected to chemo-drugs by
calculating the 1st line chemo and after that second line chemo.
2. A method of measuring chemo prediction assay as claimed in claim 1 wherein a
lesser numerical value predicts greater efficiency of chemo drug to the patient who
has already undergone first line chemotherapy and where the disease has
recurred.
3. A method of measuring chemo prediction assay as claimed in claim 1 wherein
whole procedure takes anywhere from 2 weeks to 3 weeks to complete depending
on cell growth post first line chemotherapy.
4. A method of measuring chemo prediction assay as claimed in claim 1 which is
done on a biological sample obtained as a tumor from surgery or from FNAC or
FNAB.
5. An in vitro assay for identifying a candidate agent that reduces growth of a
cancerous cell which comprises:
a) calculation for first line chemo-prediction;
b) calculation for second line chemo-prediction;
in which a lesser numerical value predicts greater efficiency of chemo drug to the
patient who has already undergone first line chemotherapy and where the disease
has recurred.
6. A method of measuring cell homing assay substantially as hereinbefore described
with particular reference.
7. An in vitro assay for identifying a candidate agent substantially as hereinbefore
described with particular reference.

ABSTRACT

The present invention relates to a method of predicting chemotherapy for first line and
recurrent patients. More particularly, the present invention relates to a method of
measuring chemo prediction assay which is done on a coated layer of matrigel. This
matrigel is an artificial basement membrane. Furthermore, this invention also relates to a
method of measuring chemo prediction assay on which a layer of mesothelial cells is
coated from the same patient. Moreover, this invention also relates to a method of
measuring chemo prediction assay which not only predicts first line chemo prediction but
also second line or recurrent Cancer Chemo Prediction Assay. In this process, this
assay therefore is a replica of the clinical situation in an ex vivo situation giving rise than
to a more authenticated replication of the behavior of cells in vivo or in vitro.