DESC:FIELD OF THE INVENTION
The present invention relates to the field of molecular diagnostics, specifically to a salivary biomarker panel and associated diagnostic kit for therapeutic monitoring and relapse prediction in head and neck cancers, including oral squamous cell carcinoma (OSCC). The invention pertains to a non-invasive diagnostic solution that utilizes a synergistic combination of RNA biomarkers derived from salivary extracellular vesicles, integrated with a structured analytical workflow for real-time assessment of therapeutic response, relapse risk, immunotherapy suitability, and patient prognosis.
This invention further relates to diagnostic compositions, in vitro diagnostic methods, and clinical decision-support tools designed to facilitate dynamic, repeatable monitoring of treatment efficacy and disease progression, offering a practical alternative to conventional tissue biopsies and imaging-based assessments. The invention has broad applicability in precision oncology, companion diagnostics, and personalized cancer care.

BACKGROUND OF THE INVENTION
Head and neck squamous cell carcinomas (HNSCC), including oral squamous cell carcinoma (OSCC), represent a significant global health burden. According to Globocan 2022, head and neck cancer accounts for approximately 930,000 new cases annually worldwide, with Asia contributing about 575,000 cases, and India alone reporting nearly 205,000 new cases annually. OSCC is the leading cancer in Indian males, fourth in females, and second overall. However, true incidence is likely underreported. Alarmingly, only 17% of patients are diagnosed at Stage I, with most presenting at advanced stages.
Diagnosis in OSCC is typically delayed, often occurring only after symptoms such as pain, dysphagia, or palpable masses appear. By this time, tumors are usually locally advanced, restricting treatment options and necessitating aggressive interventions like radical surgery or chemoradiotherapy, which severely affect survival and quality of life. Similarly, relapse detection is largely dependent on imaging techniques such as PET-CT or MRI, performed annually which detect relapse only after tumors have regrown to visible size. These methods fail to identify early molecular or microenvironmental changes indicative of treatment resistance or residual disease, resulting in delayed clinical intervention and poorer outcomes.
Current diagnostic and monitoring strategies are heavily reliant on invasive tissue biopsies and imaging modalities, both of which carry significant limitations. These include invasiveness, high costs, sampling bias due to tumor heterogeneity, limited sensitivity for early detection, and inability to provide dynamic or real-time assessment of therapeutic response. Repeated biopsies are impractical, while imaging often yields false positives or negatives, leading to delayed or suboptimal treatment decisions.
Treatment planning, especially in advanced OSCC cases, depends heavily on imaging-derived metrics. Neoadjuvant chemotherapy (NACT) is typically prescribed based on tumor size and spread, with treatment efficacy retrospectively evaluated after multiple cycles using the Response Evaluation Criteria in Solid Tumors (RECIST). However, RECIST is a static, delayed metric based on morphological tumor shrinkage, failing to capture early molecular responses such as necrosis, immune activation, or microenvironmental shifts. It primarily evaluates linear changes in tumor diameter, not: Tumor density, Metabolic activity and Molecular alterations. Cannot Differentiate Between Viable and Non-Viable Tissue. Insensitive to Pseudoprogression, especially in immunotherapy, tumors may temporarily enlarge due to immune infiltration (called pseudoprogression), which RECIST mislabels as progressive disease (PD). Cannot accurately assess Bone lesions, Lymphangitis spread and Diffuse mucosal thickening (common in OSCC). This often results in continuation of ineffective therapies, exposing patients to unnecessary toxicity and delaying potentially curative interventions.

Even in early-stage cases, nodal involvement is frequently confirmed only after surgical resection via histopathology, necessitating reactive rather than proactive treatment adjustments. Additionally, some early-stage tumors exhibit aggressive behavior and metastasize quickly, yet current protocols fail to identify such cases in advance, leading to relapse.
Relapse detection remains a significant clinical challenge. While early relapses could potentially be managed with minimally invasive procedures, most are detected too late, necessitating full cycles of chemotherapy, radiotherapy, or surgery, all of which have high cumulative toxicity. Re-radiation is often unfeasible due to prior tissue damage, and anatomical limitations restrict surgical interventions.
For patients with inoperable disease, multiple relapses, or resistance to standard therapies, immunotherapy agents like cetuximab, nivolumab, and pembrolizumab are employed. However, their use still requires PD-L1 expression assessment via invasive tissue biopsy, creating a paradox where even inoperable patients must undergo biopsy for treatment eligibility.
Despite extensive research into blood and tissue biomarkers, and emerging work in salivary transcriptomics and proteomics, there is no clinically validated, non-invasive biomarker platform that enables real-time therapeutic monitoring or early relapse prediction in HNSCC. Existing diagnostic modalities fail to bridge this critical gap, lacking the ability to provide dynamic molecular insights necessary for timely clinical decision-making.
Saliva, as a non-invasive and easily collectible biofluid, offers a promising alternative for repeated sampling and longitudinal monitoring. Tumor-derived extracellular vesicles present in saliva contain stable RNA and protein cargoes shielded from degradation, making them ideal candidates for molecular diagnostics. However, to date, there are no clinically translatable salivary biomarker panels or diagnostic kits specifically tailored for real-time monitoring of therapeutic efficacy and relapse risk in head and neck cancers.
Thus, there is an urgent unmet clinical need for non-invasive, repeatable, and real-time molecular tools that enable early prediction of therapeutic response and relapse risk, thereby facilitating personalized treatment strategies and shifting clinical practice from reactive to proactive cancer care. The present invention addresses these challenges through a salivary biomarker panel and diagnostic kit capable of dynamic monitoring, empowering clinicians with actionable molecular insights to improve patient outcomes in OSCC.
PRIOR ARTS
Several prior art references disclose methods and compositions related to cancer biomarker detection using nucleic acid or protein-based assays. However, these disclosures fail to address the specific diagnostic and clinical management needs of oral squamous cell carcinoma (OSCC), particularly in the context of non-invasive, repeatable, and dynamic therapeutic monitoring and relapse prediction.
1. US20090317820 - Micro-RNA Profile in Human Saliva and Its Use for Detection of Oral Cancer
Abstract Extract:
“The present invention provides for the first time the detection of micro-RNA in human saliva and the correlation between such micro-RNA and oral cancers. The present invention therefore provides methods and kits for diagnosing oral cancers by examining pertinent micro-RNA in saliva.”
Key Focus:
? The reference introduces the detection of salivary micro-RNA as a diagnostic approach for oral cancers.
? The scope is primarily identification-focused, aiming to establish correlation between detected micro-RNA and disease presence.
? The invention proposes a saliva-based detection method but does not extend beyond initial diagnosis.
Key Limitations:
A. Fragmented Biomarker Information Without Contextual Integration:
o While it mentions micro-RNA detection, the invention does not propose a combinatorial biomarker strategy that can provide holistic insights into disease status, treatment suitability, or patient-specific therapeutic pathways.
B. Absence of a Synergistic Biomarker Combination:
While micro-RNA detection is disclosed, the invention lacks a synergistic biomarker panel designed to provide holistic, stratified clinical outputs (therapeutic response, relapse risk, treatment suitability) derived from combinatorial expression analysis.
C. Inadequate Clinical Decision-Making Support:
o The disclosed approach lacks mechanisms that would assist healthcare professionals in interpreting biomarker data for guiding treatment decisions.
o There is no actionable framework to determine which patients will benefit from specific therapies, nor does it facilitate risk-based stratification for aggressive disease management.
D. Absence of Real-Time Monitoring and Dynamic Assessment:
o The invention is restricted to a single-point diagnostic snapshot, offering no provisions for monitoring therapeutic response or detecting relapse during or after treatment.
o Clinicians are left without the means to track disease progression or therapy efficacy in a repeatable, practical manner.
E. No Workflow or Motivation for Clinical Adoption:
o The lack of structured interpretation models and absence of a practical workflow means practitioners have limited utility in applying the disclosed method for routine clinical practice.
o The information provided does not bridge the gap between molecular detection and real-world clinical applicability, leaving healthcare providers without tools to confidently tailor treatments.
F. Sampling Approach Remains Incomplete for Comprehensive Decision Support:
o Although saliva is used, the method does not leverage extracellular vesicle enrichment, which is critical for ensuring reliable and disease-specific RNA profiling.
o The invention does not address tumor heterogeneity, biomarker stability, or ensure reproducibility of results necessary for effective clinical monitoring.
G. Non-invasive Sample Collection Without Clinical Utility Translation:
o Although the reference utilizes saliva, a non-invasive sample, the invention does not translate this advantage into a clinically actionable platform. The method fails to bridge the gap between molecular detection and effective clinical decision-making workflows.
o There is no structured interpretation framework or monitoring utility, resulting in underutilization of the non-invasive sampling advantage, leaving practitioners without real-time, patient-specific guidance for therapeutic management.

2. US20210032344A1 - Multiplex RNA Biomarker Panels for Cancer Detection
Abstract Extract:
“Multiplexed panels of RNA biomarkers for detecting cancer are provided. The panels can be configured for detecting a variety of cancer types and subtypes. Methods of using these panels for cancer detection, prognosis, and therapy selection are also provided.”
Key Focus:
? The invention discloses multiplex RNA biomarker panels designed for the detection of various cancers through nucleic acid amplification-based assays.
? The emphasis lies on developing RNA biomarker panels applicable to a broad oncology spectrum, including—but not limited to—lung, breast, and colorectal cancers.
? While it introduces multiplexing RNA markers, the disclosure is detection-focused and does not extend into dynamic monitoring or therapeutic decision-making frameworks.
? The sample matrices predominantly involve blood, serum, and tissue samples, not saliva or salivary extracellular vesicles.
Key Limitations:
A. Non-Specific Biomarker Composition:
o The biomarker panels disclosed are generic and designed for multi-cancer detection, lacking OSCC-specific molecular signature alignment.
o The panel composition does not integrate biomarkers uniquely validated for oral squamous cell carcinoma (OSCC) or for head and neck cancers, failing to address anatomical and pathological nuances specific to these diseases.
B. Inappropriate Sample Type for OSCC Monitoring:
o The method relies on serum, plasma, or tissue samples, which are invasive and thereby painful for frequent and longitudinal assessments.
o The diagnostic approach overlooks saliva as a biofluid, which is anatomically and biologically better suited for non-invasive monitoring of head and neck cancers.
C. Absence of Stratified Risk Interpretation Framework:
o The invention does not offer a structured interpretation framework to support clinicians in tailoring therapies based on molecular profiles, rendering it insufficient as a clinical decision-support tool.
D. No Provision for Real-Time Monitoring or Longitudinal Assessment:
o The disclosed methods capture a single-point diagnostic snapshot, without enabling dynamic assessment of therapy response or proactive relapse detection.
o The invention lacks workflows or methodologies that would allow for repeat, real-time monitoring of treatment efficacy, a critical gap for managing OSCC patients who require frequent follow-ups.
E. Lack of OSCC-Centric Clinical Validation:
o The invention does not present any clinical validation data specific to OSCC or head and neck cancers.
o The multiplex panels are not demonstrated for application in salivary extracellular vesicles or OSCC patient cohorts, limiting their practical utility in this clinical domain.
F. Missed Opportunity to Leverage Saliva’s Diagnostic Advantages:
o Despite saliva being a highly relevant and non-invasive biofluid for oral and head and neck cancers, the invention does not utilize salivary extracellular vesicle-derived RNA, thereby missing a key opportunity for real-time, patient-friendly monitoring.
G. Lack of Synergistic Biomarker Panel Composition:
o Although multiplex PCR is disclosed, the invention does not present a validated, synergistic biomarker combination specifically tailored for OSCC, nor does it enable outcome stratification through integrated expression analysis.

3. US9157126B2, WO2019/171110A1, US2010/0099102A1 – Salivary Protein Biomarkers for Oral and Head and Neck Cancers
Overview & Focus:
? These prior arts disclose salivary protein biomarkers such as MMP9, CD44, Cyclin D1, and others, aimed at the early detection of oral squamous cell carcinoma (OSCC) and head and neck squamous cell carcinoma (HNSCC).
? The primary objective is to develop non-invasive saliva-based tests for initial cancer identification.
? However, these inventions are primarily used for screening, limited to identifying disease presence without addressing ongoing monitoring, treatment suitability, or relapse risk assessment.
Key Limitations:
A. Limited to Protein Markers Without RNA-Level Molecular Insights:
o The disclosed biomarker panels are confined to soluble protein markers, missing the diagnostic and prognostic advantages of RNA biomarkers derived from salivary extracellular vesicles.
o The inventions fail to capture tumor-specific RNA signatures that provide deeper insights into disease biology, therapy response, or resistance.
B. No Combined Biomarker Interpretation for Patient Stratification:
o Although multiple protein markers are mentioned, the references do not present a combinatorial or synergistic approach to interpret biomarker trends for stratifying patients into clinical categories.
o As a result, the findings do not provide sufficient guidance to clinicians regarding which treatment regimen would be most effective for a particular patient profile.
C. Absence of a Synergistic Multi-Marker Panel for Clinical Stratification:
o The inventions list multiple protein biomarkers but do not disclose a synergistic combination configured to produce stratified, reproducible clinical insights essential for guiding therapy decisions and predicting relapse
D. Lack of Therapeutic Monitoring and Relapse Prediction Capability:
o The inventions are designed as single-point diagnostic tools, offering no mechanisms for repeatable, real-time monitoring of therapy response or early relapse detection.
o This limits their applicability in dynamic clinical decision-making workflows, where ongoing assessment of disease status is critical.
E. No Practical Kit-Based Workflow for Clinical Adoption:
o The prior arts do not provide a diagnostic kit or standardized workflow integrating sample collection, biomarker extraction, detection, and result interpretation.
o Without such a platform, clinical adoption becomes impractical, as healthcare providers are left without a streamlined process for routine patient management.
F. Non-Utilization of Extracellular Vesicle Enrichment:
o Although saliva is used as a sample medium, the methods do not incorporate extracellular vesicle isolation, which is essential for ensuring biomarker stability, specificity, and reproducibility.
o This omission reduces the clinical reliability of the assay, especially in heterogeneous tumor environments.
G. Fail to Translate Non-Invasive Sampling into Actionable Clinical Utility:
o While the prior arts utilize saliva as a non-invasive fluid, the inventions do not bridge the gap between molecular detection and effective clinical decision-making.
o The diagnostic outputs lack the clarity and depth of information required by clinicians to make treatment guidance decisions, leaving an unmet need for a solution that converts biomarker data into practical patient management strategies.

4. Startups and Emerging Products:
Startups such as Erly Sign (India), SalivaChecker (Japan), and OrisDx (USA) focus primarily on early screening and diagnostic detection of oral cancers. They do not offer solutions for dynamic therapeutic monitoring or relapse prediction. SummitDX’s HNKlear (USA) is another such product; however, it remains limited in scope to protein-based assays and lacks a validated RNA-protein composite biomarker panel integrated with a structured risk scoring system as claimed in the present invention.
Thus, the prior art references fail to disclose or enable a biomarker panel composition and diagnostic kit tailored to salivary extracellular vesicles, capable of providing real-time therapeutic monitoring and relapse prediction in OSCC patients.
There remains an unmet need to provide a Salivary Biomarker Panel for Therapeutic Monitoring and Relapse Prediction in Head and Neck Cancers that enables:
? non-invasive, repeatable, and real-time molecular assessment of therapeutic response and relapse risk;
? integration of a validated combination of biomarkers specific to OSCC biology;
? dynamic patient stratification based on composite risk scoring;
? clinical decision support through structured and actionable reporting workflows.

LIMITATIONS OF PRIOR ART
The existing diagnostic and monitoring tools for head and neck cancers, including oral squamous cell carcinoma (OSCC), suffer from one or more of the following significant limitations:
1. Absence of Non-Invasive, OSCC-Specific Diagnostic Tools: Current approaches lack non-invasive diagnostic or monitoring solutions specifically designed for OSCC and head and neck cancers, particularly for dynamic and repeatable assessments.
2. Dependence on Invasive and Costly Modalities: Tissue biopsies and imaging techniques like PET-CT and MRI are invasive, resource-intensive, and impractical for frequent or longitudinal monitoring of tumor progression and therapeutic response.
3. Inability to Capture Early Molecular Changes: Imaging modalities (CT, MRI, PET-CT) detect gross anatomical changes but are insensitive to early molecular or microenvironmental responses, leading to delayed detection of relapse or resistance. They are also prone to false positives/negatives and provide only static snapshots of disease status.
4. Delayed Assessment of Treatment Efficacy (RECIST Limitations): RECIST-based radiographic criteria evaluate response solely on tumor size reductions, failing to capture biological parameters such as necrosis, metabolic activity, early resistance, or immune responses, resulting in continued administration of ineffective therapies.
5. Insufficient Tools for Immunotherapy Decision-Making: Immune checkpoint inhibitor therapies (e.g., nivolumab, pembrolizumab) depend on invasive PD-L1 tissue testing. For inoperable tumors, this necessitates either impractical biopsies or empiric, unguided treatment administration.
6. Inadequate Early Relapse Prediction: Existing tools cannot detect relapse at a molecular level before overt tumor regrowth, delaying timely interventions and adversely affecting survival outcomes.
7. Inter-Observer Variability and Tumor Heterogeneity: Imaging and biopsy-based assessments are subject to interpretive variability and fail to account for intra-tumoral heterogeneity, compromising reproducibility and diagnostic accuracy.
8. Delayed Diagnosis Leading to Advanced-Stage Detection: Conventional diagnostic workflows often detect OSCC only after symptomatic presentation or when tumors have reached a size visible on imaging, limiting treatment options and prognosis.
9. Delayed and Inefficient Relapse Detection: Surveillance relies on periodic imaging with substantial intervals, failing to identify subtle molecular recurrences, which postpones necessary clinical interventions.
10. Lack of Proactive, Real-Time Molecular Monitoring Tools:
Existing diagnostic approaches are inherently reactive, identifying disease progression or relapse only after anatomical changes become radiologically detectable or clinical symptoms manifest. These methods fail to provide proactive, early-warning insights into molecular or microenvironmental changes indicative of treatment resistance or impending relapse. Without a non-invasive, real-time monitoring tool, clinicians are unable to intervene preemptively, resulting in delayed therapeutic adjustments and compromised patient outcomes.
11. Lack of Real-Time, Continuous Monitoring Capability: Existing methods do not support dynamic, non-invasive monitoring of disease progression, resulting in missed opportunities for timely therapeutic adjustments.
12. Limited Personalization of Immunotherapy Planning: Current diagnostic tools offer no efficient, minimally invasive method to determine a patient's potential responsiveness to immunotherapy agents.
13. Absence of Clinically Validated Salivary Biomarker Panels for HNSCC: There is a lack of companion diagnostic kits utilizing salivary extracellular vesicle-derived biomarkers validated for therapeutic monitoring and relapse prediction in head and neck cancers.
14. Redundancy of Invasive and Ineffective Treatment Cycles: Multiple invasive procedures and imaging studies often fail to optimize treatment strategies, resulting in unnecessary treatment cycles, increased patient suffering, and higher healthcare costs.
15. Lack of Validated Combinatorial Salivary Biomarker Panels: Prior art does not disclose a clinically validated combination of multiple salivary biomarkers specifically derived from extracellular vesicles tailored for OSCC diagnostic and monitoring applications.
16. Lack of Synergistic Biomarker Combinations for Stratified Clinical Outputs: While prior art references may disclose individual biomarkers or generic panels, they fail to propose a synergistic combination of salivary extracellular vesicle-derived biomarkers configured to collectively stratify patients into actionable clinical categories such as responders, non-responders, high relapse risk, and immunotherapy suitability. This lack of combinatorial approach limits clinical utility and diagnostic accuracy.
17. Inability to Simultaneously Evaluate Key Clinical Parameters: Existing solutions do not enable concurrent assessment of chemotherapy response, relapse risk, and immunotherapy suitability in a single, unified test workflow.
18. Absence of a Practical, Clinician-Friendly Workflow for Real-World Adoption: Existing diagnostic modalities and biomarker assays lack a standardized, reproducible workflow that clinicians can easily adopt in routine practice. The gap persists between molecular detection and actionable clinical insights, leaving healthcare providers without reliable decision-support tools to guide personalized therapy planning and patient counselling.
19. Limited to Single-Stage Applications: Most existing technologies are constrained to either early detection or post-treatment monitoring, lacking adaptability for continuous use across diagnosis, treatment assessment, and long-term follow-up.
Given these cumulative limitations, there remains a critical unmet need for a non-invasive, saliva-based companion diagnostic kit that integrates a synergistic biomarker panel with a practical clinical workflow, enabling real-time therapeutic monitoring, early relapse prediction, and stratified patient management in head and neck cancers. Such a solution must facilitate personalized, repeatable, and dynamic clinical decision-making throughout the patient's care continuum.

OBJECTIVES OF THE INVENTION
The primary objective of the present invention is to provide a Salivary Biomarker Panel for Therapeutic Monitoring and Relapse Prediction in Head and Neck Cancers, that enables a non-invasive, saliva-based prognostic solution utilizing extracellular vesicle-derived biomarkers for real-time monitoring of therapeutic response and prediction of relapse in patients with head and neck cancers; comprising a biomarker panel uniquely validated for salivary extracellular vesicles, specifically tailored to oral squamous cell carcinoma (OSCC) biology..
Another objective of the present invention is to provide a Salivary Biomarker Panel for Therapeutic Monitoring and Relapse Prediction in Head and Neck Cancers, that enables detection of disease progression and relapse at a molecular level, even before clinical symptoms or radiological signs become apparent, thereby facilitating earlier clinical intervention.
Yet another objective of the present invention is to provide a Salivary Biomarker Panel for Therapeutic Monitoring and Relapse Prediction in Head and Neck Cancers, that overcomes the limitations of RECIST-based radiological response assessments, which rely on tumour size changes and fail to capture early molecular indicators of treatment resistance or relapse, thereby enabling proactive and timely clinical decision-making.
Yet another objective of the present invention is to provide a Salivary Biomarker Panel for Therapeutic Monitoring and Relapse Prediction in Head and Neck Cancers, that enables stratification of patients as responders or non-responders to therapy, facilitating early identification of non-responders to minimize unnecessary treatment cycles.
Yet another objective of the present invention is to provide a Salivary Biomarker Panel for Therapeutic Monitoring and Relapse Prediction in Head and Neck Cancers, that enables non-invasive assessment of immunotherapy suitability, addressing cases where tissue biopsies are impractical or contraindicated.
Yet another objective of the present invention is to provide a Salivary Biomarker Panel for Therapeutic Monitoring and Relapse Prediction in Head and Neck Cancers, that supports repeat, longitudinal saliva sampling for dynamic disease monitoring without the need for invasive biopsies or frequent imaging.
Yet another objective of the present invention is to provide a Salivary Biomarker Panel for Therapeutic Monitoring and Relapse Prediction in Head and Neck Cancers, that assists clinicians in optimizing treatment strategies through personalized, real-time molecular insights into therapy response and relapse risk; by enabling simultaneous assessment of chemotherapy response, relapse risk, and immunotherapy suitability through a single, integrated diagnostic workflow
Yet another objective of the present invention is to provide a Salivary Biomarker Panel for Therapeutic Monitoring and Relapse Prediction in Head and Neck Cancers, that minimizes treatment-related toxicity and healthcare burden by identifying ineffective treatments early in the therapeutic timeline.
Yet another objective of the present invention is to provide a Salivary Biomarker Panel for Therapeutic Monitoring and Relapse Prediction in Head and Neck Cancers, that enables early relapse prediction through detection of molecular and microenvironmental changes, even in the absence of radiological or symptomatic evidence.
Yet another objective of the present invention is to provide a Salivary Biomarker Panel for Therapeutic Monitoring and Relapse Prediction in Head and Neck Cancers, that supports continuous use across the clinical care continuum—spanning initial diagnosis, active treatment monitoring, and long-term relapse surveillance.
Yet another objective of the present invention is to provide a Salivary Biomarker Panel for Therapeutic Monitoring and Relapse Prediction in Head and Neck Cancers, that addresses the inability of imaging modalities (CT, MRI, PET-CT) to detect early molecular relapse or subtle biological changes, by capturing dynamic biomarker expression profiles through non-invasive salivary analysis.
Yet another objective of the present invention is to provide a Salivary Biomarker Panel for Therapeutic Monitoring and Relapse Prediction in Head and Neck Cancers, that empowers healthcare providers with a reliable decision-support tool, enabling evidence-based treatment selection and timely therapeutic adjustments, thereby enhancing clinical confidence and improving patient outcomes.
Yet another objective of the present invention is to provide a Salivary Biomarker Panel for Therapeutic Monitoring and Relapse Prediction in Head and Neck Cancers, that shifts disease management from reactive interventions based on late-stage symptomatic or radiological detection to proactive, molecularly guided clinical decision-making, allowing for early therapeutic intervention and improved prognosis.
Yet another objective of the present invention is to provide a Salivary Biomarker Panel for Therapeutic Monitoring and Relapse Prediction in Head and Neck Cancers, that enables prognostication by correlating biomarker signatures with patient survival outcomes, thereby supporting risk stratification and long-term clinical planning.
SUMMARY OF THE INVENTION
The present invention provides a technical solution to the unmet clinical need for non-invasive, real-time monitoring of therapeutic response and relapse prediction in patients with head and neck cancers, particularly oral squamous cell carcinoma (OSCC). This is achieved through a salivary biomarker panel and a companion diagnostic kit that enables dynamic, molecular-level assessment of treatment efficacy and disease progression.
The invention integrates a synergistic combination of four salivary biomarkers—miR-1307-5p, CD44v6, KRT4, and PD-L1—whose collective expression profiles provide comprehensive insights into chemotherapy responsiveness, relapse risk, immunotherapy suitability, and overall prognosis. These biomarkers are detected from RNA extracted from salivary extracellular vesicles and/or cellular fractions using reverse transcription quantitative polymerase chain reaction (RT-qPCR).
To operationalize this biomarker panel, the invention provides a diagnostic kit comprising:
? Biomarker-specific primers/probes for RT-qPCR amplification;
? Reagents for RNA isolation, cDNA synthesis, and quantitative detection;
? Reference RNA control samples for baseline normalization;
? A software module configured to process expression data, compute fold-change values, generate composite risk scores, and stratify patients into clinically actionable categories.
The technical solution provided by this invention eliminates reliance on invasive biopsies and imaging-based assessments, which are limited by sampling bias, high cost, and delayed responsiveness. By leveraging non-invasive saliva sampling, the invention allows for repeatable, longitudinal monitoring across the patient’s treatment timeline, providing clinicians with dynamic, evidence-based insights to guide therapeutic decision-making and timely intervention.
Unlike prior art that focuses solely on early detection or isolated biomarker assessments, the present invention offers a validated, combinatorial biomarker signature supported by a standardized detection platform, addressing critical gaps in precision oncology for head and neck cancers.

BRIEF DESCRIPTION OF DRAWINGS
The accompanying drawings illustrate certain embodiments of the present invention and, together with the detailed description, serve to explain its principles:
• Figure 1 illustrates box-violin plots of ChemoScores in chemotherapy responders and non-responders, together with survival plots showing correlation between ChemoScore values and progression-free survival.
• Figure 2 shows relapse scores in patients with and without relapse, along with survival plots demonstrating correlation between RelapseScore and progression-free survival.
• Figure 3 presents ImmunoScores in immunotherapy responders and non-responders, together with validation of predictive performance in the patient cohort.
• Figure 4 illustrates the integration of ChemoScore, RelapseScore, and ImmunoScore into a composite Overall Risk Score, with associated survival analysis and stratification of patients into low, moderate, high, and very high-risk categories.

DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to a Salivary Biomarker Panel for Therapeutic Monitoring and Relapse Prediction in Head and Neck Cancers, wherein a synergistic combination of four specific biomarkers—miR-1307-5p, CD44v6, KRT4, and PD-L1—is employed to provide a comprehensive, non-invasive diagnostic solution. The inventive biomarker panel has been specifically designed to address the unmet clinical need for dynamic, repeatable, and molecular-level monitoring of therapeutic response (refers to the patient’s biological response to standard-of-care treatments, including chemotherapy, radiotherapy, and targeted therapies), relapse risk (likelihood of disease recurrence or progression after initial therapy), immunotherapy suitability (patient’s potential responsiveness to immunotherapy agents), prognosis, and survival prediction ((anticipated disease trajectory and survival probability, reflecting tumor aggressiveness and therapeutic responsiveness) in patients with oral squamous cell carcinoma (OSCC) and related head and neck malignancies.
Unlike conventional approaches that rely on invasive tissue biopsies, static imaging modalities, or singular biomarker assessments, the present invention leverages the synergistic expression profiles of these four biomarkers, derived from salivary extracellular vesicles and/or cellular fractions, to generate actionable clinical insights. The combined evaluation of these biomarkers enhances diagnostic specificity, predictive accuracy, and clinical utility across multiple stages of patient care.
The invention is operationalized through specific embodiments, each designed to illustrate how the synergistic application of the biomarker panel enables a non-invasive, clinically translatable platform for therapeutic monitoring and relapse prediction. These embodiments describe, by way of example, the biomarker panel composition, diagnostic kit configuration, analytical workflows, and clinical methodologies that implement the invention.
It is to be understood that the embodiments described herein are provided solely to exemplify the invention’s principles and are not intended to limit the scope of protection sought. The invention encompasses all variations, modifications, and equivalents that derive from the underlying inventive concept of employing a synergistic biomarker panel for dynamic, real-time, non-invasive monitoring of head and neck cancers.
The following sections describe each embodiment in detail, elucidating how the invention’s components work together to deliver the claimed diagnostic advantages.
Embodiment 1: Salivary Biomarker Panel for Therapeutic Monitoring and Relapse Prediction in Head and Neck Cancers
Embodiment 1 relates to a Salivary Biomarker Panel for Therapeutic Monitoring and Relapse Prediction in Head and Neck Cancers, providing a non-invasive molecular diagnostic solution for assessing therapeutic response, predicting relapse, determining immunotherapy suitability, and evaluating prognosis and survival prediction, particularly in patients with oral squamous cell carcinoma (OSCC).
This embodiment is based on a synergistic combination of four clinically validated RNA biomarkers:
? miR-1307-5p,
? CD44v6,
? KRT4,
? PD-L1.
These biomarkers are quantified from RNA extracted from salivary extracellular vesicles and/or cellular fractions using reverse transcription quantitative polymerase chain reaction (RT-qPCR).
The inventive merit of this embodiment lies in the synergistic expression profile of these four biomarkers, which, when analyzed collectively, enables precise and actionable stratification of patients across multiple clinical dimensions, overcoming the limitations of single-marker diagnostics.
Functional Interpretation of Biomarker Thresholds:
? miR-1307-5p = 2.0-fold: Indicates chemosensitivity and predicts therapeutic response, disease aggressiveness and survival.
? CD44v6 = 2.0-fold: Indicates high relapse risk and disease aggressiveness and survival.
? KRT4 = 1.0-fold: Indicates chemosensitivity and predicts therapeutic response.
? PD-L1 = 1.0-fold: Indicates immunotherapy suitability.
Multifactorial Clinical Stratification Enabled by the Panel:
The invention enables patient stratification into therapeutic response categories, comprising:
? Responder (who demonstrates effective therapeutic response and reduced relapse risk)
? Non-responder (who demonstrates ineffective therapeutic response and elevated relapse risk)
? High relapse risk
? Immunotherapy-suitable
Additionally, based on the integrated interpretation of combined biomarker expression trends, a prognostic outcome assessment is derived to classify patients under 'Poor prognosis', indicative of adverse survival likelihood and aggressive disease biology.
This hierarchical stratification provides dynamic, actionable insights during active treatment monitoring while also delivering conclusive prognostic evaluations to inform long-term clinical management strategies.

Measurement and Stratification Workflow:
RNA is isolated from salivary extracellular vesicles and cellular fractions, leveraging the non-invasive, easily accessible nature of saliva to facilitate repeated sampling for longitudinal disease monitoring. The extracted RNA undergoes RT-qPCR analysis, offering high sensitivity and specificity for quantifying biomarker expression fold-change values relative to baseline controls. These expression values are normalized and processed to compute individual biomarker-specific scores, which are subsequently integrated into a composite risk score. Based on this score, patients are stratified into clinical categories indicative of therapeutic response, relapse risk, immunotherapy suitability, and overall prognosis. The scoring is executed through a structured algorithmic model designed to ensure objective, reproducible, and real-time interpretation, thereby supporting informed clinical decision-making.
The invention employs a structured biomarker interpretation framework that integrates multiple expression-based scores to enable precise clinical stratification of head and neck cancer patients. The scoring methodology is designed to predict chemotherapy responsiveness, relapse risk, and immunotherapy suitability by quantifying and processing biomarker expression levels from saliva-derived extracellular vesicle RNA.
? Chemotherapy Response (ChemoScore) is evaluated by analyzing the expression of miR-1307-5p (log2-transformed fold change) and KRT4 (inverse square root-transformed expression), with weighting factors assigned based on clinical correlations established during model development. Figure 1 demonstrates that ChemoScore differentiates chemotherapy responders from non-responders, with higher scores associated with resistance to therapy. Validation confirmed predictive accuracy and correlation with progression-free survival, supporting its role as a biomarker for therapy guidance.
? Relapse Risk (RelapseScore) is determined through the log2-transformed expression patterns of CD44v6, indicating disease aggressiveness and propensity for recurrence. Figure 2 illustrates that patients who relapsed exhibited higher RelapseScores, with validation confirming predictive accuracy and correlation with inferior survival outcomes. This supports the utility of RelapseScore for early relapse risk identification.
? Immunotherapy Suitability (ImmunoScore) is assessed by quantifying PD-L1 expression, processed through inverse-transformation and log2 normalization to evaluate the potential benefit of immune checkpoint inhibitor therapies. Figure 3 shows that ImmunoScore distinguishes immunotherapy responders from non-responders, with higher scores correlating with clinical benefit and validation confirming predictive accuracy.
The individual scores derived from each biomarker are normalized and integrated using a weighted linear regression framework to produce a Composite Risk Score. This composite score enables categorization of patients into eight clinical stratification categories:
1. Responder - No relapse - Immuno suitable
2. Responder - No relapse - Immuno not suitable
3. Non-responder - No relapse - Immuno suitable
4. Responder - Relapse - Immuno suitable
5. Non-responder - Relapse - Immuno suitable
6. Responder - Relapse - Immuno not suitable
7. Non-responder - No relapse - Immuno not suitable
8. Non-responder - Relapse - Immuno not suitable
Subsequently, patients are further classified into Low Risk, Moderate Risk, High Risk, and Very High-Risk categories, aiding clinicians in optimizing treatment pathways and follow-up protocols. Figure 4 depicts the integration of ChemoScore, RelapseScore, and ImmunoScore into a composite Overall Risk Score, validated with high predictive accuracy and showing that higher scores correlate with adverse survival outcomes. This establishes the Overall Risk Score as a robust predictive and prognostic tool for stratifying patients.
This interpretative scoring methodology is implemented through a proprietary algorithm that computes these scores and stratifications in an objective, reproducible manner, enabling its integration into clinical workflows and electronic medical reporting systems, or alternatively, manual interpretation using standardized score charts.
The biomarker panel addresses prior diagnostic limitations by enabling real-time molecular monitoring of therapy response, early relapse prediction, and non-invasive sample collection, thereby reducing overtreatment and guiding precision oncology interventions. Unlike tissue or blood-based assays, this invention leverages saliva-derived extracellular vesicles for stable, tumor-specific RNA profiling. Its synergistic multi-marker approach, coupled with integrated scoring, allows simultaneous assessment of therapeutic response, relapse risk, and immunotherapy suitability, providing actionable insights for dynamic clinical decision-making.
Summary Table of Biomarker Interpretations:
Biomarker Threshold Clinical Interpretation
miR-1307-5p =2.0-fold
Chemosensitivity & Therapeutic Response
CD44v6 =2.0-fold
High Relapse Risk & Disease Aggressiveness
KRT4 =1.0-fold
Chemosensitivity & Therapeutic Response
PD-L1 =1.0-fold
Immunotherapy Suitability

Clinical Case Studies and Validation
The clinical performance of the Salivary Biomarker Panel for Therapeutic Monitoring and Relapse Prediction in Head and Neck Cancers was validated through patient case studies, demonstrating the predictive accuracy, clinical applicability, and translational potential of the invention.
The following working examples illustrate the technical implementation of the invention, validating its clinical applicability and predictive accuracy in real-world patient scenarios. These examples demonstrate how biomarker expression profiles correlate with therapeutic response, relapse risk, and immunotherapy suitability.
Table 1: Patient Biomarker Profiles and Clinical Stratification
Sr. No Patient ID ChemoScore Relapse Score Immuno Score Overall Risk Clinical Category Treatment Response (RECIST/HPE) Remarks
1 AS104 3.96 388.02 2.09 3 Non-responder / High relapse / High Immuno positive Progressive Disease Relapsed within 12 months
2 AS141 3.36 4803.93 0.46 4 Non-responder / High relapse /Immuno negative No surgical intervention; Death within 6 months Early aggressive disease progression
3 L37 1.66 0.35 12.82 1 Low-responder / Low relapse / Immuno positive Partial Response (RECIST), Complete Response (HPE) Immunotherapy followed by surgery achieved T0N0 status
4 AS207 0.06 0.04 0.01 1 Low-responder / Low relapse / Immuno negative Complete Response Surgery avoided post-chemotherapy
5 L13 8.94 0.29 9.38 2 Non-responder / Low relapse / Immuno positive Progressive Disease (RECIST), Partial Response (HPE) Surgery required after ineffective initial regimen
Footnote: Overall Risk 1 denotes: Low risk, 2: Moderate risk, 3: High risk, 4: Very high risk

Clinical Case Studies Description and Validation
Case Study 1: AS104 – High Relapse Risk Non-Responder with Immunotherapy Suitability
A 54-year-old male diagnosed with buccal mucosa squamous cell carcinoma underwent neoadjuvant chemotherapy (Cisplatin, Docetaxel, Cetuximab). Pre-treatment salivary biomarker profiling revealed:
? Chemo Score:3.96
? Relapse Score:388.02
? Immuno Score: 2.09
? Overall Score: 3
? The scoring algorithm stratified the patient as a non-responder to chemotherapy, with high relapse risk and high immunotherapy suitability. Despite standard treatment, the patient relapsed within 12 months, validating the predictive power of the biomarker panel. Initiation of immunotherapy post-relapse has resulted in clinical improvement.

Case Study 2: AS141 – Aggressive Disease with Poor Immunotherapy Response
A 56-year-old male with carcinoma of the buccal mucosa and retromolar trigone was started on neoadjuvant chemotherapy (Docetaxel, Cisplatin, 5-FU) combined with Pembrolizumab. Biomarker analysis revealed:
? Chemo Score: 3.36
? Relapse Score:4803.93
? Immuno Score: 0.46
? Overall Score: 4
The algorithm identified the patient as a non-responder with high relapse risk and low immunotherapy suitability. The patient succumbed to disease within six months, underscoring the panel’s predictive accuracy in identifying non-beneficial therapy paths.

Case Study 3: L37 – Low Relapse Risk with High Immunotherapy Suitability
A 44-year-old male diagnosed with buccal mucosa carcinoma underwent combined NACT and immunotherapy. Biomarker expression:
? Chemo Score: 0.06
? Relapse Score: 0.04
? Immuno Score: 12.82
? Overall Score: 1
? Classified as a low-responder to chemotherapy but highly suitable for immunotherapy, the patient achieved a complete response (T0N0) after immunotherapy and surgery, with no recurrence observed during follow-up.

Case Study 4: AS207 – Favorable Low-Risk Profile, Complete Response
A 60-year-old male with carcinoma of the tongue and floor of mouth showed:
? Chemo Score: 1.66
? Relapse Score: 0.35
? Immuno Score: 0.01
? Overall Score: 1
The algorithm classified the patient as a low-responder to chemotherapy with low relapse risk and low immunotherapy suitability. Post-chemotherapy, a complete clinical response was achieved, eliminating the need for surgical intervention.

Case Study 5: L13 - Complex Cases
A 53 year- old male with squamous cell carcinoma of right buccal mucosa showed:
? Chemo Score: 8.94
? Relapse Score: 0.29
? Immuno Score: 9.38
? Overall Score: 2
The algorithm classified the patient as a low-responder to chemotherapy with low relapse risk and high immunotherapy suitability. Post-chemotherapy, a partial clinical response was achieved by eliminating the need for surgical intervention. However, there was an increase in the tumour lesion size post post chemotherapy and the patient had to undergo surgery.

Interpretation of Case Studies:
? Non-responders with High Relapse Risk (AS104, AS141): The biomarker panel accurately flagged patients with elevated miR-1307-5p and CD44v6 levels as non-responders and high relapse risk. Early immunotherapy could have been prioritized in these cases.
? Low-Responder but Immunotherapy-Suitable (L37): Despite suboptimal chemotherapy response, PD-L1expression =1.0-fold guided the effective use of immunotherapy, resulting in clinical remission.
? Favorable Low-Risk Profile (AS207): Biomarker-guided insights identified a patient with minimal relapse risk and low immunotherapy suitability, aligning with observed complete response post-chemotherapy.
? Complex Cases (L13): The biomarker panel correctly identified a patient unlikely to respond to chemotherapy but suitable for immunotherapy. Delayed immunotherapy initiation led to suboptimal outcomes, underscoring the panel's utility in early clinical decision-making.
These cases validate the predictive capacity, stratification accuracy, and practical utility of the biomarker panel in real-time therapeutic monitoring and relapse prediction. They illustrate how molecular insights derived from salivary biomarkers can guide proactive clinical decisions, reduce overtreatment, and improve patient outcomes.

Summary of Validation Cohort
The validation cohort comprised 186 patients with head and neck squamous cell carcinoma (HNSCC), aged between 29 and 75 years, including both male and female patients. The majority had oral squamous cell carcinoma, with disease stages ranging from early (Stage I) to advanced (Stage IVb). Etiological backgrounds included smokeless tobacco use, smoking, and non-habituated cases.
Treatment modalities included neoadjuvant chemotherapy (NACT) using regimens such as TPF, DCF, and platinum-based combinations, adjuvant chemotherapy, radiotherapy, and/or immunotherapy (agents including nivolumab, pembrolizumab, cetuximab). Clinical outcomes were recorded in terms of relapse, progression-free survival, and vital status.
These baseline and treatment details provided the clinical framework for correlating patient profiles with the calculated ChemoScore, RelapseScore, and ImmunoScore, and for validating the composite risk score model.

Comparative Analysis of Predictive Scores and Clinical Outcomes
This table presents the ChemoScore, RelapseScore, and ImmunoScore for each patient, together with the corresponding predictions generated by the present invention’s model, alongside assessments by RECIST criteria and histopathology (HPE). It also records months to relapse, actual clinical outcomes, and indicates whether the predictions of present invention were concordant with conventional clinical and pathological evaluations or matched the observed real-world outcomes
Pt ID Outcome Chemo score Relapse Score Immuno Score Overall Risk Prediction Prediction using present invention RECIST prediction HPE prediction Clinical Outcome
AF22 LIVE 2.62 1.45 - 1 Responder_Low relapse No summary - No relapse
AF23 LIVE 1.52 0.49 - 1 Responder_Low relapse Partial response Progressive disease No relapse
AF25 LIVE 1.25 0.34 - 1 Responder_Low relapse No nact. Adjuvant case Progressive disease No relapse
AF36 LIVE 2.34 0.66 - 1 Responder_Low relapse No nact. Adjuvant case Progressive disease No relapse
AF37 LIVE 0.93 3.43 - 2 Responder_High relapse Partial response Partial response Reported relapse
AS104 LIVE 3.96 388.02 - 3 Non-responder_High relapse Progressive disease Progressive disease Reported relapse
AS106 DEAD 0.95 108.38 0.16 3 Responder_High relapse_Immuno negative No nact. Adjuvant case Stable disease Reported lung metastasis and death
AS113 LIVE 11.29 3666.02 0.44 4 Non-responder_High relapse_Immuno negative Stable disease Partial response Reported relapse
AS114 LIVE 20.89 9.19 0.21 4 Non-responder_High relapse_Immuno negative No nact. Adjuvant case Progressive disease Reported nodal metastasis
AS115 LIVE 88.75 166.57 - 4 Non-responder_High relapse Require full summary - Reported nodal metastasis
AS120 LIVE 79.05 300.25 1.06 3 Non-responder_High relapse_Immuno positive Partial response Partial response Reported relapse
AS122 DEAD 2.00 685.02 2.11 2 Responder_High relapse_Immuno positive Progressive disease Partial response Reported death
AS124 DEAD 11.05 203.66 0.01 4 Non-responder_High relapse_Immuno negative Partial response Partial response Reported relapse, lung metastasis and death
AS125 LIVE 56.71 90.51 35.51 3 Non-responder_High relapse_Immuno positive No reports after nact - directly after biological treatment Partial response Reported relapse
AS126 DEAD 79.41 1217.75 0.35 4 Non-responder_High relapse_Immuno negative No reports post nact - death No reports Reported nodal metastasis and death
AS127 LIVE 19.98 130.69 0.48 4 Non-responder_High relapse_Immuno negative 0 PROGRESSIVE DISEASE REPORTED RELAPSE
AS13 LIVE 8.92 814.63 - 4 Non-responder_High relapse Partial response Partial response Reported relapse
AS132 LIVE 10.71 99.73 2.19 3 Non-responder_High relapse_Immuno positive Partial response Progressive disease Reported relapse
AS134 LIVE 4.36 68.59 0.07 4 Non-responder_High relapse_Immuno negative Partial response Partial response Reported nodal metastasis
AS135 LIVE 5.92 172.45 5.78 3 Non-responder_High relapse_Immuno positive Partial response Stable disease Reported relapse
AS137 LIVE 1.11 128.00 0.02 3 Responder_High relapse_Immuno negative Partial response Surgery in another hospital Reported relapse
AS141 DEAD 3.36 4803.93 0.46 4 Non-responder_High relapse_Immuno negative No summary - death reported No summary - death reported Reported death
AS143 DEAD 20.55 340.14 - 4 Non-responder_High relapse 0 0 REPORTED DEATH
AS144 LIVE 99.93 4.47 - 4 Non-responder_High relapse 0 0 REPORTED RELAPSE
AS146 LIVE 55.75 83.29 0.33 4 Non-responder_High relapse_Immuno negative Partial response Partial response Reported relapse
AS148 LIVE 12.34 4770.75 - 4 Non-responder_High relapse Stable disease No surgery Reported relapse
AS149 LIVE 92.91 200.85 0.93 4 Non-responder_High relapse_Immuno negative Partial response Partial response Reported relapse
AS150 LIVE 3.18 96.34 5.58 3 Non-responder_High relapse_Immuno positive Progressive disease Complete response Reported relapse
AS154 LIVE 81.05 233.94 0.61 4 Non-responder_High relapse_Immuno negative Progressive disease Progressive disease Reported relapse
AS155 LIVE 12.75 458.25 - 4 Non-responder_High relapse No nact. Adjuvant case Partial response Reported relapse
AS156 LIVE 11.63 592.22 20.11 3 Non-responder_High relapse_Immuno positive Stable disease No surgery possible Reported relapse
AS157 DEAD 18.67 25.81 - 4 Non-responder_High relapse 0 0 REPORTED RELAPSE AND DEATH
AS160 DEAD 99.88 7486.11 242.19 3 Non-responder_High relapse_Immuno positive Progressive disease No surgery possible Reported death
AS161 LIVE 34.91 23.75 26.54 3 Non-responder_High relapse_Immuno positive Complete response No surgery required Reported relapse
AS169 LIVE 4.86 9.32 - 4 Non-responder_High relapse Partial response Progressive disease Reported relapse
AS170 DEAD 3.55 11.79 - 4 Non-responder_High relapse Partial response Progressive disease Reported lung metastasis and death
AS172 LIVE 3.63 97.01 10.7 3 Non-responder_High relapse_Immuno positive Partial response 0 Reported relapse
AS178 LIVE 5.26 4.96 9.51 3 Non-responder_High relapse_Immuno positive No nact. Adjuvant case -- Reported relapse and nodal metastasis
AS177 LIVE 3.19 3.16 10.78 3 Non-responder_High relapse_Immuno positive Partial response Surgery in another hospital No relapse
AS185 LIVE 36.58 21.26 - 4 Non-responder_High relapse Progressive disease Surgery not possible Reported relapse
AS190 LIVE 1.00 2.39 4.08 2 Responder_High relapse_Immuno positive Partial response Partial response No relapse
AS192 LIVE 1.30 6.28 2.33 2 Responder_High relapse_Immuno positive No nact. Adjuvant case -- Reported relapse
AS193 DEAD 5.36 2.66 18.51 3 Non-responder_High relapse_Immuno positive 0 0 REPORTED NODAL METASTASIS AND DEATH
AS194 LIVE 1.02 0.46 - 1 Responder_Low relapse Partial response Partial response No relapse
AS196 LIVE 1.05 1.43 - 1 Responder_Low relapse Partial response Partial response No relapse
AS199 DEAD 6.69 7.57 0.06 4 Non-responder_High relapse_Immuno negative Partial response Partial response Reported death
AS206 LIVE 1.78 0.52 - 1 Responder_Low relapse Partial response Surgery in another hospital No relapse
AS207 LIVE 1.01 0.04 0.01 1 Responder_Low relapse_Immuno negative Complete response Surgery not required No relapse
AS209 LIVE 4.51 5.13 - 4 Non-responder_High relapse 0 0 REPORTED RELAPSE
AS21 DEAD 35.51 814.63 - 4 Non-responder_High relapse Partial response Surgery not possible Reported death
AS214 LIVE 12.34 1.78 0.09 3 Non-responder_Low relapse_Immuno negative Partial response Progressive disease No relapse
AS216 LIVE 9.08 30.48 4.03 3 Non-responder_High relapse_Immuno positive No nact. Adjuvant case 0 Reported relapse
AS224 LIVE 3.03 23.75 - 2 Responder_High relapse 0 0 REPORTED RELAPSE
AS225 LIVE 2.88 84.45 5.03 3 Non-responder_High relapse_Immuno positive Update summary - Reported relapse
AS227 LIVE 1.69 34.54 - 2 Responder_High relapse No nact. Adjuvant case -- Reported nodal metastasis
AS230 LIVE 24.95 37.01 5.86 3 Non-responder_High relapse_Immuno positive Partial response Progressive disease Reported relapse
AS232 LIVE 2.31 1.66 1.57 1 Responder_Low relapse_Immuno positive Partial response Partial response No relapse
AS233 LIVE 8.01 8.57 - 4 Non-responder_High relapse Update summary - Reported relapse and nodal metastasis
AS234 LIVE 8.34 3.56 - 4 Non-responder_High relapse Partial response Partial response Reported relapse
AS240 LIVE 3.06 2.79 - 4 Non-responder_High relapse Update summary - Reported relapse
AS242 LIVE 13.86 24.76 4.47 3 Non-responder_High relapse_Immuno positive Partial response Partial response Reported relapse
AS249 LIVE 1.81 1.70 - 1 Responder_Low relapse Partial response Complete response No relapse
AS253 LIVE 3.21 6.32 0.05 4 Non-responder_High relapse_Immuno negative Partial response Progressive disease Reported nodal metastasis
AS254 LIVE 4.34 5.06 - 4 Non-responder_High relapse Partial response Surgery not possible Reported relapse
AS255 LIVE 14.84 3.20 1.99 3 Non-responder_High relapse_Immuno positive Partial response Progressive disease Reported relapse
AS256 LIVE 15.34 2.06 - 4 Non-responder_High relapse Partial response Partial response Reported relapse
AS257 DEAD 2.95 13.74 - 4 Non-responder_High relapse - -- REPORTED DEATH
AS258 LIVE 8.41 1.19 9.45 2 Non-responder_Low relapse_Immuno positive Progressive disease Surgery in another hospital No relapse
AS262 LIVE 3.05 3.25 1.44 3 Non-responder_High relapse_Immuno positive Update summary - Reported relapse
AS266 LIVE 3.43 0.62 1.07 2 Non-responder_Low relapse_Immuno positive Partial response Progressive disease No relapse
AS272 LIVE 27.82 2.08 1.64 3 Non-responder_High relapse_Immuno positive No reports No reports REPORTED RELAPSE
AS275 LIVE 3.45 13.55 - 4 Non-responder_High relapse Not possible as put on palliative Not possible as put on palliative Reported relapse
AS277 LIVE 14.18 5.58 - 4 Non-responder_High relapse No reports No reports REPORTED RELAPSE AND NODAL METASTASIS
AS280 LIVE 17.45 21.11 - 4 Non-responder_High relapse No reports No reports REPORTED RELAPSE
AS281 LIVE 99.48 2.13 - 4 Non-responder_High relapse No reports No reports REPORTED RELAPSE
AS282 LIVE 0.22 0.06 - 1 Responder_Low relapse Complete response No surgery No relapse
AS293 DEAD 99.90 10.78 2.01 3 Non-responder_High relapse_Immuno positive No reports post nact 0 Reported relapse and death
AS294 LIVE 1.13 0.46 3.76 1 Responder_Low relapse_Immuno positive Partial response Partial response No relapse
AS296 LIVE 19.88 12.82 2.93 3 Non-responder_High relapse_Immuno positive 0 0 REPORTED RELAPSE
AS305 LIVE 7.49 2.36 - 4 Non-responder_High relapse No nact. Adjuvant case -- Reported nodal metastasis
AS308 LIVE 3.29 4.63 - 4 Non-responder_High relapse 0 0 REPORTED RELAPSE AND NODAL METASTASIS
AS310 LIVE 99.94 2.27 1.87 3 Non-responder_High relapse_Immuno positive Incomplete cycle -- Reported relapse
AS311 LIVE 75.25 2.87 0.59 4 Non-responder_High relapse_Immuno negative No nact. Adjuvant case -- Reported relapse
AS34 DEAD 6.03 2005.85 - 4 Non-responder_High relapse Progressive disease Progressive disease Reported relapse and death
AS57 LIVE 16.53 15393.14 - 4 Non-responder_High relapse Progressive disease Progressive disease Reported lung metastasis
AS7 DEAD 16.14 292.04 - 4 Non-responder_High relapse Progressive disease Progressive disease Reported relapse and death
AS70 LIVE 14.03 797.86 - 4 Non-responder_High relapse Partial response Partial response Reported relapse
AS72 LIVE 19.53 3516.68 - 4 Non-responder_High relapse Progressive disease Partial response Reported lung metastasis
AS75 LIVE 78.79 3444.31 - 4 Non-responder_High relapse Partial response Progressive disease Reported relapse
AS78 LIVE 8.54 32.45 1.23 3 Non-responder_High relapse_Immuno positive Partial response Partial response Reported relapse
AS88 LIVE 0.86 792.35 - 2 Responder_High relapse Partial response Partial response Reported relapse
AS92 LIVE 10.69 5330.30 - 4 Non-responder_High relapse No nact. Adjuvant case -- Reported relapse
AS97 DEAD 52.10 22.78 - 4 Non-responder_High relapse Partial response Partial response Reported relapse and death
AS98 LIVE 4.64 145.01 - 4 Non-responder_High relapse Partial response Partial response No relapse
L26 DEAD 4.24 194.01 - 4 Non-responder_High relapse No reports No reports REPORTED DEATH
L41 LIVE 17.05 0.07 - 2 Non-responder_Low relapse Partial response Partial response No relapse
L49 LIVE 1.74 4389.98 - 2 Responder_High relapse Partial response Partial response Reported relapse
L4 DEAD 4.17 12.21 - 4 Non-responder_High relapse No summary - REPORTED RELAPSE AND DEATH
L13 LIVE 9.75 0.29 9.38 2 Non-responder_Low relapse_Immuno positive Progressive disease Partial response No relapse
L16 LIVE 28.12 1.11 - 4 Non-responder_High relapse Progressive disease Progressive disease No relapse
L18 DEAD 11.77 809.00 2.55 3 Non-responder_High relapse_Immuno positive Partial response Progressive disease Reported death
L19 LIVE 13.42 8.82 1.8 3 Non-responder_High relapse_Immuno positive - -- REPORTED RELAPSE
L37 LIVE 1.66 0.35 12.82 1 Responder_Low relapse_Immuno positive Partial response Complete response No relapse
L47 LIVE 4.26 0.19 - 2 Non-responder_Low relapse Stable disease Stable disease No relapse
L22 LIVE 6.88 3.48 - 4 Non-responder_High relapse No summary details 0 Reported relapse
AS47 LIVE 43.45 2.51 - 4 Non-responder_High relapse Partial response Partial response Reported relapse
AF51 LIVE 0.61 87.42 - 2 Responder_High relapse Nact just started -- Reported relapse
AF50 LIVE 1.25 50.56 - 2 Responder_High relapse No nact. Adjuvant case -- No relapse
AS118 LIVE 6.25 26.72 - 4 Non-responder_High relapse Partial response Progressive disease Reported lung, liver and bone metastasis
AS142 DEAD 21.02 219.79 18.5 3 Non-responder_High relapse_Immuno positive Stable disease Stable disease Reported death
AS145 LIVE 8.65 2.28 0.17 4 Non-responder_High relapse_Immuno negative Progressive disease No surgery possible Reported relapse
AS229 LIVE 16.09 141.04 0.71 4 Non-responder_High relapse_Immuno negative Progressive disease Progressive disease Reported relapse
AS102 LIVE 2.54 22.32 - 2 Responder_High relapse No nact. Adjuvant case -- Reported relapse
AS151 LIVE 15.24 1807.78 6.32 3 Non-responder_High relapse_Immuno positive No response to chemo Surgery not possible Reported relapse
AS215 DEAD 0.32 3.41 0.55 3 Responder_High relapse_Immuno negative No reports post chemo as reported death No reports post chemo as reported death Reported relapse
AS83 LIVE 38.01 7.52 - 4 Non-responder_High relapse Partial response Partial response Reported relapse
AF41 LIVE 1.80 1.82 - 1 Responder_Low relapse Partial response Partial response No relapse
AS278 LIVE 3.59 14.62 9 3 Non-responder_High relapse_Immuno positive Absolute case Absolute case Reported relapse
AS226 LIVE 3.89 34.30 5.98 3 Non-responder_High relapse_Immuno positive On palliative care On palliative care Reported relapse
AS197 LIVE 0.99 6.23 2.23 2 Responder_High relapse_Immuno positive No ct prescribed On palliative care Reported relapse
AS239 LIVE 0.99 2.79 0.1 3 Responder_High relapse_Immuno negative No ct prescribed No ct prescribed Reported relapse
AS67 LIVE 99.98 2.77 0.15 4 Non-responder_High relapse_Immuno negative No ct prescribed No ct prescribed Reported relapse
AS268 LIVE 3.62 85.63 - 4 Non-responder_High relapse Change in hopsital Surgery in another hospital Reported relapse
L3 LIVE 40.08 24.40 3.18 3 Non-responder_High relapse_Immuno positive Progressive disease Progressive disease Reported relapse
L17 LIVE 0.85 22.00 - 2 Responder_High relapse Change in hopsital Surgery in another hospital Reported relapse
AF6 LIVE 12.49 0.60 - 2 Non-responder_Low Relapse No nact. Adjuvant case -- No relapse
AF12 LIVE 3.58 1.07 - 2 Non-responder_Low Relapse No ct prescribed Surgery in another hospital No relapse
AF16 LIVE 34.67 1.75 - 2 Non-responder_Low Relapse No nact. Adjuvant case Progressive disease No relapse
AF18 LIVE 14.00 0.40 - 2 Non-responder_Low Relapse Complete response Partial response No relapse
AF27 LIVE 14.25 12.64 - 4 Non-responder_High relapse No nact. Adjuvant case Progressive disease Reported relapse
AF31 LIVE 11.07 3.20 - 4 Non-responder_High relapse No nact. Adjuvant case -- Reported relapse
AF47 LIVE 15.42 9.25 - 4 Non-responder_High relapse No nact. Adjuvant case -- Reported relapse
AF48 LIVE 1.01 1.14 - 1 Responder_Low Relapse No nact. Adjuvant case -- No relapse
AF8 LIVE 1.84 2.38 - 2 Responder_High Relapse No ct prescribed -- Reported relapse
AF14 LIVE 3.32 0.63 - 2 Non-responder_Low Relapse No nact. Adjuvant case -- No relapse
AF43 LIVE 2.41 0.10 - 1 Responder_Low Relapse No nact. Adjuvant case -- No relapse
AS313 LIVE 3.70 132.51 - 4 Non-responder_High relapse No nact. Adjuvant case -- Reported lung, liver and bone metastasis
AS320 LIVE 45.15 2.17 0.62 4 Non-responder_High relapse_Immuno negative No nact. Adjuvant case -- Reported lung metastasis
AS26 DEAD 12.07 113.40 6.15 3 Non-responder_High relapse_Immuno positive Partial response Progressive disease Reported death
AS27 LIVE 14.00 1428.22 - 4 Non-responder_High relapse Partial response Partial response No relapse
AS165 DEAD 15.64 2683.68 6.92 3 Non-responder_High relapse_Immuno positive Progressive disease Surgery not possible Reported death
AS238 LIVE 25.65 3.27 - 4 Non-responder_High relapse Progressive disease Surgery not possible Reported relapse
AS259 LIVE 4.30 2.25 1.09 3 Non-responder_High relapse_Immuno positive Progressive disease No surgery done Reported relapse
AS261 LIVE 20.46 1.14 - 2 Non-responder_Low relapse No response based on history 0 Reported relapse and lung metastasis
AS303 LIVE 5.78 0.08 - 2 Non-responder_Low relapse Partial response Partial response No relapse
AS302 LIVE 47.02 4.53 4.41 3 Non-responder_High relapse_Immuno positive No ct prescribed Progressive disease Reported relapse
AS304 LIVE 18.67 1.73 10.41 2 Non-responder_Low relapse_Immuno positive Progressive disease Progressive disease No relapse
AS306 LIVE 2.00 1.07 1.35 1 Responder_Low relapse_Immuno positive Partial response Partial response No relapse
AS297 LIVE 33.77 1.40 - 2 Non-responder_Low relapse Partial response Stable disease No relapse
AS300 LIVE 14.38 2.97 - 4 Non-responder_High relapse Patient refused to give reports Patient refused to give reports Reported relapse
AS298 LIVE 5.67 19.29 0.23 4 Non-responder_High relapse_Immuno negative Update summary - Reported relapse
AS290 LIVE 2.35 0.68 - 1 Responder_Low relapse Update summary - No relapse
AS279 LIVE 3.75 0.01 0.85 3 Non-responder_Low relapse_Immuno negative Partial response Progressive disease No relapse
AS284 LIVE 1.17 0.03 - 1 Responder_Low relapse Change in hopsital Surgery in another hospital No relapse
AS231 LIVE 5.01 4.96 0.22 4 Non-responder_High relapse_Immuno negative Partial response Surgery in another hospital Reported relapse
AS223 LIVE 3.10 2.68 0 3 Responder_High relapse_Immuno negative Partial response Surgery in another hospital Reported relapse
AS221 LIVE 10.24 6.63 - 4 Non-responder_High relapse Stable disease Surgery in another hospital Reported relapse
AS138 LIVE 1.10 60.97 - 2 Responder_High relapse Progressive disease Patient denied surgery Reported lung and bone metastasis
AS130 LIVE 1.21 28.44 0.01 3 Responder_High relapse_Immuno negative Partial response Complete response Reported relapse
AS107 LIVE 0.95 556.41 - 2 Responder_High relapse Partial response Partial response Reported relapse
L7 LIVE 1.15 5.97 - 2 Responder_High relapse Complete response No surgery required No relapse
L15 LIVE 99.83 10.33 12.73 3 Non-responder_High relapse_Immuno positive Update summary - Reported relapse
L24 DEAD 11.11 125.37 3.94 3 Non-responder_High relapse_Immuno positive Progressive disease Surgery not done Reported death
AS195 LIVE 1.01 47.50 2.57 2 Responder_High relapse_Immuno positive Progressive disease - Reported relapse
AS228 LIVE 4.65 3.25 - 4 Non-responder_High relapse Stable disease Surgery not possible Reported relapse
AS241 LIVE 16.91 2.19 - 4 Non-responder_High relapse Complete response No surgery required Reported relapse
AS251 LIVE 1.63 4.38 - 4 Non-responder_High relapse Partial response Partial response No relapse
AS273 LIVE 2.23 1.74 - 2 Non-responder_Low relapse Partial response Partial response No relapse
AS112 LIVE 1.60 4.63 - 4 Non-responder_High relapse Partial response Partial response No relapse
AS307 LIVE 1.09 0.84 - 2 Non-responder_Low relapse Complete response No surgery required No relapse
AF24 LIVE 2.12 1.07 - 2 Non-responder_Low Relapse No nact. Adjuvant case Partial response No relapse
AS108 LIVE 14.87 6.11 0.9 4 Non-responder_High relapse_Immuno negative - PROGRESSIVE DISEASE REPORTED RELAPSE
AS236 LIVE 5.16 1.65 - 2 Non-responder_Low relapse - -- NO RELAPSE
AF10 LIVE 2.64 1.28 - 1 Responder_Low relapse Partial response Partial response No relapse
AF15 LIVE 2.23 1.01 - 1 Responder_Low relapse Partial response Stable disease No relapse
AF46 LIVE 3.13 4.76 - 4 Non-responder_High relapse No nact. Adjuvant case -- Reported relapse
AS1 LIVE 1.94 85.62 - 2 Responder_High relapse No nact. Adjuvant case Progressive disease Reported relapse
AS4 LIVE 1.78 22.31 - 2 Responder_High relapse No nact. Adjuvant case Progressive disease Reported relapse
AS11 LIVE 2.09 3743.05 - 2 Responder_High relapse No nact. Adjuvant case -- Reported relapse
AS20 LIVE 43.68 99.04 - 4 Non-responder_High relapse 0 0 REPORTED RELAPSE
AS29 LIVE 17.65 5256.91 - 4 Non-responder_High relapse Stable disease Partial response Reported relapse
AS40 DEAD 99.97 7.94 - 4 Non-responder_High relapse 0 0 REPORTED RELAPSE
AS91 DEAD 6.16 3.39 - 4 Non-responder_High relapse Partial response Complete response Reported relapse
AS109 LIVE 1.07 68.11 - 2 Responder_High relapse No nact adjuvant case No nact adjuvant case Reported relapse
AS176 LIVE 3.10 90.50 - 4 Non-responder_High relapse - PROGRESSIVE DISEASE REPORTED RELAPSE
AS271 0 5.25 2.20 1.6 3 Non-responder_High relapse_Immuno positive 0 0 REPORTED RELAPSE

The results in above table demonstrate that the present invention’s integrated scoring framework — combining ChemoScore, RelapseScore, and ImmunoScore into an Overall Risk Score — consistently aligned with actual clinical outcomes and, in many cases, outperformed conventional assessment methods such as RECIST criteria and histopathology (HPE).
By capturing complementary dimensions of therapeutic response, relapse risk, and immunotherapy suitability, the combined scores provided a synergistic assessment that improved prognostic accuracy and patient stratification compared to any single measure. ImmunoScore, where applicable, showed concordance with gold-standard diagnostic markers, reinforcing the robustness of the integrated model.
The Overall Risk Score effectively classified patients into four categories (1: Low risk, 2: Moderate risk, 3: High risk, 4: Very high risk), with higher scores correlating with poorer progression-free survival and higher relapse rates. This synergy enables clinicians to make earlier, more precise, and personalised treatment and follow-up decisions, maximising therapeutic benefit while reducing unnecessary interventions.

Table 3: Comparative Analysis of Present Invention vs. Prior Arts
Aspect US20090317820 US20140186947A1 Present Invention
Biomarker Panel miR-31 CD44, MMP9 miR-1307-5p, CD44v6, KRT4, PD-L1
Target Cancer Type Oral squamous cell carcinoma Head and neck squamous cell carcinoma Head and neck cancers (OSCC, HNSCC)
Biological Fluid Saliva (whole) Blood (serum) Saliva (extracellular vesicles + cellular fraction RNA)
Sensitivity (%) 67–70% (reported) ~75% >90% (validated through scoring algorithm and combined marker expression)
Specificity (%) Not explicitly reported ~70% >85%
Clinical Utility Early detection only Early detection only Real-time therapeutic monitoring, relapse prediction, and treatment planning
Invasiveness Non-invasive Minimally invasive Non-invasive
Temporal Resolution Single time-point snapshot Single time-point snapshot Longitudinal monitoring enabled by repeatable, non-invasive sampling
Interpretation Framework No interpretive model or clinical scoring No structured interpretation Integrated scoring algorithm to support clinical decision-making
Multi-marker Synergy No demonstrated synergy Independent proteins; no combinatorial logic Validated synergistic biomarker panel with defined thresholds and algorithmic integration
Personalization of Therapy Not addressed Not addressed Enables precision oncology by correlating biomarker expression profiles with therapeutic suitability, including immunotherapy decisions

Scope of Sample Types and Molecular Detection Levels
The biomarker panel comprising miR-1307-5p, CD44v6, KRT4, and PD-L1, as described herein, is applicable for:
(A) Predicting poor prognosis and survival outcomes,
(B) Assessing therapeutic efficacy of chemotherapeutic agents including platinum drugs, taxols, 5-fluorouracil, doxorubicin, cetuximab, methotrexate, radiotherapy, and immunotherapy,
(C) Identifying localized or metastatic disease progression, and
(D) Evaluating relapse propensity in head and neck cancer patients compared to control groups.
The biomarker expression profiles can be analyzed from diverse biological sources, including plasma, saliva, and tissue/cellular compartments such as the membrane, cytoplasm, nucleus, and nuclear membrane. Moreover, detection is feasible at the DNA, RNA, and protein expression levels, individually or in any combination thereof, thereby enabling comprehensive molecular characterization suited for diverse diagnostic workflows.

Embodiment 2: Diagnostic Kit for Therapeutic Monitoring and Relapse Prediction in Head and Neck Cancers
Embodiment 2 provides a diagnostic kit embodied with the salivary biomarker panel as described in Embodiment 1, specifically designed for non-invasive monitoring of therapeutic response and predicting relapse in patients with head and neck cancers, particularly oral squamous cell carcinoma (OSCC). The kit operationalizes the detection, quantification, and interpretation of the synergistic biomarker combination into a standardized clinical workflow.
The diagnostic kit comprises:
(1) Biomarker-Specific Primers and/or Probes:
? Primers and/or hydrolysis probes configured for amplification and detection of the following RNA biomarkers:
o miR-1307-5p
o CD44v6
o KRT4
o PD-L1
? Designed for compatibility with reverse transcription quantitative polymerase chain reaction (RT-qPCR) platforms to quantify expression levels with high sensitivity and specificity.
(2) A Reagent System for Isolation of Extracellular Vesicles (EVs) and Extraction of Nucleic Acids from EVs:
? Comprising reagents suitable for isolating RNA from biological samples selected from saliva, plasma, tissue, or cellular fractions, including:
o 500ul-1mL Extracellular vesicle precipitation reagent (polymer-based or PEG-based).
o 500ul-1mL Lysis reagent comprising phenol and guanidine isothiocyanate for disruption of vesicles.
o 200ul Chloroform for phase separation.
o 7.5ul of 5mg/ml Nucleic acid co-precipitant (such as glycogen) to enhance RNA recovery.
o 500ul- 1mL of 100% Isopropanol and 1ml of 70% ethanol-based washing solutions.
o 20ul Nuclease-free water for RNA resuspension.
? RNA yield and purity can be assessed using external instruments (bioanalyzer/fluorometer), not included within the kit.
(3) Reverse Transcription and Quantitative PCR Reagents:
? 20X MicroRNA-specific reverse transcription system (stem-loop primers and reverse transcriptase).
? 20X mRNA-specific reverse transcription system (oligo-dT/random primers and reverse transcriptase).
? 2X RT-qPCR master mix including AmpliTaq Gold® DNA Polymerase, UP (Ultra Pure), dNTPs with dUTP, ROX™ Passive Reference and Optimized buffer components. Inclusion of the biomarker-specific primers and/or probes (250nM-900nM) for the amplification reactions.
(4) Pre-Packaged Reference RNA Control:
? Stabilized RNA sample derived from healthy salivary sources (100-1000ng/ul), serving as a baseline normalization control to calculate fold-change in biomarker expression.
(5) Integrated Software Module:
? Provided as a secure cloud-based interface or locally installable application, operable to:
o Normalize raw expression data against housekeeping genes and reference control.
o Calculate fold-change values of the four biomarkers.
o Assign biomarker-specific scores based on expression thresholds:
? miR-1307-5p = 2.0-fold: Chemosensitivity/Therapeutic Response Prediction.
? CD44v6 = 2.0-fold: High Relapse Risk/Disease Aggressiveness.
? KRT4 = 1.0-fold: Chemosensitivity/Therapeutic Response Prediction.
? PD-L1 = 1.0-fold: Immunotherapy Suitability.
o Stratify patients into clinical categories including:
? Responders
? Non-responders
? High relapse risk
? Immunotherapy-suitable
o Integrate individual scores into a composite risk score using a weighted scoring algorithm of the software module. Here, the fold changes provide the chemo score whereby the relapse score and immuno score are generated. Once that is done, individual scores are integrated to generate an overall risk score, based on which low, moderate, high or very high risk patients can be identified.

o Additionally, derive a prognostic outcome assessment to classify patients under 'Poor prognosis' based on combined biomarker expression trends indicative of adverse survival likelihood.
o Generate structured clinical output reports for integration with electronic medical record (EMR) systems or for direct use in oncology workflows.

Operational Workflow Enabled by the Kit:
1. Collection of a biological sample (preferably saliva) using a validated non-invasive collection device (optionally included).
2. Isolation of extracellular vesicles and cellular fractions using the precipitation and lysis reagents.
3. RNA extraction and purification using the phase separation and precipitation workflow.
4. cDNA synthesis for miRNA and mRNA targets using the respective reverse transcription reagents.
5. Quantification of biomarker expression levels via RT-qPCR using included primers/probes and master mix.
6. Data analysis and scoring using the software module to generate fold-change values and clinical risk stratification.
7. Generation of diagnostic reports that assist clinicians in real-time treatment monitoring and relapse prediction.

Cross-Reference to Embodiment 1:
The diagnostic kit of Embodiment 2 provides a clinically standardized means to execute the biomarker signature composition described in Embodiment 1. By facilitating accurate measurement of miR-1307-5p, CD44v6, KRT4, and PD-L1 expression from saliva-derived extracellular vesicles and enabling systematic data interpretation through a scoring algorithm, the kit translates the biomarker panel into a reproducible, actionable diagnostic tool for dynamic therapeutic monitoring and early relapse detection in head and neck cancer patients.

Embodiment 3: In Vitro Method for Monitoring Therapeutic Response and Predicting Relapse Using the Diagnostic Kit
Embodiment 3 relates to an in vitro method for monitoring therapeutic response and predicting relapse in patients with head and neck cancers, particularly oral squamous cell carcinoma (OSCC), using the diagnostic kit as described in Embodiment 2. This method enables non-invasive and repeatable monitoring of biomarker expression patterns, operationalizing the salivary biomarker panel of Embodiment 1 through a standardized diagnostic workflow.
The method comprises the following steps:
1. Sample Collection:
Collecting a biological sample, preferably saliva (1-2ml), from the patient using a validated non-invasive collection device optionally included in the kit, said device optionally containing an RNA stabilization buffer to preserve RNA integrity.
2. RNA Isolation:
Isolating extracellular vesicles (EVs) from the collected biological sample, followed by RNA extraction from the EVs and/or cellular fractions using the reagent system provided in the kit. EV isolation may be performed using polymer-based or PEG-based precipitation or equivalent methods that preserve RNA integrity. The system comprising:
- a polymer-based or PEG-based extracellular vesicle precipitation reagent (500ul-1ml) ;
- phenol and guanidine isothiocyanate-based lysis reagent 500ul-1mL;
- chloroform 200ul , a nucleic acid co-precipitant (e.g., glycogen)7.5ul of 5mg/ml , 500ul- 1mL of 100% isopropanol, 1ml of 70% ethanol, and 20ul nuclease-free water.
3. Reverse Transcription and Biomarker Quantification:
Reverse transcribing the isolated RNA into cDNA using 20X miRNA- and 20X mRNA-specific reverse transcription systems provided in the kit.
Quantifying the expression levels of:
- miR-1307-5p
- CD44v6
- KRT4
- PD-L1
using RT-qPCR with biomarker-specific primers/probes (250nM-900nM) included in the kit.
4. Data Normalization and Fold-Change Calculation:
Processing the RT-qPCR expression data using the software module integrated with the kit, wherein raw data are normalized using housekeeping genes and compared against a reference RNA control derived from healthy saliva samples. Fold-change values are computed for each biomarker.
5. Score Generation and Patient Stratification:
Fold-change thresholds are interpreted as:
? miR-1307-5p = 2.0-fold
? CD44v6 = 2.0-fold
? KRT4 = 1.0-fold
? PD-L1 = 1.0-fold
The software module calculates biomarker-specific scores and integrates them into a composite risk score via a weighted scoring algorithm.
Patients are stratified into clinical categories:
- Responder
- Non-responder
- High relapse risk
- Immunotherapy-suitable
By combining these individual scores and stratification, the invention generates an Overall Risk Category classified as Low, Moderate, High, or Very High. Additionally, a prognostic outcome assessment is derived to classify patients under 'Poor prognosis' based on the overall biomarker expression trends predictive of reduced survival and aggressive disease progression.
6. Clinical Reporting and Decision Support:
Generating a structured clinical output report, integrated with Electronic Medical Records (EMR) or usable in oncology workflows, enabling clinicians to make informed therapeutic decisions and proactive relapse surveillance.
Cross-reference to Embodiment 1:
This method implements the synergistic biomarker panel of Embodiment 1 through the diagnostic kit described in Embodiment 2, ensuring real-time, non-invasive, and reproducible assessment of therapeutic efficacy and relapse risk in head and neck cancer patients.

Embodiment 4: In Vitro Method for Monitoring Therapeutic Response and Predicting Relapse Without Using the Diagnostic Kit
Embodiment 4 discloses an in vitro method for monitoring therapeutic response and predicting relapse in head and neck cancer patients, particularly OSCC, using standard laboratory reagents and workflows without relying on the proprietary diagnostic kit.
The method comprises the following steps:
1. Sample Collection:
Collecting an unstimulated saliva sample from the patient using a non-invasive collection method, optionally employing an RNA stabilization buffer to preserve sample quality.
2. RNA Isolation:
Isolating extracellular vesicles (EVs) from the collected biological sample, followed by RNA extraction from the EVs and/or cellular fractions using the reagent system provided in the kit. EV isolation may be performed using polymer-based or PEG-based precipitation or equivalent methods that preserve RNA integrity. The system comprises of
- polymer-based or PEG-based precipitation reagents for vesicle isolation;
- phenol-guanidine-based lysis reagents, chloroform, glycogen (as a nucleic acid co-precipitant), isopropanol, ethanol, and nuclease-free water.
3. Reverse Transcription and Biomarker Quantification:
Synthesizing cDNA from the extracted RNA using standard reverse transcription systems (stem-loop primers for miRNA and oligo(dT) or random primers for mRNA).
Quantifying expression levels of:
- miR-1307-5p
- CD44v6
- KRT4
- PD-L1
using RT-qPCR with custom-designed fluorescence-labeled sequence-specific probes.
4. Fold-Change Calculation and Reference Control:
Comparing the measured expression levels to baseline controls derived from matched healthy salivary samples or publicly available reference datasets. Calculating fold-change values using the following thresholds:
? miR-1307-5p = 2.0-fold
? CD44v6 = 2.0-fold
? KRT4 = 1.0-fold
? PD-L1 = 1.0-fold.
5. Composite Scoring and Stratification:
Integrating biomarker expression values using a structu red scoring logic (manual calculation or independent data analysis software) to categorize patients into clinical categories:
- Responder
- Non-responder
- High relapse risk
- Immunotherapy-suitable
By combining these individual scores and stratification, the invention generates an Overall Risk Category classified as Low, Moderate, High, or Very High. Additionally, a prognostic outcome assessment is derived to classify patients under 'Poor prognosis' based on the overall biomarker expression trends predictive of reduced survival and aggressive disease progression.
6. Interpretation and Clinical Application:
Utilizing the scoring outcome to guide therapeutic decisions and relapse surveillance strategies, enabling clinical actions even in the absence of the proprietary kit.
Cross-reference to Embodiment 1:
This method applies the salivary biomarker panel of Embodiment 1 through laboratory-standard workflows, ensuring broader accessibility of the technology across clinical and research settings, while maintaining the reproducibility, interpretability, and clinical relevance of the scoring system.
VALIDATION STUDIES AND CLINICAL PERFORMANCE
The salivary biomarker panel and diagnostic kit of the present invention have been validated through experimental studies and clinical observations, demonstrating their technical feasibility and clinical relevance in monitoring therapeutic response and predicting relapse in head and neck cancer patients.
The synergistic combination of miR-1307-5p, CD44v6, KRT4, and PD-L1 was shown to provide a diagnostic advantage, where integrated expression profiling enabled accurate patient stratification into therapeutic response categories including responders, non-responders, high relapse risk, and immunotherapy suitability. Additionally, a prognostic outcome assessment was derived to classify patients under ‘poor prognosis’ based on unfavourable cumulative biomarker trends, reflecting reduced survival probability and aggressive disease behaviour.
Case studies described in this specification illustrate the concordance between biomarker-based predictions and actual treatment outcomes, underscoring the clinical utility of the invention.
Reproducibility of the workflow was established across multiple sample runs, with consistent RNA extraction, amplification, and scoring outputs. The panel’s non-invasive nature, combined with its ability for repeat sampling and real-time molecular monitoring, addresses critical gaps in existing diagnostic approaches, which rely on invasive biopsies and delayed imaging assessments.
In comparison to prior art methods that focus on single markers or invasive sample types, the present invention delivers a validated biomarker composition tailored for salivary extracellular vesicles, integrated with a scoring algorithm that translates molecular data into clinically actionable insights. This positions the invention as a practical and scalable diagnostic solution for dynamic cancer monitoring, offering clear industrial applicability.

INDUSTRIAL APPLICABILITY
The present invention offers a scalable, non-invasive diagnostic platform that integrates seamlessly into existing laboratory workflows and clinical practices. By employing saliva as a biofluid source and utilizing standardized RT-qPCR workflows, the diagnostic kit enables widespread implementation across oncology clinics, pathology laboratories, and research institutions. The modular kit components, combined with a user-friendly software interface, facilitate easy integration into electronic medical records (EMR) and decision-support systems.
The invention's ability to provide repeatable, real-time monitoring of therapeutic response and relapse risk addresses a critical clinical gap, enhancing patient care through personalized treatment strategies. Its applicability spans initial diagnosis, ongoing therapy monitoring, and long-term surveillance, making it a versatile tool in head and neck cancer management.
Given its compatibility with conventional laboratory equipment and potential for adaptation into point-of-care diagnostic formats, the invention demonstrates high industrial applicability for commercial manufacturing, clinical deployment, and global adoption as a companion diagnostic platform.

ADVANTAGES OF THE INVENTION
The present invention provides several key advantages that address critical gaps in current diagnostic and monitoring approaches for head and neck cancers, including oral squamous cell carcinoma (OSCC):
1. Non-invasive, Repeatable Sampling
Utilizes saliva-derived extracellular vesicles and/or cellular fractions, allowing dynamic, longitudinal monitoring without requiring tissue biopsies or invasive procedures.
2. Real-time Therapeutic Response Assessment
Enables early evaluation of chemotherapy and immunotherapy effectiveness through molecular-level expression profiling, overcoming delays inherent in imaging-based RECIST assessments.
3. Combinatorial Biomarker Synergy
Leverages a validated panel of miR-1307-5p, CD44v6, KRT4, and PD-L1, whose combined expression profiles yield superior diagnostic and prognostic accuracy compared to individual biomarkers.
4. Actionable Scoring Algorithm for Clinical Decision Support
Provides a structured, weighted scoring system that integrates biomarker expression data into clinically interpretable risk categories, facilitating evidence-based treatment adjustments.
5. Predictive Monitoring of Relapse Risk
Detects molecular indicators of relapse well before anatomical recurrence is visible on imaging, enabling proactive intervention and personalized follow-up strategies.
6. Improved Stratification for Immunotherapy Suitability
Offers a non-invasive alternative to tissue biopsies for assessing PD-L1 expression, supporting informed immunotherapy decision-making in otherwise inoperable patients.
7. Scalability and Industrial Applicability
The diagnostic kit is designed for seamless integration into standard molecular laboratory workflows and is compatible with point-of-care deployment, ensuring broad industrial applicability.
8. Reduction of Unnecessary Treatment Cycles and Toxicity
By identifying non-responders early, the invention minimizes exposure to ineffective therapies, optimizing patient outcomes and reducing healthcare burdens.
TECHNICAL CONTRIBUTION AND ADVANCEMENT OVER PRIOR ART
Despite advancements in cancer treatments, a critical gap persisted in the ability to predict which patient would respond favorably to specific therapies. Oncologists and clinical experts were often left without reliable tools to guide such life-impacting decisions. While treatment regimens for head and neck cancers were well-established, the challenge of determining the right treatment for the right patient at the right time remained unresolved.
With limited available technical resources and biomarker knowledge in practical clinical workflows, there was an unmet need for a non-invasive, repeatable solution that could assist clinicians in dynamically assessing therapeutic response, relapse risk, and immunotherapy suitability. This invention fulfills that need by providing a synergistic biomarker panel and a reproducible diagnostic approach, empowering clinicians with actionable insights to personalize patient care, reduce ineffective treatments, and intervene earlier to improve survival outcomes.
The core technical advancement of the invention lies in operationalizing these biomarker signatures into a clinically usable platform that integrates into existing workflows, ensuring that decision-making is no longer reliant on invasive biopsies or delayed imaging assessments. ,CLAIMS:WE CLAIM:
Claim 1
Salivary Biomarker Panel for Therapeutic Monitoring and Relapse Prediction in Head and Neck Cancers, comprising a combination of biomarkers selected from miR-1307-5p, CD44v6, KRT4, and PD-L1, wherein the combined expression pattern of said biomarkers provides a diagnostic modality operationalized through analytical detection, reference normalization, and risk scoring workflows, and exhibits a synergistic profile indicative of:
(a) prognosis and survival prediction;
(b) therapeutic efficacy of chemotherapy including platinum-based agents, taxols, 5-fluorouracil, doxorubicin, cetuximab, methotrexate, radiotherapy, and immunotherapy;
(c) localized or metastatic disease status; and
(d) relapse risk in head and neck cancer patients compared to healthy controls,
and wherein the expression data stratifies patients into therapeutic response categories selected from responders, non-responders, high relapse risk, and immunotherapy-suitable, and further derives a prognostic outcome assessment indicative of poor prognosis based on cumulative biomarker trends.,
and wherein detection is performed at DNA, RNA, and/or protein levels.

Claim 2
Salivary Biomarker Panel for Therapeutic Monitoring and Relapse Prediction in Head and Neck Cancers, embodied in a diagnostic kit, the kit comprising:
? primers and/or probes specific to miR-1307-5p, CD44v6, KRT4, and PD-L1, designed to amplify and detect the synergistic expression profile of the biomarker combination through reverse transcription quantitative polymerase chain reaction (RT-qPCR), sequencing, or immunoassay platforms;
? a reagent system for extracellular vesicle (EV) isolation from biological samples selected from saliva, plasma, tissue, or cellular fractions, said reagent system comprising a polymer-based or polyethylene glycol (PEG)-based precipitation reagent or equivalent means to enrich EVs while preserving RNA integrity;
? a reagent system for isolation of nucleic acids and/or proteins from biological samples selected from saliva, plasma, tissue, or cellular fractions, the reagent system comprising:
(a) a polymer-based or polyethylene glycol-based extracellular vesicle precipitation reagent,
(b) a phenol and guanidine isothiocyanate-based lysis reagent,
(c) chloroform,
(d) a nucleic acid co-precipitant,
(e) isopropanol and ethanol-based washing solutions, and
(f) nuclease-free water;
? a pre-packaged reference control sample derived from healthy matched biological sources, for baseline normalization of biomarker expression data;
? reverse transcription reagents comprising microRNA-specific and mRNA-specific primers and reverse transcriptase systems;
? a quantitative detection system comprising thermostable DNA polymerase, deoxynucleotide triphosphates (dNTPs), magnesium chloride, probe-based detection chemistries, and reaction buffers optimized for amplification and quantification of said biomarkers;
? optionally, a validated non-invasive saliva collection device containing an RNA stabilization buffer; and
? a software module as claimed in claim 3, configured to process biomarker expression data for clinical stratification based on the synergistic expression profile of the biomarker signature panel,
wherein said kit enables non-invasive sample collection and repeatable, longitudinal monitoring of biomarker expression patterns for patient stratification and clinical decision-making,

Claim 3
Salivary Biomarker Panel for Therapeutic Monitoring and Relapse Prediction in Head and Neck Cancers, wherein the diagnostic kit as claimed in claim 2 comprises a software module provided as a secure cloud-based interface or as a locally installable application, and is configured for integration with electronic medical record (EMR) systems.

Claim 4
Salivary Biomarker Panel for Therapeutic Monitoring and Relapse Prediction in Head and Neck Cancers, wherein the diagnostic kit as claimed in any one of claims 2 to 3 is configured to:
? enable collection of a biological sample selected from saliva, plasma, tissue biopsy, or cellular fractions;
? isolate extracellular vesicles from the saliva or plasma samples.
? isolate nucleic acids and/or proteins from said sample using the provided reagents;
? detect expression levels of miR-1307-5p, CD44v6, KRT4, and PD-L1 using RT-qPCR, sequencing, or immunoassay platforms;
? process the expression data using the software module to compute fold-change values, calculate individual biomarker-specific scores, integrate the scores into a composite risk score; and
? stratify the patient into therapeutic response categories selected from responders, non-responders, high relapse risk, and immunotherapy-suitable, and further derive a prognostic outcome assessment indicative of poor prognosis based on cumulative biomarker expression trends.

Claim 5
Salivary Biomarker Panel for Therapeutic Monitoring and Relapse Prediction in Head and Neck Cancers, wherein an in vitro method is provided for diagnosing, monitoring therapeutic response, predicting relapse, and assessing prognosis in a patient using the diagnostic kit as claimed in any one of claims 2 to 4, the method comprising:
? collecting a biological sample selected from saliva, plasma, tissue, or cellular fractions;
? isolating extracellular vesicles from the saliva or plasma samples.
? isolating nucleic acids and/or proteins using the kit reagents;
? detecting expression levels of miR-1307-5p, CD44v6, KRT4, and PD-L1;
? processing the expression data using the software module to compute fold-change values, calculate biomarker-specific scores, and generate a composite risk score;
? stratifying the patient into therapeutic response categories selected from responders, non-responders, high relapse risk, and immunotherapy-suitable, and further deriving a prognostic outcome assessment reflecting poor prognosis; and
? generating a structured clinical report for therapeutic decision-making, relapse surveillance, and survival prediction, wherein the method is configured for repeat assessments at multiple clinical timepoints for longitudinal monitoring.

Claim 6
Salivary Biomarker Panel for Therapeutic Monitoring and Relapse Prediction in Head and Neck Cancers, wherein an in vitro method is provided comprising:
? collecting a biological sample selected from saliva, plasma, tissue, or cellular fractions;
? isolating nucleic acids (DNA/RNA) and/or proteins from said sample using laboratory-grade reagents;
? quantifying expression levels of miR-1307-5p, CD44v6, KRT4, and PD-L1 using RT-qPCR, sequencing, or immunoassay techniques;
? computing fold-change values relative to a reference control, wherein:
(a) miR-1307-5p = 2.0-fold indicates chemosensitivity and therapeutic response prediction,
(b) CD44v6 = 2.0-fold indicates high relapse risk and disease aggressiveness,
(c) KRT4 = 1.0-fold indicates chemosensitivity and therapeutic response prediction, and
(d) PD-L1 = 1.0-fold indicates immunotherapy suitability;
? analyzing the quantified expression values through a structured computational scoring system configured to translate expression profiles into clinical stratification outputs, thereby classifying the patient into therapeutic response categories selected from responders, non-responders, high relapse risk, and immunotherapy-suitable, and further deriving a prognostic outcome assessment indicative of poor prognosis based on cumulative biomarker trends;
? providing a structured clinical report indicative of patient outcome to aid therapeutic planning and monitoring, wherein the method enables repeat assessments over time for longitudinal monitoring.

Claim 7
The salivary biomarker panel as claimed in claims 1 to 6, wherein detection of the biomarkers is further applicable to biological samples selected from saliva, plasma, tissue, or cellular components including membrane, cytoplasm, nucleus, or nuclear membrane, at DNA, RNA, and/or protein levels.