TMB (Tumor Mutational Burden) Test at Chughtai Lab

TMB (Tumor Mutational Burden) Test at Chughtai Lab

Tumor Mutational Burden (TMB) has emerged as a critical genomic biomarker in modern oncology, playing a pivotal role in predicting a patient’s response to immune checkpoint inhibitors (ICIs). The TMB test, offered at Chughtai Lab in collaboration with Genetech, is an advanced molecular diagnostic assay that measures the quantity of somatic mutations present within a tumor’s genome. Specifically, it calculates the number of non-synonymous somatic mutations per megabase (Mut/Mb) of sequenced DNA. By identifying tumors with a high mutational load, this test helps oncologists design highly personalized immunotherapy regimens, offering new hope to patients with advanced, metastatic, or refractory malignancies.

The clinical importance of TMB lies in its direct correlation with the immune system’s ability to recognize and destroy cancer cells. When a tumor accumulates a high number of somatic mutations, it produces a greater variety of novel, mutated proteins known as neoantigens. These neoantigens are displayed on the surface of tumor cells, making them highly visible to the patient’s immune system, particularly T-cells. However, tumors often exploit immune checkpoints, such as the PD-1/PD-L1 pathway, to evade immune destruction. Immune checkpoint inhibitors block these inhibitory pathways, unleashing the T-cells to attack the cancer. Patients with a high TMB score are significantly more likely to benefit from these immunotherapies because their tumors present an abundance of targetable neoantigens. Conversely, tumors with low TMB scores typically present fewer neoantigens and are less responsive to immunotherapy, directing clinicians to explore alternative therapeutic pathways such as chemotherapy, targeted therapy, or radiation.

The TMB test at Chughtai Lab utilizes state-of-the-art Next-Generation Sequencing (NGS) technology outsourced to Genetech, a leader in specialized molecular diagnostics. NGS allows for the comprehensive, high-throughput sequencing of hundreds of cancer-related genes simultaneously, ensuring an accurate assessment of the mutational landscape. This molecular evaluation can be performed on formalin-fixed paraffin-embedded (FFPE) tumor tissue obtained from a biopsy or surgical resection, or via a liquid biopsy using peripheral blood (circulating tumor DNA or ctDNA) when tissue is insufficient or inaccessible. By evaluating the genomic profile of the tumor, the TMB test provides invaluable diagnostic and prognostic insights, helping to optimize treatment efficacy, minimize unnecessary toxicity from ineffective therapies, and improve overall survival outcomes for cancer patients across Pakistan.

Clinical Procedure: What to Expect

Patient Preparation

Proper preparation is essential to ensure sample viability and the accuracy of the TMB test results. Depending on whether the test is performed on tissue or blood, the preparation guidelines vary:

• Tissue-Based TMB Test: If the test is being performed on an existing biopsy sample, no direct patient preparation is required. The patient or their oncologist must arrange for the transfer of the Formalin-Fixed Paraffin-Embedded (FFPE) tissue block or freshly cut unstained slides to Chughtai Lab. The tissue sample must meet specific quality criteria, including a minimum tumor cellularity (typically at least 20% to 30% tumor content) and adequate tissue volume.
• Liquid Biopsy (Blood-Based TMB): If a blood sample is being used to analyze circulating tumor DNA (ctDNA), no fasting is required. Patients should remain well-hydrated prior to the blood draw. It is important to inform the healthcare provider of any recent blood transfusions, active chemotherapy, or surgical procedures, as these can temporarily alter the concentration of circulating tumor DNA in the bloodstream.
• Medical Documentation: Patients must provide a complete clinical history, previous pathology reports (such as histopathology and immunohistochemistry results), and the oncologist’s prescription. This documentation is crucial for the molecular pathologists to interpret the genomic findings in the correct clinical context.

During the Procedure

The procedure for the TMB test depends on the specimen type utilized for the genomic analysis:

• Tissue Specimen Processing: For tissue-based testing, the pathology team at Chughtai Lab receives the FFPE block. A consultant pathologist reviews a hematoxylin and eosin (H&E) stained slide to confirm the diagnosis, assess the percentage of tumor cells, and select the optimal areas for DNA extraction. The selected tissue is then carefully microdissected, and genomic DNA is extracted using specialized molecular biology reagents.
• Blood Specimen Collection (Liquid Biopsy): If a liquid biopsy is ordered, a trained phlebotomist at Chughtai Lab will perform a standard venipuncture. Approximately 10 to 20 mL of peripheral blood is collected in specialized cell-free DNA (cfDNA) preservative tubes. The sample is processed via centrifugation to separate the plasma, from which the cell-free circulating tumor DNA is extracted.
• Next-Generation Sequencing (NGS): The extracted DNA is prepared for sequencing through library construction, target enrichment, and amplification. The prepared libraries are then sequenced on high-throughput NGS platforms at Genetech. The sequencing process generates millions of data points, capturing the precise nucleotide sequence of the target genomic regions.
• Bioinformatics Analysis: Advanced bioinformatics pipelines are employed to analyze the raw sequencing data. These algorithms filter out germline variants (inherited mutations) and focus exclusively on somatic mutations (acquired tumor mutations). The total number of eligible somatic mutations is calculated and normalized to the size of the sequenced genomic region to determine the final TMB score, expressed as mutations per megabase (Mut/Mb).

When is a TMB (Tumor Mutational Burden) Test Performed?

Advanced or Metastatic Non-Small Cell Lung Cancer (NSCLC)

In patients diagnosed with advanced or metastatic non-small cell lung cancer, determining the TMB status is highly recommended. NSCLC is often associated with a high mutational load, particularly in patients with a history of tobacco use. Identifying a high TMB score helps clinicians determine if the patient is an ideal candidate for first-line or subsequent-line immunotherapy, either as a monotherapy or in combination with chemotherapy, significantly improving progression-free survival.

Metastatic Melanoma

Melanoma is known for having one of the highest mutation rates among all cancers, primarily driven by ultraviolet (UV) radiation damage. A TMB test is performed in patients with metastatic melanoma to evaluate the genomic mutational load. This information, combined with BRAF mutation status, guides the selection of dual immunotherapy agents (such as ipilimumab and nivolumab) or targeted therapies, allowing for a highly tailored therapeutic approach.

Urothelial Carcinoma (Bladder Cancer)

Patients with advanced or metastatic urothelial carcinoma who have progressed after standard platinum-based chemotherapy are prime candidates for TMB testing. High TMB in urothelial tumors correlates strongly with durable clinical responses to anti-PD-1 or anti-PD-L1 therapies. The test assists oncologists in identifying which patients are most likely to benefit from these second-line immunotherapeutic options.

Microsatellite Instability-High (MSI-H) or dMMR Solid Tumors

TMB testing is frequently performed alongside or as a follow-up to Microsatellite Instability (MSI) and Deficient Mismatch Repair (dMMR) testing. Tumors that are MSI-H or dMMR typically exhibit exceptionally high TMB due to the loss of DNA repair mechanisms. Performing a TMB test helps confirm the hypermutated phenotype in various solid tumors, including colorectal, endometrial, and gastric cancers, qualifying patients for pembrolizumab or other pan-cancer immunotherapy indications.

Refractory Solid Tumors Seeking Immunotherapy Options

For patients with advanced solid tumors that have exhausted standard-of-care treatment options, a TMB test is performed as part of comprehensive genomic profiling. The FDA has approved pembrolizumab for the treatment of any unresectable or metastatic solid tumor with a TMB score of 10 mutations per megabase (Mut/Mb) or higher, making this test a crucial tool for identifying salvage immunotherapy options across a wide range of cancer types.

What Does a TMB (Tumor Mutational Burden) Test Detect?

The TMB test evaluates several molecular and genomic parameters within the tumor DNA to provide a comprehensive mutational profile. The key parameters and findings detected by this advanced assay include:

• Somatic Single Nucleotide Variants (SNVs): Detects point mutations where a single nucleotide is replaced by another in the tumor DNA.
• Small Insertions and Deletions (Indels): Identifies the insertion or deletion of small segments of DNA, which can disrupt the reading frame of proteins.
• Mutations per Megabase (Mut/Mb): Calculates the final quantitative score representing the density of somatic mutations across the sequenced genomic region.
• Synonymous vs. Non-Synonymous Mutations: Filters out synonymous mutations (which do not alter amino acid sequences) and focuses on non-synonymous mutations that generate neoantigens.
• Germline Variant Filtering: Distinguishes inherited genetic variations from acquired somatic mutations to ensure accurate tumor-specific profiling.
• Hypermutation Status: Identifies tumors with exceptionally high mutation rates, often exceeding 20 or 30 Mut/Mb.
• Mismatch Repair (MMR) Gene Mutations: Detects mutations in MLH1, MSH2, MSH6, and PMS2 genes, which lead to high mutational burden.
• Polymerase Epsilon (POLE) Mutations: Identifies mutations in the proofreading domain of DNA polymerase epsilon, causing ultra-hypermutated tumor profiles.
• Polymerase Delta 1 (POLD1) Mutations: Detects alterations in DNA polymerase delta 1, another driver of extreme genomic instability.
• Tumor Mutational Signature: Analyzes the pattern of mutations to identify underlying causes, such as UV exposure, tobacco smoke, or alkylating agents.
• Targetable Driver Mutations: Identifies concurrent actionable mutations in genes like EGFR, ALK, ROS1, or BRAF that may dictate targeted therapy over immunotherapy.
• Resistance Mutations: Detects genomic alterations that may confer resistance to specific targeted therapies or immunotherapies.
• Tumor Cellularity Assessment: Evaluates the proportion of tumor cells in the sample to ensure the genomic data is representative of the malignancy.
• DNA Degradation Index: Measures the quality of the extracted DNA to ensure the sequencing data meets stringent diagnostic standards.
• Sequencing Coverage Depth: Confirms that the target genomic regions were sequenced with sufficient depth to detect low-frequency mutations.
• Variant Allele Frequency (VAF): Measures the percentage of sequencing reads observing a specific mutation, reflecting tumor heterogeneity.
• Microsatellite Instability (MSI) Status: Often co-analyzed to determine if genomic instability is localized to repetitive DNA sequences.
• Copy Number Variations (CNVs): Detects amplifications or deletions of large chromosomal segments containing oncogenes or tumor suppressors.
• Tumor Mutational Load Classification: Categorizes the tumor into Low, Intermediate, or High TMB brackets based on clinical cutoffs.
• Immunotherapy Eligibility: Provides a clear indication of whether the genomic profile supports the clinical use of immune checkpoint inhibitors.

Turnaround Time and Report Access at Chughtai Lab

Due to the highly sophisticated nature of Next-Generation Sequencing and the outsourcing workflow to Genetech, the turnaround time for the TMB test is typically between 14 to 21 working days. This period allows for meticulous pathology review, DNA extraction, high-depth sequencing, complex bioinformatics analysis, and clinical correlation by a multidisciplinary team of molecular pathologists and geneticists.

Chughtai Lab ensures that patients and referring oncologists can access these critical reports seamlessly. Once the genomic analysis is complete and verified, the report is uploaded directly to the Chughtai Lab online portal and mobile application. Patients receive an automated SMS notification with a secure link to download their PDF report. Hard copies of the comprehensive molecular report can also be collected from any main diagnostic center of Chughtai Lab across Pakistan or delivered via courier upon request.

TMB Findings Overview

Structure / Parameter Evaluated	Normal Findings	Possible Abnormal Findings
Tumor Mutational Burden (TMB) Score	Low TMB (< 10 mutations/megabase)	High TMB (≥ 10 mutations/megabase), indicating high neoantigen load and potential immunotherapy benefit.
Mismatch Repair (MMR) Status	Proficient Mismatch Repair (pMMR)	Deficient Mismatch Repair (dMMR), leading to genomic instability and hypermutated tumor states.
Microsatellite Status	Microsatellite Stable (MSS)	Microsatellite Instability-High (MSI-H), frequently associated with high TMB and hereditary cancer syndromes.
POLE/POLD1 Gene Status	Wild-type (No pathogenic mutations)	Pathogenic mutations in POLE or POLD1, leading to ultra-hypermutated phenotypes (>100 Mut/Mb).
Tumor Cellularity	Adequate tumor content (≥ 20-30%)	Inadequate tumor content (< 20%), which may lead to false-negative results or test failure.
Somatic Driver Mutations	No actionable driver mutations detected	Detection of targetable mutations (e.g., EGFR, ALK, BRAF V600E) which may prioritize targeted therapies.
DNA Quality and Quantity	High-quality genomic DNA extracted	Highly degraded DNA or low yield, common in old or poorly fixed FFPE blocks, requiring sample recollection.

Note: Diagnostic findings should always be interpreted by a qualified healthcare professional together with the patient’s symptoms, medical history, physical examination, laboratory investigations, previous imaging studies, and other relevant clinical information. Additional investigations or specialist consultation may be recommended depending on the findings.

Why Choose Chughtai Lab for TMB?

• Strategic Collaboration with Genetech: Combines Chughtai Lab’s extensive diagnostic infrastructure with Genetech’s advanced molecular and genomic expertise.
• State-of-the-Art NGS Technology: Utilizes high-throughput Next-Generation Sequencing platforms to ensure maximum accuracy and analytical sensitivity.
• Expert Molecular Pathologists: Reports are reviewed and interpreted by highly qualified molecular pathologists and clinical geneticists.
• Comprehensive Genomic Reports: Provides detailed, easy-to-interpret reports outlining TMB scores, clinical significance, and potential therapeutic options.
• Rigorous Quality Control: Adheres to international standards of quality assurance and proficiency testing for molecular diagnostics.
• Convenient Sample Collection: Offers tissue block pick-up services and specialized blood collection at Chughtai Lab centers nationwide.
• Seamless Digital Access: Patients and oncologists can access reports online via the Chughtai Lab portal or mobile app.
• Dedicated Patient Support: Provides professional guidance throughout the testing process, from sample submission to report delivery.