Molecular Testing Updates. Karen Rasmussen, PhD, FACMG Clinical Molecular Genetics Spectrum Medical Group, Pathology Division Portland, Maine

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1 Molecular Testing Updates Karen Rasmussen, PhD, FACMG Clinical Molecular Genetics Spectrum Medical Group, Pathology Division Portland, Maine

2 Keeping Up with Predictive Molecular Testing in Oncology: Technical and Clinical Which tests have immediate clinical utility? What are the new tests on the horizon? For any particular biomarker or panel, what is the best methodology? or preferred lab? What if there s a designated Companion Diagnostic? Is liquid biopsy testing of ctdna a suitable replacement for molecular testing of a tumor biopsy

3 Current State of Predictive Molecular Markers Some well characterized molecular biomarkers have become standard of care in certain clinical situations some others have a good deal of supporting evidence, but not yet standard of care still others with little or no clinical evidence, but may be of interest when out of options Positive or negative evidence may accumulate over time

4 Few or Many? Molecular markers may be ordered as singles/small sets or as very large panels (hundreds; profiling) Standard of care markers are typically one or a few, used to direct 1 st or 2 nd line therapy In this situation, mutational profiling with large panels is generally overkill Profiling does include standard of care markers but is comprised mostly of markers associated with clinical trials, with prognosis, and/or for which there s little or no clinical data only preclinical information

5 FAQs How do I stay on top of standard of care markers? How do I know what new biomarkers are coming along? And how do I manage the growing list? What do I do with all the info that comes from large scale molecular profiling? What about Companion Diagnostics? What about reimbursement? (Why) Do I need to use an NGS (next generation sequencing) assay? Can I use a liquid biopsy instead of obtaining tumor tissue via an invasive procedure?

6 Exploring the Questions and Issues Colorectal Cancer and Non small Cell Lung Cancer

7 Colorectal Cancer expanded RAS testing Note: Currently labs may or may not include BRAF in their expanded RAS assay double check yours What are the current best practice recommendations? Guidelines (ex: NCCN) Multidisciplinary position papers

8 Colorectal Cancer expanded RAS testing Evolution: KRAS exon 2 mutations (codons 12 & 13) > KRAS exon 2 & BRAF V600 > More KRAS mutations > no BRAF > add NRAS > include BRAF > more BRAF

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10 Consensus Statement 1: Advanced colorectal cancer patients should receive expanded RAS testing on their tumors KRAS & NRAS codons 12 & 13 of exon 2, codons 59 & 61 of exon 3, and codons 117 & 146 of exon 4 Turnaround Time of days Consensus Statement 2A & 2B: BRAF activating mutation analysis should be performed in late stage colorectal cancers for prognostic stratification BRAF V600 is also useful in MMR deficient tumors with loss of MLH1 protein to evaluate probability of Lynch Syndrome

11 Additional Consensus Statements Metastatic or recurrent colorectal carcinoma tissues are the preferred specimens for therapy predictive biomarker testing and should be used if available and adequate. In their absence, primary tumor tissue is an acceptable alternative There is insufficient evidence to recommend HER2 analysis for therapy selection outside of a clinical trial There is insufficient evidence to recommend PIK3CA mutational analysis for therapy selection outside of a clinical trial There is insufficient evidence to recommend PTEN analysis for therapy selection outside of a clinical trial

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15 Colon Cancer NCCN Guidelines Version

16 NGS is a massively parallel process aligning & comparing many short sequences

17 NGS requires sophisticated computer analytics REPORT

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19 As with position papers, NCCN comments on biomarkers not ready for prime time

20 SOC Biomarker Use: NSCLC

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22 Need some graphics in here Lung driver mutations pie chart etc

23 Question 1: Which Patients Should Be Tested for Mutations and Gene Rearrangements? 1.1: Recommendation: Molecular testing should be used to select patients for targeted therapy, and patients with lung adenocarcinoma should not be excluded from testing on the basis of clinical characteristics. 1.2: Recommendation: In the setting of lung cancer resection specimens, Molecular testing is recommended for adenocarcinomas and mixed lung cancers with an adenocarcinoma component, regardless of histologic grade. In the setting of fully excised lung cancer specimens, molecular testing is not recommended in lung cancers that lack any adenocarcinoma component, such as pure squamous cell carcinomas, pure small cell carcinomas, or large cell carcinomas lacking any immunohistochemistry (IHC) evidence of adenocarcinoma differentiation. 1.3: Recommendation: In the setting of more limited lung cancer specimens (biopsies, cytology) where an adenocarcinoma component cannot be completely excluded, molecular testing may be performed in cases showing squamous or small cell histology but clinical criteria (eg, young age, lack of smoking history) may be useful in selecting a subset of these samples for testing. 1.4: Recommendation: To determine mutational status for initial treatment selection, primary tumors or metastatic lesions are equally suitable for testing.

24 Key Question 2: What methods should be used to perform molecular testing? IHC is an equivalent alternative to FISH for ALK testing. Multiplexed genetic sequencing panels are preferred over multiple single gene tests to identify treatment options beyond EGFR, ALK, and ROS1. (Expert consensus opinion)* Laboratories should ensure test results that are unexpected, discordant, equivocal, or otherwise of low confidence are confirmed or resolved using an alternative method or sample. (Expert consensus opinion) * The logic behind this recommendation is centered on TAT and conservation of small specimens. Testing individual markers by different methodologies increases turnaround time and also requires more tissue as sections have to be cut for testing each individual marker, for example, by FISH, by sequencing, by IHC etc. as opposed to extracting total DNA from the specimen and then testing for multiple (ideally all) markers by NGS.

25 Specimens may be insufficient to test markers individually Diagnostic Stain(s) Sequencing: EGFR FISH: ALK & ROS1 IHC: PD L1

26 Question 2: When Should a Patient Specimen Be Tested? 2.1a: Recommendation: Molecular testing should be ordered at the time of diagnosis for patients presenting with advanced stage disease (stage IV according to the 7th edition TNM staging system) who are suitable for therapy or at time of recurrence or progression in patients who originally presented with lower stage disease but were not previously tested. 2.2a: Expert consensus opinion: Molecular testing of tumors at diagnosis from patients presenting with stage I, II, or III disease is encouraged but the decision to do so should be made locally by each laboratory, in collaboration with its oncology team. 2.3: Recommendation: Tissue should be prioritized for molecular testing. Question 3: How Rapidly Should Test Results Be Available? 3.1: Expert consensus opinion: Molecular test results should be available within 2 weeks (10 working days) of receiving the specimen in the pathology laboratory.

27 ctdna analysis can rule in a mutation but can not rule out a mutation Incongruence between Liquid Biopsy Mutational Assays: Liquid Biopsy Mutational Assays miss many tumor tissue mutations

28 Liquid Biopsy : mutational analysis of circulating (cell free) tumor DNA

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30 NGS Assays vary in their ability to detect anomalies other than point mutations

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37 Assessing Specimens for Adequacy

38 Develop an Algorithm

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40 Tumor Mutational Burden (TMB) A measure of the mutational load in a tumor; the frequency of acquired point mutations Originally determined by whole exome sequencing Appears to correlate with response to immune modulator therapy Ex: nivolumab and ipilumumab in NSCLC TMB is believed to represent the relative amount of neoantigen presentation TMB is completely independent of PD L1 expression level TMB does not correlate well with MSI (Microsatellite Instability)

41 Problems with TMB No standardization no validated, accepted cut off originally developed by WES, not clear how many genes or how much DNA should be sequenced to truly assess mutational burden 75 80% correlation when genes are used Shortage of studies across tumor types and immune modulator therapies

42 When in Doubt: Check Drug Labels for Approved/Appropriate Biomarkers Ex: Keytruda/pembrolizumab NSCLC: PD L1 IHC, Tumor Proportion Score Gastric/GEJ, Urothelial, Cervical: PD L1 IHC, Combined Positive Score Melanoma, HNSCC, Classic Hodgkins Lymphoma: No biomarker needed All other solid tumors: MSI/DNA Mismatch Repair Deficiency

43 Conclusion Policy/Position papers and NCCN guidelines can answer most if not all FAQ s Forming interdisciplinary working groups locally has great benefits Development of local algorithms & standard practices Molecular Tumor Boards provide a forum for discussion, education and best practices