Managing Colon cancer in the era of molecular markers Dr B.R.Das President, Research and Innovation SRL Ltd, Mumbai INDIA 1 1

Top 3 cancers Four Solid Tumors (Lung, Breast, Colorectal and Stomach) account for almost 40% of total cancer incidences.

Alarming cancer stats in India

Mortality Rate In India, 70% of diagnosed patients die in first year due to late diagnostics. India also has some of the highest incidences in the world for certain cancers including: cervical, gall bladder, oral and pharynx.

Molecular markers: Role

Molecular markers in colon cancer K/N RAS BRAF PIK3CA

Let’s understand their role in CRC

What happens to the remaining patients? Why they do not respond??? Anti-EGFR Therapy is given to patient’s with Metastatic Colorectal Carcinoma ? ? When Kras is found then what is the therapy? ? ? ? ? What happens to the remaining patients? Why they do not respond??? 8

KRAS, NRAS, BRAF, PIK3CA : Clinical Significance 3-10% BRAF Gene Mutations – Non Responders/Worst Prognosis 30-50% KRAS Gene Mutations / 3% NRAS mutations – Non Responders 12% PIK3CA and other Gene Mutations – Non Responders Combined KRAS, NRAS, BRAF and PIK3CA gene mutations explains ~ 62% of the non-responders The American Society of Clinical Oncology (ASCO) and the NCCN) both recommend KRAS mutation testing prior to prescribing EGFR antagonist therapy for patients with mCRC and state that alternative therapy should be prescribed when mutations are detected. J Clin Oncol. 2009;27:2091-2096 9

Suggested Algorithm for Testing KRAS mutations Positive Negative No Response NRAS mutations Positive Negative No Response BRAF mutations Positive Negative No Response PIK3CA mutations Positive Negative No Response Response to anti-EGFR likely 

Specimen & Processing Steps Paraffin Block Unstained Sections on Slides 3-4 nos Tumor cells not present Cytology Sufficient Tumor cells present Test Not Performed DNA Extraction & PCR Pyrosequencing (kras) BRAF, PIK3CA, MSI

Most Important critical point!!! (Tumor heterogeneity) Recommendation: - Pathologists should determine the adequacy of specimens for molecular testing by assessing cancer cell content and DNA quantity and quality. -Laboratories should test methods that are able to detect mutations in specimens with at least 30-40% cancer cell content,

Tumor % and mutation detection Expected mutation Level (10%) Expected mutation Level (15%) Expected mutation Level (20%)

Technologies available - 96 samples - 20% mutant limit - Laborious - 9-12 hours - Cheaper Sanger Sequencing - 96 samples - 5% mutant limit - Easy handling - 3-4 hours - Expensive Real Time PCR - 24 samples - 5-10% mutant limit - Easy workflow - 10 to 40 mins - Cost effective Pyrosequencing

DNA quality assessment Nucleic acid

PCR and Pyrosequencing

Pyrogram Codon 12,13 (Not Detected) GGT>GAT codon 12 (Detected)

Use of conventional molecular markers: Is there a need to go beyond? - KRAS extended panel - NRAS extended panel - MMR and MSI testing - OncotypeDX (Upcoming) - CTC/Liquid Biopsy (Upcoming)

KRAS/NRAS extended panel What’s New and Different? Currently, KRAS exon 2 (codon 12, 13) ; exon 3 (codon 61). Extended-KRAS exons 3 (codon 59) and 4 (codons 117 and 146) Currently, NRAS exon 2 (codon 12, 13) ; exon 3 (codon 61). Extended -NRAS exons 3 (codon 59), and 4 (codons 117 and 146)

ASCO – 2015 Recommendations KRAS/NRAS extended mutation screening is a must before giving anti-EGFR therapy

Progression Free Survival Why extended panels? 20% of the KRAS exon 2 non-mutated tumors harbored one of the extended RAS mutations Anti-EGFR therapy Progression Free Survival Overall Survival J Clin Oncol;33 (2015)

Multigene expression profile and cancer treatment decision The challenge: Which stage II colon cancer patients should receive adjuvant chemotherapy?

Some facts to be known for stage II colon cancer Generally, Not all colon cancer patients are given chemotherapy, especially early stage (II and IIIA,B) Because, they can be managed by simple surgery and chemotherapy is not required (low risk of recurrence) However, some of them do need chemotherapy because they have high risk of metastasis (high risk of recurrence) Chemo has significant toxicity and impacts quality of life ….This is the treatment dilemma High Risk is based on MMR (Proficient), T stage (T4), Tumor grade (High), No. of nodes examined (>12)

Low risk The continuous RS will have the greatest clinical utility for T3, MMR-proficient patients, who constitute the majority of stage II colon cancer (~70% of pts) New paradigm for quantitative assessment of recurrence risk in stage II colon cancer, emphasizing the role of three measures, Recurrence Score, MMR/MSI, and T stage

Circulating Tumour DNA (ctDNA) ctDNA is tumour DNA that has been shed into the bloodstream ctDNA can be present in 0.01% - >90% of the total Cell Free DNA (cfDNA) The amount of ctDNA is related to the tumour burden and varies between patients with different clinical presentations Circulating tumour DNA is a component of cell free DNA, this is DNA present in the blood stream and this is the same sample as the free fetal DNA The tumour DNA is released into the blood stream by secretion, apoptosis and necrosis, however most cfDNA fragments are between 180-200bp suggesting apoptosis produces the majority of cfDNA in circulation. Diaz and Bardelli, 2014 Journal of Clincial Oncology 32

Workflow Sample arrives in lab and spun to isolate the plasma Plasma is stored at -80ºc Blood sample taken in Cell Save preservative tubes Sample is extracted on the same day as the downstream process set up due to ctDNA instability Set up: Pyrosequencing Next-generation sequencing Quantative PCR BEAMing Digital PCR ctDNA is extracted from the plasma using the QIAamp Circulating Nucleic Acid on the QIAVac system

Concluding remarks Molecular markers plays a critical role in therapeutic decision making (~ 70% of non-responders) RAS mutational testing of colorectal carcinoma tissue should be performed for patients who are being considered for anti-EGFR therapy. This analysis must include KRAS and NRAS codons 12, 13 of exon 2; 59, 61 of exon 3; and 117 and 146 of exon 4 ("expanded" or "extended" RAS). dMMR/MSI testing must be performed in all colorectal cancers for prognostic stratification and identification of Lynch syndrome patients.

And we are here.. Many more to explore….

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