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

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

3. Alarming cancer stats in India

4. 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.

5. Molecular markers: Role

6. Molecular markers in colon cancer
K/N RAS
BRAF
PIK3CA

7. Let’s understand their role in CRC

8. 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

9. 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: 9

10. 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 

11. 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

12. 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,

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

15. Technologies available
- 96 samples
- 20% mutant limit
- Laborious
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

16. DNA quality assessment
Nucleic acid

17. PCR and Pyrosequencing

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

19. 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)

20. 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)

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

22. 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)

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

24. 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)

25. 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

27. 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 bp suggesting apoptosis produces the majority of cfDNA in circulation.
Diaz and Bardelli, 2014 Journal of Clincial Oncology 32

29. 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

30. 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.

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

32. Thank you