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Issue 02 | February 2017
Easy Test for Cancer??How About a Simple Blood Draw!
Strand Center at Pinkathon Bengaluru 2017 – Let’s go Pink Sisters!
In the News
Genetic testing and Counselling Among Patients With Newly Diagnosed Breast Cancer
Strand Life Sciences expands oncogenomic portfolio in India with globally benchmarked 152-gene test
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Welcome to Strand Genomics-A Monthly E-zine from Strand Life Sciences
Strand Life Sciences welcomes you to Strand Genomics, our monthly E-zine that includes articles of interest to physicians. This e-zine brings the latest news in the world of genetic diagnostics, to your doorstep. We present carefully crafted articles as well as curated news in the field of cancer therapy and genetic analyses to support the implementation of personalized medical care. We invite you to peruse as well as share these articles. Please also feel free to write back to us with comments and questions at strandlive@strandls.com
Liquid Biopsy For Detection of Mutations in Lung Cancer Patients
– Dr. Ramesh Hariharan, Dr. Rashmi Updhayay & Dr. Shefali Sabharanjak
Strand Life Sciences
Abstract
- Strand Life Sciences introduces Strand Liquid Biopsy – a portfolio of tests, based on the liquid biopsy technique, for detecting genetic changes in blood samples of cancer patients.
- Liquid Biopsy is a minimally invasive technique that enables identification of tumor mutations from tumor DNA fragments in the blood.
- Our first liquid biopsy tests are designed to detect mutations in EGFR from lung cancer patients, specifically, EGFR del 19 and L858R mutations for sensitivity towards TKI therapy and T790M mutation for resistance to TKI therapy.
- These tests have been rigorously validated, both analytically and clinically, and perform at the highest sensitivity in the industry.
- The liquid biopsy-based tests are highly significant in cases where excision of sufficient quantity of tissue samples by conventional biopsies is not feasible.
- Longitudinal monitoring of cancer patients is facilitated by the use of these liquid biopsy tests. Our test can also help detect resistance to EGFR-TKIs, months before clinical progression of lung cancer is evident.
Introduction
Dying cells all over the body shed their DNA into the blood; these DNA fragments in the blood plasma are called cfDNA or circulating free DNA. A fraction of these fragments come from tumor cells; this fraction is accordingly called circulating tumor DNA or ctDNA. Liquid biopsy is a minimally-invasive blood test that aims to detect this ctDNA.
ctDNA is usually distinguished from cfDNA by the presence of tumor-specific mutations. A measure of tumor burden can be obtained by identifying and quantifying fragments with these mutations. Repeating this exercise over time can help monitor tumor progression and response to therapy.
Figure 1: Tumor cells and normal cells release DNA into the bloodstream
Work Flow of Liquid Biopsy
A natural question that comes to mind now is how well sequencing of ctDNA compares with direct sequencing of solid tumor biopsies. Several studies have shown that mutations found in a tumor can also be detected in ctDNA in 40-50% of stage I and II patients, and 80% of stage II and IV patients (Bettegowda et al. 2014). Studies have also shown detection of ctDNA across a range of tumor types, with colorectal and bladder showing the highest detection, and gliomas showing the lowest (the latter probably on account of the blood brain barrier)(Bettegowda et al. 2014).
Further, multiple studies have shown how ctDNA can predict recurrence earlier and better than other clinico-pathological parameters such as protein markers and CT-Scans (Pereira et al. 2015; Ma et al. 2016; Tie et al. 2016). Coupled with its non-invasiveness, this makes ctDNA an exciting frontier for cancer management.
A key challenge in liquid biopsies is that ctDNA comprises a minuscule fraction of the entire cfDNA. Care is therefore needed at multiple steps to detect this ctDNA. It is essential that blood be collected in special tubes that prevent blood cells from breaking and releasing their DNA into the plasma, thus diluting ctDNA even more. It is also essential that as much of the cfDNA present in the plasma be extracted as possible. Finally, it is essential that a very sensitive method be used for detecting mutated fragments in cfDNA.
Indeed, conventional methods cannot provide the sensitivity that is required for ctDNA detection. Instead, Digital droplet PCR (ddPCR) and adaptations of Next Generation Sequencing (NGS) where the assay and bioinformatics are specially designed and validated, can be used for ctDNA detection.
This e-zine article describes the release of the first in a series of liquid biopsy products from Strand Life Sciences.
Strand’s Liquid Biopsy Product for Lung Cancer
Strand’s Lung Cancer product for liquid biopsies can detect EGFR Exon 19 deletions, the L858 mutation, and the well-known resistance mutation, T790M. As is the case with all of Strand’s genetic diagnostic tests, this release is brought to you after rigorous analytics and clinical validation described below.
First, we have standardized procedures for maintaining the integrity of blood samples and ensure that no additional cell lysis is evident during our workflow.
Second, by way of analytical validation, we spiked in cfDNA with mutated fragments of the EGFR gene at a 0.1% concentration and showed that we could detect the mutation with 100% sensitivity and specificity, as described in the following table.
Table 1. Analytical Validation of the Strand Liquid Biopsy Tests
| Samples | Analytical Sensitivity* at 0.1% spike in conc | Analytical Specificity* at 0.1% spike in conc |
| TKI sensitizing EGFR Exon 19 del | 100 % | 100 % |
| TKI sensitizing EGFR L858R | 100 % | 100 % |
| TKI resistant EGFR T790M | 100 % | 100 % |
We would like to draw your attention to the fact that the limit of detection for other similar tests (data available with Strand Life Sciences) stands at 6-10%, as against 0.1% for the Strand Liquid Biopsy tests as established above.
Finally, we performed clinical validation for 10 lung cancer samples spanning multiple stages. In each case, we obtained a tumor biopsy samples and a simultaneous plasma sample. The tumor sample was sequenced to identify key mutations, and the ctDNA test was used to detect these mutations. This effort was successful in 9 out of the 10 samples, yielding a sensitivity of 90%, as in the table below.
| Clinical Validation Samples | Concordance between Tumor DNA & ctDNA | Failure |
| 10 | 90 % | 10 % |
Applications of Strand’s Lung Cancer Products
These tests are expected to simplify the biopsy of lung cancer and can help to determine the eligibility of lung cancer patients for tyrosine kinase inhibitor therapies. With increasing adoption of these test in diagnostic protocols, these tests have the potential to help in longitudinal surveillance of each cancer patient and make cancer therapy truly a personalized experience.
Molecular testing for EGFR from tumor biopsies is recommended by treatment guidelines to determine if treatment with EGFR TKIs (Tyrosine Kinase Inhibitors) may be applicable. Our experience with profiling hundreds of lung tumor biopsies in India shows that >30% show EGFR mutations and are thus eligible for TKI therapy. Of these, nearly 95% of these are exon 19 deletions or the exon 21 L858R mutation, covered by our liquid biopsy test.
It is well established that about 23% of lung biopsies provide insufficient material for histopathological analysis. Strand’s liquid biopsy tests can help by assessing for EGFR mutations from plasma instead. Indeed, this convenience is not restricted to difficult cases but can be adopted across the whole spectrum of lung cancer cases.
In case you are wondering whether assessment of EGFR mutation from ctDNA is acceptable for treatment decisions, a meta-analysis of 7 studies has shown that the presence of EGFR activating mutation in ctDNA was associated with a greater chance of response to EGFR-TKIs (OR, 1.96; 95% CI, 1.59-2.42) (Ai et al. 2016). In addition, an analysis of 12 studies has shown that patients with higher ctDNA concentrations had shorter median PFS and OS (Ai et al. 2016).
Further, it is well known that resistance to first-generation EGFR TKI’s like Erlotinib appears on account of the T790M mutation (Sullivan and Planchard 2017). Studies show that in 50% of the patients, T790M can be detected from ctDNA itself, a median of 2.2 months prior to clinically progressive disease (Zheng et al. 2016). If this mutation is detected in ctDNA, unnecessary biopsies can be avoided and possibly treatment switched to later generation TKIs like Osimertinib earlier than it would be if one were to wait for clinical signs of resistance and follow-up tumor biopsies (Remon et al. 2017; Pao et al. 2010).
The Liquid Biopsy Workflow
Strand will send a special tube for collecting blood to cancer patients, called Streck Tubes. This tube will carry special reagents that will stabilize blood cells for more than 72 hours. The sample will reach our lab in the meantime where the plasma will be separated out after centrifugation, as shown below.
A simple 10-minute procedure to collect blood by a trained nurse or phlebotomist is all it will take to conduct a ‘biopsy’ on a cancer patient!
cfDNA will then be extracted from the plasma and then tested for the specified EGFR mutation. We will provide a report of the genetic profile of the tumor within 5 business days.
These tests are expected to simplify the biopsy of lung cancer and can help to determine the eligibility of lung cancer patients for tyrosine kinase inhibitor therapies. With increasing adoption of these tests in diagnostic protocols, these tests have the potential to help in longitudinal surveillance of each cancer patient and make cancer therapy truly a personalized experience.
Summary
- Strand Life Sciences introduces Strand Liquid Biopsy – a portfolio of tests, based on the liquid biopsy technique, for detecting genetic changes in ctDNA isolated from blood samples of cancer patients.
- Liquid Biopsy is a minimally invasive technique that allows for collection of circulating tumor DNA, from a blood sample, in order to assess the genetic changes that cause cancer.
- The mutation status of cancer, at various stages of treatment of a patient, can be accessed with the help of Strand Liquid Biopsy tests.
- These tests have been rigorously validated, both analytically and clinically, and perform at the highest sensitivity in the industry.
- Longitudinal surveillance of cancer patients is possible using tests based on the liquid biopsy technique. This development has the potential to create new windows of therapeutic interventions for each cancer patient, with significant clinical and research advantages.
References
Ai, Bo, Huiquan Liu, Yu Huang, and Ping Peng. 2016. “Circulating Cell-Free DNA as a Prognostic and Predictive Biomarker in Non-Small Cell Lung Cancer.” Oncotarget 7 (28). Impact Journals, LLC: 44583–95. doi:10.18632/oncotarget.10069.
Bettegowda, Chetan, Mark Sausen, Rebecca J Leary, Isaac Kinde, Yuxuan Wang, Nishant Agrawal, Bjarne R Bartlett, et al. 2014. “Detection of Circulating Tumor DNA in Early- and Late-Stage Human Malignancies.” Science Translational Medicine 6 (224). NIH Public Access: 224ra24. doi:10.1126/scitranslmed.3007094.
Ma, Fei, Wenjie Zhu, Yanfang Guan, Ling Yang, Xuefeng Xia, Shanshan Chen, Qiao Li, et al. 2016. “ctDNA Dynamics: A Novel Indicator to Track Resistance in Metastatic Breast Cancer Treated with Anti-HER2 Therapy.” Oncotarget 7 (40): 66020–31. doi:10.18632/oncotarget.11791.
Pao, William, Juliann Chmielecki, Delong Liu, W Pao, J Chmielecki, SV Sharma, DW Bell, et al. 2010. “Rational, Biologically Based Treatment of EGFR-Mutant Non-Small-Cell Lung Cancer.” Nature Reviews Cancer 10 (11). BioMed Central: 760–74. doi:10.1038/nrc2947.
Pereira, Elena, Olga Camacho-Vanegas, Sanya Anand, Robert Sebra, Sandra Catalina Camacho, Leopold Garnar-Wortzel, Navya Nair, et al. 2015. “Personalized Circulating Tumor DNA Biomarkers Dynamically Predict Treatment Response and Survival In Gynecologic Cancers.” Edited by Goli Samimi. PLOS ONE 10 (12). Public Library of Science: e0145754. doi:10.1371/journal.pone.0145754.
Remon, J., C. Caramella, C. Jovelet, L. Lacroix, A. Lawson, S. Smalley, K. Howarth, et al. 2017. “Osimertinib Benefit in EGFR -Mutant NSCLC Patients with T790M -Mutation Detected by Circulating Tumour DNA.” Annals of Oncology, January, mdx017. doi:10.1093/annonc/mdx017.
Sullivan, Ivana, and David Planchard. 2017. “Next-Generation EGFR Tyrosine Kinase Inhibitors for Treating EGFR-Mutant Lung Cancer beyond First Line.” Frontiers in Medicine 3 (January): 76. doi:10.3389/fmed.2016.00076.
Tie, J., Y. Wang, C. Tomasetti, L. Li, S. Springer, I. Kinde, N. Silliman, et al. 2016. “Circulating Tumor DNA Analysis Detects Minimal Residual Disease and Predicts Recurrence in Patients with Stage II Colon Cancer.” Science Translational Medicine 8 (346): 346ra92-346ra92. doi:10.1126/scitranslmed.aaf6219.
Zheng, D, X Ye, M Z Zhang, Y Sun, J Y Wang, J Ni, H P Zhang, et al. 2016. “Plasma EGFR T790M ctDNA Status Is Associated with Clinical Outcome in Advanced NSCLC Patients with Acquired EGFR-TKI Resistance.” Scientific Reports 6 (February). Nature Publishing Group: 20913. doi:10.1038/srep20913.
Hereditary Breast Cancer in India
At Strand Life Sciences, we have analyzed close to 2000 cases of hereditary breast and ovarian cancer and have found that approximately 40% of the cases are familial. This is also reflected in the finding that the age of onset of breast cancer in India is a full decade lower than that seen in the West.
In Pursuit of A VUS Mutation
– Dr. Shefali Sabharanjak
Strand Life Sciences
Abstract
- A 56-year-old female patient with recurrent ovarian cancer was referred for genetic diagnosis to Strand Life Sciences.
- Patient was found to be heterozygous for a BRCA2 mutation-initially classified as a ‘variant of uncertain significance (VUS)’.
- Co-segregation and in silico analyses were performed to understand whether the VUS mutation could be responsible for causing cancer.
- Additional analyses indicated the eligibility of the patient for PARP inhibitor therapy.
- Patient has responded well to PARP inhibitors with significant improvement in quality of her life.
- Therapeutic options were created for the patient by tenacious and persistent follow-up of a BRCA2 VUS
Introduction
Genomic analyses of patient DNA samples yield novel and interesting information as more and more people opt for molecular diagnostics for their health issues. At Strand Life Sciences, we have analyzed nearly 2000 patient samples for presence of germline mutations in genes associated with breast and ovarian cancer. Some cases yield straightforward results of pathogenic mutations, while other present confounding scenarios, such as ‘variants of uncertain significance’ or VUS mutations.
Case Description
One recent case was that of Mrs. Meena Nandal, a resident of Delhi (name disclosed after obtaining patient and physician consent). She is a 56- year- old patient who was diagnosed with ovarian cancer at the age of 52 years. As per international guidelines, Mrs. Nandal was prescribed platinum-based chemotherapy to treat ovarian cancer. In the first couple of rounds of treatment, her cancer responded to the therapy but then she suffered a relapse. Since the cancer recurred, Mrs. Nandal was advised to undergo genetic diagnosis to understand the genes responsible for her cancer, by a prominent oncologist based in Delhi.
Identification of a BRCA2 VUS
One recent case was that of Mrs. Meena Nandal, a resident of Delhi (name disclosed after obtaining patient and physician consent). She is a 56- year- old patient who was diagnosed with ovarian cancer at the age of 52 years. As per international guidelines, Mrs. Nandal was prescribed platinum-based chemotherapy to treat ovarian cancer. In the first couple of rounds of treatment, her cancer responded to the therapy but then she suffered a relapse. Since the cancer recurred, Mrs. Nandal was advised to undergo genetic diagnosis to understand the genes responsible for her cancer, by a prominent oncologist based in Delhi.
Co-Segregation Analysis
In this case, scientists at Strand decided to undertake a co-segregation analysis in order to understand the inheritance pattern as well as potential pathogenicity of this VUS of BRCA2. Accordingly, germline testing was advised to Mrs. Nandal’s brother as well as sister. Incidentally, Mrs. Nandal’s younger sister had been diagnosed with breast cancer, an year after Mrs. Nandal’s diagnosis of ovarian cancer. Germline analyses of DNA samples provided by Mrs. Nandal’s siblings showed the presence of the same VUS mutation in her sister as well as her brother. Although her brother has not been diagnosed with cancer, the incidence of cancer as well as the co-segregation analysis of this variant in this family, suggested that the identified VUS of BRCA2 is likely to be pathogenic.In Silico Analysis
Is this proof enough of the pathogenicity of the identified gene VUS? In order to ensure that the analyses are accurate, an in silico analysis of the predicted protein that is likely to be produced by the mutant BRCA2 gene was undertaken.
The identified heterozygous missense substitution (p.Lys2657Asn) was found to alter a conserved residue in the protein. The variant lies in the DNA binding domain (2470-3200 residues), which is involved in the physical interaction of BRCA2 to both single-stranded DNA and double-stranded DNA. In silico missense prediction tools (SIFT, Mutation Taster, Polyphen-2, FATHMM, Mutation Assessor and Align-GVGD) suggested that this variant is probably damaging to protein function.
Taken together, these results indicated that the identified BRCA2 VUS is likely to be pathogenic and accordingly her oncologist was advised that the gene variant is most likely to have been the cause of Mrs. Nandal’s ovarian cancer.
Benefits to Patient
Mrs. Nandal has ovarian cancer that is resistant to platinum-based chemotherapy. Additionally, she is found to be heterozygous for a variant of unknown significance the BRCA2 gene. The same variant was identified in the genomic DNA of her borther and sister as well. Mrs. Nandal’s sister had also been diagnosed with breast cancer. Taken together, these facts indicate that the heritable BRCA2 mutation is most likely to be pathogenic. These facts, indicative about the potential pathogenicity of the identified VUS were communicated to Mrs. Nandal’s oncologist. These factors made her eligible for treatment with PARP inhibitor therapy (Jenner, Sood, and Coleman 2016; Swisher et al. 2017; Crafton, Bixel, and Hays 2016; Oza et al. 2015; Mirza et al. 2016). Mrs. Nandal has been on PARP inhibitor therapy and is able to lead a very normal life that includes travel and leisure activities, as well.
Summary
- A 56-year-old female patient with recurrent ovarian cancer was referred for genetic diagnosis to Strand Life Sciences.
- Patient was found to be heterozygous for a BRCA2 mutation-initially classified as a ‘variant of uncertain significance (VUS)’.
- Co-segregation and in silico analyses were performed to understand whether the VUS mutation could be responsible for causing cancer.
- Additional analyses indicated the eligibility of the patient for PARP inhibitor therapy.
- Patient has responded well to PARP inhibitors with significant improvement in quality of her life.
- Therapeutic options were created for the patient by tenacious and persistent analyses of a BRCA2 VUS.
References
Crafton, S.M., K. Bixel, and J.L. Hays. 2016. “PARP Inhibition and Gynecologic Malignancies: A Review of Current Literature and on-Going Trials.” Gynecologic Oncology 142 (3): 588–96. doi:10.1016/j.ygyno.2016.05.003.
Jenner, Zachary B, Anil K Sood, and Robert L Coleman. 2016. “Evaluation of Rucaparib and Companion Diagnostics in the PARP Inhibitor Landscape for Recurrent Ovarian Cancer Therapy.” Future Oncology 12 (12): 1439–56. doi:10.2217/fon-2016-0002.
Mirza, Mansoor R., Bradley J. Monk, Jørn Herrstedt, Amit M. Oza, Sven Mahner, Andrés Redondo, Michel Fabbro, et al. 2016. “Niraparib Maintenance Therapy in Platinum-Sensitive, Recurrent Ovarian Cancer.” New England Journal of Medicine 375 (22). Massachusetts Medical Society: 2154–64. doi:10.1056/NEJMoa1611310.
Oza, Amit M, David Cibula, Ana Oaknin Benzaquen, Christopher Poole, Ron H J Mathijssen, Gabe S Sonke, Nicoletta Colombo, et al. 2015. “Olaparib Combined with Chemotherapy for Recurrent Platinum-Sensitive Ovarian Cancer: A Randomised Phase 2 Trial.” The Lancet Oncology 16 (1): 87–97. doi:10.1016/S1470-2045(14)71135-0.
Swisher, Elizabeth M, Kevin K Lin, Amit M Oza, Clare L Scott, Heidi Giordano, James Sun, Gottfried E Konecny, et al. 2017. “Rucaparib in Relapsed, Platinum-Sensitive High-Grade Ovarian Carcinoma (ARIEL2 Part 1): An International, Multicentre, Open-Label, Phase 2 Trial.” The Lancet Oncology 18 (1): 75–87. doi:10.1016/S1470-2045(16)30559-9.