Issue 11 | November 2017
StrandGenomics Issue 11 starburst

Welcome to Strand Genomics

Strand Life Sciences welcomes you to Strand Genomics, our monthly E-zine that includes articles of interest to physicians. We have a new version of this e-zine!! Strand Genomics 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.

Lung Cancer in India: Leveraging Liquid Biopsy For Optimal Treatment

 Dr. Shefali Sabharanjak
Strand Life Sciences

Abstract

  • Lung cancer is one of the leading causes of cancer-related mortality in India.
  • The prevalence to incidence ratio for lung cancer is the lowest amongst cancers at all sites, in the Indian population.
  • Owing to the rapid progression and high fatality of lung cancer, techniques for frequent evaluation of the status of the disease can help to manage therapeutic decisions.
  • Liquid biopsies can be used to monitor patients’ responses to chosen therapies and understand the changing genetic profile of lung cancer.
  • Liquid biopsies are amenable to analysis using various molecular techniques (NGS, ddPCR, etc) that can provide a comprehensive genetic profile of the tumor, at chosen frequencies.
  • Preliminary results indicate that liquid biopsies can yield prognostic information in NSCLC patients, as early as 2 weeks into the therapy regimen.

Introduction

Lung cancer has the dubious distinction of being the most fatal cancer disease, causing 1.38 million deaths, worldwide, in 2016. In India, this is the second most common cancer in men and sixth most frequent cancer in women (Noronha et al. 2016). In fact, analysis of 5-year survival trends in data collected from several Indian registries shows that lung and stomach cancers have the poorest survival rates amongst various cancers (Figure 1) (Takiar & Jayant 2013).

Figure 1. Prevalence of cancer types in India

Histopathological profiling has shown that the incidence of adenocarcinoma is higher in India (43.8%) than that of squamous cell carcinoma 26.2%. Although the use of tobacco-related smoking products is a strong causative factor of lung cancer in India, the proportion of non-smoking lung cancer patients is also as high as 52% in some cohorts (Parikh et al. 2016; Noronha et al. 2016). Secondary smoking and environmental pollution are the most likely reasons for the incidence of lung cancers in non-smoking individuals.

India also has a high prevalence of germline BRCA1 and BRCA2 mutations, evident in the 3-times higher incidence of hereditary breast and ovarian cancer (HBOC) cases (Mannan et al. 2016). An Italian study has highlighted the increased risk of incidence of lung cancer in families bearing germline BRCA1 and BRCA2 mutations (Digennaro et al. 2017). Although similar correlations have not been tested in Indian studies, the incidence of lung cancer in non-smoking individuals can perhaps be explained by a hereditary predisposition such as a germline BRCA1 / BRCA2 mutation, in addition to other factors.

Given that lung cancer has high and fast mortality, there is very little room for trial-and-error methods to understand a patient’s response to therapy and modify it in subsequent rounds. As such, the availability of prognostic markers that can provide fast and accurate indications of a patient’s progress would be highly valuable in the treatment of lung cancer. In fact, the availability of prognostic indicators for all solid tumors can allow for rapid and accurate modification of therapy, assessed at regular intervals.

Novel PET-CT assay for Cancer Proliferation

PET-CT scans using 18F-FDG (fluorodeoxyglucose) uptake to identify sites of progression of cancer have been the gold standard for assessing the status of the disease in cancer patients. A modified PET-CT scan using 68Ga-DOTA-A2B1, as a tracer for identifying expression of α2β1integrin as a marker for growth of non-small cell lung cancer has been recently described (Huang et al. 2017). The integrin-specific tracer is expected to tide over problems of 18F-FDG uptake in normal tissues as well as at sites of inflammation.

The technique needs to be validated in other studies, nonetheless, the specificity of the tracer is a positive development. It is also unclear whether the new tracer can be used at a frequency higher than that for 18F-FDG, which is once in 6 months.

ctDNA for monitoring Lung Cancer

An alternative monitoring technique is ‘Liquid Biopsy’ that allows rapid and repetitive assessment of circulating tumor DNA (ctDNA) in the blood samples obtained from cancer patients. Isolation of cell-free DNA(cfDNA) and identification of ctDNA to understand disease prognosis has been demonstrated with breast cancer samples, initially (Garcia-Murillas et al. 2015). Following the progress of breast cancer patients over a period of two years, these researchers have shown that detection of ctDNA can precede the emergence of radiologically detectable lesions in the body, by a period of 2 months.

The utility of ctDNA as a prognostic marker has been shown in other studies as well (Pécuchet et al. 2016). Detection of ctDNA in lung cancer patients at the time of initial diagnosis and throughout therapy was correlated with poor survival.

Tumor marker assessment can be performed using several DNA amplification and sequencing techniques such as Next-generation sequencing (NGS), deep sequencing methods, digital PCR, Beads, Emulsion, Amplification and Magnetics (BEAMing) and real-time PCR (Vendrell et al. 2017).

Development of highly sensitive probes for specific mutations like EGFR-del19, EGFR-L858R and EGFR-T790M has also set the stage of periodic monitoring of lung cancer patients who have mutations in this transmembrane receptor. Strand Life Sciences has developed these ultrasensitive assays that can track the changing mutation profile of EGFR in NSCLC patients.

Figure 2. ctDNA for Monitoring Progression of Lung Cancer

The feasibility of using multi-gene panels and NGS sequencing on cell-free DNA to obtain a comprehensive genetic profile, has been described in a recent study in lung cancer patients (Hou et al. 2017). In a cohort of 119 patients, resistant to TKI-therapy, NGS analysis of cfDNA helped to identify 25 actionable mutations in EGFR, 5 in BRAF and 1 in MET, along with copy number variants (CNVs) and small indels in other genes (Hou et al. 2017).

Patient response to targeted therapies can also be understood at very early stages, providing good indications for the predicted response of a patient. A pilot study of 14 lung cancer patients, treated with nivolumab, evaluation of ctDNA within ‘2 weeks’ of initiation of treatment helped to identify patients who are most likely to respond to treatment or not (Iijima et al. 2017). Seven out of fourteen patients were identified as ‘responders’ owing to a decrease in the allelic frequency of mutations that had been identified from the solid tumor biopsy.

Liquid biopsies can also be performed frequently, based on a physician’s evaluation of a patient, without limitations such as use of radio-labelled tracers and access to imaging facilities.

Summary

  • Lung cancer is one of the leading causes of cancer-related mortality in India.
  • The prevalence to incidence ratio for lung cancer is the lowest amongst cancers at all sites, in the Indian population.
  • Owing to the rapid progression and high fatality of lung cancer, techniques for frequent evaluation of the status of the disease can help to manage therapeutic decisions.
  • Liquid biopsies can be used to monitor patients’ responses to chosen therapies and understand the changing genetic profile of lung cancer.
  • Liquid biopsies are amenable to analysis using various molecular techniques (NGS, ddPCR, etc) that can provide a comprehensive genetic profile of the tumor, at chosen frequencies.
  • Preliminary results indicate that liquid biopsies can yield prognostic information in NSCLC patients, as early as 2 weeks into the therapy regimen.

References

Digennaro, M. et al., 2017. Hereditary and non-hereditary branches of family eligible for BRCA test: cancers in other sites. Hereditary cancer in clinical practice, 15, p.7. Available at: http://www.ncbi.nlm.nih.gov/pubmed/28559958 [Accessed June 15, 2017].

Garcia-Murillas, I. et al., 2015. Mutation tracking in circulating tumor DNA predicts relapse in early breast cancer. Science Translational Medicine, 7(302), p.302ra133-302ra133. Available at: http://www.ncbi.nlm.nih.gov/pubmed/26311728 [Accessed July 5, 2017].

Hou, H. et al., 2017. Discovery of targetable genetic alterations in advanced non-small cell lung cancer using a next-generation sequencing-based circulating tumor DNA assay. Scientific reports, 7(1), p.14605. Available at: http://www.ncbi.nlm.nih.gov/pubmed/29097733 [Accessed November 21, 2017].

Huang, C.-W. et al., 2017. The Use of PET Imaging for Prognostic Integrin α2β1 Phenotyping to Detect Non-Small Cell Lung Cancer and Monitor Drug Resistance Responses. Theranostics, 7(16), pp.4013–4028. Available at: http://www.ncbi.nlm.nih.gov/pubmed/29109795 [Accessed November 21, 2017].

Iijima, Y. et al., 2017. Very early response of circulating tumour–derived DNA in plasma predicts efficacy of nivolumab treatment in patients with non–small cell lung cancer. European Journal of Cancer, 86, pp.349–357. Available at: http://www.ncbi.nlm.nih.gov/pubmed/29078173 [Accessed November 21, 2017].

Mannan, A.U. et al., 2016. Detection of high frequency of mutations in a breast and/or ovarian cancer cohort: implications of embracing a multi-gene panel in molecular diagnosis in India. Journal of Human Genetics, 61(6), pp.515–22. Available at: http://www.ncbi.nlm.nih.gov/pubmed/26911350.

Noronha, V. et al., 2016. Lung cancer in the Indian subcontinent. South Asian journal of cancer, 5(3), pp.95–103. Available at: http://www.ncbi.nlm.nih.gov/pubmed/27606290 [Accessed June 12, 2017].

Parikh, P.M. et al., 2016. Lung cancer in India: Current status and promising strategies. South Asian journal of cancer, 5(3), pp.93–5. Available at: http://www.ncbi.nlm.nih.gov/pubmed/27606289 [Accessed June 12, 2017].

Pécuchet, N. et al., 2016. Base-Position Error Rate Analysis of Next-Generation Sequencing Applied to Circulating Tumor DNA in Non-Small Cell Lung Cancer: A Prospective Study. M. Ladanyi, ed. PLoS medicine, 13(12), p.e1002199. Available at: http://www.ncbi.nlm.nih.gov/pubmed/28027313 [Accessed April 24, 2017].

Takiar, R. & Jayant, K., 2013. A model approach to calculate cancer prevalence from 5 year survival data for selected cancer sites in India. Asian Pacific journal of cancer prevention: APJCP, 14(11), pp.6899–903. Available at: http://www.ncbi.nlm.nih.gov/pubmed/24377623 [Accessed November 20, 2017].

Vendrell, J. et al., 2017. Circulating Cell Free Tumor DNA Detection as a Routine Tool forLung Cancer Patient Management. International Journal of Molecular Sciences, 18(2), p.264. Available at: http://www.mdpi.com/1422-0067/18/2/264 [Accessed March 13, 2017].

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Genetic Predisposition to Cancer Assessed for 25 Family Members

Family of 25 gets germline genetic testing done to assess cancer risk

Ashok Gholap (in pink shirt) got his entire family together, from different parts of the state, to undergo a Germ Line Cancer Test. (Express Photo/Sandip Daundkar)

One of the advantages of genetic counselling is easing a patient as well as his family members into the concept of cancer risk assessment. Owing to the high fatality of this disease, a halo of fear and avoidance is always associated with each and every diagnosis of cancer. Genetic counselling sessions conducted by Strand’s expert genetic counsellors help in allaying patients’ fears and queries about cancer. In a recent case, Mr. Gholap from Pune was counselled and tested at Strand Life Sciences. Mr. Gholap was diagnosed with Pancreatic cancer (2008) and later with Male Breast cancer (2011- right breast and 2013- left breast). The incidence of cancer in two or more distinct sites of origin is a sure sign of a hereditary mutation at work.

The Strand Germline Cancer Test helped to identify a likely pathogenic mutation in exon 21 of the BRCA2 gene, most likely to be responsible for his recurrent multiple cancers. Once Mr. Gholap understood the hereditary angle of his health problems, vide the post-test genetic counselling session, he convinced 24 other people from his extended family to undergo testing and understand their personal risk, before they develop symptoms. The entire cohort is being analyzed at Strand’s laboratory in Bangalore and we expect a comprehensive family report to be published soon.

You can read the whole story, here.

Genetic Counselling: A Pivotal Role in Identifying Families At Risk For Hereditary Breast and Ovarian Cancer Syndrome

Ms. Payal Manek & Dr. Shefali Sabharanjak
Strand Life Sciences

Abstract

  • The proband could make an informed choice of testing herself and counselling helped to resolve her phobia about the breast MRI procedure.
  • The proband’s cousin was given adequate counselling about her genetic status and was prescribed PARP inhibitor therapy without any further delay. This has improved her quality of life tremendously as a cancer survivor.
  • The proband’s sister was counselled against undergoing unnecessary surgical interventions. Her genetic status as a non-BRCA1 mutation carrier was clearly established vide the Strand Germline Risk Assessment Test. Her anxiety about suffering from cancer was eliminated after hearing the options of surveillance and other preventive measures in the counselling session.
  • The family’s attention was drawn to the fact that the proband’s unaffected mother was also at risk for hereditary breast and ovarian cancer, paving the way for pre-emptive medical surveillance.
  • Other relatives from the proband’s family were tested and their BRCA1-mutation negative status was clearly established.

Introduction

Genetic testing is set to become the cornerstone of cancer therapy in the next decade. Cancer develops owing to the progressive accumulation of one or more genetic mutations that produce overactive / loss-of-function proteins which bring about aberrant cell growth (Lawrence et al. 2014). Although most cancers develop sporadically, ~ 5-10% of cancers are hereditary. Notably, the inheritance of breast and ovarian cancers amongst blood relations (also known as Hereditary Breast and Ovarian Cancer syndrome [HBOC]) is closely associated with the inheritance of mutations in BRCA1, BRCA2, TP53 and 16 other genes (McPherson et al. 2000; Wong et al. 2016; Mannan et al. 2016).

International guidelines have been published in order to identify the incidence of HBOC as well as to prescribe genetic tests to assess the prevalence of germline mutations in HBOC-associated genes. These guidelines, based on family health history and age of the proband, can be used to identify individuals at risk from HBOC as well as those that can benefit from targeted therapies for breast cancer. Since this is an involved procedure, Strand’s genetic counsellors (GCs) are trained to perceive socio-psychological cues and employ genetic knowledge to identify the prevalence of HBOC in Indian patients. GCs can also provide significant benefits to cancer patients by counselling them about the implications of their genetic test results.

Patient Background

  • Uma* and Rama*, two sisters were simultaneously diagnosed with breast and ovarian cancer, respectively.
  • They had a family history of pancreatic cancer in an uncle and ovarian cancer in a maternal aunt and cousin sister Padma*. Uma’s uncle and aunt had succumbed to the disease while their cousin sister Padma was seeking treatment for her ovarian cancer.
  • Anandita*, the third sibling of Uma and Rama, was unaffected but with her sister’s diagnosis she was suffering from carcinophobia. She had already taken appointment for preventive prophylactic surgeries.
  • Uma was referred for genetic counselling. She was reluctant to participate in genetic testing or counselling because of a misconception of regular MRIs required for medical surveillance. She had several reservations about the MRI procedure.
  • All three independent cases of the same family were managed by different oncologists; however all of them were referred to the same Genetic Counsellor (GC), affiliated with Strand Life Sciences.

Genetic Counselling Session #1

Uma, along with her husband met a Strand GC. The GC explained to her that her personal and family history is highly suggestive of HBOC. In the counselling session, the GC explained the procedure of undergoing a germline risk assessment test. The fact that benefits of this tests can be extended to other family members as well was also emphasized. During the session, they also had a detailed discussion regarding her phobia of MRI. She was informed regarding the option of preventive surgery vs radiological surveillance for her risk management. At the end of the session, she was convinced and made an informed decision to get herself tested.

Genetic Test Result and Implications

 

Gene Variation Zygosity Clinical significance
BRCA1 chr17:41228504C>T
c.4484+1G>A
Heterozygous Pathogenic

 

Uma was positive for a ‘pathogenic’ (disease-causing) variant, which was detected in the splice donor site at the junction of exon 13-intron 13 of the BRCA1 gene. The individual carries one copy (heterozygous) of a pathogenic variant in the BRCA1 gene, which has been associated with HBOC. Women with BRCA1 germline pathogenic variations are at an increased lifetime risk for breast cancer (40%-80%) and ovarian cancer (11%-40%).
Identification of a known pathogenic mutation suggests the following for this family:

  • Uma has an increased risk for contralateral breast cancer and ovarian cancer.
  • Rama is affected with ovarian cancer, probably due to the same mutation.
  • Anandita and Uma’s daughters are at a 50% risk of having inherited the same mutation. Since BRCA1 mutations are inherited in an autosomal dominant manner.
  • Uma’s mother, who is an obligate carrier is at high risk to several cancers associated with HBOC.
  • Other unaffected family members have a 50% chance of having inherited the mutation.

Genetic Counselling Session #2

Anandita had already made her decision regarding prophylactic bilateral risk-reducing salpingo-oophorectomy (RRSO) to reduce her risk for ovarian cancer. In the genetic counselling session, she was informed that she only has a 50% chance of inheriting the mutation. Therefore, she might not be a carrier of the same BRCA1 mutation as her sister. A genetic test could ascertain her exact status and it would be wise for her to undergo genetic testing before undergoing RRSO or risk-reducing bilateral mastectomy (RRBM), purely out of fear of cancer. She agreed to reconsider her decision regarding prophylactic surgery and to get a genetic test done.
The three sisters had recently lost their father to heart disease and now the diagnosis of cancer and hereditary syndrome had taken a toll on the family. In the midst of all this, naturally, her mother’s risk was not addressed. Our GC highlighted the fact that although there is no incidence of cancer in their mother, until the age of 70 years, she is a carrier of the gene mutation and is at increased risk for cancer. She needs to be offered a surveillance program of regular mammographies or breast MRIs.

Genetic Counselling Session #3

Meanwhile, Padma was referred for comprehensive BRCA genetic testing in order to make the right therapeutic choices for her. The family was initially referred to a leading genetic testing company in the US, where the test would have cost them approximately Rs 2Lakh. However, Uma’s family advised them to speak to our GC and get the same test done, quite economically in comparison. They were reassured by Uma’s family that BRCA genetic testing is done with the same precision in India as well.
During the consultation, the GC explained that Padma does not need to undergo whole panel testing. Since a mutation is already identified in the family, a specific point mutation test would be sufficient. During further discussion, the family revealed that they were referred for a genetic test, to another genetic diagnostics laboratory in India, long back but they never understood the report and nor did any physician make use of that knowledge.
On reading the report, the GC advised, the report already suggests that she is a carrier of BRCA1 mutation, same variant as her cousin Uma. She further advised that no additional testing is required and they can begin the use a PARP inhibitor as a maintenance therapy, right away.
Padma’s sister and their maternal uncle were also referred for genetic counselling and testing.

Figure 1. Pedigree Chart of the Family

Pedigree Chart of the Family

Conclusions

  • Uma (proband in this case) underwent bilateral mastectomy with reconstruction and prophylactic bilateral salpingo-ooph0rectomy to reduce her risk of recurring breast and ovarian cancer.
  • Padma was prescribed PARP inhibitor therapy.
  • Unaffected family members were tested for their carrier status to assess their personal risk.
  • Anandita was tested negative and did not undergo preventive surgery.
  • Uma’s daughters will be tested shortly.

Summary

The genetic counselling sessions were beneficial to the proband’s family in multiple ways:

  • The proband could make an informed choice of testing herself and genetic counselling helped to resolve her phobia about the breast MRI procedure.
  • The proband’s cousin was given adequate counselling about her genetic status and was prescribed PARP inhibitor therapy without any further delay. This has improved her quality of life tremendously as a cancer survivor.
  • Proband’s sister was counselled against undergoing unnecessary surgical interventions. Her genetic status as a non-BRCA1 mutation carrier was clearly established vide the Strand Germline Risk Assessment Test. Her anxiety about suffering from cancer was eliminated after hearing the option of surveillance and other preventive measures in the session.
  • The family’s attention was drawn to the fact that the proband’s unaffected mother was also at risk for HBOC, paving the way for pre-emptive medical surveillance.
  • Other relatives from the proband’s family were tested and their BRCA1-mutation negative status was clearly established.

*- Patient names changed to protect privacy

References

Lawrence, M.S. et al., 2014. Discovery and saturation analysis of cancer genes across 21 tumour types. Nature, 505(7484), pp.495–501. Available at: http://www.ncbi.nlm.nih.gov/pubmed/24390350 [Accessed November 20, 2017].

Mannan, A.U. et al., 2016. Detection of high frequency of mutations in a breast and/or ovarian cancer cohort: implications of embracing a multi-gene panel in molecular diagnosis in India. Journal of Human Genetics, 61(6), pp.515–22. Available at: http://www.ncbi.nlm.nih.gov/pubmed/26911350.

McPherson, K., Steel, C.M. & Dixon, J.M., 2000. ABC of breast diseases. Breast cancer-epidemiology, risk factors, and genetics. BMJ (Clinical research ed.), 321(7261), pp.624–8. Available at: http://www.ncbi.nlm.nih.gov/pubmed/10977847 [Accessed October 4, 2017].

Wong, E.S.Y. et al., 2016. Inherited breast cancer predisposition in Asians: multigene panel testing outcomes from Singapore. npj Genomic Medicine, 1, p.15003. Available at: http://www.nature.com/articles/npjgenmed20153 [Accessed December 6, 2016].

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Advanced genomic analysis for answers not found before

Adherence to Rigorous Quality and Process Control Procedures

Strand Center for Genomics and Personalized Medicine CAP

We are happy to announce that in the wake of a mandatory inspection by officials from CAP, the accreditation granted to SCGMP has been reaffirmed, recently. The concordance between standards set by CAP and those put in practice at SCGPM have been commended.

Strand Center for Genomics and Personalized Medicine (SCGPM) is India’s first Next-Generation Sequencing  (NGS) laboratory to receive the College of American Pathologists (CAP) accreditation in 2015. The CAP accreditation is awarded to diagnostic laboratories that follow stringent quality and process control norms at every stage. Adoption of these quality control measures enhance the accuracy of the results obtained from the analytical processes. In keeping with this vision, SCGPM participates in blind, comparative exercises wherein unknown samples are sent to us and the results are validated against those provided by other NGS laboratories, worldwide. Results provided by SCGPM have been consistently found to be at par with those obtained from other renowned laboratories.

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