Issue 13 | January 2018
Welcome to Strand Genomics
Strand welcomes you to Strand Genomics, our monthly E-zine that includes articles of interest to physicians like you. 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. A new feature is the section that provides updates on clinical trials in India as well as on recently-approved drugs for cancer. Now that Strand and Triesta Sciences have joined hands to create a synergistic, end-to-end diagnostic services company, ask our sales representatives about all the tests we can provide for your specialty! Feel free to write back to us with comments and questions at firstname.lastname@example.org.
Targeted Therapies: Genetic Analysis-led Expansion of Scope of Usage in Other Solid Tumors
– Dr. Shefali Sabharanjak
Strand Life Sciences
The US Food and Drug Administration (USFDA) has recently approved the use of a PARP inhibitor for treatment of metastatic breast cancer, if a patient bears a germline BRCA1 or BRCA2 mutation. This is a significant development in expanding the scope of proven and approved therapeutics from one solid tumor to other cancers, as long as the genetic signature of both tumors is similar for the genes / proteins that the therapy is designed against. Metastatic breast cancer, with a background germline BRCA1/BRCA2 mutation can now be treated with a PARP inhibitor that AstraZeneca has developed for treatment of ovarian cancer.The transitivity of therapeutic molecules to genetically similar tumors, irrespective of tissue of origin, is an important paradigm shift in the management of solid tumors. The trendsetter development, in this direction, was the approval of Keytruda (Pembrolizumab, Anti-PD-L1 Ab) for therapeutic use in tumors that express the PD-L1 receptor.
The first approval for this humanized antibody was obtained for its role in blocking the growth of metastatic melanoma cells that express PD-L1. Engagement of PD-L1 with PD-1, its natural ligand, leads to the suppression of the innate immune response of the body against the developing tumor. Antibody-mediated prevention of interaction between PD-1 and PD-L1 /PD-L2 releases this inhibitory block, thereby resulting in the death of malignant cells. Pembrolizumab was granted FDA approval against advanced melanoma in 2014 (Tsai et al. 2014). Clinical trials with other solid tumors expressing PD-L1 receptors ensued, resulting in the approval of Pembrolizumab for other tumors that also express adequate levels of the PD-L1 receptor (Navarro & Felip 2017; Joshi et al. 2017; Tahara et al. 2017; Ramos-Esquivel et al. 2017).
A similar expansion of the use of PARP inhibitors (such as Lynparza) to breast cancers is now possible based on the results from the OlympiAD trial (Robson et al. 2017).
Exploring the ‘BRCAness’ of other solid tumors
One of the targeted therapies developed for treatment of ovarian cancer patients who have germline mutations in BRCA1 and BRCA2 genes is a class of drugs that can stop the activity of an enzyme called poly-ADP-ribose polymerase, or PARP for short. PARP is an enzyme engaged in the repair of breaks in double-stranded DNA. Therefore, inhibition of this enzyme can actually promote the action of chemotherapy drugs that induce damage by breaking DNA strands in actively growing cancer cells. PARP inhibitors have been approved for use in ovarian cancer patients bearing BRCA1 and BRCA2 mutations (Jenner et al. 2016; Crafton et al. 2016; Swisher et al. 2017; Mirza et al. 2016; Oza et al. 2015). PARP inhibitors are also thought to be effective in prostate cancer cells where stimulation of androgen-receptor mediated signalling leads to reduced expression of the BRCA1&2 genes, thereby creating conditions that mimic the presence of BRCA1&2 mutations (also termed as ‘BRCAness’ of tumor cells)(Li et al. 2017). There is some evidence for a combinatorial use of inhibitors of PI3K and PARP, for the treatment of breast and ovarian cancer (Condorelli & André 2017).
BRCA1&2 and other genes that play a role in the development of Fanconi anemia are engaged in double-stranded DNA break repair. A state of functional BRCA1 and BRCA2 deficiency can be induced in solid tumors by using treatments like tumor-treatment fields (low-intensity, intermediate frequency, alternating electric fields) that result in the downregulation of genes involved in the BRCA-mediated DNA repair mechanisms (Karanam et al. 2017).
Similarly, triple-negative breast cancers have been classified into BRCA-like and non-BRCA-like groups, based on MLPA ligation-based tests to understand the status of BRCAness in TNBC patients (Severson et al. 2015; Lips et al. 2011). Comparative gene expression analysis of these TNBC subpopulatons have helped to identify other genes that are strongly associated with the BRCAness status of these tumors. Genes such as TP53 were seen to be more frequently mutated in BRCA-like TNBC whereas mutations in PIK3CA were associated with non-BRCA-like TNBC (Severson et al. 2015). Extending the logic further, the sensitivity of BRCA-like tumors to a veliparib-cisplatin combination has also been demonstrated (Severson et al. 2015). Promoter methylation of BRCA1 is another mechanism by which the expression of this gene can be downregulated (Kumar et al. 2017).
Genetic Diagnostic Tests
The identification of the BRCA1&2 status of an individual can be accomplished with the help of accurate, large-panel genetic tests like the Strand Germline Cancer test. Additionally, genetic analyses of tumors facilitated by pan-cancer tests like the StrandAdvantage 152-gene test can help to identify the mutation status of other genes (eg. TP53 and PIK3CA, amongst others) that may serve as proxy-indicators of the BRCAness of most solid tumors. Amplification of genes that methylate the BRCA1 promoter sequence is also detected with the StrandAdvantage 152-gene test. Moreover, the test is capable of identifying mutations in genes engaged in the Fanconi anemia double-strand repair mechanisms. Hence, it is possible to understand the BRCAness of many solid tumors using this genetic test.
Although the clinical utility of PARP inhibitors has not yet been demonstrated in solid tumors barring prostate, ovarian, and breast cancers, the transition to using synthetic lethality of PARP inhibitors and platinum-based chemotherapy may be possible in the future. The strategy may prove useful in tumors that are resistant to first-line Standard-of-Care chemotherapy.
Condorelli, R. & André, F., 2017. Combining PI3K and PARP inhibitors for breast and ovarian cancer tratement. Annals of Oncology. Available at: https://academic.oup.com/annonc/article-lookup/doi/10.1093/annonc/mdx218 [Accessed May 29, 2017].
Crafton, S.M., Bixel, K. & Hays, J.L., 2016. PARP inhibition and gynecologic malignancies: A review of current literature and on-going trials. Gynecologic Oncology, 142(3), pp.588–596. Available at: https://www.ncbi.nlm.nih.gov/pubmed/27168003 [Accessed January 21, 2017].
Jenner, Z.B., Sood, A.K. & Coleman, R.L., 2016. Evaluation of rucaparib and companion diagnostics in the PARP inhibitor landscape for recurrent ovarian cancer therapy. Future Oncology, 12(12), pp.1439–1456. Available at: https://www.ncbi.nlm.nih.gov/pubmed/27087632 [Accessed January 20, 2017].
Joshi, S.S., Maron, S.B. & Catenacci, D. V, 2017. Pembrolizumab for treatment of advanced gastric and gastroesophageal junction adenocarcinoma. Future Oncology, p.fon-2017-0436. Available at: https://www.ncbi.nlm.nih.gov/pubmed/29094609 [Accessed January 16, 2018].
Karanam, N.K. et al., 2017. Tumor-treating fields elicit a conditional vulnerability to ionizing radiation via the downregulation of BRCA1 signaling and reduced DNA double-strand break repair capacity in non-small cell lung cancer cell lines. Cell Death & Disease, 8(3), pp.e2711–e2711. Available at: https://www.ncbi.nlm.nih.gov/pubmed/28358361 [Accessed January 16, 2018].
Kumar, M. et al., 2017. BRCA1 Promoter Methylation and Expression – Associations with ER+, PR+ and HER2+ Subtypes of Breast Carcinoma. Asian Pacific journal of cancer prevention : APJCP, 18(12), pp.3293–3299. Available at: https://www.ncbi.nlm.nih.gov/pubmed/29286222 [Accessed January 16, 2018].
Li, L. et al., 2017. Androgen receptor inhibitor–induced “BRCAness” and PARP inhibition are synthetically lethal for castration-resistant prostate cancer. Science Signaling, 10(480), p.eaam7479. Available at: https://www.ncbi.nlm.nih.gov/pubmed/28536297 [Accessed May 29, 2017].
Lips, E.H. et al., 2011. Quantitative copy number analysis by Multiplex Ligation-dependent Probe Amplification (MLPA) of BRCA1-associated breast cancer regions identifies BRCAness. Breast Cancer Research, 13(5), p.R107. Available at: https://www.ncbi.nlm.nih.gov/pubmed/22032731 [Accessed January 16, 2018].
Mirza, M.R. et al., 2016. Niraparib Maintenance Therapy in Platinum-Sensitive, Recurrent Ovarian Cancer. New England Journal of Medicine, 375(22), pp.2154–2164. Available at: https://www.nejm.org/doi/10.1056/NEJMoa1611310 [Accessed January 16, 2017].
Navarro, A. & Felip, E., 2017. Pembrolizumab in advanced pretreated small cell lung cancer patients with PD-L1 expression: data from the KEYNOTE-028 trial: a reason for hope? Translational lung cancer research, 6(Suppl 1), pp.S78–S83. Available at: https://www.ncbi.nlm.nih.gov/pubmed/29299418 [Accessed January 16, 2018].
Oza, A.M. et al., 2015. Olaparib combined with chemotherapy for recurrent platinum-sensitive ovarian cancer: a randomised phase 2 trial. The Lancet Oncology, 16(1), pp.87–97. Available at: https://www.ncbi.nlm.nih.gov/pubmed/25481791 [Accessed January 16, 2017].
Ramos-Esquivel, A. et al., 2017. Anti-PD-1/anti-PD-L1 immunotherapy versus docetaxel for previously treated advanced non-small cell lung cancer: a systematic review and meta-analysis of randomised clinical trials. ESMO Open, 2(3), p.e000236. Available at: https://www.ncbi.nlm.nih.gov/pubmed/29181191 [Accessed January 16, 2018].
Robson, M. et al., 2017. Olaparib for Metastatic Breast Cancer in Patients with a Germline BRCA Mutation. New England Journal of Medicine, 377(6), pp.523–533. Available at: https://www.ncbi.nlm.nih.gov/pubmed/28578601 [Accessed January 16, 2018].
Severson, T.M. et al., 2015. BRCA1-like signature in triple negative breast cancer: Molecular and clinical characterization reveals subgroups with therapeutic potential. Molecular oncology, 9(8), pp.1528–38. Available at: https://www.ncbi.nlm.nih.gov/pubmed/26004083 [Accessed January 16, 2018].
Swisher, E.M. 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), pp.75–87. Available at: https://www.ncbi.nlm.nih.gov/pubmed/27908594 [Accessed January 21, 2017].
Tahara, M. et al., 2017. Pembrolizumab in Asia-Pacific patients with advanced head and neck squamous cell carcinoma: analyses from KEYNOTE-012. Cancer Science. Available at: https://www.ncbi.nlm.nih.gov/pubmed/29284202 [Accessed January 16, 2018].
Tsai, K.K., Zarzoso, I. & Daud, A.I., 2014. PD-1 and PD-L1 antibodies for melanoma. Human Vaccines & Immunotherapeutics, 10(11), pp.3111–3116. Available at: https://www.ncbi.nlm.nih.gov/pubmed/25625924 [Accessed January 16, 2018].
Multi-Gene NGS Tests For Accurate Risk Assessment in Hereditary Breast Cancer
he clinical utility of genetic analysis of breast cancer biopsies with a single-gene (or a set of 3-5 genes) test or with a multi-gene panel is an ongoing debate. Mini-panel tests are sometimes preferred over multi-gene panel NGS test, keeping in mind the affordability factor. However, a multi-gene panel NGS test can provide better insights into the predisposition of a person, towards hereditary cancer. In a recent publication, NGS analysis of a person using a multi-gene panel helped to identify a new gene – MRE11A– that can be associated with familial breast and endometrial cancer. A nonsense mutation in the MRE11A gene can result in the production of a truncated protein, a form that is bereft of the DNA_binding domain of the MRE11A protein.
MRE11A is a part of the MRN (MRE11A, RAD50-NBS1) complex that is engaged in binding to and repairing breaks in double-stranded DNA. The mutation (NM_005590 c.1090C>T: p.Arg364Ter) results in the loss of the DNA binding domain as well as the domain that binds to the RAD50 protein. The truncated protein, therefore, increases a carrier’s risk of familial breast cancer (https://www.ncbi.nlm.nih.gov/pubmed/28559769). Interestingly, the tested patient had normal BRCA1 and BRCA2 genes. Similar results have been obtained with the Strand Hereditary Cancer Test, wherein patients opting for the multi-gene panel germline test, have been found to have mutations in NBN, MLH1 and other genes although they had normal copies of the BRCA1, BRCA2 and TP53 genes. Although the predominance of BRCA1 and BRCA2 mutations in Indian hereditary breast and ovarian cancer (HBOC) patients is high, a comprehensive risk assessment can be obtained only with a muti-gene test. Pre-emptive testing of first- and second-degree relatives of breast and ovarian cancer patients with multi-gene panel NGS tests can be used very effectively for risk prediction. Going further, knowledge about ones genetic predisposition can help to adopt frequent surveillance measures and perhaps help to prevent the actual incidence of cancer, in susceptible individuals.
Differential Diagnosis Between 3-MCC Deficiency and Holocarboxylase Synthetase Deficiency Facilitated by the Strand Clinical Exome Test
– Dr. Shefali Sabharanjak
Strand Life Sciences
Inborn errors of metabolism (IEM) are congenital disorders that are caused by mutations within a single gene. Some IEM disorders are characterized by inability to breakdown and absorb nutrients, resulting in delayed development and malnutrition. One such IEM disorder is 3-methylcrotonyl-CoA carboxylase deficiency (also known as 3-MCC deficiency), wherein the breakdown of proteins containing leucine is impaired.
Symptoms of 3-MCC deficiency include vomiting and diarrhea in infants, weak muscles and feeding problems in children. Sometimes, the deficiency can be fatal as well (Grünert et al. 2012).
A deficiency in the holocarboxylase synthetase enzyme is evident if mutations in the HLCS gene are present in a person’s genome. This enzyme is required for the regulation of other enzymes involved in the catabolism of fats and proteins. Another important function of this enzyme is regulation of expression of other genes (Bao et al. 2011; Liu & Zempleni 2014). Symptoms of the deficiency of this enzyme include feeding problems in infants, breathing difficulties, skin rashes and alopecia. In some cases, a deficiency of this enzyme can be fatal to infants.
Recently, a couple was referred to Strand Life Sciences for genetic diagnosis. The unfortunate couple had lost two children, one at the age of 3 years and the other just 4 days old. Unable to conceive, they consulted a renowned geneticist in Hyderabad and discussed the symptoms that their young son (age 3 years) had suffered from. The geneticist- pediatrician suspected the incidence of either a genetic mutation causing either 3-MCC deficiency or holocarboxylase synthetase deficiency, in the lost children (Tammachote et al. 2009; Fonseca et al. 2016). She recommended that the young boy’s DNA sample be analyzed to understand the reason for his symptoms and the resultant fatality. The Strand Clinical Exome test was used to differentiate between these two possibilities in order to provide an accurate diagnosis.
Rohit*, a 3-year-old boy, suffered from issues like feeding problems, malnutrition, vomiting and diarrhea. He remained sickly throughout his infancy and finally succumbed to his health issues at the age of 3 years. Medical investigations to understand the causes of his problems had been inconclusive. However, a dried blood spot from this child was obtained and preserved during these investigations.
Treatment OptionsTripti was advised treatment with Afatinib, a targeted therapy molecule. This drug inhibits the activity of the EGFR protein, which is mutated in most adenocarcinoma cases. However, her oncologist noted that the tumor persisted and response to afatinib therapy waned, a few months into the therapy. A fresh biopsy of the lung tumor was advised to understand the genetic profile of the persistent NSCLC, in a second attempt. The StrandAdvantage 48-Gene Tissue Specific Test (Lung) was prescribed for identification of mutant genes as well as other molecular markers.
Tripti’s oncologist also advised her to take advantage of novel liquid biopsy tests which can facilitate tracking of the tumor via a blood sample, at any point of time, during the therapy.
Rohit’s parents, Preetha* and Jayachandra*, are a consanguineous couple. Preetha’s grandmother and Jayachandra’s mother are sisters. The couple had lost their first child at the age of 4 days and Rohit at the age of 3 years.
Family Tree: Pre-Genetic Test Counselling
Figure 1. Pedigree Chart- Rohit, Preetha and Jayachandra
Considering the consanguinity in the family, the Strand Clinical Exome test was prescribed to understand whether inheritable mutations that could cause an IEM were present in the family.
DNA extracted from the dried blood sample from Rohit was used to understand if the child had 3-MCC deficiency or holocarboxylase synthetase deficiency.
Results of Genetic Testing (Rohit)
A new mutation- a Variant of Unknown Significance – was found in exon 11 of the HLCS gene in Rohit’s DNA. The HLCS gene codes for an enzyme – holocarboxylase synthetase- that adds a molecular tag- biotin- to various enzymes that are engaged in the breakdown of fats and proteins. When the HLCS gene is mutated, this tagging function is inefficient and results in reduced breakdown of fats and proteins for production of energy.
Analysis of the mutant gene sequence using bioinformatics tools suggested that the mutation in Rohit’s DNA is likely to change an essential amino acid in the holocarboxylase synthetase protein. Additionally, since infant mortality is evident in the family, the variant has been re-classified as ‘Variant of Unknown Significance with a Probable Damaging Effect (VUSD)’.
Rohit is homozygous for this new VUSD.
Results of Genetic Testing (Preetha)
Figure 2. Electrophoregram of Sanger sequencing data (electrophoregram) from the individual showing a heterozygous nucleotide change ‘G>A’ at position c.1912 in the HLCS gene (RefSeq id: NM_000411).This variation was confirmed by sequencing with reverse primer in two independent experiments.
Strand offers a Mutation-Specific Test (MST) which is designed to identify specific mutations in the genomes of family members of probands (index patients). These tests are fast and highly specific.
An MST for the identified VUSD mutation in the HLCS gene showed that Preetha is heterozygous for this mutation. Essentially, she has one normal copy of this gene and one mutant copy.
Results of Genetic Testing (Jayachandra)
Figure 3. Electrophoregram of Sanger sequencing data from the individual showing a heterozygous nucleotide change ‘G>A’ at position c.1912 in the HLCS gene (RefSeq id: NM_000411).This variation was confirmed by sequencing with reverse primer in two independent experiments.
Counselling Post Genetic Analysis
Preetha and Jayachandra are heterozygous for the VUSD mutation in exon 11 of the HLCS gene identified in their son. They were advised that their chances of having another child with a similar homozygous inheritance as Rohit were 25%.
*- Patient names changed to protect privacy
Bao, B. et al., 2011. Human holocarboxylase synthetase with a start site at methionine-58 is the predominant nuclear variant of this protein and has catalytic activity. Biochemical and biophysical research communications, 412(1), pp.115–20. Available at: https://www.ncbi.nlm.nih.gov/pubmed/21802411 [Accessed November 13, 2017].
Fonseca, H. et al., 2016. 3-Methylcrotonyl-CoA carboxylase deficiency: Mutational spectrum derived from comprehensive newborn screening. Gene, 594(2), pp.203–210. Available at: https://www.ncbi.nlm.nih.gov/pubmed/27601257 [Accessed November 13, 2017].
Grünert, S.C. et al., 2012. 3-methylcrotonyl-CoA carboxylase deficiency: Clinical, biochemical, enzymatic and molecular studies in 88 individuals. Orphanet Journal of Rare Diseases, 7(1), p.31. Available at: https://www.ncbi.nlm.nih.gov/pubmed/22642865 [Accessed November 13, 2017].
Liu, D. & Zempleni, J., 2014. Holocarboxylase synthetase interacts physically with nuclear receptor co-repressor, histone deacetylase 1 and a novel splicing variant of histone deacetylase 1 to repress repeats. Biochemical Journal, 461(3), pp.477–486. Available at: https://www.ncbi.nlm.nih.gov/pubmed/24840043 [Accessed November 13, 2017].
Tammachote, R. et al., 2009. Holocarboxylase synthetase deficiency: novel clinical and molecular findings. Clinical Genetics, 78(1), pp.88–93. Available at: https://www.ncbi.nlm.nih.gov/pubmed/20095979 [Accessed November 13, 2017].
Advanced genomic analysis for answers not found before
In the News!
Clinical Trials Update
Study of 1st Line Therapy Study of Durvalumab With Tremelimumab Versus SoC in Non Small-Cell Lung Cancer (NSCLC) (NEPTUNE)
Clinical Study Identifier: NCT02542293
Recruitment is open for a lung cancer clinical trial in Mumbai. This is to understand the clinical efficacy of Durvalumab combined with Tremelimumab as a first line therapy for Non-Small Cell Lung Cancer (NSCLC) compared against SoC therapy.
Patients with the following profile are eligible for this study:
More information about the trial: https://clinicaltrials.gov/ct2/show/NCT02542293
Call us now to arrange a free genetic counselling session!