Issue 14 | February 2018
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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. The news headlines have been buzzing for a month now. Strand and Triesta Sciences have joined hands to create a synergistic, end-to-end diagnostic services company. We now offer a complete bouquet of tests based on biochemistry, immunohistochemistry as well as genetic analyses! Please also feel free to write back to us with comments and questions at strandlive@strandls.com.

Expanding the Scope of Liquid Biopsy

Dr. Shefali Sabharanjak
Strand Life Sciences

Abstract

  • Liquid biopsy has added a new dimension to cancer therapy by enabling periodic, no-fuss and unlimited access to the genetic profile of solid tumors.
  • Monitoring tumor progression by periodic evaluation of ctDNA present in blood can serve as an accessory prognostic tool, in addition to clinical evaluations and medical imaging techniques.
  • Liquid biopsies can present a comprehensive picture of clonal subpopulations of tumors, in combination with the solid primary tissue biopsy.
  • Detection of mRNA from exosomes in circulation is possible, thereby extending the reach of liquid biopsy for early stage tumors as well.

Introduction

Liquid biopsy for solid tumorsLiquid biopsy for solid tumors is fast becoming an actionable technique to understand tumor status as well as patient response to therapy. Simply put, a liquid biopsy provides a snapshot of the molecular profile of a patient’s cancer.

 

 

 

 

 

 

 

 

 

Molecular Assessments

Tumor cells as well as aged normal tissues shed cells and cell fragments into the bloodstream. Intact whole cells as well as cellular fragments like exosomes and free nucleic acids from tumor as well as normal tissues are the breadcrumb trail that can be picked up and identified. Circulating tumor cells (CTCs) can be harvested to isolate enriched tumor DNA. However, technological challenges with harvesting and labeling CTCs, coupled with the scarcity of CTCs make these a difficult target. In contrast, the ease of extraction of cell-free DNA (cfDNA) coupled with specific probes to identify mutations in circulating tumor DNA (ctDNA), make ctDNA-based tumor assessment a very attractive technique.
Identification of ctDNA to assess the molecular profile of solid tumors has been demonstrated as a viable measure of tumor progression in breast, colorectal and lung cancer patients (Ai, Liu, Huang, & Peng, 2016; Bettegowda et al., 2014; Ma et al., 2016; Olsson et al., 2015; Pécuchet et al., 2016).

Clinical Applications of Liquid Biopsy

Monitoring Tumor Progression

At Strand, we have developed and standardized liquid biopsy assays to pick up ctDNA from cfDNA at a ten-fold higher sensitivity than other off-the-shelf liquid biopsy kits. These liquid biopsy-based detection assays have been successfully used in colorectal and breast cancer patients.

A 55-year-old patient presented with colorectal carcinoma that was amenable to surgical removal. Prior to surgery, a baseline liquid biopsy (10 ml blood) was obtained. Post-surgery, tumor tissue embedded in FFPE blocks and the baseline LB sample was sent to Strand. A KRASG13D mutation was identified from the FFPE embedded solid tumor tissue. The same mutation was identified in the LB sample as well (1100 copies of ctDNA / ml plasma). Upon completion of adjuvant therapy, 3 more LB samples were drawn at 4-month intervals in order to check for tumor progression. The subsequent LB samples were assessed for presence of ctDNA at 4-month intervals, with zero detection of ctDNA. These findings were corroborated by other clinical evaluations as well. Moreover, the patient has the option to continue undergoing LB, since there are no dependencies on radiolabeled tracers and specialized equipment.

Clonal Evolution of Tumors

Metastatic tumors are prone to changing their genetic profile by acquiring new mutations. Alternatively, a subpopulation of tumors that has remained subdued in the primary tissue may thrive at another site in the body. A 61-year-old woman presented with non-small cell lung cancer (NSCLC) at a hospital in Bhubaneshwar. Genetic analysis of the primary tumor biopsy indicated the predominance of an EGFRL858R mutation and hence the patient was prescribed Afatinib therapy.

Although the patient fared well initially, the cancer relapsed and Afatinib therapy turned out to be inefficient.

A second tumor biopsy from the lung and a concurrent liquid biopsy was obtained, for fresh genetic analysis. The genetic profile of the lung biopsy indicated a higher predominance of the EGFRL858R mutation in the lung but the liquid biopsy showed a greater predominance of the EGFRT790M mutation in ctDNA. Clearly, two different subpopulations of NSCLC were prevalent in the patient’s system. A clear picture of the clonal diversity of NSCLC was possible only with a combination of solid and liquid biopsies. The patient was switched to Osimertinib therapy and is now living with cancer.

Is DNA the Only Viable Tracer?

A significant limitation of detection of ctDNA is the fact that a concordance between solid tumor status and detection of ctDNA has only been established, by and large, for late stage tumors (Bettegowda et al., 2014; Ilié et al., 2018; Xie, Li, Gong, Tan, & Ma, 2017; Yu et al., 2017). Early stage tumors may or may not shed enough cells and nuclei for adequate presence of tumor DNA in blood.

In a recent case analyzed at Strand, mRNA from exosomes (small membrane bound fragments of cells containing cytoplasm) has been isolated from a liquid biopsy sample. A 59-year-old patient was diagnosed with NSCLC, with a solid tissue biopsy from the lung as well as a liquid biopsy sample sent for genetic analysis. A characteristic fusion of the EML4 and ALK genes, known to cause NSCLC, was identified in the solid tumor.

Exosomes were isolated from the liquid biopsy sample and mRNA retrieved from these was transcribed to cDNA followed by amplification by PCR. The EML4-ALK fusion construct was detected in the amplified tracer as well.
Availability of nucleic acid tracers from exosomes is a breakthrough in extending the use of liquid biopsy for diagnosis and assessment of early stage tumors.

Summary

  • Liquid biopsy has added a new dimension to cancer therapy by enabling periodic, no-fuss and unlimited access to the genetic profile of solid tumors.
  • Monitoring tumor progression by periodic evaluation of ctDNA present in blood can serve as an accessory prognostic tool, in addition to clinical evaluations and medical imaging techniques.
  • Liquid biopsies can present a comprehensive picture of clonal subpopulations of tumors, in combination with the solid primary tissue biopsy.
  • Detection of mRNA from exosomes in circulation is possible, thereby extending the reach of liquid biopsy for early stage tumors as well.

References

Ai, B., Liu, H., Huang, Y., & Peng, P. (2016). Circulating cell-free DNA as a prognostic and predictive biomarker in non-small cell lung cancer. Oncotarget, 7(28), 44583-44595. http://doi.org/10.18632/oncotarget.10069

Bettegowda, C., Sausen, M., Leary, R. J., Kinde, I., Wang, Y., Agrawal, N., … Jr. (2014). Detection of circulating tumor DNA in early- and late-stage human malignancies. Science Translational Medicine, 6(224), 224ra24. http://doi.org/10.1126/scitranslmed.3007094

Ilié, M., Szafer-Glusman, E., Hofman, V., Chamorey, E., Lalvée, S., Selva, E., … Hofman, P. (2018). Detection of PD-L1 in circulating tumor cells and white blood cells from patients with advanced non-small-cell lung cancer. Annals of Oncology, 29(1), 193-199. http://doi.org/10.1093/annonc/mdx636

Ma, F., Zhu, W., Guan, Y., Yang, L., Xia, X., Chen, S., … Xu, B. (2016). ctDNA dynamics: a novel indicator to track resistance in metastatic breast cancer treated with anti-HER2 therapy. Oncotarget, 7(40), 66020-66031. http://doi.org/10.18632/oncotarget.11791

Olsson, E., Winter, C., George, A., Chen, Y., Howlin, J., Tang, M. E., … Saal, L. H. (2015). Serial monitoring of circulating tumor DNA in patients with primary breast cancer for detection of occult metastatic disease. EMBO Molecular Medicine, 7(8), 1034-47. http://doi.org/10.15252/emmm.201404913

Pécuchet, N., Zonta, E., Didelot, A., Combe, P., Thibault, C., Gibault, L., … Fabre, E. (2016). Base-Position Error Rate Analysis of Next-Generation Sequencing Applied to Circulating Tumor DNA in Non-Small Cell Lung Cancer: A Prospective Study. PLoS Medicine, 13(12), e1002199. http://doi.org/10.1371/journal.pmed.1002199

Xie, F., Li, P., Gong, J., Tan, H., & Ma, J. (2017). Urinary cell-free DNA as a prognostic marker for KRAS-positive advanced-stage NSCLC. Clinical and Translational Oncology. http://doi.org/10.1007/s12094-017-1754-7

Yu, Q., Huang, F., Zhang, M., Ji, H., Wu, S., Zhao, Y., … Guo, W. (2017). Multiplex picoliter-droplet digital PCR for quantitative assessment of EGFR mutations in circulating cell-free DNA derived from advanced non-small cell lung cancer patients. Molecular Medicine Reports, 16(2), 1157-1166. http://doi.org/10.3892/mmr.2017.6712

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A Snapshot of Rare Inherited Genetic Disorders in India

Rare Inherited Genetic Disorders in India

Rare genetic disorders occupy a special niche in the category of non-communicable diseases. This section of the population is especially underserved by genetic tests, although accurate identification of the causative genetic mutations is possible.

References : http://www.rarediseasesindia.org/

https://www.ncbi.nlm.nih.gov/pubmed/26029702

https://www.amrita.edu/news/new-nampoothiri-syndrome-described

Diagnosis of LAMA2-Dystrophy Provided by the Strand Clinical Exome Test

Dr. Shefali Sabharanjak
Strand Life Sciences

Abstract

  • Aditya* (6 years) was found to be compound heterozygous for two distinct mutations in the LAMA2 gene, thus confirming the diagnosis of LAMA2-related muscular dystrophy.
  • Mutation-specific testing (MST) was performed on the parents’ DNA samples, thereby establishing their status as carriers of one variation each.
  • The probability of the unborn fetus inheriting both mutations is 25%. Hence, chorionic villus sampling was done in the current pregnancy to enable genetic testing of the fetus.
  • The fetus had not inherited the c.7147C>T variation and was heterozygous for the c.8767G>T mutation.
  • Since congenital muscular dystrophy resulting from mutations in the LAMA2 gene is inherited in autosomal recessive manner, the fetus found to be heterozygous for one of the two variations is likely to be unaffected with the same condition.
  • Radhika’s pregnancy was allowed to reach full-term and a healthy baby girl was born to the couple.

Introduction

Inherited muscular dystrophies are characterized by progressive muscle degeneration that results in several health problems. Early onset inherited muscular dystrophies can have high mortality and guarded prognosis.

 

 

 

 

 

 

 

Patient Profile

Radhika* and Sundar, a couple from a southern state in India, had lost two children, one at age 8 years and the other within a few days after birth. Their third child, 6-year-old Aditya*, had been diagnosed with congenital muscular dystrophy. In their subsequent pregnancy, Radhika and Sundar were anxious to understand whether the newborn was likely to survive or not. Even if the child survived, they were not sure if the child would have the same kind of health problems that Aditya has.

Sundar and Radhika were referred to a prominent clinical geneticist in the country. Going by Aditya’s symptoms, their doctor suspected the incidence of early-onset, heritable muscular dystrophy and advised them to get genetic tests done to confirm the diagnosis.

Family Tree: Pre-Genetic Test Counselling

Pedigree Chart

Figure 1. Pedigree Chart- Radhika, Sundar and Aditya

Aditya had an older brother who had also suffered from muscular dystrophy and was lost to the disease. Another sibling had expired within a few days of birth.

The Strand Clinical Exome Test was prescribed, in order to understand the genetic mutations that caused his muscular dystrophy.

Results of Genetic Testing (Aditya’s DNA)

 

Results of Genetic Testing

Figure 2. LAMA2 mutations in compound heterozygous state

 

Two distinct mutations were identified in the LAMA2 gene during Aditya’s genetic testing. Both variants are known to cause an inherited muscular dystrophy known as LAMA2 dystrophy.

Interestingly, Aditya was found to be compound heterozygous for these two mutations. This suggested that he had inherited one gene variant each, from either parent. Given this status, Radhika and Sundar decided to undergo genetic testing in order to ascertain their status as carrier individuals. Radhika was also pregnant with another baby and they were anxious to understand the risk of transmitting these variants to the unborn child.

Genetic Analyses of DNA from Sundar and Radhika

 

 SG-Iss14-Art2-Fig3-new.png February 22, 2018127 KB 1181 × 737 Edit Image Delete Permanently URL http://strandls.com/wp-content/uploads/2018/02/SG-Iss14-Art2-Fig3-new.png Title SG-Iss14-Art2-Fig3-new Caption Figure 3. Radhika and Sundar are heterozygous for one LAMA2 mutation, each.

Figure 3. Radhika and Sundar are heterozygous for one LAMA2 mutation, each.

Mutation-specific tests were offered to Sundar and Radhika to understand whether they were carriers for these LAMA2 variants.

Genetic analyses show that Radhika is a carrier (heterozygous) for the c.7147C>T mutation and does not have the c.8767G>T variant. Conversely, Sundar is heterozygous for the c.8767G>T mutation but does not have the c.7147C>T variant in his genome. Aditya inherited one variant each from either of his parents.

It is possible that the deceased children of this couple had been compound heterozygous for these variations in the LAMA2 gene. Since the couple was pregnant with another child, chorionic villus sampling was advised, in order to obtain tissue which essentially has the same genetic makeup as the fetus, for genetic testing.

Genetic Analysis of DNA from Radhika’s Fetus

 

Genetic Analyses of DNA from Sundar and Radhika

Figure 4. Radhika’s fetus is heterozygous for only one LAMA2 mutation- c.8767G>T

The fetus had not inherited the c.7147C>T variant of the LAMA2 gene. However, the fetus was heterozygous for the c.8767G>T variant in the same gene.

 

Inheritance of LAMA2 Mutations

Disease-causing variations in the LAMA2 gene can cause dystrophy in skeletal muscles. These are muscles that are required for voluntary movements such as eating, swallowing, walking and holding objects. LAMA2 dystrophy is manifested in two forms: early-onset and late-onset dystrophies. Early-onset LAMA2 dystrophy can result in severe retardation of growth because of inability to swallow and in some cases may be fatal(Quijano-Roy et al. 1993). Mutations in the LAMA2 gene are inherited in an autosomal recessive manner. Therefore, heterozygous or carrier individuals are usually not affected by the disease. However, homozygous or compound heterozygous individuals, like Aditya, manifest symptoms like dystonia, scoliosis and lordosis of the spine, problems with speech, cardiovascular issues and seizures.

Conclusions

  • Aditya (6 years) was found to be compound heterozygous for two distinct mutations in the LAMA2 gene, thus confirming the diagnosis of LAMA2-related muscular dystrophy.
  • Mutation-specific testing (MST) was performed on the parents’ DNA samples, thereby establishing their status as carriers of one variation each.
  • The probability of the unborn fetus inheriting both mutations is 25%. Hence, chorionic villus sampling was done in the current pregnancy to enable genetic testing of the fetus.
  • The fetus had not inherited the c.7147C>T variation and was heterozygous for the c.8767G>T mutation.
  • Since congenital muscular dystrophy resulting from mutations in the LAMA2 gene is inherited in autosomal recessive manner, the fetus found to be heterozygous for one of the two variations is likely to be unaffected with the same condition.
  • Radhika’s pregnancy was allowed to reach full-term and a healthy baby girl was born to the couple.

*- Patient names changed to protect privacy

References

Quijano-Roy, S., Sparks, S.E. & Rutkowski, A., 1993. LAMA2-Related Muscular Dystrophy, University of Washington, Seattle. Available at: http://www.ncbi.nlm.nih.gov/pubmed/22675738 [Accessed October 31, 2017].

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Advanced genomic analysis for answers not found before

Clinical Trials Update

 

 

 

 

 

List Of Drugs Approved Against Various Types of Cancers in 2017

  • Alunbrig (Brigatinib); Ariad Pharmaceuticals; For the treatment of advanced ALK-positive metastatic non-small cell lung cancer, Approved April 2017
  • Imfinzi (Durvalumab); AstraZeneca; For the treatment of advanced or metastatic urothelial carcinoma, Approved May 2017
  • Kisqali (Ribociclib); Novartis; For the treatment of breast cancer, Approved March 2017
  • Nerlynx (Neratinib); Puma Biotech; For the treatment of HER2 breast cancer, Approved July 2017
  • Verzenio (Abemaciclib); Eli Lilly; For the treatment of HR+, HER2- breast cancer, Approved September 2017
  • Zejula (Niraparib); Tesaro; For the treatment of recurrent epithelial ovarian, fallopian tube, or primary peritoneal cancer,Approved March 2017

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