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Table of Contents
ORIGINAL ARTICLE - REAL WORLD DATA
Year : 2020  |  Volume : 3  |  Issue : 4  |  Page : 742-747

Biomarker testing in non-small cell lung carcinoma – More is better: A case series


1 Department of Medical Oncology, Molecular Diagnostics and Transfusion Medicine, Rajiv Gandhi Cancer Institute and Research Centre, New Delhi, India
2 Department of Molecular Diagnostics, Molecular Diagnostics and Transfusion Medicine, Rajiv Gandhi Cancer Institute and Research Centre, New Delhi, India
3 Department of Radiology, Molecular Diagnostics and Transfusion Medicine, Rajiv Gandhi Cancer Institute and Research Centre, New Delhi, India
4 Department of Laboratory Services, Molecular Diagnostics and Transfusion Medicine, Rajiv Gandhi Cancer Institute and Research Centre, New Delhi, India

Date of Submission10-Sep-2020
Date of Decision09-Dec-2020
Date of Acceptance09-Dec-2020
Date of Web Publication25-Dec-2020

Correspondence Address:
Ullas Batra
Sir Chhotu Ram Marg, Sector 5, Rohini, New Delhi - 110 085
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/crst.crst_285_20

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  Abstract 


Background: Biomarker-driven lung adenocarcinomas involving alterations in oncogenic drivers such as EGFR, ALK, ROS1, and NTRK have witnessed a dramatic shift in the therapeutic and prognostic landscape owing to the development of molecular targeted therapies. The recent approval of the selective RET inhibitor, selpercatinib, has led to an ardent interest in RET-rearranged non-small cell lung carcinoma (NSCLC). However, sequential single-gene testing cannot detect RET rearrangements accurately or characterize the fusion partners.
Objectives: We aimed to determine the incidence and types of RET alterations in our patients with NSCLC, and to describe the demographic and clinical profile of our patients with RET-driven NSCLC. In addition, our aim was to highlight the advantages of broader panel-based testing by the next generation sequencing (NGS) over single-gene assays.
Materials and Methods: This is a retrospective, case series of patients with advanced NSCLC who underwent testing by NGS between December 2018 and August 2020 at our center, with a focus on the cases who were found to have the RET gene rearrangement. The demographic, clinicopathological profiles, and treatment details were retrieved from the medical record archives. Statistical analysis was performed using the Statistical Package for the Social Sciences software version 23 for Windows.
Results: A total of 169 patients were enrolled in the study. RET rearrangement was detected in 2.9% (n = 5) of the patients in our cohort. Four cases had the KIF5B-RET fusion, and one case had the CCDC6-RET fusion. The median age of the patients was 55 years (range, 45–82), with a slight female preponderance (men: 2 and women: 3). The RET fusions were detected using an NGS-based assay. Four out of the five patients were administered pemetrexed-carboplatin-based chemotherapy and are alive and on regular follow-up. One patient did not receive any treatment and has succumbed to the disease. One patient has been administered selpercatinib after failing many lines of chemotherapy.
Conclusions: The emergence of newer molecular targets necessitates the use of an broader panel-based NGS testing to detect oncogene addiction in NSCLC. This case series highlights the importance of NGS-based testing in the light of the recent approval of selpercatinib for RET-rearranged NSCLC.

Keywords: Next-generation sequencing, non-small cell lung carcinoma, RET, selpercatinib


How to cite this article:
Batra U, Sharma M, Nathany S, Soni S, Bansal A, Jain P, Mehta A. Biomarker testing in non-small cell lung carcinoma – More is better: A case series. Cancer Res Stat Treat 2020;3:742-7

How to cite this URL:
Batra U, Sharma M, Nathany S, Soni S, Bansal A, Jain P, Mehta A. Biomarker testing in non-small cell lung carcinoma – More is better: A case series. Cancer Res Stat Treat [serial online] 2020 [cited 2021 Jan 21];3:742-7. Available from: https://www.crstonline.com/text.asp?2020/3/4/742/304965




  Introduction Top


Precision medicine has reshaped the therapeutic scenario of patients with oncogene-addicted lung carcinomas. Sensitizing mutations in oncogenes such as EGFR, ALK, BRAF (p. V600E), ROS1, and NTRK have been identified as molecular targets, and the use of novel targeted therapies has resulted in robust response rates and improved overall survival (OS).[1]

Rearrangements involving the RET oncogene have been previously described in many malignancies such as papillary and medullary (both sporadic and hereditary) carcinomas of the thyroid gland,[2],[3] and in rare cases of colorectal, breast, and pancreatic cancers.[4] RET fusions have also been reported in 1%–2% cases of non-small cell lung carcinoma (NSCLC).[5] The recent approval of selpercatinib for RET-rearranged NSCLC[6],[7] has led to a keen interest in the detailed characterization of the clinicopathological features and response outcomes of this disease. The current practice of performing single-gene testing does not incorporate RET fusion detection, and hence, it may be imperative to now move from sequential testing to a broader panel-based testing. Next-generation sequencing (NGS)-based panel testing has shown promise in detecting uncommon and complex variants in the canonical genes, even at a low mutation load. Sequential single gene testing is limited not only by the sensitivity and throughput, but also the premise that the amount of tissue in lung core biopsies is limited, rendering it inadequate for further panel-based testing.

Prior to the development of selective RET inhibitors, platinum-based chemotherapy was the recommended treatment approach with a best response rate of 52% as reported by Gautschi et al.[8] Since 2012, multiple clinical trials have been launched with multi-kinase inhibitors such as cabozantinib, vandetanib, lenvatinib, and sunitinib, which have shown diffident anti-RET activity, but with an increased toxicity profile.[8],[9],[10] The new-generation, highly selective RET inhibitors, selpercatinib and pralsetinib, have shown robust clinical results with low toxicity. Here, we report a series of five cases of RET-rearranged NSCLC, their clinicopathological characteristics, and our experience of selpercatinib use along with a review of literature on this entity.


  Materials and Methods Top


General study details

This is a retrospective, case series of patients with advanced NSCLC who underwent testing by NGS at our center between December 2018 and August 2020. The study was approved by the institutional scientific committee, and a blanket informed consent was obtained from the patients. The study was not registered in a public clinical trials registry. No direct or indirect identifiers/clinical photographs have been used, and the study adhered to the ethical principles outlined in the Declaration of Helsinki.

Participants

A total of 169 patients with advanced-stage NSCLC who underwent panel-based genomic profiling by NGS during the course of their treatment were enrolled in the study. Patients who tested negative for alterations in the EGFR, ALK, and ROS1 genes by single-gene assays were not subjected to NGS-based testing. RET rearrangements were detected in five cases. Clinical data pertaining to the demographics, clinical presentation, radiologic evaluation, histopathology, and findings of molecular testing were retrieved from the electronic medical records of the hospital. Patients who did not undergo NGS-based molecular profiling were excluded from the study.

Variables

Our primary objective was to determine the incidence of RET alterations in our patients with NSCLC who underwent NGS-based tumor profiling. Our secondary objectives were to understand the types of RET fusions that occur in our Indian patients with NSCLC and to evaluate the demographic, clinical, and treatment-related profiles of Indian patients with RET-driven NSCLC.

Study methodology

Next-generation sequencing

DNA and RNA were extracted using the Recover All Total Nucleic Acid Isolation kit (Thermo Fisher Scientific, San Francisco, USA) from formalin-fixed, paraffin-embedded (FFPE) tumor blocks after macro-dissection to include maximum tumor content. The library was prepared using the Oncomine Focus assay, and its quality was checked using the Tape Station with High sensitivity DNA kits. Sequencing was performed on the Ion PGM sequencer (Thermo Fisher Scientific) or the Ion S5 sequencer using a 520/530 chip. Data analysis and variant calling were done using the Torrent Suite Browser version 5.10 and the Oncomine Knowledge Reporter (both from Thermo Fisher Scientific). The NGS-based Oncomine Focus Assay targets single nucleotide variations, gene rearrangements, and copy number variations in 52 genes.

Fluorescence in situ hybridization

Four-micron sections from the FFPE tumor blocks were used for the fluorescence in situ hybridization (FISH) assay. RET fusions that were detected on NGS were validated by FISH using the RET dual-color break-apart fusion probe (Cytocell). The kit consists of two probes, RET, 10q11.21, Green and RET, 10q11.21, Red. Signal evaluation was done using a fluorescence microscope (Leica DM6000 B) equipped with three filters (4',6-diamidino-2-phenylindole, green, red). A minimum of 50 tumor cells were evaluated for the detection of RET rearrangement. Fused, split, or isolated green/orange signals were detected and enumerated.

Statistics

No formal sample size calculation was performed for this study, and all patients who underwent testing by NGS were included. The Statistical Package for the Social Sciences (IBM Corp. Released 2015. IBM SPSS Statistics for Windows, Version 23.0. Armonk, NY, USA: IBM Corp) was used for the statistical analysis. The data are presented as simple descriptive statistics.


  Results Top


A total of 169 patients with NSCLC who underwent NGS-based tumor profiling for the detection of genomic alterations at our institute were enrolled in the study. Of these, RET rearrangements were detected in five patients (2.9%). Four patients harbored a KIF5B-RET fusion, and one had a CCDC6-RET fusion [Figure 1].
Figure 1: Flow chart for patient recruitment and types of RET fusions observed in the study

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The median age of the patients was 55 years (range, 45–82), and the male-to-female ratio was 2:3. All the patients with RET-rearranged NSCLC were never smokers and presented with advanced (stage IV) disease. All five cases had an adenocarcinoma histology on morphologic evaluation, and the tumor of the patient with CCDC6-RET fusion also had extracellular mucin [Figure 2]. Four of the five patients had evidence of extrathoracic metastases at diagnosis, and two patients had brain metastases (one at diagnosis and one developed later).
Figure 2: (a) Lung core biopsy from the patient with CCDC6-RET fusion showing solid cribriform architecture lined by malignant cells with intraluminal mucin. (b) Positive periodic acid schiff (PAS) staining in the intraluminal mucin, (c) Periodic acid Schiff with diastase digestion showing periodic acid schiff-positivity and diastase-resistance in the intraluminal mucin. (d) Immunopositivity for TTF1 in the tumor cells, favoring adenocarcinoma histology. H and E: Hematoxylin and eosin, PAS: Periodic Schiff, DAB: Diaminobenzidine

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Four out of the five patients who were administered pemetrexed-carboplatin-based chemotherapy are alive (40.9 months, 58.6 months, 3.9 months, and 31.6 months since initial diagnosis, respectively) and on regular follow-up, whereas one patient did not receive any treatment and has succumbed to the disease 1 month after initial diagnosis. One patient has been administered selpercatinib after failing many lines of chemotherapy. The radiologic response to selpercatinib, both intracranial and extracranial, is depicted in [Figure 3] and [Figure 4], respectively. A detailed description of clinicopathological features, treatment details, and response outcomes is depicted in [Table 1].
Figure 3: Upper panel: T1 postcontrast magnetic resonance imaging axial images (a) before and (b) after the initiation of selpercatinib showing reduction in the size of nodular enhancing lesion in the left cerebellar hemisphere. Lower panel: T1 postcontrast magnetic resonance imaging axial images (c) before and (d) after the initiation of selpercatinib showing reduction in size of the nodular-enhancing lesion in the left frontal lobe

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Figure 4: Upper panel: Fused fluorodeoxyglucose positron emission tomography–computed tomography axial images (a) before and (b) after the initiation of selpercatinib showing partial response to treatment with reduction in size and activity of the right lung lesion and right pleural thickening. Lower panel: Fused fluorodeoxyglucose positron emission tomography–computed tomography coronal images (c) before and (d) after the initiation of selpercatinib showing partial response to treatment with reduction in size and activity of the right lung lesion and right pleural thickening

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Table 1: The clinicopathologic features and treatment details in five cases of RET-rearranged rearranged non-small cell lung carcinoma

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  Discussion Top


RET rearrangements have been reported in 1%–2% cases of NSCLC, whereas in our early experience, it has been observed in 2.9% of our Indian patients with NSCLC. This however may not be the true prevalence, as NGS-based testing was not performed in all the patients with NSCLC. The most commonly encountered fusion partner for RET was KIF5B, which was detected in four cases, whereas one case showed a CCDC6-RET fusion. All the patients were never-smokers and depicted adenocarcinoma histology on morphologic evaluation. One patient received treatment with the selective RET inhibitor, selpercatinib, following multiple cycles of chemotherapy.

RET is a proto-oncogene which was first identified in 1985[11] and has been mapped to chromosome 10q11.2. It codes for a receptor tyrosine kinase consisting of three domains: an extracellular domain, a transmembrane domain, and an intracellular kinase domain.[12] Ligand binding to RET activates the signaling cascades which trigger the activation of the downstream signals like the PI3K-AKT and mitogen-activated protein kinase pathways. Activating mutations and rearrangements lead to uncontrolled cell growth and malignant transformation.

Mutations in RET can be loss-of-function in nature, which have been described in Hirschsprung disease,[4] or they can be gain-of-function mutations which are associated with myriad of malignancies. Germline RET mutations involving codons 609, 611, 618, 620, and 634 have been identified in 92% cases of multiple endocrine neoplasia type 2 by the international RET mutation consortium (n = 477).[3] Sporadic RET mutations have been reported in 50% cases of medullary thyroid carcinoma[3] and in rare cases of small cell lung carcinoma. RET/papillary histotype (PTC) was the first rearrangement identified in papillary carcinoma of the thyroid gland associated with ionizing radiation, and till date 13 different RET/PTC fusion proteins have been characterized.[2],[13]

In NSCLC, RET rearrangement was first identified in 2011[14] in a young nonsmoker male with lung adenocarcinoma. It was identified as an in-frame fusion between KIF5B and RET, which is now reported to be the most common fusion partner, as is also evident from our case series. Other upstream fusion partners identified in NSCLC include CCDC6, NCOA4, TRIM33, MYO5C, EPHA5, CLIP1, ERC1, PICALM, FRMD4A, RUFY2, and TRIM24.[11],[15]

Wang et al. tested for RET fusions in 936 patients with NSCLC[16] using a reverse transcriptase polymerase chain reaction (PCR). Prominent clinical features noted were young, never-smoker patients with early lymph node metastases, poor differentiation, and solid predominant subtype of adenocarcinoma. Wang et al. also demonstrated that RET fusions were mutually exclusive of other oncogenic drivers.[16] However, in one study on 12 RET-rearranged patients,[17] 4 showed concomitant genetic alterations in EGFR, MAP2K1, and CTNNB1 genes. The largest RET registry from the Global, Multicenter RET Registry (GLORY)[8] includes 165 RET-rearranged NSCLC cases, and Gautschi et al. have described this disease in never smokers with adenocarcinoma histology and advanced stage (III-IV) at diagnosis. The cases in our study have similar attributes. The median age of the patients in Gautschi et al.'s study was 61 years (range, 29–89, whereas in ours it was 55 years (range, 45–82). Pathologically, RET fusions have been found to be associated with papillary, lepidic, and solid histologic patterns with extracellular/intracellular mucin being associated with CCDC6-RET fusion. This was also observed in our case with CCDC6-RET fusion.

RET rearrangement has been classified as Tier-1A alteration as per the American College of Medical Genetics and Genomics/Association for Molecular Pathology guidelines[18] for the reporting of somatic variants in cancer, with the therapeutic level of evidence as 1[12],[19] (as per MSKCC-IMPACT OncoKB). Detection of this rearrangement can be done using orthogonal modalities such as immunohistochemistry, break-apart FISH, or PCR for various fusion partners; however, these are all limited by sensitivity and low throughput. Sophisticated RNA sequencing employing NGS technologies has emerged as a promising tool, with the advantages of high throughput, sensitivity, accurate detection of the fusion breakpoints, detection of a wider spectrum of fusion partners, and potential use in response monitoring.

RET-rearranged NSCLCs have been reported to be sensitive to platinum-based chemotherapy with a median OS of 23.6 months and a median progression-free survival (PFS) of 6.6 months.[20] In another study by Shen et al.,[21] patients who received pemetrexed-based chemotherapy showed a higher PFS (9.2 months). Drilon et al. also reported a better objective response rate (ORR) and PFS (45%; 19 months) with pemetrexed-based chemotherapy in 18 patients with RET-rearranged NSCLC.

Since 2012, multi-kinase inhibitors such as cabozantinib, vandetanib, sunitinib, and lenvatinib have been tried in RET-rearranged NSCLC with modest activity but have shown increased off-target toxicity. However, the emergence of the highly selective RET inhibitor, selpercatinib, has changed the therapeutic landscape of RET-rearranged NSCLC. Drilon et al. in 2019 reported the groundwork data from the LIBRETTO-001 trial, depicting an ORR of 68% and a PFS of 18.4 months in patients who received selpercatinib after pretreatment with platinum-based chemotherapy.[6] Selpercatinib has been shown to have intracranial activity, and this was evident by the 91% intracranial objective response detected in 10 out of 11 patients who had brain metastases at diagnosis in this trial. In patients who received selpercatinib as first-line therapy (n = 39), the ORR was 85% and the median PFS was not reached. In our series, selpercatinib-induced partial response in both intracranial and extracranial sites. The drug was very well tolerated with no side effects.[6],[7]


  Conclusions Top


Classifying NSCLC as oncogene-addicted or nononcogene -addicted tumors is important in order to render the optimal first-line therapy. Our study highlights this with an emphasis on RET-rearranged NSCLC in the light of the recent approval of selective RET inhibitors such as selpercatinib. With the identification of more molecular targets, it is essential to move to a broader panel-based testing using the sophisticated NGS based-technologies for the diagnosis. This will increase the chances of detection of potential targets, thus improving the final survival outcomes for patients.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Network NCC. Non-Small Cell Lung Cancer (Version 3.2020). Available from: https://www.nccn.org/professionals/physician_gls/pdf/nscl.pdf. Published 2020. [Last accessed on 2020 Apr 10].  Back to cited text no. 1
    
2.
Santoro M, Melillo RM, Fusco A. RET/PTC activation in papillary thyroid carcinoma: European Journal of Endocrinology Prize Lecture. Eur J Endocrinol 2006;155:645-53.  Back to cited text no. 2
    
3.
Cerrato A, Falco V De, Santoro M. Molecular genetics of medullary thyroid carcinoma: The quest for novel therapeutic targets. J Mol Endocrinol 2008;43:143-55.  Back to cited text no. 3
    
4.
Kato S, Subbiah V, Marchlik E, Elkin SK, Carter JL, Kurzrock R. RET aberrations in diverse cancers: Next-generation sequencing of 4, 871 patients. Clin Cancer Res 2017;23:1988-98.  Back to cited text no. 4
    
5.
Bronte G, Ulivi P, Verlicchi A, Cravero P, Delmonte A, Crinò L. Targeting RET-rearranged non-small-cell lung cancer: Future prospects. Lung Cancer (Auckl) 2019;10:27-36.  Back to cited text no. 5
    
6.
Drilon A, Oxnard GR, Tan DSW, Loong HHF, Johnson M, Gainor J, et al. Efficacy of Selpercatinib in RET Fusion-Positive Non-Small-Cell Lung Cancer. N Engl J Med 2020;383:813-24.  Back to cited text no. 6
    
7.
Osta BE, Ramalingam SS. RET fusion: Joining the ranks of targetable molecular drivers in non-small cell lung cancer. JTO Clin Res Rep 2020;1:1–11.  Back to cited text no. 7
    
8.
Gautschi O, Milia J, Filleron T, Wolf J, Carbone DP, Owen D, et al. Targeting RET in patients with RET -Rearranged lung cancers: Results from the global, multicenter RET registry. J Clin Oncol 2017;35:1403-10.  Back to cited text no. 8
    
9.
Drilon A, Rekhtman N, Arcila M, Wang L, Ni A, Albano M, et al. Cabozantinib in patients with advanced RET-rearranged non-small-cell lung cancer: an open-label, single-centre, phase 2, single-arm trial. The Lancet. Oncology 2016;17:1653-60.   Back to cited text no. 9
    
10.
Lee SH, Lee JK, Ahn MJ, Kim DW, Sun JM, Keam B, et al. Vandetanib in pretreated patients with advanced non-small cell lung cancer-harboring RET rearrangement: a phase II clinical trial. Ann Oncol. 2017;28:292-7.   Back to cited text no. 10
    
11.
Ferrara R, Auger N, Auclin E, Besse B. Clinical and translational implications of RET rearrangements in non – small cell lung cancer. J Thorac Oncol 2017;13:27-45.  Back to cited text no. 11
    
12.
Chakravarty D, Gao J, Phillips S, Kundra R, Zhang H, Wang J, et al. OncoKB: A precision oncology knowledge base. JCO Precis Oncol 2017;2017:1-16.  Back to cited text no. 12
    
13.
Richardson DS, Gujral TS, Peng S, Asa SL, Mulligan LM. Transcript level modulates the inherent oncogenicity of RET/PTC oncoproteins. Cancer Res 2009;69:4861-9.  Back to cited text no. 13
    
14.
Ju YS, Lee W, Shin J, Lee S, Bleazard T, Won JK, et al. A transforming KIF5B and RET gene fusion in lung adenocarcinoma revealed from whole-genome and transcriptome sequencing. Genome Res 2012:436-45.  Back to cited text no. 14
    
15.
Kohno T, Ichikawa H, Totoki Y, Yasuda K, Hiramoto M, Nammo T, et al. KIF5B-RET fusions in lung adenocarcinoma. Nat Med 2012;18:375-7.   Back to cited text no. 15
    
16.
Wang R, Hu H, Pan Y, Li Y, Ye T, Li C, et al. RET fusions define a unique molecular and clinicopathologic subtype of non – small-cell lung cancer. J Clin Oncol 2020;30:4352-9.  Back to cited text no. 16
    
17.
Song Z, Yu X, Zhang Y. Clinicopathologic characteristics, genetic variability and therapeutic options of RET rearrangements patients in lung adenocarcinoma. Lung Cancer 2016;101:16-21.  Back to cited text no. 17
    
18.
Richards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J, et al. Standards and guidelines for the interpretation of sequence variants: A joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology Sue. Genet Med 2015;17:405-24.  Back to cited text no. 18
    
19.
Kopanos C, Tsiolkas V, Kouris A, Chapple CE, Albarca Aguilera M, Meyer R, et al. VarSome: The human genomic variant search engine. Bioinformatics 2019;35:1978-80.  Back to cited text no. 19
    
20.
Drilon A, Bergagnini I, Delasos L, Sabari J, Woo KM, Plodkowski A, et al. Clinical outcomes with pemetrexed-based systemic therapies in RET-rearranged lung cancers. Ann Oncol 2016;27:1286-91.  Back to cited text no. 20
    
21.
Shen T, Pu X, Wang L, Yu Z, Li J, Zhang Y, et al. Association between RET fusions and efficacy of pemetrexed-based chemotherapy for patients with advanced NSCLC in China: A multicenter retrospective study. Clin Lung Cancer 2020;21:e349-54.  Back to cited text no. 21
    


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