Letter to the Editor

Dear Editor

I read with interest the original article titled “A study of therapy targeted EGFR/ALK mutations in Indian patients with lung adenocarcinoma: A clinical and epidemiological study” by Rana et al. published in Med J Armed Forces India 2018;74:148–153.¹ This topic is close and dear to me because during my study period in the Tata Memorial Center, Mumbai, and Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), I was involved in molecular studies and followed up the whole procedure of mutational analysis and sequencing of KRAS in lung adenocarcinomas.

In this particular article, it was stated that the molecular studies of 152 cases were outsourced; therefore, I want to highlight the salient features of the type and adequacy of specimens; the type of biopsy such as trucut biopsy, the use of bronchoscopic biopsy or transthoracic needle biopsy in bigger specimens, or resection specimens in some cases; and difficulties encountered in DNA extraction, and the overall mutation rate was found in literature to be high in resection specimens.² We had studied lobectomy blocks in lung adenocarcinomas, and DNA extraction was very difficult; there was noise in signals, and repeated analysis was required in almost all the cases.

The genetic abnormalities in non–small-cell lung carcinoma (NSCLC; adenocarcinoma and squamous carcinoma) include EGFR^{3, 4, 5, 6, 7, 8} (exon 21 L858R mutation and exon 19 deletion) mutations; HER2 mutations (1%); BRAF mutations (2%); exon 14 MET mutations (3.3%); and ALK,^{9, 10, 11} ROS1, RET, and NTRK rearrangements. Targeted therapy with gefitinib, erlotinib, and afatinib was used for EGFR-mutated advanced NSCLC; the ALK inhibitors crizotinib and ceritinib were used for ALK-positive NSCLC, and for ROS1 rearrangement–positive NSCLC, crizotinib was used.

In small centers, where lobectomies and metastasectomies are not performed for lung cancers, small biopsies and cytology specimens are the only material available for almost 80% of patients with NSCLC, and usually, the molecular tests are outsourced. In that case, the tissue should be optimal and it is essential that the tissue sample size is maximized. The biopsy specimen while performing bronchoscopy or transthoracic needle biopsy should not be one or two haphazard cores. At least six biopsy specimens should be taken and many blocks prepared in the laboratory, which can be used separately for EGFR, ALK, KRAS mutations, and so forth. For polymerase chain reaction–based next-generation sequencing (NGS) technique, a minimum of 5–10 ng of DNA (1000 cells; including malignant and normal DNA) is required. Hybrid capture–based NGS technologies, encompassing RNA sequencing, require 100–200 ng of RNA or DNA. There is always a possibility of false-negative and false-positive results that occur because of artificial base pair changes. Whole-genome sequencing analyses require more DNA (10⁶ tumor cells) and are not adapted for small samples.

In this era of targeted therapy, small samples (biopsy and cell blocks from fine needle aspiration cytology (FNAC) or fluid cytology) may play an important role in the management and prioritization of tumor material for multiple predictive marker testing in NSCLC and are preferable to obtain the largest biopsy possible for better molecular results and correct targeted treatment.