After obtaining Institutional Review Board approval from the University of Tennessee we queried the Caris Life Sciences database for patients profiled by Caris Molecular Intelligence using the keywords “small cell” to search in the clinical history and diagnosis fields for cases of SCCO, SCCO-HT (hypercalcemic type), neuroendocrine tumors of the ovary (NET-O) from 2007–2015. Comparative data was pulled for SCLC from April 2015- September 2015. Caris registry is a proprietary database to which the investigators have access for research purposes, which collates and categorizes molecular alterations and cancer types across patients who have had cancer molecular testing done throughout The United States using a multi-platform tumor profiling service that includes gene sequencing with NGS and Sanger, protein expression analysis with IHC, and gene copy number and translocation analysis with CISH or FISH.
Clinicopathologic characteristics of patients with SCCO, SCCO-HT, NET-O and SCLC patients were identified, including age and whether metastatic disease was present at the time of profiling. IHC analysis was performed using commercially available detection kits and automated staining techniques (Benchmark XT, Ventana, Tucson, AZ; and AutostainerLink 48, Dako, Carpinteria, CA). Tumors were assessed with up to 25 IHC stains (ALK, AR, BCRP, c-KIT, ER, PR, cMET, EGFR, HER2, IGF1R, PTEN, PD-1, PDGFR, PD-L1, ERCC1, TS, MGMT, RRM1, TLE3, TUBB3, SPARC, TOP2A, TOPO1, MRP1, PGP). Gene copy number alterations of cMET, EGFR, HER2, PIK3CA, and TOP2A were analyzed by DNA ISH using (FISH and/or CISH probes as part of automated staining techniques (Benchmark XT, Ventana, Tucson, AZ) and automated imaging systems (BioView, Billerica, MA). The ratio of gene to pericentromeric regions of chromosome 7 (EGFR, cMET), 17 (HER2, TOP2A) and 3 (PIK3CA) were used to determine increases in gene copy number. Tumors also underwent NGS analysis, which is a form of parallel sequencing that greatly enhances the efficiency of identifying both somatic and germline mutations [
11]. NGS sequencing was performed on genomic DNA isolated from tumor tissue using the Illumina MiSeq platform. PCR products were bi-directionally sequenced using the BigDye Terminator v1.1 chemistry, analyzed using the 3730 DNA Analyzer (Applied Biosystems, Grand Island, NY). Sequence traces were analyzed using Mutation Surveyor software v3.25 (Soft Genetics, State College, PA). NGS and Sanger sequencing of a 47-gene panel (ABL, AKT, ALK, APC, ATM, BRAF, BRCA1, BRCA2, CDH1, cKIT, cMET, CSF1R, CTNNB1, EGFR, ERBB4, FBXW7, FGFR1, FGFR2, FLT3, GNA11, GNAQ, GNAS, HER2, HNF1A, HRAS, IDH1, JAK2, JAK3, KDR, KRAS, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RB, RET, SMAD4, SMARCB1, SMO, STK11, TP53, VHL) was evaluated for somatic mutations. Coding changes were accessed for pathogenicity using PolyPhen-2 [
10,
12].
Retrospective analysis of biomarker frequency distributions was attained using standard descriptive statistics evaluating the incidence of the aforementioned genomic alterations in these tumors by Caris Molecular Intelligence profiling. The two-tail Fisher’s exact test analyzed whether frequencies differed by subgroup, specifically to determine if any non-random associations between SCCO and SCLC existed. A p-value <0.05 was considered statistically significant and all p-values were 2-sided.