Jack Hsiang

Large-Scale Screening and Molecular Characterization of EML4-ALK Fusion Variants in Archival Non–Small-Cell Lung Cancer Tumor Specimens Using Quantitative Reverse Transcription Polymerase Chain Reaction Assays

Introduction: The objective of this study was to identify and characterize echinoderm microtubule-associated protein-like 4 anaplastic lymphoma kinase fusion (EML4-ALK+) cancers by variant-specific, quantitative reverse transcription polymerase chain reaction (RT-PCR) assays in a large cohort of North American non–small-cell lung cancer (NSCLC) patients. Methods: We developed a panel of single and multiplex RT-PCR assays suitable for rapid and accurate detection of the eight most common EML4-ALK+ variants and ALK gene expression in archival formalin-fixed, paraffin-embedded NSCLC specimens. EGFR and KRAS genotyping and thymidylate synthase RNA level by RT-PCR assays were available in a subset of patients. Results: Between December 2009 and September 2012, 7344 NSCLC specimens were tested. An EML4-ALK+ transcript was detected in 200 cases (2.7%), including 109 V1 (54.5%), 20 V2 (10.0%), 68 V3 (34.0%), and three V5a (1.5%) variants. Median age was 54.5 years (range, 23–89), and 104 patients (52.0%) were women. The great majority (n=188, 94.0%) of EML4-ALK+ NSCLC tumors had adenocarcinoma histology. ALK expression level varied significantly among different EML4-ALK+ variants and individual tumors. Only one case each of concurrent EGFR or KRAS mutation was detected. The median thymidylate synthase RNA level from 85 EML4-ALK+ cancers was significantly lower compared with that of EML4-ALK-negative lung adenocarcinomas (2.02 versus 3.29, respectively, p<0.001). Conclusions: This panel of variant-specific, quantitative RT-PCR assays detects common EML4-ALK+ variants as well as ALK gene expression level in archival formalin-fixed paraffin-embedded NSCLC specimens. These RT-PCR assays may be useful as an adjunct to the standard fluorescence in situ hybridization assay to better understand biologic variability and response patterns to anaplastic lymphoma kinase inhibitors.

Histology-Related Associations of ERCC1, RRM1, and TS Biomarkers in Patients with Non–Small-Cell Lung Cancer: Implications for Therapy

Introduction: On the basis of the results of recent clinical trials, histology-based decision-making for therapy of non–small-cell lung cancer has been advocated. We hypothesized associations of the biomarkers excision repair cross-complementing 1 (ERCC1), ribonucleotide reductase M1 (RRM1), and thymidylate synthase (TS) with histology as a contributing factor to reported differences in chemotherapy outcomes between squamous cell carcinoma (SCCA) and adenocarcinoma (AC) subtypes. Here, we report analysis of the Response Genetics Inc., database and implications for histology-based therapy. Methods: RNA from microdissected formalin-fixed paraffin-embedded tumors was extracted and analyzed as previously described. Specimens from 2540 individual non–small-cell lung cancer patients were analyzed for one or more biomarkers, of which 1457 were categorized as AC or SCCA. Results: For each biomarker, gene expression was lower in AC compared with SCCA (<0.001), although there was a wide range between individual patients. Gene expression was higher in men versus women: ERCC1: 2.51 versus 2.22 (p = 0.005); RRM1: 1.41 versus 1.24 (p = 0.004); TS: 3.23 versus 2.83 (p < 0.001). However, SCCA was more frequent in men versus women (30%/19%; p < 0.001). When AC and SCCA were assessed separately, the statistical significance between gene expression and sex was lost (in SCCA: ERCC1, p = 0.14; RRM1, p = 0.26; TS, p = 0.11). Conclusions: This analysis represents the largest data set for gene expression of these biomarkers reported so far. Significant histology-related associations for ERCC1, RRM1, and TS are seen. However, marked heterogeneity exists in individual patient tumor expression levels. Randomized phase III trials assessing the predictive value of these chemotherapy-related biomarkers are warranted.

Distinct gene expression profiles of proximal and distal colorectal cancer: implications for cytotoxic and targeted therapy.

Colorectal cancer (CRC) is a heterogeneous disease with genetic profiles and clinical outcomes dependent on the anatomic location of the primary tumor. How location has an impact on the molecular makeup of a tumor and how prognostic and predictive biomarkers differ between proximal versus distal colon cancers is not well established. We investigated the associations between tumor location, KRAS and BRAF mutation status, and the messenger RNA (mRNA) expression of proteins involved in major signaling pathways, including tumor growth (epidermal growth factor receptor (EGFR)), angiogenesis (vascular endothelial growth factor receptor 2 (VEGFR2)), DNA repair (excision repair cross complement group 1 (ERCC1)) and fluoropyrimidine metabolism (thymidylate synthase (TS)). Formalin-fixed paraffin-embedded tumor specimens from 431 advanced CRC patients were analyzed. The presence of seven different KRAS base substitutions and the BRAF V600E mutation was determined. ERCC1, TS, EGFR and VEGFR2 mRNA expression levels were detected by reverse transcriptase-PCR. BRAF mutations were significantly more common in the proximal colon (P<0.001), whereas KRAS mutations occurred at similar frequencies throughout the colorectum. Rectal cancers had significantly higher ERCC1 and VEGFR2 mRNA levels compared with distal and proximal colon tumors (P=0.001), and increased TS levels compared with distal colon cancers (P=0.02). Mutant KRAS status was associated with lower ERCC1, TS, EGFR and VEGFR2 gene expression in multivariate analysis. In a subgroup analysis, this association remained significant for all genes in the proximal colon and for VEGFR2 expression in rectal cancers. The mRNA expression patterns of predictive and prognostic biomarkers, as well as associations with KRAS and BRAF mutation status depend on primary tumor location. Prospective studies are warranted to confirm these findings and determine the underlying mechanisms.

KRAS mutations in non-small-cell lung cancer and colorectal cancer: Implications for EGFR-targeted therapies

BACKGROUND KRAS mutations are associated with diverse biologic functions as well as prognostic and predictive impact in non-small cell-lung cancer (NSCLC) and colorectal cancer (CRC). In CRC, benefit from monoclonal antibody therapies targeting EGFR is generally limited to patients whose tumors have wild-type (WT) KRAS, whereas data suggest that this association is not present for NSCLC. We hypothesized that the unique tobacco-related carcinogenesis of NSCLC results in a divergence of KRAS MT genotype compared with CRC, contributing to differences in outcomes from EGFR-targeted therapies. MATERIAL AND METHODS Tumor from 2603 patients (838 CRC and 1765 NSCLC) was analyzed for KRAS mutations. DNA was extracted from microdissected formalin-fixed-paraffin-embedded specimens (FFPE) and 7 different base substitutions in codons 12 and 13 of KRAS were determined. RESULTS KRAS mutation genotype differed significantly between NSCLC and CRC in frequency (25% vs. 39%; p<0.001), smoking-associated G>T transversions (73% versus 27%; p<0.001), and ratio of transversions to transitions (3.5 vs. 0.79; p<0.001). In NSCLC GLY12Cys mutations, resulting from a codon 12 GGT>TGT substitution, were observed in 44% compared to 10% for CRC. In contrast, codon 12 or 13 GLY>ASP substitutions (resulting in a G>A transition) were more frequent in CRC (42%) compared with NSCLC (21%). CONCLUSION In this large dataset, KRAS mutation patterns are quantitatively and qualitatively distinct between NSCLC and CRC, reflecting in part differences in tobacco-related carcinogenesis. In light of differences in predictive value for EGFR-directed monoclonal antibody therapy and prognosis for specific KRAS mutations between NSCLC and CRC, these data provide an underlying biologic rationale.

Immune-related Genes to Dominate Neutrophil-lymphocyte Ratio (NLR) Associated With Survival of Cetuximab Treatment in Metastatic Colorectal Cancer

Background Few clinical studies have investigated the association between neutrophil‐lymphocyte ratio (NLR) and treatment with cetuximab‐based chemotherapy in metastatic colorectal cancer (mCRC). The NLR may reflect immune cells modulating specific cytokine signals in the tumor microenvironment; however, which immune‐related genes affect the NLR remain unclear. Patients and Methods In 77 patients with KRAS exon2 wild‐type mCRC from prospective trials of first‐line chemotherapy with cetuximab, expression levels of 354 immune‐related genes were measured in tissue samples obtained from all patients by the HTG EdgeSeq Oncology Biomarker Panel. The association between the NLR and clinical outcomes was evaluated using the Spearman rank correlation coefficient. In addition, 2‐sample t tests were performed to investigate which genes among the top 100 genes associated with survival had significantly different expression levels between the NLR‐low and NLR‐high groups among all measured genes. Results NLR data were available for 71 patients. The NLR was associated with progression‐free survival and overall survival (r = −0.24; P = .040 and r = −0.29; P = .010, respectively). When stratified by the median value of the NLR, the Kaplan‐Meier curve of NLR‐low versus NLR‐high differed significantly for both progression‐free survival (median, 11.8 vs. 9.1 months; P = .036) and overall survival (median, 42.8 vs. 26.7 months; P = .029). The 2‐sample t test revealed that the expression levels of the LYZ, TYMP, and CD68 genes differed significantly between the NLR‐low and NLR‐high groups (t test P‐value < .005; false discovery rate P‐value < .15). Conclusion NLR is significantly associated with survival in patients with mCRC treated with first‐line chemotherapy with cetuximab. Genes encoding for activities on macrophages may affect the NLR. Micro‐Abstract Our study, using data of prospective trials, demonstrated that the neutrophil‐lymphocyte ratio (NLR) was associated with survival time in patients with KRAS wild‐type metastatic colorectal cancer treated with first‐line chemotherapy with cetuximab. The expression levels of the LYZ, TYMP, and CD68 genes differed significantly between the NLR‐low and NLR‐high groups. Genes encoding for activities on macrophages may affect the NLR.

Performance comparison of the Vysis automated versus manual ALK gene rearrangement assays on archived FFPE and cytology specimens.

e20056Background: The Vysis ALK Break Apart FISH Probe assay (ALK FISH) detects ALK gene chromosome 2p23 rearrangements in NSCLC FFPE tissue by fluorescence in situ hybridization (FISH) and involve...

Abstract A211: Multiethnic screening for irinotecan sensitivity in FFPE tissue from colorectal cancer patients.

The selection of chemotherapies is guided by the analysis of molecular targets in solid tumors for well-known somatic mutations identified in microdissected FFPE tissue. Combining a molecular targeting panel with select pharmacogenomic targets provides additional clinical information useful in customized patient management. We have developed a combined pharmacogenomic/molecular target approach using FFPE tissue from colon cancer patients. Our test, based on FDA guidance for Irinotecan administration to metastatic colorectal cancer (CRC) patients, is a recommended prescreening assay for UGT1A1 [TA] promoter variations to mimimize neutropenia. Meta-analysis of Irinotecan treated CRC patients has demonstrated an association between the [TA]7 allele (UGT1A1*28) and elevated levels of neutropenia in both homozygous and heterozygous carriers suggesting lowered dosing regimens (Liu, et al Pharmacogenomics J, 2013). Additional alleles, while uncommon in Caucasians, contribute to decreased levels of UGT1A1 enzymatic activity, and are restricted to genetic subgroups. Collectively these alleles influence the pharmacodynamic t1/2 of Irinotecan but are often not widely screened. Thus, a screening palate encompassing multiple ethnic-specific variants provides improved predictive information in a cosmopolitan population of cancer patients. As these polymorphisms are germline changes, UGT1A1 genotypes can be derived from either traditional whole blood assessment or more conveniently from FFPE surgical tissue at the time of diagnosis. We have screened paired blood/tumor samples from CRC patients to examine potential loss of heterozygosity in tumor specimens at this locus. In parallel, we screened microdissected tumor and adjacent normal tissue from FFPE samples to verify the potential of using tumor specimens as a routine alternative to blood. The use of FFPE tissue from CRC patients as part of a molecular work-up can provide genotypes for multiple UGT1A1 alleles of reduced activity (including *28), and may be better at predicting neutropenia in distinct Caucasian, Asian or other genetically distinct patient populations. The development of FFPE-based tests for pharmacogenomic tests such as UGT1A1 premised on genetic ancestry provides actionable information that can be used to maximize patient responses. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A211. Citation Format: Jack Hsiang, Rita El-Khoueiry, Heinz-Josef Lenz, Stephanie H. Astrow, Garrett P. Larson. Multiethnic screening for irinotecan sensitivity in FFPE tissue from colorectal cancer patients. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A211.

Correlation of messenger RNA expression patterns of ERCC1, TS, EGFR, and VEGFR2 with KRAS and BRAF mutational status in advanced colorectal cancer: Implications for targeted therapies.

383 Background: Gene expression levels of ERCC1, TS, EGFR and VEGFR2 may have predictive value for the personalized use of standard chemotherapeutics as well as agents targeting the EGFR and VEGF pathways and the efficacy of EGFR directed monoclonal antibodies like panitumumab and cetuximab has been confirmed to be dependent on wt KRAS and wt BRAF in patients with advanced colorectal cancer. We investigated the correlations between KRAS/BRAF mutational status and the mRNA expression levels of these genes. Methods: Formalin-fixed paraffin-embedded tumor specimens from 600 patients with advanced colorectal adenocarcinoma were microdissected and DNA and RNA was extracted. Specifically designed primers and probes were used to detect 7 different base substitutions in codon 12 and 13 of KRAS, V600E mutations in BRAF and the expression levels of ERCC1, TS, EGFR and VEGFR2 by RT-PCR. Results: Mt KRAS tumors had significantly lower TS and EGFR gene expression levels compared with wt KRAS (p<0,001), whereas mt BRAF...