Stephanie H. Astrow

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.

Combined assessment of EGFR-related molecules to predict outcome of 1st-line cetuximab-containing chemotherapy for metastatic colorectal cancer.

ABSTRACT Several studies have reported that epidermal growth factor receptor (EGFR)-related molecules may serve as predictors of cetuximab treatment for metastatic colorectal cancer (mCRC), such as EGFR gene copy number (GCN), expression of 2 ligands of EGFR, amphiregulin (AREG) and epiregulin (EREG), and EGFR CA simple sequence repeat 1 (CA-SSR1) polymorphism; however, these biomarkers still remain not useful in clinical practice since they have been evaluated using cohorts with patients treated in various settings of chemotherapy. We therefore analyzed associations of mRNA expression of AREG and EREG, EGFR GCN, and CA-SSR1 polymorphism [short (S;≤ 19) / long (L; ≥ 20)] with clinical outcomes in 77 Japanese patients with KRAS exon 2 wild-type mCRC enrolled in phase II trials of FOLFOX (n = 28/57, UMIN000004197) or SOX (n = 49/67, UMIN000007022) plus cetuximab as first-line therapy. High AREG expression correlated with significantly better progression-free survival (median 11.6 vs. 66 months, HR 0.52, P = 0.037); moreover, it remained statistically significant in multivariate analysis (HR: 0.48, P = 0.027). S/S genotype of CA-SSR1 predicted severe skin toxicity (P = 0.040). Patients with both AREG-low and EGFR low-GCN had significantly shorter overall survival than the others (median 22.2 vs. 42.8 months, HR 2.34, P = 0.042). The multivariate analysis showed that molecular status with both AREG-low and EGFR low-GCN was a predictor of worse survival (P = 0.006). In conclusion, AREG mRNA expression and EGFR CA-SSR1 polymorphism predict survival and skin toxicity, respectively, of initial chemotherapy with cetuximab. Our results also suggest potential prognostic value of the combined assessment of AREG and EGFR GCN for first-line cetuximab treatment.

Prognostic Value of ACVRL1 Expression in Metastatic Colorectal Cancer Patients Receiving First-line Chemotherapy With Bevacizumab: Results From the Triplet Plus Bevacizumab (TRIBE) Study.

Micro‐Abstract: ACVRL1 (activin receptor like‐protein 1) mediates tumor angiogenesis; however, its utility as a predictive or prognostic biomarker in colorectal cancer is unclear. We present the first evidence from a phase III study that intratumoral ACVRL1 expression is associated with tumor response and survival among metastatic colorectal cancer patients undergoing first‐line bevacizumab chemotherapy. These results could guide the selection of colorectal cancer patients for chemotherapy and antiangiogenic therapy. Background: No biomarkers exist to predict benefit from antiangiogenic therapy in metastatic colorectal cancer patients. ACVRL1 (activin receptor like‐protein 1) encodes for ALK1, a member of the transforming growth factor‐&bgr; receptor family, which directs pathologic angiogenesis. We examined the intratumoral expression of ACVRL1 and other angiogenesis pathway‐related genes to identify molecular markers in the TRIBE study. Materials and Methods: Of 503 randomized patients, 228 had sufficient tissue for analysis. Formalin‐fixed paraffin‐embedded specimens were examined for expression of VEGF‐A, VEGF‐B, VEGF‐C, VEGFR1, VEGFR2, ACVRL1, EphB4, and EGFL7 using reverse transcription polymerase chain reaction. A maximal χ2 approach was used to determine the messenger RNA levels associated with progression‐free survival (PFS), overall survival (OS), response rate, early tumor shrinkage, and depth of response. Recursive partitioning trees were constructed to identify composite prognostic biomarker profiles. External validation was conducted in silico using the Oncomine database. Results: High ACVRL1 expression was associated with superior OS in both treatment arms (FOLFOXIRI [5‐fluorouracil, leucovorin, oxaliplatin, irinotecan]‐bevacizumab, 32.7 vs. 13.5 months, hazard ratio [HR], 0.38, P = .023; FOLFIRI [5‐fluorouracil, leucovorin, irinotecan]‐bevacizumab, 35.1 vs. 22.0 months, HR, 0.36, P = .006) and prolonged PFS (11.7 vs. 5.9 months, multivariate HR, 0.17; P = .001) for patients receiving FOLFOXIRI‐bevacizumab on univariate and multivariate analyses. In recursive partitioning analysis, ACVRL1 was the strongest discriminator of the response rate, PFS, and OS in patients receiving FOLFOXIRI‐bevacizumab and of OS in patients receiving FOLFIRI‐bevacizumab. In silico validation revealed significant associations between ACVRL1 expression, disease recurrence, and 1‐year survival (P < .05) among all colorectal cancer stages. Conclusion: ACVRL1 expression could serve as a prognostic biomarker in metastatic colorectal cancer patients receiving chemotherapy and bevacizumab and warrants further evaluation in prospective studies.

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.

Expression of genes involved in vascular morphogenesis and maturation predicts efficacy of bevacizumab-based chemotherapy in patients undergoing liver resection

Angiogenesis-related gene expression is associated with the efficacy of anti-VEGF therapy. We tested whether intratumoral mRNA expression levels of genes involved in vascular morphogenesis and early vessel maturation predict response, recurrence-free survival (RFS), and overall survival (OS) in a unique cohort of patients with colorectal liver metastases (CLM) treated with bevacizumab-based chemotherapy followed by curative liver resection. Intratumoral mRNA was isolated from resected bevacizumab-pretreated CLM from 125 patients. In 42 patients, a matching primary tumor sample collected before bevacizumab treatment was available. Relative mRNA levels of 9 genes (ACVRL1, EGFL7, EPHB4, HIF1A, VEGFA, VEGFB, VEGFC, FLT1, and KDR) were analyzed by RT-PCR and evaluated for associations with response, RFS, and OS. P values for the associations between the individual dichotomized expression level and RFS were adjusted for choosing the optimal cut-off value. In CLM, high expression of VEGFB, VEGFC, HIF1A, and KDR and low expression of EGFL7 were associated with favorable RFS in multivariable analysis (P < 0.05). High ACVRL1 levels predicted favorable 3-year OS (P = 0.041) and radiologic response (PR = 1.093, SD = 0.539, P = 0.002). In primary tumors, low VEGFA and high EGFL7 were associated with radiologic and histologic response (P < 0.05). High VEGFA expression predicted shorter RFS (10.1 vs. 22.6 months; HR = 2.83, P = 0.038). High VEGFB (46% vs. 85%; HR = 5.75, P = 0.009) and low FLT1 (55% vs. 100%; P = 0.031) predicted lower 3-year OS rates. Our data suggest that intratumoral mRNA expression of genes involved in vascular morphogenesis and early vessel maturation may be promising predictive and/or prognostic biomarkers. Mol Cancer Ther; 15(11); 2814–21. ©2016 AACR.

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...

Expanded KRAS and NRAS assays increase detection of mutations that predict for resistance to therapy in colorectal cancer patients.

519 Background: The selection of targeted therapies is guided by the analysis of somatic mutations. The identification of RAS activating mutations can be used to examine tyrosine kinase inhibitor therapeutic eligibility and prognosis. Beyond known mutations in RAS exon 2 (codons 12 and 13), the identification of additional mutations in RAS exons 3 and 4 (codons 61, 117 and 146) also predict for resistance to EGFR therapy in colorectal cancer (CRC). Meta-analysis supports screening for these additional mutations in any screening strategy prior to administration of EGFR mAb therapy in metastatic CRC patients (Sorich, MJ, et al., Ann Oncol, Aug 12, 2014). Methods: We expanded our existing allele-specific KRAS and sequencing-based NRAS assays to include codons 61, 117, and 146 and analytically validated these assays to CAP/CLIA standards. DNA, from microdissected colon tumor tissue that was wild-type for RAS exon 2, was tested for exon 3 and 4 mutations and included over 15 additional mutations. Results: Fort...