Paula N. Friedman

Predictors of Resistance to Preoperative Trastuzumab and Vinorelbine for HER2-Positive Early Breast Cancer

Purpose: To assess pathologic complete response (pCR), clinical response, feasibility, safety, and potential predictors of response to preoperative trastuzumab plus vinorelbine in patients with operable, human epidermal growth factor receptor 2 (HER2)–positive breast cancer. Experimental Design: Forty-eight patients received preoperative trastuzumab and vinorelbine weekly for 12 weeks. Single and multigene biomarker studies were done in an attempt to identify predictors of response. Results: Eight of 40 (20%) patients achieved pCR (95% confidence interval, 9-36%). Of 9 additional patients recruited for protocol-defined toxicity analysis, 8 were evaluable; 42 of 48 (88%) patients had clinical response (16 patients, clinical complete response; 26 patients, clinical partial response). T1 tumors more frequently exhibited clinical complete response (P = 0.05) and showed a trend to exhibit pCR (P = 0.07). Five (13%) patients experienced grade 1 cardiac dysfunction during preoperative treatment. Neither HER2 nor estrogen receptor status changed significantly after exposure to trastuzumab and vinorelbine. RNA profiling identified three top-level clusters by unsupervised analysis. Tumors with extremes of response [pCR (n = 3) versus nonresponse (n = 3)] fell into separate groups by hierarchical clustering. No predictive genes were identified in pCR tumors. Nonresponding tumors were more likely to be T4 stage (P = 0.02) and express basal markers (P < 0.00001), growth factors, and growth factor receptors. Insulin-like growth factor-I receptor membrane expression was associated with a lower response rate (50% versus 97%; P = 0.001). Conclusions: Preoperative trastuzumab plus vinorelbine is active and well tolerated in patients with HER2-positive, operable, stage II/III breast cancer. HER2-overexpressing tumors with a basal-like phenotype, or with expression of insulin-like growth factor-I receptor and other proteins involved in growth factor pathways, are more likely to be resistant to this regimen.

A Genome-Wide Association Study Identifies Novel Loci for Paclitaxel-Induced Sensory Peripheral Neuropathy in CALGB 40101

Purpose: Sensory peripheral neuropathy is a common and sometimes debilitating toxicity associated with paclitaxel therapy. This study aims to identify genetic risk factors for the development of this toxicity. Experimental Design: A prospective pharmacogenetic analysis of patients with primary breast cancer, randomized to the paclitaxel arm of CALGB 40101, was used to identify genetic predictors of the onset and severity of sensory peripheral neuropathy. A genome-wide association study in 855 subjects of European ancestry was conducted and findings were replicated in additional European (n = 154) and African American (n = 117) subjects. Results: A single nucleotide polymorphism in FGD4 was associated with the onset of sensory peripheral neuropathy in the discovery cohort [rs10771973; HR, 1.57; 95% confidence interval (CI), 1.30–1.91; P = 2.6 × 10−6] and in a European (HR, 1.72; 95% CI, 1.06–2.80; P = 0.013) and African American (HR, 1.93; 95% CI, 1.13–3.28; P = 6.7 × 10−3) replication cohort. There is also evidence that markers in additional genes, including EPHA5 (rs7349683) and FZD3 (rs10771973), were associated with the onset or severity of paclitaxel-induced sensory peripheral neuropathy. Conclusions: A genome-wide association study has identified novel genetic markers of paclitaxel-induced sensory peripheral neuropathy, including a common polymorphism in FGD4, a congenital peripheral neuropathy gene. These findings suggest that genetic variation may contribute to variation in development of this toxicity. Validation of these findings may allow for the identification of patients at increased risk of peripheral neuropathy and inform the use of an alternative to paclitaxel and/or the clinical management of this toxicity. Clin Cancer Res; 18(18); 5099–109. ©2012 AACR.

Biologic Determinants of Tumor Recurrence in Stage II Colon Cancer: Validation Study of the 12-Gene Recurrence Score in Cancer and Leukemia Group B (CALGB) 9581

PURPOSE A greater understanding of the biology of tumor recurrence should improve adjuvant treatment decision making. We conducted a validation study of the 12-gene recurrence score (RS), a quantitative assay integrating stromal response and cell cycle gene expression, in tumor specimens from patients enrolled onto Cancer and Leukemia Group B (CALGB) 9581. PATIENTS AND METHODS CALGB 9581 randomly assigned 1,713 patients with stage II colon cancer to treatment with edrecolomab or observation and found no survival difference. The analysis reported here included all patients with available tissue and recurrence (n = 162) and a random (approximately 1:3) selection of nonrecurring patients. RS was assessed in 690 formalin-fixed paraffin-embedded tumor samples with quantitative reverse transcriptase polymerase chain reaction by using prespecified genes and a previously validated algorithm. Association of RS and recurrence was analyzed by weighted Cox proportional hazards regression. RESULTS Continuous RS was significantly associated with risk of recurrence (P = .013) as was mismatch repair (MMR) gene deficiency (P = .044). In multivariate analyses, RS was the strongest predictor of recurrence (P = .004), independent of T stage, MMR, number of nodes examined, grade, and lymphovascular invasion. In T3 MMR-intact (MMR-I) patients, prespecified low and high RS groups had average 5-year recurrence risks of 13% (95% CI, 10% to 16%) and 21% (95% CI, 16% to 26%), respectively. CONCLUSION The 12-gene RS predicts recurrence in stage II colon cancer in CALGB 9581. This is consistent with the importance of stromal response and cell cycle gene expression in colon tumor recurrence. RS appears to be most discerning for patients with T3 MMR-I tumors, although markers such as grade and lymphovascular invasion did not add value in this subset of patients.

Ethics of Mandatory Research Biopsy for Correlative End Points Within Clinical Trials in Oncology

Clinical investigators in oncology are increasingly interested in using molecular analysis of cancer tissue to understand the biologic bases of response or resistance to novel interventions and to develop prognostic and predictive biomarkers that will guide clinical decision making. Some scientific questions of this nature can only be addressed, or may best be addressed, through the conduct of a clinical trial in which research biopsies are obtained from all participants. However, trial designs with mandatory research biopsies have raised ethical concerns related to the risk of harm to participants, the adequacy of voluntary informed consent, and the potential for misunderstanding among research participants when access to an experimental intervention is linked to the requirement to undergo a research biopsy. In consideration of the ethical and scientific issues at stake in this debate, the Cancer and Leukemia Group B Ethics Committee proposes guidelines for clinical trials involving mandatory research biopsies. Any cancer clinical trial that requires research biopsies of participants must be well designed to address the scientific question, obtain the biopsy in a way that minimizes risk, and ensure that research participants are fully informed of the risks, rationale, and requirements of the study, as well as of treatment alternatives. Further guidelines and discussions of this issue are specified in this position paper. We feel that if these principles are respected, an informed adult with cancer can both understand and voluntarily consent to participation in a clinical trial involving mandatory research biopsy for scientific end points.

A genome-wide association study of overall survival in pancreatic cancer patients treated with gemcitabine in CALGB 80303

Background and Aims: Cancer and Leukemia Group B 80303 was a randomized, phase III study in patients with advanced pancreatic cancer treated with gemcitabine plus either bevacizumab or placebo. We prospectively collected germline DNA and conducted a genome-wide association study (GWAS) using overall survival (OS) as the endpoint. Experimental Design: DNA from 351 patients was genotyped for more than 550,000 single-nucleotide polymorphisms (SNP). Associations between OS and SNPs were investigated using the log-linear 2-way multiplicative Cox proportional hazards model. The subset of 294 genetically European patients was used for the primary analysis. Results: A nonsynonymous SNP in interleukin (IL)17F (rs763780, H161R) and an intronic SNP in strong linkage disequilibrium (rs7771466) were associated with OS using genome-wide criteria (P ≤ 10−7). Median OS was significantly shorter (P = 2.61 × 10−8) for the rs763780 heterozygotes [3.1 months; 95% confidence interval (CI), 2.3–4.3] than for the patients without this variant (6.8 months; 95% CI, 5.8–7.3). After adjustment by stratification factors, the P value for the association was 9.51 × 10−7. Conclusions: The variant 161R form of IL-17F is a natural antagonist of the antiangiogenic effects of wild-type 161H IL-17F, and angiogenesis may play an important role in the metastatic spread of pancreatic cancer. In this preliminary study, we hypothesize that the angiogenetic potential of pancreatic cancers in patients with variant IL-17F is higher than that of tumors in patients with wild-type IL-17F, conferring worse prognosis. This exploratory GWAS may provide the foundation for testing the biology and clinical effects of novel genes and their heritable variants through mechanistic and confirmatory studies in pancreatic cancer. Clin Cancer Res; 18(2); 577–84. ©2011 AACR.

Prognostic and Predictive Blood-Based Biomarkers in Patients with Advanced Pancreatic Cancer: Results from CALGB80303 (Alliance)

Purpose: CALGB80303 was a phase III trial of 602 patients with locally advanced or metastatic pancreatic cancer comparing gemcitabine/bevacizumab versus gemcitabine/placebo. The study found no benefit in any outcome from the addition of bevacizumab to gemcitabine. Blood samples were collected and multiple angiogenic factors were evaluated and then correlated with clinical outcome in general (prognostic markers) and with benefit specifically from bevacizumab treatment (predictive markers). Experimental Design: Plasma samples were analyzed via a novel multiplex ELISA platform for 31 factors related to tumor growth, angiogenesis, and inflammation. Baseline values for these factors were correlated with overall survival (OS) using univariate Cox proportional hazard regression models and multivariable Cox regression models with leave-one-out cross validation. Predictive markers were identified using a treatment by marker interaction term in the Cox model. Results: Baseline plasma was available from 328 patients. Univariate prognostic markers for OS were identified including: Ang2, CRP, ICAM-1, IGFBP-1, TSP-2 (all P < 0.001). These prognostic factors were found to be highly significant, even after adjustment for known clinical factors. Additional modeling approaches yielded prognostic signatures from multivariable Cox regression. The gemcitabine/bevacizumab signature consisted of IGFBP-1, interleukin-6, PDGF-AA, PDGF-BB, TSP-2; whereas the gemcitabine/placebo signature consisted of CRP, IGFBP-1, PAI-1, PDGF-AA, P-selectin (both P < 0.0001). Finally, three potential predictive markers of bevacizumab efficacy were identified: VEGF-D (P < 0.01), SDF1 (P < 0.05), and Ang2 (P < 0.05). Conclusion: This study identified strong prognostic markers for pancreatic cancer patients. Predictive marker analysis indicated that plasma levels of VEGF-D, Ang2, and SDF1 significantly predicted for benefit or lack of benefit from bevacizumab in this population. Clin Cancer Res; 19(24); 6957–66. ©2013 AACR.

Topoisomerase IIα Amplification Does Not Predict Benefit From Dose-Intense Cyclophosphamide, Doxorubicin, and Fluorouracil Therapy in HER2-Amplified Early Breast Cancer: Results of CALGB 8541/150013

PURPOSE We have demonstrated that patients with HER2-amplified tumors derive more benefit from higher doses of doxorubicin-containing chemotherapy (cyclophosphamide, doxorubicin, and fluorouracil [CAF]). Because topoisomerase IIalpha (Topo-IIalpha) is a target for doxorubicin and is coamplified in 20% to 50% of HER2-amplified tumors, we postulated that Topo-IIalpha copy number might account for the benefit from CAF dose escalation in HER2-positive tumors. To address this hypothesis, we examined Topo-IIalpha and HER2 copy number, CAF dose, and clinical outcomes in Cancer and Leukemia Group B (CALGB) 8541. PATIENTS AND METHODS Topo-IIalpha and HER2 copy number were measured by fluorescent in situ hybridization (FISH) using a triple-probe system, which includes Topo-IIalpha, HER2, and chromosome 17 (CEP17). Topo-IIalpha and/or HER2 were classified as amplified (> or = two copies/CEP17, deleted (< or = 0.67 copies/CEP17) and normal copy number (> .67 to < 2.0 copies/CEP17). Results Topo-IIalpha/HER2/CEP17 measurement was successful in 624 of 687 cases. HER2 was amplified in 117 cases (19%). Topo-IIalpha was amplified in 41 cases (7%) and deleted in 69 cases (11%). Topo-IIalpha amplification was highly correlated with HER2 amplification (39 of 41; P < .0001), HER2 by immunohistochemistry, and by dual-probe FISH. Topo-IIalpha was deleted in both the HER2-amplified (30 of 69; 43%), normal (22 of 69; 32%) and HER2-deleted tumors (17 of 69; 25%). Although Topo-IIalpha-amplified tumors were nearly always HER2 amplified, these tumors did not receive benefit from increasing the dose of CAF (P = .15). CONCLUSION The correlative companion study CALGB 8541-150013 does not support the hypothesis that Topo-IIalpha amplification is the mechanism behind benefit from increased dose of anthracyclines in HER2-positive breast cancer.

Identification and Immunohistochemical Characterization of a Mucin-Like Glycoprotein Expressed in Early Stage Breast Carcinoma

In this report we describe a cDNA sequence, BS106, identified from Incyte Genomics LifeSeq® Expressed Sequence Tag database. A multi-tissue mRNA expression array, northern blots, and RT-PCR assays demonstrate the expression of BS106 in mammary, salivary and prostate glands, but not in other tissue types. BS106 mRNA was detected in 90% of the breast tissues examined. The cDNA encodes a 90-amino acid protein characterized as a small, mucin-like protein based on amino acid composition, extensive O-linked glycosylation, and expression profile. BS106 protein was recombinantly expressed in human embryonic kidney 293 cells and the secreted product was purified from the culture media. Monoclonal antibodies were prepared and used for immunohistochemical analysis of early stage breast cancer. BS106 protein was detected in the vast majority of carcinomas (70–100%) and overexpressed in approximately 30% of the 22 specimens analyzed. BS106 protein was not detected in other solid tumor types including bladder carcinoma, colon carcinoma, endometrial carcinoma, gastric carcinoma, squamous cell lung carcinoma, adenocarcinoma of the lung, ovarian carcinoma, pancreatic and prostatic carcinoma.

Ethical Aspects of Participation in the Database of Genotypes and Phenotypes of the National Center For Biotechnology Information The Cancer and Leukemia Group B Experience

The rapid pace of genetics research, coupled with evolving standards for informed consent, can create ethical challenges regarding future use of tissue or information from completed clinical trials. The Cancer and Leukemia Group B (CALGB) Oncology Cooperative Group was faced with an ethical dilemma regarding sharing genetic data from a completed genome‐wide association study (GWAS) that was conducted as part of a large, multicenter breast cancer clinical trial with a national database: the Database of Genotypes and Phenotypes National Center for Biotechnology Information (dbGaP).

Detection of GB virus C by the RT-PCR LCx® system

The recent publication of representative genomic sequences of GBV-C has permitted the selection of PCR primers for detection of GBV-C in clinical samples by PCR techniques. Traditional amplification methodologies which couple reverse transcription polymerase chain reaction (RT-PCR) and Southern blot detection are slow, cumbersome, and can be technique dependent. This has hampered studies to determine the clinical significance of GBV-C. We report the selection of highly conserved PCR primers and a probe useful for semi-automated RT-PCR using the Abbott LCx system. This adaptation of the LCx system expands its capabilities to include the detection of RNA by RT-PCR, in addition to DNA detection by ligase chain reaction (LCR).