Ann E. McCullough

Integrated genomic characterization reveals novel, therapeutically relevant drug targets in FGFR and EGFR pathways in sporadic intrahepatic cholangiocarcinoma.

Advanced cholangiocarcinoma continues to harbor a difficult prognosis and therapeutic options have been limited. During the course of a clinical trial of whole genomic sequencing seeking druggable targets, we examined six patients with advanced cholangiocarcinoma. Integrated genome-wide and whole transcriptome sequence analyses were performed on tumors from six patients with advanced, sporadic intrahepatic cholangiocarcinoma (SIC) to identify potential therapeutically actionable events. Among the somatic events captured in our analysis, we uncovered two novel therapeutically relevant genomic contexts that when acted upon, resulted in preliminary evidence of anti-tumor activity. Genome-wide structural analysis of sequence data revealed recurrent translocation events involving the FGFR2 locus in three of six assessed patients. These observations and supporting evidence triggered the use of FGFR inhibitors in these patients. In one example, preliminary anti-tumor activity of pazopanib (in vitro FGFR2 IC50≈350 nM) was noted in a patient with an FGFR2-TACC3 fusion. After progression on pazopanib, the same patient also had stable disease on ponatinib, a pan-FGFR inhibitor (in vitro, FGFR2 IC50≈8 nM). In an independent non-FGFR2 translocation patient, exome and transcriptome analysis revealed an allele specific somatic nonsense mutation (E384X) in ERRFI1, a direct negative regulator of EGFR activation. Rapid and robust disease regression was noted in this ERRFI1 inactivated tumor when treated with erlotinib, an EGFR kinase inhibitor. FGFR2 fusions and ERRFI mutations may represent novel targets in sporadic intrahepatic cholangiocarcinoma and trials should be characterized in larger cohorts of patients with these aberrations.

Adjuvant Lapatinib and Trastuzumab for Early Human Epidermal Growth Factor Receptor 2–Positive Breast Cancer: Results From the Randomized Phase III Adjuvant Lapatinib and/or Trastuzumab Treatment Optimization Trial

BACKGROUND Lapatinib (L) plus trastuzumab (T) improves outcomes for metastatic human epidermal growth factor 2-positive breast cancer and increases the pathologic complete response in the neoadjuvant setting, but their role as adjuvant therapy remains uncertain. METHODS In the Adjuvant Lapatinib and/or Trastuzumab Treatment Optimization trial, patients with centrally confirmed human epidermal growth factor 2-positive early breast cancer were randomly assigned to 1 year of adjuvant therapy with T, L, their sequence (T→L), or their combination (L+T). The primary end point was disease-free survival (DFS), with 850 events required for 80% power to detect a hazard ratio (HR) of 0.8 for L+T versus T. RESULTS Between June 2007 and July 2011, 8,381 patients were enrolled. In 2011, due to futility to demonstrate noninferiority of L versus T, the L arm was closed, and patients free of disease were offered adjuvant T. A protocol modification required P ≤ .025 for the two remaining pairwise comparisons. At a protocol-specified analysis with a median follow-up of 4.5 years, a 16% reduction in the DFS hazard rate was observed with L+T compared with T (555 DFS events; HR, 0.84; 97.5% CI, 0.70 to 1.02; P = .048), and a 4% reduction was observed with T→L compared with T (HR, 0.96; 97.5% CI, 0.80 to 1.15; P = .61). L-treated patients experienced more diarrhea, cutaneous rash, and hepatic toxicity compared with T-treated patients. The incidence of cardiac toxicity was low in all treatment arms. CONCLUSION Adjuvant treatment that includes L did not significantly improve DFS compared with T alone and added toxicity. One year of adjuvant T remains standard of care.

Genomic Analysis Reveals That Immune Function Genes Are Strongly Linked to Clinical Outcome in the North Central Cancer Treatment Group N9831 Adjuvant Trastuzumab Trial

PURPOSE To develop a genomic signature that predicts benefit from trastuzumab in human epidermal growth factor receptor 2-positive breast cancer. PATIENTS AND METHODS DASL technology was used to quantify mRNA in samples from 1,282 patients enrolled onto the Combination Chemotherapy With or Without Trastuzumab in Treating Women With Breast Cancer (North Central Cancer Treatment Group N9831 [NCCTG-N9831]) adjuvant trastuzumab trial. Cox proportional hazard ratios (HRs), adjusted for significant clinicopathologic risk factors, were used to determine the association of each gene with relapse-free survival (RFS) for 433 patients who received chemotherapy alone (arm A) and 849 patients who received chemotherapy plus trastuzumab (arms B and C). Network and pathway analyses were used to identify key biologic processes linked to RFS. The signature was built by using a voting scheme. RESULTS Network and functional ontology analyses suggested that increased RFS was linked to a subset of immune function genes. A voting scheme model was used to define immune gene enrichment based on the expression of any nine or more of 14 immune function genes at or above the 0.40 quantile for the population. This model was used to identify immune gene-enriched tumors in arm A and arms B and C. Immune gene enrichment was linked to increased RFS in arms B and C (HR, 0.35; 95% CI, 0.22 to 0.55; P < .001), whereas arm B and C patients who did not exhibit immune gene enrichment did not benefit from trastuzumab (HR, 0.89; 95% CI, 0.62 to 1.28; P = .53). Enriched immune function gene expression as defined by our predictive signature was not associated with increased RFS in arm A (HR, 0.90; 95% CI, 0.60 to 1.37; P = .64). CONCLUSION Increased expression of a subset of immune function genes may provide a means of predicting benefit from adjuvant trastuzumab.

Impact of PTEN Protein Expression on Benefit From Adjuvant Trastuzumab in Early-Stage Human Epidermal Growth Factor Receptor 2–Positive Breast Cancer in the North Central Cancer Treatment Group N9831 Trial

PURPOSE It has been suggested that PTEN, a negative regulator of PI3K/AKT signaling, is involved in tumor sensitivity to trastuzumab. We investigated the association between tumor PTEN protein expression and disease-free survival (DFS) of patients randomly assigned to receive chemotherapy alone (arm A) or chemotherapy with sequential (arm B) or concurrent trastuzumab (arm C) in the phase III early-stage human epidermal growth factor receptor 2 (HER2) -positive trial-North Central Cancer Treatment Group (NCCTG) N9831. PATIENTS AND METHODS The intensity and percentage of invasive cells with cytoplasmic PTEN staining were determined in tissue microarray sections containing three cores per block (n = 1,286) or in whole tissue sections (WS; n = 516) by using standard immunohistochemistry (138G6 monoclonal antibody). Tumors were considered positive for PTEN (PTEN-positive) if any core or WS had any invasive cells with ≥ 1+ staining. Median follow-up was 6.0 years. RESULTS Of 1,802 patients included in this analysis (of 3,505 patients registered to N9831), 1,342 (74%) had PTEN-positive tumors. PTEN positivity was associated with hormone receptor negativity (χ(2) P < .001) and nodal positivity (χ(2) P = .04). PTEN did not have an impact on DFS within the various arms. Comparing DFS of arm C to arm A, patients with PTEN-positive and PTEN-negative tumors had hazard ratios (HRs) of 0.65 (P = .003) and 0.47 (P = .005), respectively (interaction P = .16). For arm B versus arm A, patients with PTEN-positive and PTEN-negative tumors had HRs of 0.70 (P = .009) and 0.85 (P = .44), respectively (interaction P = .47). CONCLUSION In contrast to selected preclinical and limited clinical studies suggesting a decrease in trastuzumab sensitivity in patients with PTEN-negative tumors, our data show benefit of adjuvant trastuzumab for patients with HER2-positive breast cancer, independent of tumor PTEN status.

MR Imaging of Hypervascular Liver Masses: A Review of Current Techniques

Major technologic advances in magnetic resonance (MR) imaging, including the advent of novel pulse sequences (eg, diffusion-weighted and steady-state free precession sequences) and the use of hepatocyte-specific contrast agents, have led to better image quality and shorter acquisition times, resulting in dramatic improvements in the noninvasive detection and characterization of hepatic lesions, particularly hypervascular neoplasms. However, as the role of MR imaging in clinical evaluation of the liver continues to evolve, keeping abreast of new developments can be daunting as well as confusing. A systematic approach that makes use of a simple decision algorithm can help differentiate hypervascular hepatic lesions on the basis of their distinguishing MR imaging characteristics and related clinical information.

Predictability of Adjuvant Trastuzumab Benefit in N9831 Patients Using the ASCO/CAP HER2-Positivity Criteria

The 2007 American Society of Clinical Oncology and College of American Pathologists (ASCO/CAP) joint guidelines defined criteria for HER2 positivity of tumors that modified those of the US Food and Drug Administration (FDA), causing some confusion and uncertainty among clinicians. Using data from the HER2-positive breast cancer adjuvant trial N9831, we compared eligibility for patients who met both criteria, and disease-free survival (DFS) was assessed by Cox proportional hazards regression. The number of patients in the N9831 trial retrospectively eligible for trastuzumab therapy was decreased when ASCO/CAP criteria vs FDA criteria were applied to immunohistochemistry and/or fluorescence in situ hybridization results (107 [3.7%] of 2904 patients with immunohistochemistry results, 37 [1.3%] of 2809 patients with fluorescence in situ hybridization results, and 47 [1.7%] of 2809 patients with both results). Improvement in DFS was similar among patients treated with trastuzumab under either set of criteria (concurrent trastuzumab and chemotherapy compared with chemotherapy alone: by ASCO/CAP criteria, hazard ratio of DFS = 0.59, 95% confidence interval = 0.48 to 0.73; by FDA criteria but not ASCO/CAP criteria, hazard ratio = 0.60, 95% confidence interval = 0.12 to 3.13; number needed to treat to prevent one additional DFS event at 5 years: 10 and 11.2 patients, respectively). Following the 2007 ASCO/CAP criteria for HER2 positivity would negate the option of potentially life-saving trastuzumab therapy for a small but meaningful group of patients. We recommend using FDA-approved HER2 criteria for therapeutic decision making.

Genomic amplification of 9p24.1 targeting JAK2, PD-L1, and PD-L2 is enriched in high-risk triple negative breast cancer

We used DNA content flow cytometry followed by oligonucleotide array based comparative genomic hybridization to survey the genomes of 326 tumors, including 41 untreated surgically resected triple negative breast cancers (TNBC). A high level (log2ratio ≥1) 9p24 amplicon was found in TNBC (12/41), glioblastomas (2/44), and colon carcinomas (2/68). The shortest region of overlap for the amplicon targets 9p24.1 and includes the loci for PD-L1, PD-L2, and JAK2 (PDJ amplicon). In contrast this amplicon was absent in ER+ (0/8) and HER2+ (0/15) breast tumors, and in pancreatic ductal adenocarcinomas (0/150). The PDJ amplicon in TNBCs was correlated with clinical outcomes in group comparisons by two-sample t-tests for continuous variables and chi-squared tests for categorical variables. TNBC patients with the PDJ amplicon had a worse outcome with worse disease-free and overall survival. Quantitative RT-PCR confirmed that the PDJ amplicon in TNBC is associated with elevated expression of JAK2 and of the PD-1 ligands. These initial findings demonstrate that the PDJ amplicon is enriched in TNBC, targets signaling pathways that activate the PD-1 mediated immune checkpoint, and identifies patients with a poor prognosis.

C-MYC Alterations and Association With Patient Outcome in Early-Stage HER2-Positive Breast Cancer From the North Central Cancer Treatment Group N9831 Adjuvant Trastuzumab Trial

PURPOSE Findings from the human epidermal growth factor receptor 2 (HER2) -positive National Surgical Adjuvant Breast and Bowel Project (NSABP) B31 trial suggested that MYC/HER2 coamplification (> 5.0 copies/nucleus) was associated with additional benefit from adjuvant trastuzumab in patients with early-stage breast cancer. To further explore this relationship, we investigated associations between MYC amplification and disease-free survival (DFS) in a similar adjuvant trastuzumab HER2-positive breast cancer trial-North Central Cancer Treatment Group (NCCTG) N9831. PATIENTS AND METHODS This analysis included 799 patients randomly assigned to receive chemotherapy alone or with concurrent trastuzumab on N9831. Fluorescence in situ hybridization (FISH) was performed by using a dual-probe mixture for MYC and centromere 8 (MYC:CEP8) on tissue microarrays. MYC amplification was prespecified as MYC:CEP8 ratio > 2.2 or average MYC copies/nucleus > 5.0. Exploratory variables included polysomy 8. RESULTS In comparing DFS (median follow-up, 4.0 years) between treatments, patients with MYC:CEP8 ratio ≤ 2.2 (n = 618; 77%) and > 2.2 (n = 181; 23%) had hazard ratios (HRs) of 0.46 (P < .001) and 0.67 (P = .33), respectively (interaction P = .38). Patients with MYC copies/nucleus ≤ 5.0 (n = 534; 67%) and > 5.0 (n = 265; 33%) had HRs of 0.52 (P = .002) and 0.48 (P = .02), respectively (interaction P = .94). Patients with MYC:CEP8 ratio < 1.3 with normal chromosome 8 copy number (n = 141; 18%) and ≥ 1.3 or < 1.3 with polysomy 8 (n = 658; 82%) had HRs of 0.66 (P = .28) and 0.44 (P < .001), respectively (interaction P = .23). Patients with MYC copies/nucleus < 2.5 (n = 130; 16%) and ≥ 2.5 (n = 669; 84%) had HRs of 1.07 (P = .87) and 0.42 (P < .001), respectively (interaction P = .05). CONCLUSION We did not confirm the B31 association between MYC amplification and additional trastuzumab benefit. Exploratory analyses revealed potential associations between alternative MYC/chromosome 8 copy number alterations and differential benefit of adjuvant trastuzumab.

Deep Clonal Profiling of Formalin Fixed Paraffin Embedded Clinical Samples

Formalin fixed paraffin embedded (FFPE) tissues are a vast resource of annotated clinical samples. As such, they represent highly desirable and informative materials for the application of high definition genomics for improved patient management and to advance the development of personalized therapeutics. However, a limitation of FFPE tissues is the variable quality of DNA extracted for analyses. Furthermore, admixtures of non-tumor and polyclonal neoplastic cell populations limit the number of biopsies that can be studied and make it difficult to define cancer genomes in patient samples. To exploit these valuable tissues we applied flow cytometry-based methods to isolate pure populations of tumor cell nuclei from FFPE tissues and developed a methodology compatible with oligonucleotide array CGH and whole exome sequencing analyses. These were used to profile a variety of tumors (breast, brain, bladder, ovarian and pancreas) including the genomes and exomes of matching fresh frozen and FFPE pancreatic adenocarcinoma samples.

Progression of Pelvic Implants to Complex Atypical Endometrial Hyperplasia After Uterine Morcellation

BACKGROUND: Supracervical hysterectomy, a surgical technique that involves removal of the uterus while preserving the cervix, places women at an increased risk for future morbidity. CASE: The patient presented 7 years after laparoscopic supracervical hysterectomy for benign disease, with pelvic pain, urinary frequency, abdominal bloating, and tenderness. Surgical exploration revealed myometrial implants, along with a larger mass on the mesentery of the sigmoid composed of endometrium with complex atypical hyperplasia in a patient without a prior history of complex atypical hyperplasia. The patient was symptom-free 6 months postoperatively. CONCLUSION: Histology found during reoperation after laparoscopic supracervical hysterectomy includes endometriosis, residual endometrium, and leiomyomas. In this case, a woman with no prior history of endometrial atypia later developed complex adenomatous endometrial hyperplasia in a peritoneal implant. This finding adds to the list of possible complications after laparoscopic-assisted supracervical hysterectomy and uterine morcellation.