Lydia Usha

A Gynecologic Oncology Group phase II trial of the protein kinase C-beta inhibitor, enzastaurin and evaluation of markers with potential predictive and prognostic value in persistent or recurrent epithelial ovarian and primary peritoneal malignancies

OBJECTIVES Protein kinase C (PKC) activation contributes to proliferation and angiogenesis in epithelial ovarian or primary peritoneal carcinoma (EOC/PPC). A multi-institutional phase II trial was conducted to evaluate the efficacy and safety of PKCβ inhibitor enzastaurin in persistent or recurrent EOC/PPC and to explore potential prognostic and predictive biomarkers. METHODS Eligible women with measurable platinum-sensitive and resistant EOC/PPC were treated with continuous administration of oral enzastaurin until disease progression or unacceptable toxicity. A two-stage sequential design was used to evaluate progression-free survival (PFS) ≥6-months, tumor response, and toxicity. Translational studies included sequencing of the TP53, PTEN, PIK3CA and PKCβII genes for somatic mutations, quantitative PCR assays for AKT2 and PTEN copy number alterations, and measurement of circulating VEGF-A plasma levels. RESULTS Among 27 eligible and evaluable patients, 3 women with PFS≥6-months (11%) and 2 women with partial responses (7%) were observed. One of them achieved a durable response and remains on the study. No grade 4 adverse events were observed. Most common grade 3 adverse events were constitutional (4) and gastrointestinal (3). Mutations in the TP53 gene and abnormal copy number in the PTEN gene were common (56% and 48% of cases, respectively). CONCLUSIONS Enzastaurin was tolerable but had insufficient activity to proceed with the second stage of accrual. However, 1 patient has been progression-free for 44 months. No association between a biomarker and response to enzastaurin has been found. Exploratory analysis suggested an association between survival and PTEN copy number losses.

Effects of ERBB2 amplicon size and genomic alterations of chromosomes 1, 3, and 10 on patient response to trastuzumab in metastatic breast cancer

Trastuzumab is widely used for advanced breast cancer patients with ERBB2‐amplified tumors. Nevertheless, over half of these patients do not have an objective response. One reason may be altered expression of genes that might compensate for ERBB2 inhibition. We previously mapped the gene‐rich region of chromosome 17 telomeric to ERBB2, and reported considerable variability in the telomeric extent of the ERBB2 amplicon. Here we examined whether the variable amplicon size may be associated with patient response to trastuzumab. In addition, we looked at associations between response and several signaling pathway‐related genes unrelated to the ERBB2 amplicon, including AKT3, PTEN, PIK3CA, and PTGS2. In 35 patients with ERBB2‐amplified metastatic breast cancer, with 40% overall response to trastuzumab, fluorescence in situ hybridization identified the telomeric extent of the ERBB2 amplicon and the status of the several pathway‐related genes. Objective response strongly correlated with the telomeric amplicon size, with 62% of patients with shorter amplicons responding, compared with only 7% of patients with longer amplicons (P = 0.0015). Abnormal copy number of PTGS2 was marginally associated with objective response (P = 0.066), while abnormal copy numbers of two reference loci, 1q25 and the chromosome 10 centromere, were significantly associated with response. Pairwise combinations of copy number status of these loci and ERBB2 amplicon size provided stronger associations and identified a group of patients without responders. These results suggest that patient selection for trastuzumab may be improved by considering ERBB2 amplicon size and genomic status of the 1q25, PTGS2, and centromere 10 loci.

Ribonucleotide reductase inhibition restores platinum-sensitivity in platinum-resistant ovarian cancer: a Gynecologic Oncology Group Study

BackgroundThe potent ribonucleotide reductase (RNR) inhibitor 3-aminopyridine-2-carboxyaldehyde-thiosemicarbazone (3-AP) was tested as a chemosensitizer for restored cisplatin-mediated cytotoxicity in platinum-resistant ovarian cancer.MethodsPreclinical in vitro platinum-resistant ovarian cancer cell survival, RNR activity, and DNA damage assays were done after cisplatin or cisplatin plus 3-AP treatments. Six women with platinum-resistant ovarian cancer underwent four-day 3-AP (96 mg/m2, day one to four) and cisplatin (25 mg/m2, day two and three) infusions every 21 days until disease progression or adverse effects prohibited further therapy. Pre-therapy ovarian cancer tissues were analyzed by immunohistochemistry for RNR subunit expression as an indicator of cisplatin plus 3-AP treatment response.Results3-AP preceding cisplatin exposure in platinum-resistant ovarian cancer cells was not as effective as sequencing cisplatin plus 3-AP together in cell survival assays. Platinum-mediated DNA damage (i.e., γH2AX foci) resolved quickly after cisplatin-alone or 3-AP preceding cisplatin exposure, but persisted after a cisplatin plus 3-AP sequence. On trial, 25 four-day overlapping 3-AP and cisplatin cycles were administered to six women (median 4.2 cycles per patient). 3-AP-related methemoglobinemia (range seven to 10%) occurred in two (33%) of six women, halting trial accrual.ConclusionsWhen sequenced cisplatin plus 3-AP, RNR inhibition restored platinum-sensitivity in platinum-resistant ovarian cancers. 3-AP (96 mg/m2) infusions produced modest methemoglobinemia, the expected consequence of ribonucleotide reductase inhibitors disrupting collateral proteins containing iron.Trial registryClinicalTrials.gov NCT00081276

Inhibition of p70 S6 Kinase (S6K1) Activity by A77 1726 and Its Effect on Cell Proliferation and Cell Cycle Progress

Leflunomide is a novel immunomodulatory drug prescribed for treating rheumatoid arthritis. It inhibits the activity of protein tyrosine kinases and dihydroorotate dehydrogenase, a rate-limiting enzyme in the pyrimidine nucleotide synthesis pathway. Here, we report that A77 1726, the active metabolite of leflunomide, inhibited the phosphorylation of ribosomal protein S6 and two other substrates of S6K1, insulin receptor substrate-1 and carbamoyl phosphate synthetase 2, in an A375 melanoma cell line. A77 1726 increased the phosphorylation of AKT, p70 S6 (S6K1), ERK1/2, and MEK through the feedback activation of the IGF-1 receptor–mediated signaling pathway. Invitro kinase assay revealed that leflunomide and A77 1726 inhibited S6K1 activity with IC50 values of approximately 55 and 80 μM, respectively. Exogenous uridine partially blocked A77 1726–induced inhibition of A375 cell proliferation. S6K1 knockdown led to the inhibition of A375 cell proliferation but did not potentiate the antiproliferative effect of A77 1726. A77 1726 stimulated bromodeoxyuridine incorporation in A375 cells but arrested the cell cycle in the S phase, which was reversed by addition of exogenous uridine or by MAP kinase pathway inhibitors but not by rapamycin and LY294002 (a phosphoinositide 3-kinase inhibitor). These observations suggest that A77 1726 accelerates cell cycle entry into the S phase through MAP kinase activation and that pyrimidine nucleotide depletion halts the completion of the cell cycle. Our study identified a novel molecular target of A77 1726 and showed that the inhibition of S6K1 activity was in part responsible for its antiproliferative activity. Our study also provides a novel mechanistic insight into A77 1726–induced cell cycle arrest in the S phase.

Topoisomerase II alpha gene copy loss has adverse prognostic significance in ERBB2-amplified breast cancer: a retrospective study of paraffin-embedded tumor specimens and medical charts

BackgroundAmplification of the ERBB2 (Her-2/neu) oncogene, which occurs in approximately 25% of breast carcinomas, is a known negative prognostic factor. Available data indicate that a variable number of nearby genes on chromosome 17q may be co-amplified or deleted, forming a continuous amplicon of variable size. In approximately 25% of these patients, the amplicon extends to the gene for topoisomerase II alpha (TOP2A), a target for anthracyclines. We sought to understand the significance of these associated genomic changes for breast cancer prognosis and predicting response to therapy.Methods and patientsArchival tissue samples from 63 breast cancer patients with ERBB2 amplification, stages 0–IV, were previously analyzed with FISH probes for genes located near ERBB2. In the present study, the clinical outcome data were determined for all patients presenting at stages I–III for whom adequate clinical follow up was available.ResultsFour amplicon patterns (Classes) were identified. These were significantly associated with the clinical outcome, specifically, recurrence of breast cancer. The Amplicon class IV with deleted TOP2A had 67% (6/9) cases with recurrence, whereas the other three classes combined had only 12% (3/25) cases (p-value = 0.004) at the time of last follow-up. TOP2A deletion was also significantly associated with time to recurrence (p-value = 0.0002). After adjusting for age in Cox regression analysis, the association between TOP2A deletion and time to recurrence remains strongly significant (p-value = 0.002) whereas the association with survival is marginally significant (p-value = 0.06).ConclusionTOP2A deletion is associated with poor prognosis in ERBB2-amplified breast carcinomas. Clarification of the mechanism of this association will require additional study.

Mesenchymal Stem Cells Develop Tumor Tropism but Do Not Accelerate Breast Cancer Tumorigenesis in a Somatic Mouse Breast Cancer Model

The role of mesenchymal stem cells (MSCs) on breast cancer progression, growth and tumorigenesis remains controversial or unknown. In the present study, we investigated the role of MSCs on breast tumor induction and growth in a clinically relevant somatic breast cancer model. We first conducted in vitro studies and found that conditioned media (CM) of RCAS-Neu and RCAS-PyMT breast cancer cell lines and tumor cells themselves dramatically increased the proliferation and motility of MSCs and induced morphological changes of MSCs and differentiation into fibroblast-like cells. In contrast, the CM of MSCs inhibited the proliferation of two breast cancer cell lines by arresting the cell cycle at the G0/G1 phase. In vivo studies revealed that fluorescence dye-labeled MSCs migrated into tumor tissues. Unexpectedly, single or multiple intravenous injections of MSCs did not affect the latency of breast cancer in TVA- transgenic mice induced by intraductal injection of the RCAS vector encoding polyoma middle-T antigen (PyMT) or Neu oncogenes. Moreover, MSCs had no effect on RCAS-Neu tumor growth in a syngeneic ectopic breast cancer model. While our studies consistently demonstrated the ability of breast cancer cells to profoundly induce MSCs migration, differentiation, and proliferation, the anti-proliferative effect of MSCs on breast tumor cells observed in vitro could not be translated into an antitumor activity in vivo, probably reflecting the antagonizing or complex effects of MSCs on tumor environment and tumor cells themselves.

Radiosensitizing effect of anti-HER2/neu agents: Report of 2 cases and review of the literature

The human epidermal growth factor receptors (HERs), including HER1, HER2, HER3, and HER4, are transmembrane tyrosine kinase receptors involved in the regulation of cell growth and survival. Overexpression of HER type 2 (HER2) is seen in approximately 20% to 30% of invasive breast carcinomas. Trastuzumab and pertuzumab (Herceptin and Perjeta, Genentech-Roche, South San Francisco, CA) are humanized recombinant monoclonal antibodies that bind to the extracellular domain of the HER2 receptor and prevent activation of the downstream signaling cascade.1 Pertuzumab and trastuzumab bind to different HER2 epitopes, resulting in a greater antitumor effect through complementary mechanisms of action.2 Multiple randomized clinical trials have demonstrated the efficacy of trastuzumab, pertuzumab, and other antiHER2 agents in early-stage and metastatic HER2-positive breast cancer3-6; therefore, their use has become standard of care in the management of this breast cancer subtype. Trastuzumab remains the first and the best-studied agent in the class of anti-HER2 drugs. Adverse effects of trastuzumab and pertuzumab have been observed primarily when these antibodies were coadministered with chemotherapy. These adverse effects include infusion-

Tumor Screening and DNA Testing in the Diagnosis of Lynch Syndrome

A 43-year-old woman presented with abnormal vaginal bleeding. Endometrial carcinoma was found on dilation and curettage. Hysterectomy and surgical staging confirmed stage IA endometrial cancer. Family history was significant for prostate cancer in her father and paternal grandfather. Her tumor was screened for Lynch syndrome by immunohistochemistry staining for DNA mismatch repair proteins (mutL homolog 1 [MLH1]; MutS homologs 1 and 6 [MSH1, MSH6]; PMS 1 homolog 2 [PMS2]) and microsatellite instability testing (MSI)1 (Table 1).

Abstract 1730: Expression of serum biomarkers related to epithelial-to-mesenchymal transition in non-small cell lung cancer recurrence

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Objective: Recurrent disease in stage I non-small cell lung cancer (NSCLC) is primarily attributed to metastatic dissemination at the time of surgery undetected by current staging practices. We hypothesized that metastatic progression is driven by epithelial-to-mesenchymal transition (EMT) resulting in differences in tumor-shed protein biomarkers. The objective of this study was to evaluate the difference in expression of these biomarkers in patients with recurrent stage 1 NSCLC. Methods: We used the Luminex immunobead platform, including the MILLIPLEX map human angiogenesis/growth factor magnetic bead panel, to evaluate 80 biomarkers related to EMT against a total of 75 patients who underwent a complete anatomic resection. Patients were divided into the following cohorts: a) stage I NSCLC without recurrence in 2 years (n=50), and b) stage I NSCLC with recurrence within 2 years of follow up (n=25). Peripheral blood was collected and processed using standard phlebotomy techniques. Specimens were obtained in compliance with institutional IRB standards and consent. The Mann-Whitney test and receiver operator characteristics (ROC) curves were used to assess differences in biomarker concentrations between cohorts. Results: Univariate analysis revealed 19 biomarkers with significant (ROC >0.6) differences in expression between the patient cohorts including: angiopoietin 2, MCP-1, MIP-IB, TNF-R1, IGFBP-5, VEGF-D, IGF-1, IGFBP-3, follistatin, sICAM-1, sE-SELECTIN, CYFRA 21.1, RANTES, IL-Ira, M-CSF, IGFBP-1, IGFBP-6, HB-EGF, and PGF. The Mann-Whitney test revealed five biomarkers highly significant (p<0.05) for recurrence in stage 1 NSCLC, including MCP-1, VEGF-D, follistatin, sICAM-1, and placental growth factor (PGF). Evaluation of these biomarkers with the Ingenuity Pathway Analysis (IPA) suite identified several highly significant (p<1x10−5) biological themes, including ten IPA-defined processes associated with development (e.g. embryonic development and cardiovascular system development), seven processes associated with pathological processes (e.g. cancer, cell death, and respiratory disease), and seven processes associated with metastasis (e.g. cellular movement, immune cell trafficking, and cell-to-cell signaling and interaction). Random Forest analysis generated a 6-analyte panel consisting of MCP-1, IP-10, sICAM-1, IGFBP2, RANTES, and IGFBP3 that provided 71.1% classification accuracy with 66.1% sensitivity and 73.3% specificity. Conclusions: Here we report observations concerning the expression of EMT pathway members that may provide key insights into the role of circulating biomarkers related to recurrence in stage 1 NSCLC. Upon further validation, these biomarkers may serve as convenient surrogates to help guide molecular diagnostics and treatment strategies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1730. doi:1538-7445.AM2012-1730

Can chimerism explain breast/ovarian cancers in BRCA non-carriers from BRCA-positive families?

Hereditary breast and ovarian cancer syndrome (HBOC) is most frequently caused by mutations in BRCA1 or BRCA2 (in short, BRCA) genes. The incidence of hereditary breast and ovarian cancer in relatives of BRCA mutation carriers who test negative for the familial mutation (non-carriers) may be increased. However, the data is controversial, and at this time, these individuals are recommended the same cancer surveillance as general population. One possible explanation for BRCA phenocopies (close relatives of BRCA carriers who have developed cancer consistent with HBOC but tested negative for a familial mutation) is natural chimerism where lack of detectable mutation in blood may not rule out the presence of the mutation in the other tissues. To test this hypothesis, archival tumor tissue from eleven BRCA phenocopies was investigated. DNA from the tumor tissue was analyzed using sequence-specific PCR, capillary electrophoresis, and pyrosequencing. The familial mutations were originally detected in the patients’ first-degree relatives by commercial testing. The same testing detected no mutations in the blood of the patients under study. The test methods targeted only the known familial mutation in the tumor tissue. Tumor diagnoses included breast, ovarian, endometrial and primary peritoneal carcinoma. None of the familial mutations were found in the tumor samples tested. These results do not support, but do not completely exclude, the possibility of chimerism in these patients. Further studies with comprehensive sequence analysis in a larger patient group are warranted as a chimeric state would further refine the predictive value of genetic testing to include BRCA phenocopies.