Katherine Alpaugh

Defects in DNA Repair Genes Predict Response to Neoadjuvant Cisplatin-based Chemotherapy in Muscle-invasive Bladder Cancer

BACKGROUND Cisplatin-based neoadjuvant chemotherapy (NAC) before cystectomy is the standard of care for muscle-invasive bladder cancer (MIBC), with 25-50% of patients expected to achieve a pathologic response. Validated biomarkers predictive of response are currently lacking. OBJECTIVE To discover and validate biomarkers predictive of response to NAC for MIBC. DESIGN, SETTING, AND PARTICIPANTS Pretreatment MIBC samples prospectively collected from patients treated in two separate clinical trials of cisplatin-based NAC provided the discovery and validation sets. DNA from pretreatment tumor tissue was sequenced for all coding exons of 287 cancer-related genes and was analyzed for base substitutions, indels, copy number alterations, and selected rearrangements in a Clinical Laboratory Improvements Amendments-certified laboratory. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS The mean number of variants and variant status for each gene were correlated with response. Variant data from the discovery cohort were used to create a classification tree to discriminate responders from nonresponders. The resulting decision rule was then tested in the independent validation set. RESULTS AND LIMITATIONS Patients with a pathologic complete response had more alterations than those with residual tumor in both the discovery (p=0.024) and validation (p=0.018) sets. In the discovery set, alteration in one or more of the three DNA repair genes ATM, RB1, and FANCC predicted pathologic response (p<0.001; 87% sensitivity, 100% specificity) and better overall survival (p=0.007). This test remained predictive for pathologic response in the validation set (p=0.033), with a trend towards better overall survival (p=0.055). These results require further validation in additional sample sets. CONCLUSIONS Genomic alterations in the DNA repair-associated genes ATM, RB1, and FANCC predict response and clinical benefit after cisplatin-based chemotherapy for MIBC. The results suggest that defective DNA repair renders tumors sensitive to cisplatin. PATIENT SUMMARY Chemotherapy given before bladder removal (cystectomy) improves the chance of cure for some but not all patients with muscle-invasive bladder cancer. We found a set of genetic mutations that when present in tumor tissue predict benefit from neoadjuvant chemotherapy, suggesting that testing before chemotherapy may help in selecting patients for whom this approach is recommended.

Phase I Dose Escalation, Pharmacokinetic and Pharmacodynamic Study of Naptumomab Estafenatox Alone in Patients With Advanced Cancer and With Docetaxel in Patients With Advanced Non–Small-Cell Lung Cancer

PURPOSE Two phase I studies were conducted of ABR-217620 alone or in combination with docetaxel. This is a recombinant fusion protein consisting of a mutated variant of the superantigen staphylococcal enterotoxin E (SEA/E-120) linked to fragment antigen binding moiety of a monoclonal antibody recognizing the tumor-associated antigen 5T4. PATIENTS AND METHODS Patients with non-small-cell lung cancer (NSCLC), pancreatic cancer (PC), and renal cell cancer (RCC) received 5 daily boluses of ABR-217620 (3-month cycles) in escalating doses to determine the maximum-tolerated dose (MTD; ABR-217620 dose escalation monotherapy [MONO] study). Doses were selected based on individual patient anti-SEA/E-120 titers pretreatment. Patients with NSCLC received 4 daily, escalating doses of ABR-217620 followed by docetaxel in 21-day cycles (ABR-217620 dose escalation combination with docetaxel [COMBO] study). RESULTS Thirty-nine patients were enrolled in the MONO study and 13 were enrolled in the COMBO study. The monotherapy MTD was 26 microg/kg (NSCLC and PC) and 15 microg/kg (RCC). Dose-limiting toxicities (DLTs) in the MONO study were fever, hypotension, acute liver toxicity, and vascular leak syndrome. In the COMBO study, the MTD was 22 microg/kg (neutropenic sepsis). Adverse events included grade 1 to 2 fever, hypotension, nausea, and chills. Treatment caused a systemic increase of inflammatory cytokines and selective expansion of SEA/E-120 reactive T-cells. Tumor biopsies demonstrated T-cell infiltration after therapy. Fourteen patients (36%) had stable disease (SD) on day 56 of the MONO study. Two patients (15%) in the COMBO study had partial responses, one in a patient with progressive disease on prior docetaxel, and five patients (38%) had SD on day 56. CONCLUSION ABR-217620 was well tolerated with evidence of immunological activity and antitumor activity.

Circulating Tumor Cells: Evolving Evidence and Future Challenges

Circulating tumor cells (CTCs) are rare malignant cells found in the peripheral blood that originate from the primary tumor or metastatic sites. New techniques have been developed to isolate and characterize these cells. CTC enumeration has been incorporated into different fields of oncology as a prognostic marker, a tool to monitor therapy response, and a method to understand basic tumor characteristics. This review covers the different techniques available for isolation of CTCs, the clinical utility of CTCs in breast, prostate, and colon cancer, and future directions in this field.

TP53 mutations detected in circulating tumor cells present in the blood of metastatic triple negative breast cancer patients

IntroductionCirculating tumor cells (CTCs) are tumor cells shed from either primary tumors or its metastases that circulate in the peripheral blood of patients with metastatic cancers. The molecular characterization of the CTCs is critical to identifying the key drivers of cancer metastasis and devising therapeutic approaches. However, the molecular characterization of CTCs is difficult to achieve because their isolation is a major technological challenge.MethodsCTCs from two triple negative breast cancer patients were enriched using CellSearch and single cells selected by DEPArray™. A TP53 R110 fs*13 mutation identified by next generation sequencing in the breast and chest skin biopsies of both patients was studied in single CTCs.ResultsFrom 6 single CTC isolated from one patient, 1 CTC had TP53 R110 delC, 1 CTC showed the TP53 R110 delG mutation, and the remaining 4 single CTCs showed the wild type p53 sequence; a pool of 14 CTCs isolated from the same patient also showed TP53 R110 delC mutation. In the tumor breast tissue of this patient, only the TP53 R110 delG mutation was detected. In the second patient a TP53 R110 delC mutation was detected in the chest wall skin biopsy; from the peripheral blood of this patient, 5 single CTC and 6 clusters of 2 to 6 CTCs were isolated; 3 of the 5 single CTCs showed the TP53 R110 delC mutation and 2 CTCs showed the wild type TP53 allele; from the clusters, 5 showed the TP53 R110 delC mutation, and 1 cluster the wild type TP53 allele. Single white blood cells isolated as controls from both patients only showed the wild type TP53 allele.ConclusionsWe are able to isolate uncontaminated CTCs and achieve single cell molecular analysis. Our studies showed the presence of different CTC sub-clones in patients with metastatic breast cancer. Some CTCs had the same TP53 mutation as their matching tumor samples although others showed either a different TP53 mutation or the wild type allele. Our results indicate that CTCs could represent a non-invasive source of cancer cells from which to determine genetic markers of the disease progression and potential therapeutic targets.

EZH2 knockdown suppresses the growth and invasion of human inflammatory breast cancer cells

IntroductionInflammatory breast cancer (IBC) is the most metastatic variant of breast cancer with the poorest survival in all types of breast cancer patients and presently therapeutic targets for IBC are very limited. Enhancer of zeste homolog 2 (EZH2) is frequently expressed in human IBC and its expression positively correlates with worse clinical outcome. However, the molecular basis for EZH2 promoting IBC has not been explored. Here, we investigated the functional role of EZH2 in IBC cells by examining the effects of its knockdown on the formation of tumor spheroids and invasion of these cells in vitro and in vivo in an orthotopic xenograft model.MethodsSUM149 and a new IBC cell line-FC-IBC-02 derived from pleural effusion fluid of an IBC patient were used in this study. Specific knockdown of EZH2 was performed using short hairpin RNA (shRNA) specific to the human EZH2 gene. Cell growth and the formation of tumor spheroids were examined in vitro. The effects of EZH2 knockdown on IBC cell migration and invasion were examined by a Boyden chamber assay. For the in vivo tumor growth studies, IBC cells were orthotopically transplanted into the mammary fat pads of immunodeficient mice.ResultsThe results showed that EZH2 is expressed at higher levels in human IBC cell lines compared with normal human mammary epithelial cells, and the knockdown of EZH2 expression significantly suppressed cell growth and tumor spheroid formation of human IBC cells in vitro. In addition, EZH2 knockdown inhibited the migration and invasion of IBC cells. Significantly, EZH2 knockdown suppressed the angiogenesis and tumor growth of IBC cells in vivo.ConclusionsOur results provide direct evidence that EZH2 is critical for the formation of tumor spheroids and invasion of human IBC cells and could be a potential target for developing novel therapeutic strategies for human IBC.

Monoclonal Antibodies in Cancer Treatment

Research advances and promising clinical outcomes with immunotherapeutics has led to a resurgence of incorporating monoclonal antibodies in cancer treatment. Unconjugated, conjugated and multi-target constructs are emerging as a conventional form of therapy along with the classical trio of surgery, radiation and chemotherapy. The recent major accomplishments in monoclonals include: first, the development of human and chimeric structures negating the induction of humoral responses to murine counterparts which limited use; second, protein engineering has improved the affinity and specificity of the antibody to its target; third, technics have been designed to select monoclonal antibodies imparting a biological consequence (function) following binding; and, lastly, recombinant proteins are being created with multiple epitopic specificities and/or fusion with other biologically active proteins such as toxins and cytokines/growth factors. Clinical efficacy in the treatment of haematological malignancies has secured a role for monoclonals in routine treatment. Evidence of clinical responses in patients with metastatic solid tumours is leading to the next generation of trials in the adjuvant setting. This paper presents an overview of the clinical experience with monoclonal antibodies in cancer treatment over the past 5 years. Our aim is to highlight the successes and advances, as well as noting limitations of antibody therapeutics. The advances seen support a continued effort to optimise the creation, selection and use of immunotherapeutics in the battle against cancer.

Association of Circulating Tumor Cells With Late Recurrence of Estrogen Receptor–Positive Breast Cancer: A Secondary Analysis of a Randomized Clinical Trial

Importance Late recurrence 5 or more years after diagnosis accounts for at least one-half of all cases of recurrent hormone receptor–positive breast cancer. Objective To determine whether the presence of circulating tumor cells (CTCs) in a peripheral blood sample obtained approximately 5 years after diagnosis was associated with late clinical recurrence of operable human epidermal growth factor receptor 2–negative breast cancer. Design, Setting, and Participants This per-protocol secondary analysis of the Double-Blind Phase III Trial of Doxorubicin and Cyclophosphamide Followed by Paclitaxel With Bevacizumab or Placebo in Patients With Lymph Node Positive and High Risk Lymph Node Negative Breast Cancer enrolled patients from 2007 to 2011 who were without clinical evidence of recurrence between 4.5 and 7.5 years after primary surgical treatment of human epidermal growth factor receptor 2–negative stage II-III breast cancer followed by adjuvant systemic therapy. Patients were enrolled in a subprotocol for secondary analysis from February 25, 2013, to July 29, 2016, after signing consent for the subprotocol. The analysis was performed in April 2018. Interventions A blood sample was obtained for identification and enumeration of CTCs. Main Outcome and Measures The association between a positive CTC assay result (at least 1 CTC per 7.5 mL of blood) and clinical recurrence. Results Among 547 women included in this analysis, the results of the CTC assay were positive for 18 of 353 with hormone receptor–positive disease (5.1% [95% CI, 3.0%-7.9%]); 23 of 353 patients (6.5% [95% CI, 4.2%-9.6%]) had a clinical recurrence. The recurrence rates per person-year of follow-up in the CTC-positive and CTC-negative groups were 21.4% (7 recurrences per 32.7 person-years) and 2.0% (16 recurrences per 796.3 person-years), respectively. In multivariate models including clinical covariates, a positive CTC assay result was associated with a 13.1-fold higher risk of recurrence (hazard ratio point estimate, 13.1; 95% CI, 4.7-36.3). Seven of 23 patients (30.4% [95% CI, 13.2%-52.9%]) with recurrence had a positive CTC assay result at a median of 2.8 years (range, 0.1-2.8 years) before clinical recurrence. The CTC assay result was also positive for 8 of 193 patients (4.1% [95% CI, 1.8%-8.0%]) with hormone receptor–negative disease, although only 1 patient (0.5% [95% CI, 0%-2.9%]) experienced disease recurrence (this patient was CTC negative). Conclusions and Relevance A single positive CTC assay result 5 years after diagnosis of hormone receptor–positive breast cancer provided independent prognostic information for late clinical recurrence, which provides proof of concept that liquid-based biomarkers may be used to risk stratify for late recurrence and guide therapy. Trial Registration ClinicalTrials.gov identifier: NCT00433511

Abstract 4593: Genomic profiling of cell free DNA (cfDNA) from patients with inflammatory breast cancer (IBC)

Inflammatory breast cancer (IBC) currently accounts for 2% to 6% of all breast cancer cases in the United States and up to 20% of all breast cancer cases globally. IBC exhibits distinctively aggressive clinical features compared to all breast cancers, and accounts for a disproportionally high mortality rate—15% of breast cancer-related deaths in U.S. In addition, the survival rates between stage-matched IBC and non-IBC differ drastically. Therefore, we are in need of better understanding the molecular abnormalities driving IBC aggressive phenotype. We performed a study to evaluate the genomic alterations in cell free DNA (cfDNA) from plasma from 13 IBC patients, including 9 with triple-negative disease. In 6 patients, mutation analyses were also studied in tumor samples (tumor tissue or tumor cells from pleural effusions). Mutation analysis was performed by next-generation sequencing (NGS) using a unique molecular identifiers (UMI) assay and a panel of 93 breast cancer-related genes (Qiagen). The data were analyzed using the Qiagen9s GeneGlobe portal and Biomedical Genomics Workbench and interpretation was performed with Qiagen9s QCI. Somatic mutations detected in cfDNA samples were seen in: TP53 (7/13), RB1 (2/13), GEN1 (2/13) and EP300 (2/13); additional somatic mutations were found in PIK3CA (1/13), ERBB2 (1/13), PALB2 (1/13) and MUC16 (1/13). In 4 patients with plasma and tumor samples taken at the same time of the disease progression, complete concordance was not found in the somatic mutations detected in cfDNA and tumor cells DNA. Interestingly, in 12 of 13 IBC patients some variants were observed with high variant allele frequencies (VAF), ~50% or ~100% at a total coverage depth ≥230 reads, in the following genes: BRCA2 (1/13), RAD51D (2/13), PALB2 (1/13), RAD51C (1/13), AR (1/13) and MUTYH (1/13), which were classified as pathogenic or likely pathogenic, and BARD1 (3/13), SYNE1 (2/13), KMT2C (2/13), BRIP1 (1/13), XRCC3 (1/13), RET (1/13), APC (1/13), RAD50 (1/13) and MUC16 (1/13), classified as variants of uncertain significance. Moreover, 11 of these patients had a family history of different cancers including breast, colon, prostate, stomach, bladder, cervical cancers, and melanoma and myeloma. In the 6 patients where cfDNA and tumor samples were available, mutations with high VAF were found in both samples (100% concordance), suggesting that variants with high VAF are germline variants. These results suggest that the described germline variants could increase the risk of IBC and somatic mutation information obtained from cfDNA is complementary to that obtained from tissue samples. Studies on more samples are in progress. Citation Format: Jianming Pei, Jacqueline Talarchek, Jennifer S. Winn, Katherine Alpaugh, Massimo Cristofanilli, Sandra V. Fernandez. Genomic profiling of cell free DNA (cfDNA) from patients with inflammatory breast cancer (IBC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4593.

Abstract 5165: Cancer associated macrophage-like cells as a blood-based biomarker for the screening of solid tumors

and analyze Circulating Tumor Cells (CTCs), and Circulating Cancer Associated Macrophage-Like cells (CAMLs), from the blood of cancer patients for numerous clinical implications. However, while the clinical utility of CTCs is a well studied field, CAMLs are a largely unstudied event. Here, we studied the peripheral blood of cancer patients to ascertain the prevalence, specificity and sensitivity of CAMLs in relation to disease status at presentation in benign and malignant diseases. We supply evidence that this previously unidentified circulating immune cell may be used as a screening tool to detect solid tumors in various malignancies, irrespective of disease stage. Cancer associated macrophage-like cells as a blood-based biomarker for the screening of solid tumors

Abstract 1115: A discrete tuning of α5β1 integrin activity sustains the tumor-ECM induced fibroblastic activation in pancreatic cancer stroma

Pancreatic adenocarcinoma (PDAC) presents one of the highest mortality rates amongst all neoplasias. The desmoplastic microenvironment characteristic of PDAC plays a pivotal role in this aggressive cancer progression. Using an in vivo-mimetic 3D human stroma system, we have shown that quiescent fibroblasts become activated, undergoing a desmoplastic (i.e., myofibroblastic) phenotypic switch in response to tumor-associated (TA) but not to normal fibroblast-derived extracellular matrix (ECM). Nevertheless, the molecular mechanisms responsible for this process remain unclear. Using syngeneic human fibroblasts harvested from patient-matched normal and tumor samples, we produced a human 3D pancreatic stromal system. The system was used to investigate the role of integrins during the TA-ECM-induced phenotypic switch, focusing on two integrins associated with myofibroblastic differentiation, αvβ5 and α5β1. We evaluated the role of well-known integrin signaling effectors, i.e., non-receptor tyrosine kinases FAK and Src family kinases (SFK), in the observed TA-ECM-induced phenotypic switch. Approaches included the use of specific inhibitors or activators of integrins and SFK or FAK chemical or genetic inhibitors. Our data strongly suggest a αvβ5/α5β1-integrin cross-talk necessary to maintain the TA-ECM-induced phenotypic switch. This fine-tuned integrin cross-talk participates in the TA-ECM-induced overexpression and stress fiber localization of desmoplastic stroma marker, α-smooth muscle actin (α-SMA). Our findings correlate with clustering of α5β1-integrin at TA-ECM-altered 3D adhesion structures, together with differences in α5β1 activation. Interestingly, we uncovered a mechanism whereby αvβ5-integrin activity, in a SFK/FAK-dependent manner, inhibits a FAK-independent α5β1-integrin activity, preventing it from inhibiting the TA-ECM-induced phenotypic switch. Finally, using patient samples, we verified our in vitro generated hypothesis, while results suggested that αvβ5-integrin inhibition may constitute a valid inhibitory PDAC-associated stroma clinical treatment/approach. Likewise, this work also concluded that the TA-ECM-regulated mechanism studied may comprise a clinically relevant occurrence, suggestive of a noteworthy value in assessing stromal activity in PDAC patients. Funding provided by The Commonwealth of Pennsylvania, The Bucks County Chapter Board of Associates and NCI/NIH grants CA113451 and CA06927 Citation Format: Janusz Franco-Barraza, Tiffany Luong, Neelima Shah, Raj Madhani, Katherine Alpaugh, John Hoffman, Edna Cukierman. A discrete tuning of α5β1 integrin activity sustains the tumor-ECM induced fibroblastic activation in pancreatic cancer stroma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1115. doi:10.1158/1538-7445.AM2014-1115