Michael A. Gorin

BRCA1 Mutation Analysis of 41 Human Breast Cancer Cell Lines Reveals Three New Deleterious Mutants

Germ line mutations of the BRCA1 gene confer a high risk of breast cancer and ovarian cancer to female mutation carriers. The BRCA1 protein is involved in the regulation of DNA repair. How specific tumor-associated mutations affect the molecular function of BRCA1, however, awaits further elucidation. Cell lines that harbor BRCA1 gene mutations are invaluable tools for such functional studies. Up to now, the HCC1937 cell line was the only human breast cancer cell line with an identified BRCA1 mutation. In this study, we identified three other BRCA1 mutants from among 41 human breast cancer cell lines by sequencing of the complete coding sequence of BRCA1. Cell line MDA-MB-436 had the 5396 + 1G>A mutation in the splice donor site of exon 20. Cell line SUM149PT carried the 2288delT mutation and SUM1315MO2 carried the 185delAG mutation. All three mutations were accompanied by loss of the other BRCA1 allele. The 185delAG and 5396 + 1G>A mutations are both classified as pathogenic mutations. In contrast with wild-type cell lines, none of the BRCA1 mutants expressed nuclear BRCA1 proteins as detected with Ab-1 and Ab-2 anti-BRCA1 monoclonal antibodies. These three new human BRCA1 mutant cell lines thus seem to be representative breast cancer models that could aid in further unraveling of the function of BRCA1.

Polyclonal breast cancer metastases arise from collective dissemination of keratin 14-expressing tumor cell clusters

Significance Conventional models of cancer progression propose that single cells leave the primary tumor, enter the circulation, and seed clonal metastases. However, metastases can contain multiple clones, raising the question: How do polyclonal metastases form? We demonstrate that cancer cells seed distant organs as cohesive clusters, composed of two molecularly distinct subpopulations, whose proportions vary systematically during metastasis. We establish that collective dissemination is a frequent mechanism for metastasis and identify a molecular program in the most invasive, keratin 14+ (K14+) cancer cells, regulating cell–cell adhesion, cell–matrix adhesion, and immune evasion. We demonstrate that this metastatic phenotype is dependent upon K14 expression. Understanding the molecular basis of collective dissemination may therefore enable novel prognostics and therapies to improve patient outcomes. Recent genomic studies challenge the conventional model that each metastasis must arise from a single tumor cell and instead reveal that metastases can be composed of multiple genetically distinct clones. These intriguing observations raise the question: How do polyclonal metastases emerge from the primary tumor? In this study, we used multicolor lineage tracing to demonstrate that polyclonal seeding by cell clusters is a frequent mechanism in a common mouse model of breast cancer, accounting for >90% of metastases. We directly observed multicolored tumor cell clusters across major stages of metastasis, including collective invasion, local dissemination, intravascular emboli, circulating tumor cell clusters, and micrometastases. Experimentally aggregating tumor cells into clusters induced a >15-fold increase in colony formation ex vivo and a >100-fold increase in metastasis formation in vivo. Intriguingly, locally disseminated clusters, circulating tumor cell clusters, and lung micrometastases frequently expressed the epithelial cytoskeletal protein, keratin 14 (K14). RNA-seq analysis revealed that K14+ cells were enriched for desmosome and hemidesmosome adhesion complex genes, and were depleted for MHC class II genes. Depletion of K14 expression abrogated distant metastases and disrupted expression of multiple metastasis effectors, including Tenascin C (Tnc), Jagged1 (Jag1), and Epiregulin (Ereg). Taken together, our findings reveal K14 as a key regulator of metastasis and establish the concept that K14+ epithelial tumor cell clusters disseminate collectively to colonize distant organs.

Protein kinase Cε: an oncogene and emerging tumor biomarker

Members of the protein kinase C (PKC) family have long been studied for their contributions to oncogenesis. Among the ten different isoforms of this family of serine/threonine kinases, protein kinase Cε (PKCε) is one of the best understood for its role as a transforming oncogene. In vitro, overexpression of PKCε has been demonstrated to increase proliferation, motility, and invasion of fibroblasts or immortalized epithelial cells. In addition, xenograft and transgenic animal models have clearly shown that overexpression of PKCε is tumorigenic resulting in metastatic disease. Perhaps most important in implicating the epsilon isoform in oncogenesis, PKCε has been found to be overexpressed in tumor-derived cell lines and histopathological tumor specimens from various organ sites. Combined, this body of work provides substantial evidence implicating PKCε as a transforming oncogene that plays a crucial role in establishing an aggressive metastatic phenotype. Reviewed here is the literature that has led to the current understanding of PKCε as an oncogene. Moreover, this review focuses on the PKCε-mediated signaling network for cell motility and explores the interaction of PKCε with three major PKCε signaling nodes: RhoA/C, Stat3 and Akt. Lastly, the emerging role of PKCε as a tumor biomarker is discussed.

Five-year Analysis of a Multi-institutional Prospective Clinical Trial of Delayed Intervention and Surveillance for Small Renal Masses: The DISSRM Registry ☆

BACKGROUND A growing body of retrospective literature is emerging regarding active surveillance (AS) for patients with small renal masses (SRMs). There are limited prospective data evaluating the effectiveness of AS compared to primary intervention (PI). OBJECTIVE To determine the characteristics and clinical outcomes of patients who chose AS for management of their SRM. DESIGN, SETTING, AND PARTICIPANTS From 2009 to 2014, the multi-institutional Delayed Intervention and Surveillance for Small Renal Masses (DISSRM) registry prospectively enrolled 497 patients with solid renal masses ≤4.0cm who chose PI or AS. INTERVENTION AS versus PI. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS The registry was designed and powered as a noninferiority study based on historic recurrence rates for PI. Analyses were performed in an intention-to-treat manner. Primary outcomes were overall survival (OS) and cancer-specific survival (CSS). RESULTS AND LIMITATIONS Of the 497 patients enrolled, 274 (55%) chose PI and 223 (45%) chose AS, of whom 21 (9%) crossed over to delayed intervention. AS patients were older, had worse Eastern Cooperative Oncology Group scores, total comorbidities, and cardiovascular comorbidities, had smaller tumors, and more often had multiple and bilateral lesions. OS for PI and AS was 98% and 96% at 2 yr, and 92% and 75% at 5 yr, respectively (log rank, p=0.06). At 5 yr, CSS was 99% and 100% for PI and AS, respectively (p=0.3). AS was not predictive of OS or CSS in regression modeling with relatively short follow-up. CONCLUSIONS In a well-selected cohort with up to 5 yr of prospective follow-up, AS was not inferior to PI. PATIENT SUMMARY The current report is among the first prospective analyses of patients electing for active surveillance of a small renal mass. Discussion of active surveillance should become part of the standard discussion for management of small renal masses.

Positive Surgical Margins in Robot-Assisted Partial Nephrectomy: A Multi-Institutional Analysis of Oncologic Outcomes (Leave No Tumor Behind)

PURPOSE Expanding indications for robot-assisted partial nephrectomy raise major oncologic concerns for positive surgical margins. Previous reports showed no correlation between positive surgical margins and oncologic outcomes. We report a multi-institutional experience with the oncologic outcomes of positive surgical margins on robot-assisted partial nephrectomy. MATERIALS AND METHODS Pathological and clinical followup data were reviewed from an institutional review board approved, prospectively maintained joint database from 5 institutions. Tumors with malignant pathology were isolated and statistically analyzed for demographics and oncologic followup. The log rank test was used to compare recurrence-free and metastasis-free survival between patients with positive and negative surgical margins. The proportional hazards method was used to assess the influence of multiple factors, including positive surgical margins, on recurrence and metastasis. RESULTS A total of 943 robot-assisted partial nephrectomies for malignant tumors were successfully completed. Of the patients 21 (2.2%) had positive surgical margins on final pathological assessment, resulting in 2 groups, including the 21 with positive surgical margins and 922 with negative surgical margins. Positive surgical margin cases had higher recurrence and metastasis rates (p<0.001). As projected by the Kaplan-Meier method in the population as a whole at followup out to 63.6 months, 5-year recurrence-free and metastasis-free survival was 94.8% and 97.5%, respectively. There was a statistically significant difference in recurrence-free and metastasis-free survival between patients with positive and negative surgical margins (log rank test<0.001), which favored negative surgical margins. Positive surgical margins showed an 18.4-fold higher HR for recurrence when adjusted for multiple tumors, tumor size, tumor growth pattern and pathological stage. CONCLUSIONS Positive surgical margins on final pathological evaluation increase the HR of recurrence and metastasis. In addition to pathological and molecular tumor characteristics, this should be considered to plan appropriate management.

Factors That Influence Patient Enrollment in Active Surveillance for Low-risk Prostate Cancer

OBJECTIVES To learn from patients their rationale for enrollment in active surveillance (AS) for low-risk prostate cancer as an alternative to primary treatment. METHODS A rank-order survey was designed to assess the relative influence of factors that contributed to the decision to elect AS. The survey was mailed to 185 patients enrolled in AS at our university-based urologic oncology practice. Participants were also asked whether they had been offered AS as an alternative to primary treatment by the urologist who had initially diagnosed their cancer. RESULTS The survey was returned by 105 (57%) of 185 patients. AS was offered to 38 (36%) of 105 patients by the physician who had made the initial diagnosis. Patients most frequently reported physician influence as the greatest contributor to their decision to elect AS (73%). Patients also cited concerns regarding the potential side effects of incontinence (48%) and erectile dysfunction (44%) associated with therapy as reasons for choosing AS. CONCLUSIONS The results of the present study have shown that patients are heavily influenced by physicians in their decision to elect AS. Notably, the majority of our sampled patients were not offered AS at diagnosis. Evidence has indicated that AS is an appropriate approach for low-risk prostate cancer and should be discussed with patients in this risk category.

Grade Heterogeneity in Small Renal Masses: Potential Implications for Renal Mass Biopsy

PURPOSE Understanding the degree of phenotypic heterogeneity in a small renal mass may have implications for interpreting renal mass biopsy data. In this study we quantify nuclear grade heterogeneity in small renal masses. MATERIALS AND METHODS Our institutional renal mass database was queried for patients with T1a (less than 4 cm) renal masses stratified by the criteria of imaging diameter less than 2 cm or 2 cm or greater, clear cell or papillary histology, low grade (Fuhrman 1-2) or high grade (Fuhrman 3-4) with tissue available for review. Four consecutive specimens were chosen from each of the 8 strata for a total of 32. All specimens were reanalyzed and the highest Fuhrman grade present in each 10× powered field was recorded. A case was classified as heterogeneous if multiple grades were present and as discordant if the highest Fuhrman grade was present in less than 50% of the specimen. RESULTS A median of 5 slides (IQR 3.5-7.5) and 59, 10× powered fields (IQR 34-109) were examined per patient. Overall 26 samples (81.3%) were heterogeneous, including 15 of 16 (93.8%) high grade specimens. Among all cases 10 (31.3%) were discordant and of high grade specimens 4 (25%) were discordant. Median fraction of low grade tissue in high grade specimens was 38.9% (IQR 12.2-57.2). CONCLUSIONS The majority of small renal masses demonstrated considerable nuclear grade heterogeneity. The greatest degree of heterogeneity and discordance was observed in high grade tumors. One should consider these findings when interpreting renal mass biopsy data as the risk of under sampling high grade tumors may not be insignificant.

PET imaging of prostate-specific membrane antigen in prostate cancer: current state of the art and future challenges

Background:Prostate-specific membrane antigen (PSMA) is a cell surface enzyme that is highly expressed in prostate cancer (PCa) and is currently being extensively explored as a promising target for molecular imaging in a variety of clinical contexts. Novel antibody and small-molecule PSMA radiotracers labeled with a variety of radionuclides for positron emission tomography (PET) imaging applications have been developed and explored in recent studies.Methods:A great deal of progress has been made in defining the clinical utility of this class of PET agents through predominantly small and retrospective clinical studies. The most compelling data to date has been in the setting of biochemically recurrent PCa, where PSMA-targeted radiotracers have been found to be superior to conventional imaging and other molecular imaging agents for the detection of locally recurrent and metastatic PCa.Results:Early data, however, suggest that initial lymph node staging before definitive therapy in high-risk primary PCa patients may be limited, although intraoperative guidance may still hold promise. Other examples of potential promising applications for PSMA PET imaging include non-invasive characterization of primary PCa, staging and treatment planning for PSMA-targeted radiotherapeutics, and guidance of focal therapy for oligometastatic disease.Conclusions:However, all of these indications and applications for PCa PSMA PET imaging are still lacking and require large, prospective, systematic clinical trials for validation. Such validation trials are needed and hopefully will be forthcoming as the fields of molecular imaging, urology, radiation oncology and medical oncology continue to define and refine the utility of PSMA-targeted PET imaging to improve the management of PCa patients.

Outcomes and predictors of clinical T1 to pathological T3a tumor up-staging after robotic partial nephrectomy: a multi-institutional analysis.

PURPOSE We evaluated the early oncological end point of recurrence-free survival in patients with renal cell carcinoma up-staged from cT1 to pT3a after partial nephrectomy. We also aimed to establish preoperative factors associated with pathological tumor up-staging. MATERIALS AND METHODS A prospective database of robotic partial nephrectomy cases performed at 5 academic centers was queried for patients who underwent surgery for a solitary cT1 renal mass. Patients with pT1-2 renal cell carcinoma were compared to those with pT3a tumors to determine the difference in recurrence-free survival. Preoperative factors associated with cT1 to pT3a up-staging were studied using multivariate logistic regression analysis. RESULTS A total of 1,096 patients underwent robotic partial nephrectomy for a cT1 renal mass. At final pathological evaluation 855 tumors (78.0%) were found to be renal cell carcinoma, of which 41 (4.8%) were up-staged to pT3a. The 24-month recurrence-free survival estimates for pT1-2 and pT3a tumors were 99.2% and 91.8%, respectively (p=0.003). Multivariate analysis revealed that a high vs low R.E.N.A.L. (radius, exophytic/endophytic, nearness to collecting system or sinus, anterior/posterior and location relative to polar lines) nephrometry score was associated with tumor up-staging (OR 2.97, 95% CI 1.20-7.35, p=0.02). On separate multivariate analysis increasing tumor diameter (OR 1.66, 95% CI 1.32-2.08, p<0.001) and hilar location (OR 2.83, 95% CI 1.43-5.61, p=0.003) were also associated with up-staging. CONCLUSIONS At short-term followup patients with renal cell carcinoma up-staged from cT1 to pT3a have reasonable oncological outcomes after partial nephrectomy. Factors associated with tumor up-staging include high tumor complexity, increasing tumor diameter and hilar location. Further studies are needed to determine the comparative efficacy of partial vs radical nephrectomy for small pT3a tumors.

Pomegranate Fruit Extract Impairs Invasion and Motility in Human Breast Cancer

Purpose. Pomegranate fruit extracts (PFEs) possess polyphenolic and other compounds with antiproliferative, pro-apoptotic and anti-inflammatory effects in prostate, lung, and other cancers. Because nuclear transcription factor-kB (NF-kB) is known to regulate cell survival, proliferation, tumorigenesis, and inflammation, it was postulated that PFEs may exert anticancer effects at least in part by modulating NF-kB activity. Experimental design. The authors investigated the effect of a novel, defined PFE consisting of both fermented juice and seed oil on the NF-kB pathway, which is constitutively active in aggressive breast cancer cell lines. The effects of the PFE on NF-kB—regulated cellular processes such as cell survival, proliferation, and invasion were also examined. Results. Analytical characterization of the bioactive components of the PFE revealed active constituents, mainly ellagitannins and phenolic acids in the aqueous PFE and conjugated octadecatrienoic acids in the lipid PFE derived from seeds.The aqueous PFE dose-dependently inhibited NF-kB—dependent reporter gene expression associated with proliferation, invasion, and motility in aggressive breast cancer phenotypes while decreasing RhoC and RhoA protein expression. Conclusion. Inhibition of motility and invasion by PFEs, coincident with suppressed RhoC and RhoA protein expression, suggests a role for these defined extracts in lowering the metastatic potential of aggressive breast cancer species.