Tamar Hamburger

Cancer risks in carriers of the BRCA1/2 Ashkenazi founder mutations

Background: The risks for cancers other than breast (BC) or ovarian (OC) cancer in breast cancer gene 1 and 2 (BRCA1/2) mutation carriers were elevated in studies of carrier families. However, case–control studies did not confirm this observation. Objective: To compare the risks for other cancers in BRCA1/2 mutation carriers and non-carriers, all affected with BC and/or OC. Both groups share risk modifiers of BC/OC, which enabled assessment of the role of BRCA1/2 mutations. Methods: 1098 Ashkenazi Jewish women affected with BC and/or OC were ascertained during 1995–2003; molecular testing revealed 229 BRCA1 and 100 BRCA2 carriers and 769 non-carriers. COX proportional hazard models were used to evaluate the risk of other cancers. Analyses were conducted including all other cancers or only those diagnosed after BC/OC diagnosis. Results: The HRs for any other cancer were 2.6 (95% CI 1.7 to 4.2, p<0.001) and 1.8 (95% CI 0.95 to 3.6, p = 0.07) in BRCA1 and BRCA2 carriers, respectively. The corresponding colon cancer HRs were 3.9 (95% CI 1.3 to 12.1, p = 0.02) and 2.3 (95% CI 0.5 to 11.3, p = 0.3) in BRCA1 and BRCA2 carriers. The HR for lymphoma was 11.9 (95% CI 3.1 to 46.2, p = 0.001) in BRCA2 carriers. Risk estimates for other cancers after the onset of BC/OC were similar. Conclusion: A 2.5-fold increase in any other cancer and a fourfold risk of colon cancer were found among BRCA1 carriers. The corresponding HRs in BRCA2 carriers were non-significant, except for the markedly elevated risk of lymphoma. These results suggest a role for BRCA1/2 mutations in colorectal cancer risk in a subgroup of BC/OC-affected carriers.

Autologous cell vaccine as a post operative adjuvant treatment for high-risk melanoma patients (AJCC stages III and IV)

This study evaluates the overall survival and disease free survival of melanoma patients that were treated with an autologous melanoma cell vaccine, administered as a post-operative adjuvant. Included are 43 patients with totally resected metastatic melanoma (28-AJCC stage III, 15-AJCC stage IV), with a median follow up of 34 months (6–62). The treatment consisted of eight doses of a vaccine made of 10–25×106 autologous melanoma cells either released from the surgical specimen or grown in cell cultures. Tumour cells were conjugated with hapten dinitrophenyl, mixed with Bacille Calmette Guérin and irradiated to 110 Gy. Both disease free survival and overall survival were found to be correlated with intensity of evolving delayed type hypersensitivity to subcutaneous injection of unmodified melanoma cells. Patients with a delayed type hypersensitivity reaction of ⩾10 mm had a median disease free survival of 17 months (mean 35 months) and a mean overall survival of 63 months (median not reached). In contrast, patients with a negative or weak delayed type hypersensitivity had a median disease free survival of 9 months (relative risk of recurrence=4.5, P=0.001), and a median overall survival of 16 months (relative risk of death=15, P=0.001). Stage III patients with a positive delayed type hypersensitivity reaction had an improved disease free survival of 16 months and a mean overall survival of 38 months, whereas patients with a negative delayed type hypersensitivity had a median disease free survival of 7 months (relative risk=4.5, P=0.02) and a median overall survival of 16 months (relative risk=9.5, P=0.005). The adjuvant administration of autologous melanoma vaccine was associated with improved disease-free and overall survival to selected patients who successfully attained anti-melanoma reactivity as detected by positive delayed type hypersensitivity reactions to unmodified melanoma cells.

Control of Breast Cancer Growth and Initiation by the Stem Cell-Associated Transcription Factor TCF3

Regulatory factors controlling stem cell identity and self-renewal are often active in aggressive cancers and are thought to promote their growth and progression. TCF3 (also known as TCF7L1) is a member of the TCF/LEF transcription factor family that is central in regulating epidermal and embryonic stem cell identity. We found that TCF3 is highly expressed in poorly differentiated human breast cancers, preferentially of the basal-like subtype. This suggested that TCF3 is involved in the regulation of breast cancer cell differentiation state and tumorigenicity. Silencing of TCF3 dramatically decreased the ability of breast cancer cells to initiate tumor formation, and led to decreased tumor growth rates. In culture, TCF3 promotes the sphere formation capacity of breast cancer cells and their self-renewal. We found that in contrast to ES cells, where it represses Wnt-pathway target genes, TCF3 promotes the expression of a subset of Wnt-responsive genes in breast cancer cells while repressing another distinct target subset. In the normal mouse mammary gland, Tcf3 is highly expressed in terminal end buds, structures that lead duct development. Primary mammary cells are dependent on Tcf3 for mammosphere formation, and its overexpression in the developing gland disrupts ductal growth. Our results identify TCF3 as a central regulator of tumor growth and initiation, and a novel link between stem cells and cancer.

Application of a Novel Severity Grading System for Surgical Complications after Colorectal Resection

BACKGROUND Uniform and accurate reporting of surgical complications is the basis for quality control. We developed a computerized system for reporting and grading surgical complications in colorectal surgery. This study was conducted to evaluate this computerized reporting system. STUDY DESIGN A retrospective chart review was conducted of all surgical complications in patients who underwent resection of the colon or rectum at our institution between the years 1999 and 2004 (n = 408). All complications were recorded using the computerized reporting system and compared with complications reported in the literature. RESULTS Elective operations were performed in 75.7% of patients, and 24.3% required emergency operations. Of the 408 patients in the study, 239 (58.6%) had an uneventful recovery without complications. At least 1 complication was recorded in 169 (41.4%) patients. Grades 1 and 2 complications were recorded in 83 (20.3%) and 105 (25.7%) patients, respectively, requiring observation or medical treatment only, and 59 patients (14.5%) had grades 3 to 5 complications. The three leading complications were surgical site infection, intraabdominal abscess, and hemorrhage requiring blood transfusion. The grades 3 to 5 complication rate was within the range described in the literature, and the rate of grades 1 and 2 complications was substantially higher. These grades 1 and 2 complications were associated with a substantially longer hospital stay. CONCLUSIONS This novel complication reporting system was found feasible and proved to have a higher sensitivity for recording minor but meaningful complications that tend to prolong hospital stay.

Interleukin-2 improves tumour response to DNP-modified autologous vaccine for the treatment of metastatic malignant melanoma.

This paper is a report of response rate (RR) and survival of 34 metastatic melanoma patients who received a dinitrophenyl (DNP)-modified autologous melanoma cell vaccine. In all, 27 patients started the vaccine as a primary treatment for metastatic melanoma and seven started it as an adjuvant, with no evidence of disease at the time, but had developed new metastases. Interleukin-2 (IL-2) was administered in 24 out of the 34 patients: 19 who progressed on vaccine alone and five who had the combination from start. Interleukin-2 was administered in the intravenous, bolus high-dose regimen (seven patients) or as subcutaneous (s.c.) low-dose treatment (17). Overall response for the entire group was 35% (12 patients out of 34), 12% having a complete response (CR) and 23% a partial response (PR). However, only two patients had tumour responses while on the vaccine alone, whereas the other 10 demonstrated objective tumour regression following the combination with IL-2 (two CR, eight PR), lasting for a median duration of 6 months (range 3–50 months). Of the 12 responding patients, 11 attained strong skin reactivity to the s.c. injection of irradiated, unmodified autologous melanoma cells. None of the patients with a negative reactivity experienced any tumour response. Patients with positive skin reactions survived longer (median survival – 54 months). The results suggest enhanced RRs to the combination of IL-2 and autologous melanoma vaccine. Skin reactivity to unmodified autologous melanoma cells may be a predictor of response and improved survival, and therefore a criterion for further pursuing of immunotherapeutic strategies.

Serum thymidine kinase 1 activity in breast cancer

AIMS Thymidine kinase 1 (TK1) is an enzyme involved in DNA synthesis and an important proliferation marker. We explored the association of preoperative serum TK1 activity with clinicopathological parameters and prognosis in terms of recurrence-free survival (RFS) in breast cancer (BC) patients. PATIENTS AND METHODS TK1 activity in serum of 120 healthy women and 161 BC patients was measured by quantitative ELISA. RESULTS Serum TK1 activity in BC patients was significantly higher than in healthy women (P < 0.0001). In BC patients elevated TK1 activity was significantly associated with advanced T stage (P = 0.015), higher grade (P = 0.013), presence of tumor necrosis (P = 0.006), vascular invasion (P = 0.002), and lack of estrogen receptor (ER) and progesterone receptor (PR) expression (P = 0.0004 and P = 0.003). Higher TK1 activity was found in patients with BRCA1/2 mutations compared to those without the mutation (P = 0.004). Multivariate Cox proportional hazards analyses demonstrated that TK1, adjusted for stage, grade, necrosis, ER and PR negativity was retained as an independent predictor of disease recurrence (Hazard Ratio = 3.9, 95%CI 1.3-11.6, P = 0.013). CONCLUSION Elevated serum TK1 is an important risk factor indicating a high proliferation potential of tumors at the time of excision. In multivariate analysis TK1 activity was found to be an independent prognostic factor for RFS.

An Ashkenazi founder mutation in the MSH6 gene leading to HNPCC.

Mutations in DNA mismatch repair genes underlie lynch syndrome (HNPCC). Lynch syndrome resulting from mutations in MSH6 is considered to be attenuated in comparison to that caused by mutations in MLH1 and MSH2, thus more likely to be under diagnosed. In this study we report of a common mutation in the MSH6 gene in Ashkenazi Jews. Genetic counseling and diagnostic work-up for HNPCC was conducted in families who attended the high risk clinic for inherited cancer. We identified the mutation c.3984_3987dup in the MSH6 gene in 19 members of four unrelated Ashkenazi families. This mutation results in truncation of the transcript and in loss of expression of the MSH6 protein in tumors. Tumor spectrum among carriers included colon, endometrial, gastric, ovarian, urinary, and breast cancer. All but one family qualified for the Bethesda guidelines and none fulfilled the Amsterdam Criteria. Members of one family also co-inherited the c.6174delT mutation in the BRCA2 gene. The c.3984_3987dup in the MSH6 gene is a mutation leading to HNPCC among Ashkenazi Jews. This is most probably a founder mutation. In contrast to the c.1906G>C founder mutation in the MSH2 gene, tumors tend to occur later in life, and none of the families qualified for the Amsterdam criteria. c.3984_3987dup is responsible for 1/6 of the mutations identified among Ashkenazi HNPCC families in our cohort. Both mutations: c.3984_3987dup and c.1906G>C account for 61% of HNPCC Ashkenazi families in this cohort. These findings are of great importance for counseling, diagnosis, management and surveillance for Ashkenazi families with Lynch syndrome.

Autologous Melanoma Vaccine Induces Antitumor and Self-Reactive Immune Responses That Affect Patient Survival and Depend on MHC Class II Expression on Vaccine Cells

Purpose: Autologous melanoma cells display a broad variety of tumor antigens and were used for treatment of American Joint Committee on Cancer stages III and IV melanoma as an adjuvant or active therapy. Survival data and immune response were evaluated in vaccinated patients. Experimental Design: Forty-seven patients received 2,4-dinitrophenyl–conjugated autologous melanoma vaccine as an adjuvant (23 patients) or therapy (24 patients). CD4 and CD8 T-cell response in blood sampled before vaccination and after five or eight vaccine doses was evaluated against melanoma cells and autologous peripheral blood mononuclear cells using IFNγ enzyme–linked immunospot. Serum levels of antilivin, an inhibitor of apoptosis, and anti-gp100 IgG were determined. Results: The immunologic effect of the vaccine differed between the two groups of patients. In the adjuvant group, there was a significant increase in CD8 melanoma-reactive T cells (P = 0.035) after vaccination and an increase in antimelanoma CD4 T cells correlating with improved overall survival (P = 0.04). In the therapeutic group, there was no objective tumor regression; antimelanoma T-cell reactivity increased by a small amount, stayed the same, or in some cases decreased. In all patients, a significant increase was noted in CD4 T-cell reactivity against autologous peripheral blood mononuclear cells (P = 0.02), which did not affect survival. Increased antilivin IgG was associated with improved survival. Expression of MHC class II on melanoma cells was vital for the immunogenicity of the vaccine. Conclusion: Autologous melanoma cell vaccine is capable of inducing effective antimelanoma CD4 T-cell activity associated with improved survival. Patients with active metastatic disease generally displayed reduced immune response and gained little from active immunization.

Tissue microarray-based study of patients with lymph node-positive breast cancer shows tyrosine phosphorylation of signal transducer and activator of transcription 3 (tyrosine705-STAT3) is a marker of good prognosis.

BackgroundAlthough lymph node-positive breast cancers are associated with poorer prognosis, individual patients may have different clinical outcomes. Signal transducer and activator of transcription 3 (STAT3) is a point of convergence for numerous oncogenic signalling pathways. The goal of this study was to determine the prognostic value of phosphorylated (tyrosine705)-STAT3 in node-positive breast cancer patients.MethodsImmunohistochemical analysis of Phospho-STAT3 was performed on a tissue microarray of breast cancer specimens. The expression pattern of Phospho-STAT3 was correlated with survival outcome, and clinical and pathological parameters.ResultsOut of 125 interpretable tumours, positive Phospho-STAT3 nuclear expression was seen in 35 (28%) of tumours. There was no significant relationship between Phospho-STAT3 expression and clinical-pathological parameters including age, hormonal receptor status, grade and tumour size. Interestingly positive tumours had a significantly improved disease-free survival at 5 years (p=0.035). Additionally, positive Phospho-STAT3 nuclear expression was correlated with significantly improved survival at both 5 years (p=0.023) and 10 years (p=0.026). Finally, in multivariate analyses Phospho-STAT3 was found to be an independent prognostic marker of overall survival in node-positive breast cancer patients.ConclusionThese findings support the role of Phospho-STAT3 as an important independent prognostic marker in node-positive breast cancer patients.

Mutation spectrum in HNPCC in the Israeli population

Hereditary non-polyposis colon cancer is caused by mutations in DNA mismatch repair genes. The mutation spectrum in the Israeli population is poorly documented except for the c.1906G>C Ashkenazi founder mutation in the hMSH2 gene. To report our experience in HNPCC screening, the mutations detected and the clinical features among a cohort of Israeli patients. Diagnostic work-up was done in a multi-step process guided by clinical and ethnic information. Tumors of suspected patients were tested for microsatellite instability and immunohistochemistry. Based on tumor analyses, we proceeded to mutation screening by DHPLC followed by sequence analysis and multiplex ligase dependent probe amplification. Ashkenazi Jews were first tested for the c.1906G>C founder mutation. Of the 240 families, 24, including Arabs and Jews from different ethnic origins, were tested positive. All tumors that lost expression of mismatch repair proteins also showed microsatellite instability. There was evidence for involvement of hMSH2 (15) hMLH1 (6) and hMSH6 (3) genes. Mutations were identified in 17/24 (71%) patients: 6 Ashkenazi families harbored the c.1906G>C mutation. Eleven other mutations (2 nonsense, 3 splice site and 6 small deletions) were detected. Three of the mutations are novel. No gross deletions or insertions were detected. This is the first report that characterizes the profile of HNPCC in a cohort of patients in Israel. Tumor testing indicated that the 3 main MMR genes are involved, and that mutation spectrum is broad.