Eric R. Siegel

Thiazolidinediones and the Risk of Lung, Prostate, and Colon Cancer in Patients With Diabetes

PURPOSE Peroxisome proliferator-activated receptor gamma (PPARgamma) mediates cell cycle arrest and adipocyte differentiation; has tumor suppressor activity in liposarcoma, lung, and prostate cancers; and suppresses colonic polyp formation in adenomatous polyposis coli (APC)min/+ mice. To assess the influence of thiazolidinediones (TZDs), which are PPAR ligands used to treat diabetes mellitus, a retrospective analysis of a database from 10 Veteran Affairs medical centers was conducted. PATIENTS AND METHODS Data on male patients 40 years and older diagnosed to have diabetes mellitus between 1997 and 2003 were obtained from the Veterans Integrated Services Network 16 (VISN 16) data warehouse. Subsequent diagnoses of colorectal, lung, and prostate cancer and use of TZD, other antidiabetic agents, and insulin were identified. Cox regression with time-dependent covariates was used to estimate the association between TZD use and cancer risk. Relative risks were adjusted for confounders (age, race/ethnicity, body mass index, use of insulin, and other oral antidiabetic agents). RESULTS Of 87,678 individuals, 1,137 had colorectal cancer, 3,246 had prostate cancer, and 1,371 had lung cancer. We observed a 33% reduction in lung cancer risk among TZD users compared with nonusers after adjusting for confounder interactions (relative risk, 0.67; 95% CI, 0.51 to 0.87). The risk reduction for colorectal and prostate cancers did not reach statistical significance. CONCLUSION TZD use was associated with reduced risk of lung cancer. Further studies are warranted to confirm our findings.

Results of autologous stem cell transplant in multiple myeloma patients with renal failure

Data are presented on 81 multiple myeloma (MM) patients with renal failure (creatinine > 176·8 μmol/l) at the time of autologous stem cell transplantation (auto‐SCT), including 38 patients on dialysis. The median age was 53 years (range: 29–69) and 26% had received more than 12 months of prior chemotherapy. CD34+ cells were mobilized with granulocyte colony‐stimulating factor (G‐CSF) alone (n = 51) or chemotherapy plus G‐CSF (n = 27), yielding medians of 10 and 16 × 106 CD34+ cells/kg respectively (P = 0·003). Sixty patients (27 on dialysis) received melphalan 200 mg/m2 (MEL‐200). Because of excessive toxicity, the subsequent 21 patients (11 on dialysis) received MEL 140 mg/m2 (MEL‐140). Thirty‐one patients (38%) completed tandem auto‐SCT, including 11 on dialysis. Treatment‐related mortality (TRM) was 6% and 13% after the first and second auto‐SCT. Median times to absolute neutrophil count (ANC) > 0·5 × 109/l and to platelets > 50 × 109/l were 11 and 41 d respectively. Non‐haematological toxicities included mucositis, pneumonitis, dysrhythmias and encephalopathy. At a median follow up of 31 months, 30 patients have died. Complete remission (CR) was achieved in 21 patients (26%) after first SCT and 31 patients (38%) after tandem SCT. Two patients discontinued dialysis after SCT. Median durations of complete remission (CR) and overall survival (OS) have not been reached; probabilities of event‐free survival (EFS) and OS at 3 years were 48% and 55% respectively. Dialysis dependence and MEL dose did not affect EFS or OS. Sensitive disease prior to SCT, normal albumin level and younger age were independent prognostic factors for better OS. In conclusion, renal failure had no impact on the quality of stem cell collections and did not affect engraftment. MEL‐140 had an acceptable toxicity and appeared equally effective as MEL‐200. In the setting of renal failure, the role of auto‐SCT early in the disease course and benefits of tandem SCT require further evaluation.

Improved Outcome of Allogeneic Transplantation in High-Risk Multiple Myeloma Patients After Nonmyeloablative Conditioning

PURPOSE We present our experience with relapsed and recently diagnosed patients with high-risk multiple myeloma (MM) receiving immunosuppressive, nonmyeloablative melphalan (MEL)-based conditioning regimens (mini-allograft). PATIENTS AND METHODS Thirty-one MM patients received allografts from HLA-matched siblings (n = 25) or unrelated donors (n = 6) using a mini-allograft. Seventeen had progressive disease (PD) and 14 had responsive disease (RD) (six with primary RD and eight with responsive relapse). Thirty patients had received one (n = 13) or two or more (n = 17) prior autologous transplantations (ATs). Median age was 56 years (range, 38 to 69 years). Twenty-one patients had chromosome 13 abnormality. Two patients were hemodialysis dependent. Blood and bone marrow grafts were administered to 28 and three patients, respectively. Donor lymphocyte infusions were given to 18 patients either to attain full donor chimerism (n = 6) or to eradicate residual disease (n = 12). RESULTS By day 100, 25 (89%) of 28 patients were full donor chimeras, one was a mixed chimera, and two had autologous reconstitution. Acute graft-versus-host disease (GVHD) developed in 18 patients (58%), and 10 progressed to chronic GVHD (limited in six and extensive in four). At a median follow-up of 6 months, 19 (61%) of 31 patients achieved complete/near complete remission. Twelve patients (39%) have died: three of PD, three of early treatment-related mortality (TRM) (before day 100), and six of late TRM. Median overall survival (OS) was 15 months. At 1 year, there was a significantly longer event-free survival (86% v 31%, P =.01) and OS (86% v 48%, P =.04) when a mini-allograft was performed after one versus two or more prior ATs, respectively. When compared with historical MM controls (n = 93) receiving conventional allografts, early TRM was significantly lower (10% v 29%, P =.03), and OS at 1 year was better (71% v 45%; P =.08) in the mini-allograft MM patients. CONCLUSION Mini-allograft induced excellent disease control in MM patients with high-risk disease, but is still associated with a significant GVHD.

Autologous stem cell transplantation in elderly multiple myeloma patients over the age of 70 years

The feasibility and efficacy of autologous stem cell transplantation (auto‐SCT) in patients aged ≥ 70 years was analysed. Newly diagnosed (n = 34) and refractory multiple myeloma (n = 36) patients were studied. The median age was 72 years (range: 70–82·6). CD34+ cells were mobilized with chemotherapy and granulocyte colony‐stimulating factor (G‐CSF) (n = 35) or G‐CSF alone (n = 35), yielding medians of 11·8 × 106 versus 8 × 106cells/kg respectively (P = 0·007). Because of excessive mortality (16%) in the first 25 patients who received melphalan 200 mg/m2 (MEL‐200), the dose was subsequently decreased to 140 mg/m2 (MEL‐140). Median times to absolute neutrophil count (ANC) > 0·5 × 109/l and to platelets > 20 × 109/l were 11 and 13 d respectively. Thirty‐one patients (44%) received tandem auto‐SCT. Complete remission (CR) was 20% after the first SCT and 27% after tandem SCT. Median CR duration was 1·5 years and was significantly longer for patients with ≤ 12 months of prior chemotherapy (2·6 versus 1·0 years, P = 0·0008). The 3‐year event‐free survival (EFS) and overall survival (OS) (+ standard error, SE) were projected at 20% + 9% and 31% + 10% respectively. Tandem SCTs positively affected EFS (4·0 versus 0·7 years; P = 0·003) and OS (4·0 versus 1·4 years; P = 0·02) compared with single auto‐SCT. In conclusion, MEL‐140 is less toxic and appears equally as efficacious as MEL‐200 in elderly patients. The benefits of tandem SCT in this patient population need further evaluation in a randomized trial.

Mobilization of CD34+ cells in elderly patients (≥ 70 years) with multiple myeloma: influence of age, prior therapy, platelet count and mobilization regimen

Summary. The mobilization of peripheral blood stem cells was studied in 984 multiple myeloma patients, including 106 patients aged ≥ 70 years. Increasing age correlated inversely with CD34+ yield (P < 0·0001), but also with ≥ 12 months of prior standard chemotherapy (P = 0·0001), < 200 × 109/l platelets (P = 0·0006) premobilization and mobilization with growth factors only (P = 0·0001). After controlling for these age covariates, multivariate analysis identified ≤ 12 months standard therapy and platelet count ≥ 200 × 109/l premobilization as favourable variables (both P < 0·0001), while increasing patient age remained an unfavourable factor (P = 0·0009). With both favourable variables, 85% of elderly patients collected ≥ 4 × 106/kg CD34+ cells in a median of one collection. The effect of age was incremental with no age threshold showing acceleration in the decline of CD34+ yield. Chemotherapy significantly increased CD34+ yield compared with growth factors only. However, the subgroup of patients with > 12 months prior therapy and premobilization platelet count < 200 × 109/l mobilized as many CD34+ cells with granulocyte colony‐stimulating factor (G‐CSF) alone as with chemotherapy and haematopoietic growth factors. Increasing patient age had no effect on post‐transplant neutrophil recovery, but significantly delayed platelet recovery (≥ 50 × 109/l) if < 2 × 106/kg CD34+ cells were infused, but this effect was eliminated completely with infusion of ≥ 4 × 106/kg CD34+ cells. Increasing age adversely affected CD34+ yield even with limited premobilization therapy, indicating that early collection is important in elderly patients.

Human Papillomavirus Type 16 and 18 E7-Pulsed Dendritic Cell Vaccination of Stage IB or IIA Cervical Cancer Patients: a Phase I Escalating-Dose Trial

ABSTRACT The safety and immunogenicity of the human papillomavirus type 16 (HPV16) or HPV18 (HPV16/18) E7 antigen-pulsed mature dendritic cell (DC) vaccination were evaluated for patients with stage IB or IIA cervical cancer. Escalating doses of autologous DC (5, 10, and 15 × 106 cells for injection) were pulsed with recombinant HPV16/18 E7 antigens and keyhole limpet hemocyanin (KLH; an immunological tracer molecule) and delivered in five subcutaneous injections at 21-day intervals to 10 cervical cancer patients with no evidence of disease after they underwent radical surgery. Safety, toxicity, delayed-type hypersensitivity (DTH) reaction, and induction of serological and cellular immunity against HPV16/18 E7 and KLH were monitored. DC vaccination was well tolerated, and no significant toxicities were recorded. All patients developed CD4+ T-cell and antibody responses to DC vaccination, as detected by enzyme-linked immunosorbent spot (ELISpot) and enzyme-linked immunosorbent assays (ELISA), respectively, and 8 out of 10 patients demonstrated levels of E7-specific CD8+ T-cell counts, detected by ELISpot during or immediately after immunization, that were increased compared to prevaccination baseline levels. The vaccine dose did not predict the magnitude of the antibody or T-cell response or the time to detection of HPV16/18 E7-specific immunity. DTH responses to intradermal injections of HPV E7 antigen and KLH were detected for all patients after vaccination. We conclude that HPV E7-loaded DC vaccination is safe and immunogenic for stage IB or IIA cervical cancer patients. Phase II E7-pulsed DC-based vaccination trials with cervical cancer patients harboring a limited tumor burden, or who are at significant risk of tumor recurrence, are warranted.

Treatment of chemotherapy-resistant human ovarian cancer xenografts in C.B-17/SCID mice by intraperitoneal administration of Clostridium perfringens enterotoxin.

Ovarian cancer remains the most lethal gynecologic malignancy in the United States. Although many patients with advanced-stage disease initially respond to standard combinations of surgical and cytotoxic therapy, nearly 90% develop recurrence and inevitably die from the development of chemotherapy-resistant disease. The discovery of novel and effective therapy against chemotherapy-resistant/recurrent ovarian cancer remains a high priority. Using expression profiling, we and others have recently found claudin-3 and claudin-4 genes to be highly expressed in ovarian cancer. Because these tight junction proteins have been described as the low- and high-affinity receptors, respectively, for the cytotoxic Clostridium perfringens enterotoxin (CPE), in this study we investigated the level of expression of claudin-3 and/or claudin-4 in chemotherapy-naive and chemotherapy-resistant primary human ovarian cancers as well as their sensitivity to CPE treatment in vitro. We report that 100% (17 of 17) of the primary ovarian tumors tested overexpress one or both CPE receptors by quantitative reverse transcription-PCR. All ovarian tumors showed a dose-dependent cytotoxic effect to CPE in vitro. Importantly, chemotherapy-resistant/recurrent ovarian tumors were found to express claudin-3 and claudin-4 genes at significantly higher levels when compared with chemotherapy-naive ovarian cancers. All primary ovarian tumors tested, regardless of their resistance to chemotherapeutic agents, died within 24 hours to the exposure to 3.3 microg/mL CPE in vitro. In addition, we have studied the in vivo efficacy of i.p. CPE therapy in SCID mouse xenografts in a highly relevant clinical model of chemotherapy-resistant freshly explanted human ovarian cancer (i.e., OVA-1). Multiple i.p. administration of sublethal doses of CPE every 3 days significantly inhibited tumor growth in 100% of mice harboring 1 week established OVA-1. Repeated i.p. doses of CPE also had a significant inhibitory effect on tumor progression with extended survival of animals harboring large ovarian tumor burdens (i.e., 4-week established OVA-1). Our findings suggest that CPE may have potential as a novel treatment for chemotherapy-resistant/recurrent ovarian cancer.

Chondroitin sulfates play a major role in breast cancer metastasis: a role for CSPG4 and CHST11 gene expression in forming surface P-selectin ligands in aggressive breast cancer cells

IntroductionWe have previously demonstrated that chondroitin sulfate glycosaminoglycans (CS-GAGs) on breast cancer cells function as P-selectin ligands. This study was performed to identify the carrier proteoglycan (PG) and the sulfotransferase gene involved in synthesis of the surface P-selectin-reactive CS-GAGs in human breast cancer cells with high metastatic capacity, as well as to determine a direct role for CS-GAGs in metastatic spread.MethodsQuantitative real-time PCR (qRT-PCR) and flow cytometry assays were used to detect the expression of genes involved in the sulfation and presentation of chondroitin in several human breast cancer cell lines. Transient transfection of the human breast cancer cell line MDA-MB-231 with the siRNAs for carbohydrate (chondroitin 4) sulfotransferase-11 (CHST11) and chondroitin sulfate proteoglycan 4 (CSPG4 ) was used to investigate the involvement of these genes in expression of surface P-selectin ligands. The expression of CSPG4 and CHST11 in 15 primary invasive breast cancer clinical specimens was assessed by qRT-PCR. The role of CS-GAGs in metastasis was tested using the 4T1 murine mammary cell line (10 mice per group).ResultsThe CHST11 gene was highly expressed in aggressive breast cancer cells but significantly less so in less aggressive breast cancer cell lines. A positive correlation was observed between the expression levels of CHST11 and P-selectin binding to cells (P < 0.0001). Blocking the expression of CHST11 with siRNA inhibited CS-A expression and P-selectin binding to MDA-MB-231 cells. The carrier proteoglycan CSPG4 was highly expressed on the aggressive breast cancer cell lines and contributed to the P-selectin binding and CS-A expression. In addition, CSPG4 and CHST11 were over-expressed in tumor-containing clinical tissue specimens compared with normal tissues. Enzymatic removal of tumor-cell surface CS-GAGs significantly inhibited lung colonization of the 4T1 murine mammary cell line (P = 0.0002).ConclusionsCell surface P-selectin binding depends on CHST11 gene expression. CSPG4 serves as a P-selectin ligand through its CS chain and participates in P-selectin binding to the highly metastatic breast cancer cells. Removal of CS-GAGs greatly reduces metastatic lung colonization by 4T1 cells. The data strongly indicate that CS-GAGs and their biosynthetic pathways are promising targets for the development of anti-metastatic therapies.

Overexpression of epithelial cell adhesion molecule in primary, metastatic, and recurrent/chemotherapy-resistant epithelial ovarian cancer: implications for epithelial cell adhesion molecule-specific immunotherapy.

To evaluate the potential of epithelial cell adhesion molecule (Ep-CAM/TROP-1)-specific immunotherapy against epithelial ovarian carcinomas (EOCs), we have analyzed the expression of Ep-CAM at RNA and protein level in patients harboring primary, metastatic, and chemotherapy-resistant/recurrent EOC. Epithelial cell adhesion molecule expression was evaluated by real-time polymerase chain reaction and immunohistochemistry in 168 fresh-frozen biopsies and paraffin-embedded tissues. In addition, Ep-CAM surface expression was evaluated by flow cytometry in several freshly established ovarian carcinoma cell lines derived from patients harboring tumors resistant to chemotherapy in vivo as well as in vitro. Epithelial cell adhesion molecule transcript was found significantly overexpressed in primary, metastatic, and recurrent EOC when compared with normal human ovarian surface epithelium cell lines and fresh-frozen normal ovarian tissue (P < 0.001). Similarly, by immunohistochemistry, Ep-CAM protein expression was found significantly higher in primary, metastatic, and recurrent EOC when compared with normal ovarian tissues. Of interest, metastatic/recurrent tumors were found to express significantly higher levels of Ep-CAM protein when compared with primary ovarian carcinomas (P < 0.001). Finally, a high surface expression of Ep-CAM was found in 100% (5/5) of the chemotherapy-resistant ovarian carcinoma cell lines studied by flow cytometry. These results demonstrate high Ep-CAM overexpression in ovarian carcinoma, especially in metastatic and recurrent/chemotherapy-resistant ovarian disease. The lack of Ep-CAM expression on the chelomic epithelium in the peritoneal cavity, combined with the recent development of fully human monoclonal antibodies against this surface molecule, suggest Ep-CAM as a promising target for antibody-mediated therapies in ovarian carcinoma patients harboring tumors refractory to standard treatment modalities.

Overexpression of claudin-3 and claudin-4 receptors in uterine serous papillary carcinoma: novel targets for a type-specific therapy using Clostridium perfringens enterotoxin (CPE).

Uterine serous papillary carcinoma (USPC) represents a highly aggressive variant of endometrial cancer. Using gene expression profiling, we recently identified high expression of the claudin‐3 and claudin‐4 receptors in a limited set of USPC. These tight junction proteins represent the low‐ and high‐affinity receptors, respectively, for the cytotoxic Clostridium perfringens enterotoxin (CPE) and are sufficient to mediate CPE binding and trigger subsequent toxin‐mediated cytolysis. The potential for targeting this pathway in the treatment of USPC was explored.