Silencing of miR-148a in cancer-associated fibroblasts results in WNT10B-mediated stimulation of tumor cell motility.

The tumor microenvironment has an important role in cancer progression. Here we show that miR-148a is downregulated in 15 out of 16 samples (94%) of cancer-associated fibroblasts (CAFs) compared with matched normal tissue fibroblasts (NFs) established from patients with endometrial cancer. Laser-capture microdissection of stromal cells from normal tissue and endometrial cancer confirmed this observation. Treatment of cells with 5-aza-deoxycytidine stimulated the expression of miR-148a in the majority of CAFs implicating DNA methylation in the regulation of miR-148a expression. Investigation of miR-148a function in fibroblasts demonstrated that conditioned media (CM) from CAFs overexpressing miR-148a significantly impaired the migration of five endometrial cancer cell lines without affecting their growth rates in co-culture experiments. Among predicted miR-148a target genes are two WNT family members, WNT1 and WNT10B. Activation of the WNT/ß-catenin pathway in CAFs was confirmed by microarray analysis of gene expression and increased activity of the SuperTOPFlash luciferase reporter. We found elevated levels of WNT10B protein in CAFs and its level decreased when miR-148a was re-introduced by lentiviral infection. The 3'-UTR of WNT10B, cloned downstream of luciferase cDNA, suppressed luciferase activity when co-expressed with miR-148a indicating that WNT10B is a direct target of miR-148a. In contrast to the effect of miR-148a, WNT10B stimulated migration of endometrial cancer cell lines. Our findings have defined a molecular mechanism in the tumor microenvironment that is a novel target for cancer therapy.

Rationale for the combination of cryoablation with surgical resection of hepatic tumors.

Only 5% to 10% of metastatic and primary liver tumors are amenable to surgical resection. Hepatic cryoablation has increased the number of patients who are suitable for curative treatment. The aim of this study was to evaluate survival and intrahepatic recurrence in patients treated with cryoablation and resection. From June 1994 to July 1999, thirty-eight surgically unresectable patients underwent a total of 42 cryoablative procedures for 65 malignant hepatic lesions. Twenty patients underwent cryoablation alone, and 18 patients were treated with a combination of resection and cryoablation, with a minimum of 18 months' follow-up. The 38 patients had the following malignancies: primary hepatocellular carcinoma (n = 8) and metastases from colorectal cancer (n = 21), neuroendocrine tumors (n = 3), ovarian cancer (n = 3), leiomyosarcoma (n = 1), testicular cancer (n = 1), and endometrial cancer (n = 1). Patients were evaluated preoperatively with spiral CT scans and intraoperatively with ultrasound examinations for lesion location and cryoprobe guidance. Local recurrence was detected by CT. Major complications included bleeding in three patients and acute renal failure, transient liver insufficiency, and postoperative pneumonia in one patient each. Two patients (5%) died during the early postoperative interval; mean hospital stay was 7.1 days. Median follow-up was 28 months (range 18 to 51 months). Overall survival according to Kaplan-Meier analysis was 82%, 65%, and 54% at 12, 24, and 48 months, respectively. Forty-eight-month survival was not significantly different between those patients undergoing cryoablation alone (64%) and those treated with a combination of resection and cryoablation (42%). Disease-free survival at 45 months was 36% for patients undergoing cryoablation plus resection compared to 25% for those undergoing cryoablation alone. Local recurrences were detected at five cryosurgical sites, for a rate of 12% overall (5 of 42), 11% (2 of 18) for patients in the cryoablation plus resection group, and 12% (3 of 24) for those in the cryoablation alone group. For patients with colorectal metastases, survival was 70% at 30 months compared to 33% for hepatocellular cancer and 66% for other types of tumors. Patients with tumors larger than 5 cm or numbering more than three did not have significantly decreased survival. Cryoablation of hepatic tumors is a safe and effective treatment for some patients not amenable to resection. The combination of cryoablation and resection results in survival comparable to that achieved with cryoablation alone.

Preoperative chemoradiation for marginally resectable adenocarcinoma of the pancreas.

Only 10% to 20% of patients with pancreatic cancer are considered candidates for curative resection at the time of diagnosis. We postulated that preoperative chemoradiation therapy might promote tumor regression, eradicate nodal metastases, and allow for definitive surgical resection in marginally resectable patients. The objective of this study was to evaluate the effect of a preoperative chemoradiation therapy regimen on tumor response, resectability, and local control among patients with marginally resectable adenocarcinoma of the pancreas and to report potential treatment-related toxicity. Patients with marginally resectable adenocarcinoma of the pancreas (defined as portal vein, superior mesenteric vein, or artery involvement) were eligible for this protocol. Patients received 50.4 to 56 Gy in 1.8 to 2.0 Gy/day fractions with concurrent protracted venous infusion of 5-fluorouracil (250 mg/m2/day). Reevaluation for surgical resection occurred 4 to 6 weeks after therapy. Fifteen patients (9 men and 6 women) completed preoperative chemoradiation without interruption. One patient required a reduction in the dosage of 5-fluorouracil because of stomatitis. Acute toxicity from chemoradiation consisted of grade 1 or 2 nausea, vomiting, diarrhea, stomatitis, palmar and plantar erythrodysesthesia, and hematologic suppression. CA 19-9 levels declined in all nine of the patients with elevated pretreatment levels. Nine of the 15 patients underwent a pancreaticoduodenectomy, and all had uninvolved surgical margins. Two of these patients had a complete pathologic response, and two had microscopic involvement of a single lymph node. With a median follow-up of 30 months, the median survival for resected patients was 30 months, whereas in the unresected group median survival was 8 months. Six of the nine patients who underwent resection remain alive and disease free with follow-up of 12, 30, 30, 34, 39, and 72 months, respectively. Preoperative chemoradiation therapy is well tolerated. It may downstage tumors, sterilize regional lymph nodes, and improve resectability in patients with marginally resectable pancreatic cancer. Greater patient accrual and longer follow-up are needed to more accurately assess its future role in therapy.

Metastatic breast cancer.

The unfortunate reality of metastatic breast cancer is that all treatment is palliative in nature. This is a disease that currently has no cure and for which therapy is directed towards accentuating survival and relieving symptoms. Current technology allows the prediction and detection of metastases earlier and with greater accuracy. These achievements need to be consolidated by the discovery of innovative therapies that can alter the inevitable outcome of this disease.

Chemoradiotherapy in the management of localized tumors of the pancreas.

In western countries, carcinoma of the pancreas remains the most lethal of the common malignancies. Even the favorable "organ-confined" tumors present a considerable challenge. The lack of anatomic barriers to local infiltration and the biological propensity for early lymphatic, perineural, and vascular invasion are nearly insurmountable obstacles to complete surgical eradication of this malignancy. Various combinations of chemotherapy and radiotherapy (RT) have been used with marginal but measurable success. Earlier trials conducted by the Gastrointestinal Tumor Study Group established roles for 5-fluorouracil chemotherapy and RT in the treatment of patients with resectable or locally advanced pancreatic cancer. More recently, computed tomography-guided conformal RT and a variety of intraoperative RT techniques have enabled more reliable sterilization of the local surgical field and escalation of doses to potentially curative levels (7000 cGy) for unresectable lesions. Chemotherapy dose intensification through the use of portable programmable pumps for protracted venous infusions and the development of active systemic agents in addition to 5-fluorouracil suggest that an effective combination chemotherapeutic regimen might soon be developed. This report reviews the current standards of practice and integrates recent developments to construct a modern algorithm for the use of chemoradiotherapy in the management of localized (nonmetastatic) pancreatic cancer. The likely directions of future investigations are also discussed.

Chemo-radiotherapy for localized pancreatic cancer: increased dose intensity and reduced acute toxicity with concomitant radiotherapy and protracted venous infusion 5-fluorouracil.

PURPOSE: Although concomitant radiation therapy (RT) and bolus 5-Fluorouracil (5-FU) have been shown to improve survival in locally confined pancreatic cancer, most patients will eventually succumb to their disease. Since 1994, we have attempted to improve efficacy by administering 5-FU as a protracted venous infusion (PVI). This study compares treatment intensity and acute toxicity of consecutive protocols of concurrent RT and 5-FU by bolus injection or PVI. METHODS AND MATERIALS: Since 1986, 74 patients with resected or locally advanced pancreatic cancer were treated with continuous course RT and concurrent 5-FU by bolus injection (n = 44) or PVI throughout the course of RT (n = 30). Dose intensity was assessed for both 5-FU and radiotherapy. Toxicity endpoints which could be reliably and objectively quantified (e.g., neutropenia, weight loss, treatment interruption) were evaluated. RESULTS: Cumulative 5-FU dose (mean = 7.2 vs. 2.5 gm/m2, p < 0.001) and weekly 5-FU dose (mean = 1.3 vs. 0.5 gm/m2/wk, p < 0.001) were significantly higher for patients receiving PVI 5-FU. Following pancreaticoduodenectomy, 95% of PVI patients maintained a RT dose intensity of > or = 900 cGy/wk, compared with 63% of those receiving bolus 5-FU (p = 0.02). No difference was seen for patients with locally advanced disease (72% vs. 76%, p = n.s.). Grade II-III neutropenia was less common for patients treated with PVI (13% vs. 34%, p = 0.05). Grade II-III thrombocytopenia was uncommon (< or = 3%) in both treatment groups. Mean percent weight loss (3.8% vs. 4.1%, p = n.s.) and weight loss > or = 5% of pre-treatment weight (21% vs. 31%, p = n.s.) were similar for PVI and bolus treatment groups, respectively. Treatment interruptions for hematologic, gastrointestinal or other acute toxicities were less common for patients receiving PVI 5-FU (10% vs. 25%, p = 0.11). CONCLUSION: Concurrent RT and 5-FU by PVI was well tolerated and permitted greater chemotherapy and radiotherapy dose intensity with reduced hematologic toxicity and fewer treatment interruptions compared with RT and bolus 5-FU. Longer follow-up will be needed to assess late effects and the impact on overall survival.

Hodgkin's disease associated with neurologic paraneoplastic syndrome.

In this case of neurologic paraneoplastic syndrome in a patient with Hodgkin's disease, both the Hodgkin's disease and the associated neurologic syndrome were cured by surgical intervention and postoperative adjuvant chemotherapy. The patient's tumor was removed en bloc. Postoperative chemotherapy consisted of doxorubicin (Adriamycin), etoposide, and vinblastine. At 5-year follow-up in August 1996, the patient was active and tumor-free, without evidence of recurrent Hodgkin's disease or paraneoplastic syndrome. The possibility that this patient's syndrome was the result of a cross-reacting monoclonal idiotype produced by his tumor is suggested by the rapid recovery associated with tumor debulking. The role of surgery in the treatment of patients with Hodgkin's disease is limited, well defined, and even more infrequent in recurrent disease. Systemic chemotherapy is the treatment of choice for these patients, but surgery may play an important role in the initial cytoreduction of the recurrent tumor, especially in cases of localized bulky disease.

NCCN Colorectal Cancer Practice Guidelines. The National Comprehensive Cancer Network.

In summary, the committee believes that a multidisciplinary approach is necessary for the management of the patient with colorectal cancer. The committee endorses the concept that treatment of patients on a clinical trial has priority over standard or accepted therapy. The recommended surgical procedure for managing resectable colon cancer is an en bloc resection; laparoscopic surgery should be done only in the context of a clinical trial. For patients with stage III disease, 5-FU-based adjuvant chemotherapy is recommended. A patient who has metastatic disease in the liver or lung should be considered for surgical resection if he or she is a candidate for surgery and if surgery can extend survival. The committee advocates a conservative post-treatment surveillance program for colon and rectal cancer patients. A determination of CEA should be done only if CEA was elevated at baseline and decreased following primary resection. Abdominal and pelvic CT scans should be utilized only when there are clinical indications of possible recurrence. Patients whose disease progresses during 5-FU-based therapy should be considered for treatment with irinotecan or encouraged to participate in a phase I or II clinical trial.

National patterns of care for pancreatic cancer. Results of a survey by the Commission on Cancer.

BACKGROUND: The Commission on Cancer of the American College of Surgeons conducted a large, national survey to assess methods of diagnosis, American Joint Commission on Cancer staging, treatment, and outcome of patients with adenocarcinoma of the pancreas. STUDY DESIGN: The survey questionnaire contained 160 questions and covered two study periods, 1983 to 1985 and 1990, for time-trend analysis. Nine hundred seventy-eight institutions throughout the United States voluntarily participated, contributing 8917 case reports for 1983 to 1985 and 8025 reports for 1990, resulting in a total of 16,942 patient reports. Most, but not all, of the participating hospitals maintain approval status with the Commission on Cancer of the American College of Surgeons. RESULTS: The ratio of male-to-female cases was 1:1. Patient characteristics including age, ethnicity, neighborhood income, type of insurance coverage, and hospital characteristics--including annual caseload and type of facility (e.g., teaching, community)--appeared to influence surgical multimodality treatment patterns. The most common presenting symptom was abdominal pain. The reported history of smoking for these patients with pancreatic cancer was higher than U.S. population averages. The frequency of using abdominal computed tomography scans, endoscopic retrograde cholangiopancreatography, carcinoembryonic antigen, and CA 19-9 during patient evaluation all increased. Time trends toward lower operative mortality and more extirpative surgery were reported, as was a slightly higher survival for those patients who were resected surgically. CONCLUSIONS: Pancreatic cancer continues to be a disease of older patients. There were slight improvements in operative mortality. For a highly selective category of patients, cancer-directed surgery offers a chance for cure with excellent operative mortality and acceptable complication rates, especially when performed in institutions that have a 20 or greater case per year experience.

Production and characterization of a monoclonal antibody against Schistosoma japonicum glutathione S-transferase.

Schistosoma japonicum glutathione S-transferase (GST), expressed from a pGEX plasmid, was isolated from Escherichia coli cells and used to immunize mice in order to generate specific anti-GST monoclonal antibodies. Using a modified immunization and fusion procedure, one stable hybridoma clone secreting an anti-GST antibody (alpha GST-1) was obtained. Milligram quantities of this antibody were produced in vitro in a miniPERM bioreactor and subsequently purified by protein G affinity chromatography. The characteristics of this antibody were investigated by enzyme-linked immunosorbent assays and immunoblotting experiments. The alpha GST-1 antibody was found to react specifically with GST and GST fusion proteins and demonstrated no reactivity with normal E. coli proteins. This monoclonal antibody should be a valuable reagent for tracing the production of GST fusion proteins and possibly for affinity purification of GST fusion proteins.

The National Cancer Data Base report on pancreatic cancer.

BACKGROUND: The National Cancer Data Base (NCDB) of the Commission on Cancer gathers data on time trends in stage of disease, treatment patterns, and survival for selected cancers. The most current (1991) data for pancreatic cancer are described here. The NCDB data contain important reference information regarding patient and hospital demographics associated with the diagnosis and treatment of pancreatic cancer. METHODS: Three Calls for Data have yielded a total of 17,490 cases for 1985, 1986, and 1991, from 937 hospital cancer registries across the United States. The data for 1991 represent 32% of all pancreas cancer cases for U.S. RESULTS: A higher incidence of more advanced disease was reported for the youngest group of patients, Hispanics, African Americans, Asians, males and patients seen at smaller hospitals. For all patients combined, only 14.2% were reported to have had a pancreatectomy. Older patients, patients from lower income zip codes, and African Americans were somewhat less likely to have received a pancreatectomy. Cancer of the body of the pancreas was the anatomic subsite for which patients with pancreatic cancer were least likely to receive a pancreatectomy. Patients seen at hospitals with larger case-loads and at teaching hospitals were more frequently reported as having had a pancreatectomy. Between 1985-1986 and 1991, there was a trend from treatment with surgery only or radiation only toward more frequent use of combined chemoradiation without surgery. There was less radiation reported as received by patients at hospitals with fewer than 150 annual cancer caseloads compared with hospitals with larger caseload. For patients with resectable tumors, a moderate survival advantage was reported compared with nonresectable tumors: 1-year, 48% versus 23%; 2-year, 24% versus 9%; 3-year, 17% versus 6% respectively. CONCLUSIONS: The NCDB provides a powerful tool for examining practice patterns and outcome of cancer care in the U.S. The present report, covering one-third of all patients treated in the U.S. in 1991, confirms the advanced disease status of patients with pancreatic cancer. Minority groups present with more advanced disease and are less likely to undergo resection, currently the only potentially curative therapy. Resectability rates appear to be higher in large caseload hospitals.

Transcription of the blk gene in human B lymphocytes is controlled by two promoters.

Genomic DNA containing the first exon and 5'-flanking region of the human protein tyrosine kinase, blk, was isolated. Sequence analysis identified a TG repeat element in this region with enhancer activity, but no TATA or CCAAT sequences were found. Two blk transcripts of 2.2 and 2.5 kilobases were identified in various B-cell lines by Northern blot analyses, and primer extension experiments demonstrated two clusters of multiple transcription start sites. Subsequent promoter analyses by transient transfection assays with a reporter gene identified two promoter elements in the human blk gene. Promoter P1 contains sequences that have been shown to regulate the expression of immunoglobulin genes and promoter P2 contains elements that are highly conserved in the promoter of major histocompatibility complex class II genes, as well as a B-cell-specific activator protein- (BSAP) binding site. Electrophoretic mobility shift assays demonstrated that the binding of a protein to the BSAP-binding site was correlated with the presence of the 2.5-kilobase blk transcript. These data suggest that the two human blk RNAs arise from the transcription of the blk gene by two distinct promoters and that these promoters may be subject to regulation by different trans-acting factors.

71.67 T lymphocyte activation is accompanied by the down-regulation of three G alpha genes.

A direct role for heterotrimeric G proteins in signal transduction through the T cell receptor complex has been hypothesized, but never definitively demonstrated. Nonetheless, several lines of evidence support the importance of G proteins in lymphocyte activation and differentiation events. In this study, PCR was used to generate cDNA segments of murine G alpha genes from a lymphocyte cell line by amplifying areas of divergent sequence between conserved primer sites. These murine cDNAs are homologous with previously reported G alpha s, G alpha i2, and G alpha i3 genes from other tissues and were used as probes to determine their levels of mRNA expression in a mitogen- and antigen-stimulated T lymphocyte line, 71.67, which provides a model for T cell activation. mRNA levels for all three G alpha genes were found to be differentially down-regulated with 71.67 activation, but not with stimulation of three more immature lymphocyte cell lines. These findings suggest that an important element in signal transduction through these G proteins in lymphocytes may include regulation of G alpha mRNA steady-state levels.

Molecular cloning and chromosomal localization of the human homologue of a B-lymphocyte specific protein tyrosine kinase (blk).

A cDNA encoding the human homologue of the murine protein tyrosine kinase, blk, has been cloned from a human B-lymphocyte cDNA library by cross-species hybridization using the murine blk cDNA as a probe. The sequence of the 2608 bp human blk cDNA clone contains an open reading frame encoding a predicted 505 amino acid protein with SH3, SH2 and catalytic domains that contain consensus sequences of the src protein tyrosine kinase family. Comparison of human and murine blk sequences indicated that they share 86% amino acid identity, the most conserved region being the catalytic domain (93% identity). Like the murine blk gene human blk is expressed only in B lymphocytes. The human blk gene was mapped to chromosome 8 at p22-23.