Iván Roa

Gallbladder cancer: An analysis of a series of 139 patients with invasion restricted to the subserosal layer

The goal was to study our experience in the management of a series of patients with a potentially curative subserosal gallbladder cancer who were prospectively treated by the authors. Between April 1988 and July 2004, 139 patients were enrolled in our prospective database. Of the above, 120 were operated on with an open procedure and the rest with laparoscopic surgery. In only eight patients was the diagnosis suspected before the cholecystectomy. The majority of tumors were adenocarcinoma. Six patients had an epidermoid tumor, and one had a carcinosarcoma. Of the patients, 74 underwent reoperation, while in 55 (70.2%) it was possible to perform an extended cholecystectomy with a curative aim. Operative mortality was 0%, and operative morbidity was 16%. Lymph node metastases were found in 10 (18.8%), while in 7 (13.2%) the liver was involved. The overall survival rate was 67.7%, while in those who underwent resection, the survival rate was 77%. Through the use of a multivariate analysis, the presence of lymph node metastasis was found to be an independent factor with respect to prognosis. The feasibility of performing an extended cholecystectomy in patients with gallbladder cancer and invasion of the subserosal layer allows for a good survival rate. The presence of lymph node metastases represents the main poor prognosis factor, and some type of adjuvant therapy should be studied in this particular group.

Microsatellite instability, prognosis and metastasis in gastric cancers from a low‐risk population

We examined 169 cases of gastric adenocarcinoma for microsatellite instability (MSI), using a panel of 8 microsatellite markers. Of these cases, 142 were from the United States, a country of relatively low risk for gastric cancer. Comparing microdissected tumors to normal cells from the same patient, we classified tumors as being microsatellite‐stable (MSS) or having a low frequency of MSI (MSI‐L, up to 30% of markers different in the tumor) or a high frequency of MSI (MSI‐H, 30% or more of markers different). Among our American cases, we identified 26 (18.2%) showing MSI‐H and 15 (10.6%) showing MSI‐L. Twenty cases were from Korean patients, and they showed no significant differences in proportions of MSI‐H and MSI‐L from the American cases. MSI‐H tumors in the American patients were characterized by elevated frequencies of band shifts in repeat sequences of the BAX (50%), transforming growth factor‐β receptor type II (TGFβRII, 68.9%), β2‐microglobulin (21.4%) and E2F4 (51.7%) genes. Alterations in E2F4 in MSI‐H tumors were always integral multiples of 3 nucleotides lost or gained, which would not cause a frameshift mutation, and within the range of normal polymorphisms for this sequence. North American patients (n = 127) with MSI‐H and MSI‐L tumors had a longer median survival of 541 days and 587 days, respectively, compared to 265 days for patients with MSS tumors (p = 0.027). This survival difference may result from a significantly greater tendency for metastases in the MSS group (p = 0.031). Int. J. Cancer 89:444–452, 2000.

Microsatellite instability in preneoplastic and neoplastic lesions of the gallbladder

BackgroundGallbladder cancer is very common in Chile and is the leading cause of cancer deaths in women aged over 40 years. However, there is limited information about the molecular changes involved in its pathogenesis. Microsatellite analysis was performed using polymerase chain reaction (PCR)-based assays to identify genetic loci that were altered in neoplastic and preneoplastic conditions of early and advanced gallbladder cancer. Our findings were then correlated with clinicopathological variables and survival time.MethodsWe selected 59 surgical specimens of gallbladder adenocarcinomas (29 early cancers and 30 advanced cancers) and 22 surgical specimens from patients with chronic cholecystitis from a high-risk area for gallbladder cancer (Temuco, Chile). Laser capture microdissection (LCM) was used to harvest tumor cells and preneoplastic lesions. Microsatellite analysis was performed using 13 different markers. The tumors and preneoplastic lesions were also examined with immunohistochemistry for hMLH1, hMSH2, and hMSH6.ResultsWe found that 10% (6/59) of gallbladder cancers showed high-frequency microsatellite instability (MSI-H), with identical proportions in both early and advanced cancers. In premalignant lesions adjacent to the six MSI-H tumors, we detected instability in two of six examples of intestinal metaplasia (33%) and five of six examples of dysplasia (83%). All MSI-H cases showed an altered pattern with the antibodies studied. MSI status was not associated with survival or other clinicopathological features. No MSI or immunohistochemical alterations were found in the chronic cholecystitis group.ConclusionsMicrosatellite instability was observed in equal proportions in early and late cancers, and it was also found in premalignant lesions, indicating that inactivation of mismatch repair genes occurs early in gallbladder carcinogenesis.

Serial Analysis of Gene Expression Identifies Connective Tissue Growth Factor Expression as a Prognostic Biomarker in Gallbladder Cancer

Background: Gallbladder cancer (GBC) is an uncommon neoplasm in the United States, but one with high mortality rates. This malignancy remains largely understudied at the molecular level such that few targeted therapies or predictive biomarkers exist. Experimental Design: We built the first series of serial analysis of gene expression (SAGE) libraries from GBC and nonneoplastic gallbladder mucosa, composed of 21-bp long-SAGE tags. SAGE libraries were generated from three stage-matched GBC patients (representing Hispanic/Latino, Native American, and Caucasian ethnicities, respectively) and one histologically alithiasic gallbladder. Real-time quantitative PCR was done on microdissected epithelium from five matched GBC and corresponding nonneoplastic gallbladder mucosa. Immunohistochemical analysis was done on a panel of 182 archival GBC in high-throughput tissue microarray format. Results: SAGE tags corresponding to connective tissue growth factor (CTGF) transcripts were identified as differentially overexpressed in all pairwise comparisons of GBC (P < 0.001). Real-time quantitative PCR confirmed significant overexpression of CTGF transcripts in microdissected primary GBC (P < 0.05), but not in metastatic GBC, compared with nonneoplastic gallbladder epithelium. By immunohistochemistry, 66 of 182 (36%) GBC had high CTGF antigen labeling, which was significantly associated with better survival on univariate analysis (P = 0.0069, log-rank test). Conclusions: An unbiased analysis of the GBC transcriptome by SAGE has identified CTGF expression as a predictive biomarker of favorable prognosis in this malignancy. The SAGE libraries from GBC and nonneoplastic gallbladder mucosa are publicly available at the Cancer Genome Anatomy Project web site and should facilitate much needed research into this lethal neoplasm.

Laparoscopic Cholecystectomy: Its Effect on the Prognosis of Patients with Gallbladder Cancer

Numerous reports suggest more recurrences and a worse prognosis after laparoscopic cholecystectomy (LC) than after open cholecystectomy (OC). The objective of this study was to compare the survival rate of patients undergoing a laparoscopic procedure versus those undergoing an open operation. A series of 24 patients with gallbladder cancer detected after LC were compared with 40 consecutive patients with gallbladder cancer detected after OC. Patients were matched by wall invasion, age, and whether they underwent a reoperation or only cholecystectomy. The series included 2 patients with in situ tumors, 2 with mucosal tumors, 1 with muscular invasion, 13 with subserosal invasion, and 6 with serosal invasion. Recurrences were observed in 4 of the 10 patients with subserosal compromise who underwent reoperation. In contrast, in the OC group of 26 patients with subserosal invasion, 20 of whom were reoperated, only 2 had a recurrence. Of the six patients with serosal infiltration, three in the LC underwent reoperation, all of whom had recurrences that precluded resection. Of the 12 patients in the OC group who presented with serosal invasion, 6 were reoperated and 4 had a recurrence. Overall survival curves did not show differences when patients were compared according to the type of procedure performed. Similarly, the analysis of patients according to the level of wall invasion indicated that there was no significant difference in survival. Although multiple reports have shown a worse prognosis for patients with gallbladder cancer undergoing LC, this study did not show a significant survival difference between the two methods. Although there is a higher but insignificant recurrence rate among the patients who underwent LC, this is not translated into survival.

Molecular Characterization of Gallbladder Cancer using Somatic Mutation Profiling

Gallbladder cancer is relatively uncommon with high incidence in certain geographic locations, including Latin America, East and South Asia and Eastern Europe. Molecular characterization of this disease has been limited and targeted therapy options for advanced disease remain an open area of investigation. In the present study, surgical pathology obtained from resected gallbladder cancer cases (n=72) was examined for the presence of targetable, somatic mutations. All cases were formalin-fixed and paraffin-embedded (FFPE). Two approaches were used: a) mass spectroscopy-based profiling for 159 point (‘hot-spot’) mutations in 33 genes commonly involved in solid tumors and b) next-generation sequencing (NGS) platform that examined the complete coding sequence of in 182 cancer-related genes. Fifty-seven cases were analyzed for hotspot mutations and 15 for NGS. Fourteen hotspot mutations were identified in nine cases. Of these, KRAS mutation was significantly associated with poor survival on multivariate analysis. Other targetable mutations included PIK3CA (N=2) and ALK (N=1). On NGS, 26 mutations were noted in 15 cases. P53 and PI3 kinase pathway (STK11, RICTOR,TSC2) mutations were common. One case had FGF10 amplification while another had FGF3-TACC gene fusion, not previously described in gallbladder cancer. In conclusion, somatic mutation profiling using archival FFPE samples from gallbladder cancer is feasible. NGS, in particular may be a useful platform for identifying novel mutations for targeted therapy.

p53 tumour suppressor gene protein expression in early and advanced gallbladder carcinoma.

Gallbladder carcinoma is one of the most frequent malignant tumours occurring in Chile and the mortality rate in both sexes ranks among one of the highest in the world. Mutation of p53 tumour suppressor gene has been demonstrated in many tumours. Our aim was to determine protein expression of p53 gene in early and advanced gallbladder carcinoma.

Promoter methylation profile in gallbladder cancer.

BackgroundMethylation in the promoter region of genes is an important mechanism of inactivation of tumor suppressor genes. Our objective was to analyze the methylation pattern of some of the genes involved in carcinogenesis of the gallbladder, examining the immunohistochemical expression of proteins, clinical features, and patient survival time.MethodsTwenty cases of gallbladder cancer were selected from the frozen tumor bank. The DNA extracted was analyzed by means of a methylation-specific polymerase chain reaction test for the CDKN2A (p16), MLH1, APC, FHIT, and CDH1 (E-cadherin) genes. Morphological and clinical data and follow-up information were obtained.ResultsAll cases were in an advanced stage: histologically moderate or poorly differentiated tumors (95%). Methylation of the promoter area of genes was observed in 5%, 20%, 30%, 40%, and 65% of cases, and an altered immunohistochemical pattern (AIP) in 5%, 35%, 21%, 25%, and 66% for the MLH1, CDKN2A, FHIT, APC, and CDH1 genes, respectively. The Kappa concordance index between methylation of the promoter area and AIP for the MLH1 and CDH1 genes was very high (K > 0.75) and substantial for APC (K > 0.45). No correlation was found between survival time and the methylation of the genes studied.ConclusionsThe high frequency of gene methylation (with the exception of MLH1) and the high agreement between AIP and methylation of the gene promoter area for the MLH1, APC, and CDH1 genes suggest that the inactivation of tumor suppressor genes and of the genes related to the control of cellular proliferation through this mechanism is involved in gallbladder carcinogenesis.

Gallbladder cancer in Chile: Pathologic characteristics of survival and prognostic factors: Analysis of 1,366 cases

OBJECTIVES To explore gallbladder cancer (GBC), the second leading cause of cancer-related death in women in Chile. METHODS Analysis of macroscopic and microscopic variables, morphometry, and survival in 1,366 patients with GBC. RESULTS Patients comprised 1,138 women and 228 men; diagnoses included 213 (15.6%) cases of mucosal carcinoma, 132 (9.7%) cases of muscular carcinoma, 316 (23.1%) cases of subserosal carcinoma, 382 (28.0%) cases of serosal carcinoma, and 323 (23.6%) cases beyond the serosa. Women older than 55 years with a gallbladder length greater than 9.5 cm had a five-times-greater relative risk of cancer. Those with a gallbladder wall thickness less than 7 mm had a better 5-year survival rate than those with a gallbladder wall thickness greater than 10 mm (P = .0001). Patients who had cholesterolosis of the gallbladder had 9.2 times less probability of having cancer. The infiltration level of the gallbladder wall was the most important independent prognostic factor (P < .001), followed by differentiation and lymphatic involvement (P < .001 and P = .05, respectively). Vascular infiltration had a mortality rate of 100%. CONCLUSIONS Morphologic features are strongly associated with the prognosis of GBC and must be taken into consideration when supplementary treatment is recommended.

The low‐abundance transcriptome reveals novel biomarkers, specific intracellular pathways and targetable genes associated with advanced gastric cancer

Studies on the low‐abundance transcriptome are of paramount importance for identifying the intimate mechanisms of tumor progression that can lead to novel therapies. The aim of the present study was to identify novel markers and targetable genes and pathways in advanced human gastric cancer through analyses of the low‐abundance transcriptome. The procedure involved an initial subtractive hybridization step, followed by global gene expression analysis using microarrays. We observed profound differences, both at the single gene and gene ontology levels, between the low‐abundance transcriptome and the whole transcriptome. Analysis of the low‐abundance transcriptome led to the identification and validation by tissue microarrays of novel biomarkers, such as LAMA3 and TTN; moreover, we identified cancer type‐specific intracellular pathways and targetable genes, such as IRS2, IL17, IFNγ, VEGF‐C, WISP1, FZD5 and CTBP1 that were not detectable by whole transcriptome analyses. We also demonstrated that knocking down the expression of CTBP1 sensitized gastric cancer cells to mainstay chemotherapeutic drugs. We conclude that the analysis of the low‐abundance transcriptome provides useful insights into the molecular basis and treatment of cancer.