Micro RNA expression profiles as adjunctive data to assess the risk of hepatocellular carcinoma recurrence after liver transplantation.

Donor livers are precious resources and it is, therefore, ethically imperative that we employ optimally sensitive and specific transplant selection criteria. Current selection criteria, the Milan criteria, for liver transplant candidates with hepatocellular carcinoma (HCC) are primarily based on radiographic characteristics of the tumor. Although the Milan criteria result in reasonably high survival and low-recurrence rates, they do not assess an individual patient's tumor biology and recurrence risk. Consequently, it is difficult to predict on an individual basis the risk for recurrent disease. To address this, we employed microarray profiling of microRNA (miRNA) expression from formalin fixed paraffin embedded tissues to define a biomarker that distinguishes between patients with and without HCC recurrence after liver transplant. In our cohort of 64 patients, this biomarker outperforms the Milan criteria in that it identifies patients outside of Milan who did not have recurrent disease and patients within Milan who had recurrence. We also describe a method to account for multifocal tumors in biomarker signature discovery.

A 93 year old man with the PRSS1 R122H mutation, low SPINK1 expression, and no pancreatitis: insights into phenotypic non-penetrance.

BACKGROUND: The cationic trypsinogen (PRSS1) R122H mutation causes autosomal dominant hereditary pancreatitis (HP) with multiple attacks of acute pancreatitis, but the penetrance, frequency, and severity of attacks are highly variable. HP twins study suggests that modifier genes influence severity but not penetrance. AIM: To investigate potential trypsin associated factors in subjects with the PRSS1 R122H mutation and phenotypic non-penetrance. METHODS: Two subjects from HP families (including a 93 year old subject with PRSS1 R122H without pancreatitis), one with chronic pancreatitis and one with a normal pancreas, were studied. Relative expression of: (a) the PRSS1 R122 and H122 alleles; and (b) the PRSS1 and SPINK1 genes in pancreatitis were determined using complementary methods. RESULTS: PRSS1 wild-type (R122) and mutant (H122) allele expression was equivalent in multiple (> 3) samples from the phenotypically affected and non-penetrant subjects with R122H genotypes using allele specific quantitative reverse transcription-polymerase chain reaction (RT-PCR) and intron spanning nested RT-PCR followed by cDNA sequencing. Compared with PRSS1 mRNA levels, SPINK1 mRNA levels were low in normal appearing tissue but markedly increased in samples with chronic inflammation, independent of PRSS1 genotype. CONCLUSION: Attacks of acute pancreatitis in HP subjects appear to be independent of the relative expression of the mutant PRSS1 H122 allele or SPINK1 gene expression. The marked increase in SPINK1 gene expression with inflammation is consistent with its regulation as an acute phase protein.

Identification of genetic alterations related to chemoresistance in epithelial ovarian cancer.

OBJECTIVE: After the completion of primary chemotherapy, the majority of advanced ovarian cancer patients have persistent, chemoresistant disease. Comparative genomic hybridization (CGH) has been used to study genetic alterations that may be responsible for chemoresistance in ovarian cancer. CGH is a useful, genomewide screen but resolution is limited to 5-10 Mb. Recently, quantitative microsatellite analysis (QuMA), a TaqMan-based quantitative PCR technology, has been used for higher resolution of DNA copy number abnormalities. Our goal is to identify specific chromosomal aberrations correlated with platinum resistance. METHODS: Snap-frozen ovarian tissue samples taken from 22 patients with ovarian cancer between 1994 and 1998 were analyzed. Patients whose ovarian cancer actually demonstrated growth during platinum-combination treatment or no objective evidence of regression following four to six cycles of therapy were considered to have clinically defined platinum-resistant disease. QuMA was carried out at the following loci using the ABI Prism 7700 (TaqMan) instrument with a microsatellite repeat probe: D3S1553, D3S1617, D5S464, D5S630, D6S1581, D6S446, D8S557, D19S208, D20S196, DXS1068. Fisher's exact test, exact logistic regression, and the Cochran-Armitage trend test were used. Because of multiple hypothesis testing, the P values were adjusted with the Bonferroni procedure to limit the familywise error rate to at most 5%. RESULTS: Of the 22 patients, 12 (54.5%) were platinum-sensitive and 10 (45.5%) were platinum-resistant. When comparing sensitive and resistant patients, no statistically significant difference was noted among stage, grade, histology, and age (P = 0.1292, P = 1.0000, P = 1.0000, P = 1.0000, respectively). In the QuMA analysis, 10 of the 14 (71.4%) patients who had a low copy number of D6S1581 were platinum-resistant, while none of the patients with a normal or high copy number of D6S1581 were platinum-resistant. This was statistically significant when the marker data were treated as either a continuous or a categorical variable (P = 0.0410 and P = 0.0170, respectively). No other loci correlated significantly with platinum resistance. CONCLUSIONS: D6S1581 was the only genetic marker, of those examined, significantly related to chemoresistance. Patients with a loss of D6S1581 are more likely to be platinum-resistant. Identification of genetic alterations associated with platinum resistance detected by QuMA may contribute to a better understanding of clinical behavior and chemotherapy treatment options for patients.

The role of tumor suppressor genes in esophageal cancer.

Esophageal cancer remains one of the 10 most common cancers worldwide. Although patients with early lesions have a reasonable prognosis, most patients present with advanced disease resulting in an overall 5-year survival of 5-10%. Therefore, the current challenges in the management of esophageal cancer are to obtain a better understanding of the underlying molecular biological alterations to provide new treatment options. During the development of esophageal cancer, there is progression from a premalignant epithelium to a neoplasm that frequently demonstrates a heterogeneous mix of genetic alterations. The vast majority of esophageal cancers have inactivation of the p53 and p16 genes at an early stage followed by defects in genes such as APC, Rb and cyclin D1 at later stages of progression. There is also mounting evidence that numerous, specific regions throughout the genome are frequently lost in these cancers. As a result, we will in the next decade, likely see the discovery and characterizations of novel tumor suppressor genes that may be important in the development of esophageal cancer. The accumulating knowledge about the inactivation of the tumor suppressor genes could ultimately provide us with objective diagnostic tools, more accurate markers for prediction of malignant transformation from premalignant epithelium and facilitate the introduction of novel therapeutic options for the management of esophageal cancer.

Prognostic value of quantitative reverse transcription-polymerase chain reaction in lymph node-negative esophageal cancer patients.

PURPOSE: In esophageal cancer, lymph node metastases are the strongest predictor of recurrence and poor outcome. However, many node-negative patients still recur despite a potentially curative resection. This is probably the result of microscopically occult metastases missed by histological examination. In this study, we used both standard, gel-based reverse transcription-PCR (RT-PCR) and Taqman quantitative RT-PCR (QRT-PCR) for carcinoembryonic antigen (CEA) mRNA to detect occult micrometastases in 387 lymph nodes from 30 histologically node-negative esophageal cancer patients. EXPERIMENTAL DESIGN: CEA expression was compared with clinical outcomes to determine correlation with disease recurrence. For quantitative data, an optimum CEA expression level cutoff value was defined as the value that most accurately classified patients on the basis of disease recurrence. Kaplan-Meier survival curves were generated, and multivariate analyses were performed to evaluate the prognostic value of QRT-PCR. RESULTS: CEA expression levels were above the optimum cutoff level in 12 tissue blocks, resulting in the identification of 11 CEA-positive patients. Of these patients, 9 suffered disease recurrence and 2 remain disease free. Of the 19 CEA-negative patients, there was 1 disease recurrence. The sensitivity and specificity for predicting disease recurrence were 90 and 90%, respectively. Kaplan-Meier analysis showed that CEA positivity resulted in significantly lower disease-free and overall survival (P <0.0001 and 0.0006 respectively). In multivariate analyses, CEA positivity measured by QRT-PCR was the strongest independent predictor of disease recurrence among other clinical and pathological factors examined. CONCLUSIONS: QRT-PCR offers significant benefits over standard RT-PCR and identifies node-negative patients at high risk for recurrence.

Correlation between dysplasia and mutations of six tumor suppressor genes in Barrett's esophagus.

BACKGROUND: Barrett's esophagus (BE) may progress to adenocarcinoma through dysplastic progression. Classification of dysplasia in BE has significant interobserver variability. Our objective was to determine whether genetic alterations in BE correlate with degrees of histologic dysplasia. METHODS: Fixed tissue from 37 patients with BE and adenocarcinoma was studied for six tumor suppressor genes. Tissues were microdissected and analyzed for loss of heterozygosity. Microdissection of individual crypts showing metaplasia and dysplasia were performed and analyzed for 23 of the 37 patients whose tumors were heterozygous for at least four of the six genes studied. RESULTS: Frequency of alterations for MXI1, hOGG1, p53, MTS1, DCC, and APC were 7 of 32 (22%), 12 of 35 (34%), 12 of 26 (46%), 17 of 30 (57%), 17 of 27 (63%), and 23 of 36 (64%), respectively. Analysis of BE demonstrated that crypts with metaplasia, low-grade dysplasia, and high-grade dysplasia strongly correlated with alterations in tumor suppressor genes (p < 0.0001). CONCLUSIONS: This pilot study demonstrates that genetic analysis can be performed on individual crypts in patients with BE, and that alterations may facilitate objective classification of the severity of dysplasia.

An approach to analysis of large-scale correlations between genome changes and clinical endpoints in ovarian cancer.

This report describes analyses of associations of genome copy number abnormalities in ovarian cancers with clinical features using genome-wide graphical and analytical procedures. These studies show that tumor grade is a better indicator of the extent of genomic progression than stage, that loss of chromosome 4 occurs preferentially in high-grade tumors, and that gains of 3q26-qter, 8q24-qter, and 20q13-qter occur frequently in low-grade and low-stage tumors and thus may be early events in ovarian cancer development. In addition, loss of chromosome 16q24 and a total number of independent genome copy number aberrations >7 are associated with reduced survival duration. The association of loss of 16q24 (D16S3026) with decreased survival duration was confirmed by quantitative PCR. Regions that frequently are abnormal and associated with altered survival duration are strong candidates for higher resolution analysis and gene discovery and may be useful markers for prediction of clinical outcome.

Measurement of DNA copy number at microsatellite loci using quantitative PCR analysis.

This report describes the development and validation of quantitative microsatellite analysis (QuMA) for rapid measurement of relative DNA sequence copy number. In QuMA, the copy number of a test locus relative to a pooled reference is assessed using quantitative, real-time PCR amplification of loci carrying simple sequence repeats. Use of simple sequence repeats is advantageous because of the large numbers that are mapped precisely. In addition, all markers are informative because QuMA does not require that they be polymorphic. The utility of QuMA is demonstrated in assessment of the extent of deletions of chromosome 2 in leukemias arising in radiation-sensitive inbred SJL mice and in analysis of the association of increased copy number of the putative oncogene ZNF217 with reduced survival duration in ovarian cancer patients.

Elevated levels of FMR1 mRNA in carrier males: a new mechanism of involvement in the fragile-X syndrome.

Fragile-X syndrome is a trinucleotide-repeat-expansion disorder in which the clinical phenotype is believed to result from transcriptional silencing of the fragile-X mental retardation 1 (FMR1) gene as the number of CGG repeats exceeds approximately 200. For premutation alleles ( approximately 55-200 repeats), no abnormalities in FMR1-gene expression have been described, despite growing evidence of clinical involvement in premutation carriers. To address this (apparent) paradox, we have determined, for 16 carrier males (55-192 repeats), the relative levels of leukocyte FMR1 mRNA, by use of automated fluorescence-detection reverse transcriptase-PCR, and the percent of lymphocytes that are immunoreactive for FMR1 protein (FMRP). For some alleles with>100 repeats, there was a reduction in the number of FMRP-positive cells. Unexpectedly, FMR1 mRNA levels were elevated at least fivefold within this same range. No significant increase in FMR1 mRNA stability was observed in a lymphoblastoid cell line (160 repeats) derived from one of the carrier males, suggesting that the increased message levels are due to an increased rate of transcription. Current results support a mechanism of involvement in premutation carriers, in which reduced translational efficiency is at least partially compensated through increased transcriptional activity. Thus, diminished translational efficiency may be important throughout much of the premutation range, with a mechanistic switch occurring in the full-mutation range as the FMR1 gene is silenced.

Quantitative mRNA expression analysis from formalin-fixed, paraffin-embedded tissues using 5' nuclease quantitative reverse transcription-polymerase chain reaction.

Analysis of gene expression and correlation with clinical parameters has the potential to become an important factor in therapeutic decision making. The ability to analyze gene expression in archived tissues, for which clinical followup is already available, will greatly facilitate research in this area. A major obstacle to this approach, however, has been the uncertainty about whether gene expression analyses from routinely archived tissues accurately reflect expression before fixation. In the present study we have optimized the RNA isolation and reverse transcription steps for quantitative reverse transcription-polymerase chain reaction (RT-PCR) on archival material. Using tissue taken directly from the operating room, mRNAs with half-lives from 10 minutes to >8 hours were isolated and reverse transcribed. Subsequent real-time quantitative PCR methodology (TaqMan) on these cDNAs gives a measurement of gene expression in the fixed tissues comparable to that in the fresh tissue. In addition, we simulated routine pathology handling and demonstrate that this method of mRNA quantitation is insensitive to pre-fixation times (time from excision to fixation) of up to 12 hours. Therefore, it should be feasible to analyze gene expression in archived tissues where tissue collection procedures are largely unknown.

Allelic imbalance mapping of chromosome 16 shows two regions of common deletion in prostate adenocarcinoma.

Allelic imbalance (AI) has now been reported on the long arm of chromosome 16 in several cancers including breast, prostate, hepatocellular carcinoma, and Wilms tumor. Such nonrandom AI is commonly associated with the presence of a tumor suppressor gene (TSG) at or near the tested locus. Previous studies in our laboratory indicated that prostate cancer genomes frequently exhibit a region of allelic loss near the q terminus of chromosome 16. Here we report a detailed, PCR based, allelic imbalance study at ten polymorphic loci on 16q. The data indicate that there are two common regions of 16q AI in prostate cancer, one at 16q21-22 (50% of informative cases) and another at 16q24.2-qter (56% of informative cases). These are similar to regions of 16q previously shown to exhibit AI in breast cancer. Neither of these regions shows correlation of AI with the clinical parameters; Gleason grade, tumor stage, or metastases.

Characterization of a mammalian cell line that exhibits spontaneous and ultraviolet light-induced hypermutability while retaining resistance to cell killing by ultraviolet light.

Chronic exposure of V79 cells to 80 daily doses of 150 J/M2, 290-330-nm ultraviolet light (UVB) produced a mixed cell population that was found to be generally more resistant to cell killing by both UVB and UVC (254 nm) than the wild-type cells. Several subclones from this population were studied for their survival and mutation responses and then one was chosen for further characterization based on this data. The studies carried out on this subclone, designated N806, show that its spontaneous HPRT mutation rate is approximately 10 times higher than that of wild-type V79 cells and it is almost three times more mutable than the wild-type cells when both are induced by UVB or UVC. The mutation responses of N806 and MI2G cells to 50-kVp X-rays are different, but the N806 cells do not appear to be hypermutable as they are with UV. N806 cells are also moderately more resistant to the cytotoxic effects of UV radiation but are more sensitive than MI2G cells when exposed to X-rays. Assays to measure the removal of cyclobutane pyrimidine dimers (CPDs) and the incision step of nucleotide excision repair have revealed no detectable difference in the repair capacities of N806 and parental V79 cells. These results suggest that chronic, protracted UV irradiation may be able to induce a 'mutator phenotype' in a subpopulation of the progenitor cells.

Mitomycin C in advanced breast cancer: an update.

Mitomycin C has a documented response rate of 20% to 30% when used as treatment in advanced breast cancer. Because of its delayed and cumulative hematologic toxicity, there has been a reluctance to use this agent. However, by lowering the drug dosage, the hematologic, pulmonary, and renal toxicities have been reduced. The reduced dosage does not appear to have compromised the treatment results.

Elevation of plasma antidiuretic hormones (ADH) associated with chemotherapy-induced emesis in man.

Eleven randomly hydrated patients with metastatic malignancies received iv bolus chemotherapy. Serial observations of plasma antidiuretic hormone (ADH), serum osmolality, blood pressure, and presence of nausea or emesis were made over the next 3-4 hours. Group 1 (four patients) had no nausea or emesis and no change in ADH, osmolality, or mean blood pressure. Group 2 (seven patients) had nausea and emesis following chemotherapy, with an increase in mean ADH from a baseline level of 5.53 pg/ml to a peak after emesis of 33.83 pg/ml. Group 2 had no significant increase in osmolality or decrease in mean blood pressure before emesis. ADH levels increased 0-40 minutes before emesis and peaked 28-115 minutes (mean, 66) after emesis. Emesis caused by chemotherapy agents is associated with rapid, significant increases in plasma ADH levels, independent of changes in osmolality or blood pressure.

Mitomycin C in large infrequent doses in breast cancer.

Seventy-one women with far-advanced breast cancer resistant to standard chemotherapeutic agents were administered mitomycin C using an intermittent high dose schedule. One group consisted of 54 patients with measurable metastatic tumor; a second group consisted of 18 patients with nonmeasurable osseous metastases. Objective response rate in group 1 was 26% for an average duration of 2 1/2 months. Subjective response rate in group 2 was 44% for an average duration of 3 months. Response and toxicity data were similar to those of studies employing the less convenient protracted low-dose schedule. Prior treatment with other alkylating agents did not adversely affect response. Further investigation into the role of mitomycin in combination chemotherapy programs is recommended.

Mithramycin for hypercalcemia of malignant disease.

Mithramycin (Mithracin(R)) rapidly controlled the hypercalcemia of malignant disease in every patient. This control was temporary, and intermittent administration of the antibiotic was required.Hypercalcemia frequently responds to non-toxic maneuvers such as hydration and moderate doses of corticoids. Until the mechanism of action of Mithramycin in hypercalcemia is better understood, caution should be urged in its prolonged use for this purpose.