Different prognostic values of KRAS exon 2 submutations and BRAF V600E mutation in microsatellite stable (MSS) and unstable (MSI) stage III colon cancer: an ACCENT/IDEA pooled analysis of seven trials.

BACKGROUND: The prognostic value of KRAS and BRAFV600E mutations in stage III colon cancer (CC) remains controversial and has never been clearly analyzed in patients with microsatellite instability-high (MSI-H) tumors due to sample size limitations. Data are also lacking for KRAS submutations and prognosis. PATIENTS AND METHODS: We examined clinicopathological variables and prognosis in patients with surgically resected stage III CC who participated in seven clinical trials from the ACCENT/IDEA databases. Associations between KRAS exon 2 and BRAFV600E mutations and time to recurrence (TTR), overall survival (OS), and survival after recurrence (SAR) were assessed using a Cox model. We also analyzed the prognostic value of KRAS exon 2 submutations. RESULTS: Among 8460 patients, 11.4% had MSI-H status. In the MSI-H group, BRAFV600E, KRAS exon 2 mutants, and double-wild-type statuses were detected in 40.6%, 18.1%, and 41.3%, respectively, whereas and in the microsatellite stable (MSS) group, these were detected in 7.7%, 38.6%, and 53.8%, respectively. In the MSS group, 5-year TTR rates of 61.8%, 66.3%, and 72.9% were observed among patients with BRAFV600E, KRAS exon 2 mutants, and those who were DWT, respectively [adjusted hazard ratio (HR) = 1.58 and 1.31, both P < 0.001]. In the MSI-H group, 5-year TTR rates did not differ significantly among the mutated subgroups. Similar results were found for OS. However, survival after relapse was significantly shorter in the KRAS exon 2- and BRAFV600E-mutated patients in both MSS (adjusted HR = 2.06 and 1.15; both P < 0.05) and MSI-H (adjusted HR = 1.99 and 1.81; both P < 0.05) groups. In the MSS group, KRAS exon 2 mutations were associated with TTR, but only p.G12C, p.G12D, and p.G13D were associated with poor outcomes after disease recurrence. CONCLUSIONS: Testing for both KRAS and BRAFV600E mutations in stage III patients should be considered as they can better define individual patient prognosis, and may also enable patient selection for (neo)adjuvant trials dedicated to specific molecular subtypes with poor prognosis.

Association of tumor-infiltrating lymphocytes with survival depends on primary tumor sidedness in stage III colon cancers (NCCTG N0147) [Alliance].

BACKGROUND: Tumor-infiltrating lymphocytes (TILs) are a robust and independent prognostic variable in localized colon cancer. Given reported differences in molecular features and prognosis of right- versus left-sided tumors, we examined the association of TIL densities with patient survival by primary tumor sidedness in stage III cancers, including clinical low- (T1-3, N1) and high-risk (T4 and/or N2) groups. PATIENTS AND METHODS: In a phase III trial of FOLFOX-based adjuvant chemotherapy, TIL densities were analyzed and dichotomized in colon carcinomas (N = 1532) based on a previously determined cut point optimized for disease-free survival (DFS). Right-sided tumors were defined as proximal to the splenic flexure. Associations of TILs and sidedness with 5-year DFS were examined using Kaplan-Meier methodology along with multivariable modeling and relative contribution analysis by Cox regression. RESULTS: Lower TIL densities were found in left- versus right-sided tumors (P < 0.0001). The association of TIL densities with DFS differed significantly by tumor sidedness (Pinteraction = 0.045). Overall, patient tumors with low (versus high) TILs had significantly poorer DFS in right-sided (hazard ratio 2.02, 95% confidence interval 1.45-2.82; Padj < 0.0001), but not left-sided tumors (Padj = 0.1731). Among clinical low-risk patients, low (versus high) TILs were adversely prognostic only in right-sided tumors (Padj = 0.0058). Among high-risk patients, low TILs were prognostic independent of sidedness (Padj < 0.025). The relative contribution of TILs to DFS was substantially greater in right- versus left-sided tumors (24% versus 1.5%). In high-risk tumors, TILs had the highest relative contribution to DFS (42%) of all variables. In low-risk tumors, the contribution of TILs (16%) to DFS was second to KRAS. CONCLUSIONS: The association of TIL densities with patient survival differed by primary tumor sidedness and clinical risk group, suggesting that TILs should be interpreted in this context among stage III colon cancers. GOV IDENTIFIER: NCT00079274; https://clinicaltrials.gov/ct2/show/NCT00079274.

Analysis of tumor microenvironmental features to refine prognosis by T, N risk group in patients with stage III colon cancer (NCCTG N0147) (Alliance).

BACKGROUND: Tumor-infiltrating lymphocytes (TILs), tumor budding, and micropapillary architecture may influence tumor growth and metastatic potential, thereby enhancing prognostic stratification. We analyzed these features and their relative contribution to overall outcome and in low (T1-3 N1) and high (T4 and/or N2) risk groups that are used to inform the duration of adjuvant chemotherapy in patients with resected stage III colon cancers. PATIENTS AND METHODS: Among 1532 patients treated in a phase III adjuvant trial of FOLFOX-based therapy, intraepithelial TIL densities, tumor budding, and micropapillary features were analyzed and quantified in routine histopathological sections with light microscopy. Optimal cut-points were determined in association with disease-free survival (DFS) in training and validation sets. Associations or relative contributions of individual features or combined variables with DFS were determined using multivariable Cox regression models. RESULTS: TILs, tumor budding, and micropapillary features were shown to differ significantly by T, N risk groups and by mismatch repair (MMR) status. Low TILs, high budding, and their combined variable [hazard ratio = 2.07 (95% CI, 1.50% to 2.88%); Padj < 0.0001], but not micropapillary features, were each significantly associated with poorer DFS in a training data set and confirmed in a validation set. TILs were prognostic in proficient mismatch repair (pMMR) and deficient mismatch repair (dMMR) tumors; budding was prognostic only in pMMR tumors. The percentage relative contribution of budding/TILs to DFS was second only to nodal status overall, was second (24.4%) after KRAS in low-risk patients, and was the most important contributor (45.4%) in high-risk patients. CONCLUSIONS: TIL density and tumor budding were each validated as significant prognostic variables and their combined variable provided robust prognostic stratification by T, N risk groups, being the strongest predictor of DFS among high-risk stage III patients. CLINICALTRIALS. GOV IDENTIFIER: NCT00079274.

Prognosis of microsatellite instability and/or mismatch repair deficiency stage III colon cancer patients after disease recurrence following adjuvant treatment: results of an ACCENT pooled analysis of seven studies.

BACKGROUND: Microsatellite instable/deficient mismatch repair (MSI/dMMR) metastatic colorectal cancers have been reported to have a poor prognosis. Frequent co-occurrence of MSI/dMMR and BRAFV600E complicates the association. PATIENTS AND METHODS: Patients with resected stage III colon cancer (CC) from seven adjuvant studies with available data for disease recurrence and MMR and BRAFV600E status were analyzed. The primary end point was survival after recurrence (SAR). Associations of markers with SAR were analyzed using Cox proportional hazards models adjusted for age, gender, performance status, T stage, N stage, primary tumor location, grade, KRAS status, and timing of recurrence. RESULTS: Among 2630 patients with cancer recurrence (1491 men [56.7%], mean age, 58.5 [19-85] years), multivariable analysis revealed that patients with MSI/dMMR tumors had significantly longer SAR than did patients with microsatellite stable/proficient MMR tumors (MSS/pMMR) (adjusted hazard ratio [aHR], 0.82; 95% CI [confidence interval], 0.69-0.98; P = 0.029). This finding remained when looking at patients treated with standard oxaliplatin-based adjuvant chemotherapy regimens only (aHR, 0.76; 95% CI, 0.58-1.00; P = 0.048). Same trends for SAR were observed when analyzing MSI/dMMR versus MSS/pMMR tumor subgroups lacking BRAFV600E (aHR, 0.84; P = 0.10) or those harboring BRAFV600E (aHR, 0.88; P = 0.43), without reaching statistical significance. Furthermore, SAR was significantly shorter in tumors with BRAFV600E versus those lacking this mutation (aHR, 2.06; 95% CI, 1.73-2.46; P < 0.0001), even in the subgroup of MSI/dMMR tumors (aHR, 2.65; 95% CI, 1.67-4.21; P < 0.0001). Other factors associated with a shorter SAR were as follows: older age, male gender, T4/N2, proximal primary tumor location, poorly differentiated adenocarcinoma, and early recurrence. CONCLUSIONS: In stage III CC patients recurring after adjuvant chemotherapy, and before the era of immunotherapy, the MSI/dMMR phenotype was associated with a better SAR compared with MSS/pMMR. BRAFV600E mutation was a poor prognostic factor for both MSI/dMMR and MSS/pMMR patients. TRIAL IDENTIFICATION NUMBERS: NCT00079274, NCT00265811, NCT00004931, NCT00004931, NCT00026273, NCT00096278, NCT00112918.

Prediction of overall survival in stage II and III colon cancer beyond TNM system: a retrospective, pooled biomarker study.

Background: TNM staging alone does not accurately predict outcome in colon cancer (CC) patients who may be eligible for adjuvant chemotherapy. It is unknown to what extent the molecular markers microsatellite instability (MSI) and mutations in BRAF or KRAS improve prognostic estimation in multivariable models that include detailed clinicopathological annotation. Patients and methods: After imputation of missing at random data, a subset of patients accrued in phase 3 trials with adjuvant chemotherapy (n = 3016)-N0147 (NCT00079274) and PETACC3 (NCT00026273)-was aggregated to construct multivariable Cox models for 5-year overall survival that were subsequently validated internally in the remaining clinical trial samples (n = 1499), and also externally in different population cohorts of chemotherapy-treated (n = 949) or -untreated (n = 1080) CC patients, and an additional series without treatment annotation (n = 782). Results: TNM staging, MSI and BRAFV600E mutation status remained independent prognostic factors in multivariable models across clinical trials cohorts and observational studies. Concordance indices increased from 0.61-0.68 in the TNM alone model to 0.63-0.71 in models with added molecular markers, 0.65-0.73 with clinicopathological features and 0.66-0.74 with all covariates. In validation cohorts with complete annotation, the integrated time-dependent AUC rose from 0.64 for the TNM alone model to 0.67 for models that included clinicopathological features, with or without molecular markers. In patient cohorts that received adjuvant chemotherapy, the relative proportion of variance explained (R2) by TNM, clinicopathological features and molecular markers was on an average 65%, 25% and 10%, respectively. Conclusions: Incorporation of MSI, BRAFV600E and KRAS mutation status to overall survival models with TNM staging improves the ability to precisely prognosticate in stage II and III CC patients, but only modestly increases prediction accuracy in multivariable models that include clinicopathological features, particularly in chemotherapy-treated patients.

MiR-320e is a novel prognostic biomarker in colorectal cancer.

BACKGROUND: Advances in early detection and treatment have improved outcomes in patients with colorectal cancer (CRC). However, there remains a need for robust prognostic and predictive biomarkers. We conducted a systematic discovery and validation of microRNA (miRNA) biomarkers in two clinical trial cohorts of CRC patients. METHODS: We performed an initial 'discovery' phase using Affymetrix miRNA expression arrays to profile stage III CRC patients with and without tumour recurrence (n=50 per group) at 3-years of follow-up. All patients received adjuvant 5-fluorouracil (5-FU) plus oxaliplatin, that is, FOLFOX, treatment. During 'validation', we analysed miRNAs using qRT-PCR in an independent cohort of 237 stage II-IV CRC patients treated with 5-FU-based chemotherapy, as well as in normal colonic mucosa from 20 healthy subjects. Association with disease recurrence, disease-free survival (DFS) and overall survival (OS) was examined using Cox proportional hazard models. RESULTS: In the discovery cohort, miR-320e expression was significantly elevated in stage III colon cancers from patients with vs without recurrence (95% confidence interval (CI)=1.14-1.42; P<0.0001). These results were then independently validated in stage II and III tumours. Specifically, increased miR-320e expression was associated with poorer DFS (hazard ratio (HR)=1.65; 95% CI=1.27-2.13; P=0.0001) and OS (HR=1.78; 95% CI=1.31-2.41; P=0.0003) in stage III CRC patients. CONCLUSIONS: In two clinical trial cohorts, a systematic biomarker discovery and validation approach identified miR-320e to be a novel prognostic biomarker that is associated with adverse clinical outcome in stage III CRC patients treated with 5-FU-based adjuvant chemotherapy. These findings have important implications for the personalised management of CRC patients.

Prognostic effect of activated EGFR expression in human colon carcinomas: comparison with EGFR status.

BACKGROUND: Evidence suggests that epidermal growth factor receptor (EGFR)-activation status may better predict the clinical behaviour of colon cancers than does EGFR expression. However, the prognostic effect of phospho-EGFR in primary colon cancer remains undefined. METHODS: Phospho-EGFR (Tyr-1173) and EGFR expression were analysed by immunohistochemistry (IHC) in tissue microarrays of TNM stage II and III colon cancers from completed adjuvant therapy trials (n=388). Staining intensity was scored and correlated with clinicopathological variables, DNA mismatch repair (MMR) status, rates of cell proliferation (Ki-67), apoptosis (caspase-3), and patient survival. RESULTS: Phospho-EGFR expression was detected in 157 of 388 (40%) tumours, whereas EGFR was found in 214 of 361 (59%). Although phospho-EGFR was unrelated to clinicopathological variables, strong EGFR intensity was associated with higher tumour stage (P=0.03). Tumours overexpressing EGFR (P=0.0002) or phospho-EGFR (P=0.015) showed increased Ki-67, but not caspase-3 expression. Phospho-EGFR was not prognostic. EGFR intensity was associated with worse disease-free survival (DFS) (hazard ratio (HR): 1.21 (1.03, 1.41); P=0.019) and overall survival (OS) (HR: 1.19 (1.02, 1.39); P=0.028). Tumours expressing both EGFR and phospho-EGFR had similar survival as EGFR alone. Stage and lymph node number were prognostic for DFS and OS, and histological grade for OS. EGFR was an independent predictor of DFS (P=0.042) after adjustment for stage, histological grade, age, and MMR status. CONCLUSION: Phospho-EGFR and EGFR expression were associated with tumour cell hyperproliferation. Phospho-EGFR was not prognostic, whereas increased EGFR intensity was independently associated with poor DFS.

The consensus Immunoscore in phase 3 clinical trial (N0147) and impact on patient management decisions.

The consensus Immunoscore is a routine assay quantifying the adaptive immune response within the tumor microenvironment. It has a prognostic value that has been confirmed in a phase 3 clinical trial (NCCTG N0147) in stage III colon cancers. Moreover, results from another phase 3 randomized trial revealed the predictive value of Immunoscore for response to adjuvant chemotherapy duration. These results highlight the clinical utility of Immunoscore. In its latest edition, the World Health Organization classification of Digestive System Tumors introduced for the first time the immune response as an essential and desirable diagnostic criterion for colorectal cancer. Within the tumor microenvironment, the immune response provides an important estimate of the risk of recurrence and death in colon cancer. The international validation of the prognostic value of the consensus Immunoscore together with its prognostic value in the N0147 trial and its predictive utility for response to chemotherapy in stage III patients provide valuable information for patient management.

Impact of endoscopic ultrasonography and physician specialty on the management of patients with esophagus cancer.

While endoscopic ultrasonography (EUS) and EUS-guided fine-needle aspiration (EUS-FNA) are the most accurate techniques for locoregional staging of esophageal cancer, little evidence exists that these innovations impact on clinical care. The objective on this study was to determine the frequency with which EUS and EUS-FNA alter the management of patients with localized esophageal cancer, and assess practice variation among specialists at a tertiary care center. Three gastroenterologists, three medical oncologists, three radiation oncologists and four thoracic surgeons were asked to independently report their management recommendations as the anonymized staging information of 50 prospectively enrolled patients from another study were sequentially disclosed on-line. Compared to initial management recommendations, that were based upon history, physical examination, upper endoscopy and CT scan results, EUS prompted a change in management 24% (95% CI: 12-36%) of the time; usually to a more resource-intensive approach (71%), for example from recommending palliation to recommending neoadjuvant chemoradiation therapy. EUS-FNA plus cytology results altered management an additional 8% (95% CI: 6-15%) of the time. Agreement between specialists ranged from fair (intraclass correlation [ICC=0.32) to substantial (ICC=0.65); improving with additional information. Among specialists, agreement was greatest for patients with stage I disease. EUS and EUS-FNA changed patient management the most for patients with stages IIA, IIB or III disease. EUS, with or without FNA, significantly impacts the management of patients with localized esophageal cancer. With respect to the optimal treatment for each patient, agreement among physicians incrementally increases with endoscopic ultrasound results. Specialty training appears to influence therapeutic decision-making behavior.

Aberrant methylation of the Cyclooxygenase 2 CpG island in colorectal tumors.

Cyclooxygenases (COXs) are key enzymes that convert arachidonic acid to prostaglandins. Overexpression of one of the COX isozymes, COX2, has been shown to play an important role in colorectal cancer progression. Recently, however, low expression of COX2 has been reported in a subset of colorectal and gastric cancers. Aberrant CpG island methylation and associated transcriptional silencing are common in colorectal cancer, and we therefore investigated the potential role of methylation in the transcriptional silencing of COX2. We examined the methylation status of the COX2 5' CpG island in a series of tumor cell lines. Among the 33 cell lines examined, dense methylation (>70%) of COX2 was detected in 5 cell lines, and partial methylation was detected in 10 cell lines. Detailed methylation mapping using bisulfite genomic sequencing revealed that loss of expression of COX2 mRNA was closely correlated with methylation of a region upstream of exon 1, and expression could be restored by demethylation using the DNA methyltransferase inhibitor 5-aza-deoxycytidine. Aberrant methylation of COX2 was also detected in 12 of 92 (13%) unselected sporadic primary colorectal cancers and 7 of 50 (14%) colorectal adenomas. COX2 methylation was strongly associated with the presence of the CpG island methylator phenotype (P<0.01), inversely related to p53 gene mutation (P<0.01), and unrelated to microsatellite instability status. We propose that COX2 expression in colorectal tumors is modulated by functional factors that favor high expression and by the CpG island methylator phenotype that favors silencing in a subset of cases. These results raise the possibility that tumors with COX2 methylation may be less sensitive to treatment using specific COX2 inhibitors.

bcl-2 and p53 oncoprotein expression during colorectal tumorigenesis.

Apoptosis or programmed cell death represents a mechanism by which cells possessing DNA damage can be deleted. The bcl-2 proto-oncogene is a known inhibitor of apoptosis that may allow the accumulation and propagation of cells containing genetic alterations. To determine if and when the bcl-2 gene is activated during colorectal tumorigenesis and its relationship to p53, we analyzed normal mucosa, hyperplastic and dysplastic epithelial polyps, and carcinomas for the expression of these markers using immunohistochemistry. Whereas bcl-2 staining was restricted to basal epithelial cells in normal and hyperplastic mucosa, bcl-2 expression was detected in parabasal and superficial regions in dysplastic polyps and carcinomas. An inverse correlation was found between bcl-2 and p53 expression in adenomas, suggesting that these markers may regulate a common cell death pathway. Furthermore, carcinomas with a high percentage of bcl-2-positive cells were significantly more likely to have low rates of spontaneous apoptosis, as determined histologically, than those cancers with low or absent bcl-2 expression. Abnormal activation of the bcl-2 gene appears to be an early event in colorectal tumorigenesis that can inhibit apoptosis in vivo and may facilitate tumor progression.

Increased cyclooxygenase-2 expression in human pancreatic carcinomas and cell lines: growth inhibition by nonsteroidal anti-inflammatory drugs.

Cyclooxygenase (COX)-2 mRNA and protein expression were found to be frequently elevated in human pancreatic adenocarcinomas and cell lines derived from such tumors. Immunohistochemistry demonstrated cytoplasmic COX-2 expression in 14 of 21 (67%) pancreatic carcinomas. The level of COX-2 mRNA was found to be elevated in carcinomas, relative to histologically normal pancreas from a healthy individual, as assessed by reverse transcription-PCR. COX-2 protein expression was detected by the Western blot assay in three of five pancreatic carcinoma cell lines (BxPC-3, Capan-1, and MDAPanc-3), whereas COX-1 protein was detected in two of the five cell lines (BxPC-3 and Capan-1). Increased levels of COX-2 mRNA were found in four of five cell lines, and only in PANC-1 cells was the low level of transcript comparable to that in the normal pancreas. The level of COX-2 mRNA was positively correlated with the differentiation status of the tumor of origin for each cell line, COX-2 protein expression was up-regulated by epidermal growth factor when the cells were grown in absence of serum. Finally, two nonsteroidal anti-inflammatory drugs, sulindac sulfide and NS398, produced a dose-dependent inhibition of cell proliferation in all pancreatic cell lines tested. No correlation was found between the level of COX-2 or COX-1 expression and the extent of growth inhibition. Treatment of BxPC-3 cells with sulindac sulfide and NS398 resulted in an induction of COX-2 expression. Our findings indicate that COX-2 up-regulation is a frequent event in pancreatic cancers and suggest that nonsteroidal anti-inflammatory drugs may be useful in the chemoprevention and therapy of pancreatic carcinoma.

Phase I trial of sulindac plus 5-fluorouracil and levamisole: potential adjuvant therapy for colon carcinoma.

Epidemiological studies indicate that nonsteroidal anti-inflammatory agents may reduce colorectal cancer incidence and mortality. Moreover, sulindac has been shown to attenuate the growth and progression of colonic neoplasms in an experimental model of colon carcinoma and in patients with familial adenomatous polyposis. To determine whether sulindac (300 mg/day) would increase toxicity associated with 5-fluorouracil (5-FU) and levamisole, 15 patients with advanced colorectal cancer were treated. Median treatment duration was 3 (range, 0.6-6.0) months, and median age was 56 years (33% >/= 60 years). All patients had failed prior 5-FU-based therapy, had measurable disease, and were evaluable for toxicity. Grade III/IV granulocytopenia occurred in four patients; three patients had received prior pelvic irradiation resulting in a predisposition to myelosuppression. Two patients developed grade III anemia, and occult gastrointestinal bleeding was suspected in one. No other grade II or greater gastrointestinal or other nonhematological toxicity occurred. One patient had a partial response, 3 patients had disease stabilization, and 10 patients progressed on study. Our results indicate that sulindac does not significantly increase short-term toxicity associated with 5-FU and levamisole. To determine whether sulindac increases the efficacy of adjuvant chemotherapy, we propose a phase III randomized trial in patients with lymph node-positive colon cancer.

Prognostic value of bcl-2 oncoprotein expression in stage II colon carcinoma.

The bcl-2 proto-oncogene encodes a Mr 25,000 protein that has been shown to prevent apoptosis or programmed cell death. The bcl-2 protein is detectable in basal cells of normal colonic epithelium, and an altered topographic distribution of this protein is found in colonic neoplasms. However, the clinical significance of abnormal bcl-2 expression in colon carcinomas remains unknown. We examined the prognostic value of the bcl-2 protein in TNM stage II colon carcinomas and its relationship to DNA ploidy, cell proliferation indices, p53 expression, and clinicopathological features. We analyzed 119 resected and otherwise untreated, paraffin-embedded stage II colon carcinomas for bcl-2 and p53 protein expression using immunohistochemistry. DNA ploidy and proliferative index (% S-phase + % G2-M) were determined by flow cytometry, and tumor grade and vascular microinvasion were assessed on histological sections. Cytoplasmic expression of the bcl-2 protein was detected in 72 (66%) of 110 carcinomas, and a high level of expression was significantly correlated with diploid DNA content (P = 0.02) and low proliferative activity (P = 0.005). bcl-2 was not associated with nuclear p53 expression. In a univariate analysis, a higher fraction of bcl-2-positive tumor cells was associated with better relapse-free survival (P = 0.02) and overall survival (P = 0.05) rates. Moreover, a high level of bcl-2 expression was an independent predictor of better relapse-free survival (P = 0.04), but not overall survival (P = 0.14), after adjustment for other variables, including proliferative index, DNA ploidy, and race. In conclusion, bcl-2 overexpression is associated with favorable prognostic features and may predict clinical outcome in stage II colon carcinomas.

Apoptotic and mitotic indices predict survival rates in lymph node-negative colon carcinomas.

An imbalance between apoptosis and mitosis is believed to underlie colon cancer development and progression. These processes regulate the growth of normal and neoplastic epithelia, and in tumors, may confer prognostic information. To test this hypothesis, we determined apoptotic and mitotic indices (AI, MI) by morphology in H&E sections of 154 lymph node-negative, sporadic colon carcinomas. The relationship of these indices to genetic (p53 and Bcl-2) and biological features (DNA ploidy and cell kinetics) and patient survival rates was determined. Tumor features were compared in proximal and distal tumors, given postulated differences in their pathogenesis. Bcl-2 and p53 proteins were examined using immunohistochemistry and DNA ploidy and proliferative indices (PIs) by flow cytometry. Tumor features were dichotomized for analysis of relapse-free survival and overall survival (OS) rates using a Cox proportional hazards model. Median patient follow-up was 8.8 years. The median AI and MI were 1.2% (0-7.6) and 0.40% (0-1.8), respectively, and did not differ by tumor site. AI correlated with histological grade (P = 0.03); MI correlated with PI (P = 0.02) and inversely with Bcl-2 in distal tumors (P = 0.02). p53 and Bcl-2 expression were detected in 52 and 53% of tumors, respectively. Distal tumor site was associated with aneuploidy (P = 0.001), p53 (P = 0.001), and PI > 15% (P = 0.002). In a univariate analysis, colon cancers with high MIs (>0.5%) had a poor prognosis (P = 0.04). Bcl-2 overexpression (>20% + tumor cells) was associated with more favorable OS (P = 0.04). The association of ploidy and PI with outcome was of borderline significance for all tumors; however, diploidy predicted better survival in proximal cancers. In distal cancers, low AIs (< or = 0.25%) and high MIs (>0.5%) were adverse prognostic markers. After adjustment for other variables, an increased MI predicted shorter OS with a hazard ratio (HR) for death of 2.70; 95% confidence interval (CI) was 1.23-5.91 (P = 0.01). Expression of Bcl-2 was associated with more favorable OS (HR, 0.46; 95% CI, 0.21-1.0; P = 0.06). In proximal cancers, Bcl-2 expression was the most important predictor of OS (HR, 0.17; 95% CI, 0.03-0.85; P = 0.03). In distal tumors, low AIs (HR, 3.33; 95% CI, 1.27-9.09; P = 0.01) and high MIs predicted poor survival. In conclusion, increased mitosis and low or absent Bcl-2 expression are significant risk factors for death in node-negative colon cancers, as are low rates of apoptosis in distal tumors. If validated prospectively, our results may identify patient subsets than can benefit from adjuvant chemotherapy.

Increased apoptosis accompanies neoplastic development in the human colorectum.

A disturbance in the balance between cell proliferation and cell loss, or apoptosis, may underlie neoplastic development. Therefore, we determined spontaneous apoptotic and proliferative rates in normal, hyperplastic, adenomatous, and malignant colorectal epithelia. In paired sections, DNA strand breaks were detected using the terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling assay, and apoptotic cells were also identified in H&E-stained slides by morphological criteria. Cell proliferation, bcl-2, and p53 expression were analyzed using specific monoclonal antibodies. In normal mucosa, luminal epithelial cells demonstrated higher rates of apoptosis compared to cells in the proliferative zone. Neoplastic transformation was associated with a significant increase in rates of apoptosis and proliferation. However, apoptosis, but not proliferation, decreased at the adenoma-to-carcinoma transition coincident with expression of mutant p53. In carcinomas, both mutant p53 and bcl-2 protein levels were associated with attenuated apoptotic rates. In conclusion, apoptosis is an important regulator of growth in normal and neoplastic colorectal epithelia. Increased apoptosis and proliferation accompany neoplastic transformation, suggesting that an alteration in apoptotic rates is an important event in colorectal carcinogenesis. Furthermore, the imbalance in these processes found in carcinomas may facilitate tumor growth and progression.

Loss of p21WAF1/Cip1 protein expression accompanies progression of sporadic colorectal neoplasms but not hereditary nonpolyposis colorectal cancers.

p21 (p21WAF1/Cip1), a cyclin-dependent kinase inhibitor, induces G1 arrest and can inhibit the activity of the proliferating cell nuclear antigen (PCNA). We analyzed p21 expression during colorectal tumorigenesis, its association with its transcriptional regulator p53, and its relationship to rates of cell proliferation and apoptosis. p21 and p53 protein expression were examined in sporadic tumors and hereditary nonpolyposis colorectal cancers (HNPCCs) by immunohistochemistry (IHC) and immunoblotting. Apoptosis was examined using a DNA nick end-labeling assay, and cell proliferation was examined by PCNA staining. In normal colorectal epithelia, nuclear p21 staining was uniformly detected in crypt cells of the superficial compartment (upper one-third) that stained negatively for PCNA. p21 and PCNA expression were, therefore, mutually exclusive. In sporadic cases, a decrease in the frequency of p21 expression accompanied adenoma development and progression to carcinoma. Specifically, p21 was detected in 12 of 16 (75%) adenomas and 10 of 32 (31%) carcinomas. In contrast to sporadic cases, HNPCCs with known mutations in DNA mismatch repair genes expressed p21 in 12 of 15 (80%) carcinomas. An inverse relationship between p21 and p53 was observed wherein mutant p53 proteins were detected in 4 of 15 (27%) HNPCCs versus 22 of 32 (69%) sporadic carcinomas. Although p21+ carcinoma cells were generally negative for p53, IHC revealed that some carcinoma cells expressed both p21 and p53 proteins. Furthermore, p53-mutated SW480 colon carcinoma cells were found to coexpress p21 and p53, suggesting that p21 can also be activated by a p53-independent mechanism. No association was found between p21 or PCNA and apoptotic labeling indices in adenomas or carcinomas. In conclusion, a decrease in p21 expression accompanies neoplastic progression in sporadic cases but not in HNPCCs. This finding appears related to p53 status in that the frequency of p53 expression was significantly reduced in HNPCCs compared to sporadic cases, suggesting a difference in their molecular pathways of tumorigenesis.

bcl-2 and p53 expression in resectable pancreatic adenocarcinomas: association with clinical outcome.

The bcl-2 proto-oncogene and the p53 tumor suppressor gene are important determinants of tumor cell susceptibility to apoptosis. bcl-2 and mutant p53 proteins inhibit apoptosis in vitro and can provide prognostic information in certain tumor types. We analyzed bcl-2 and p53 expression in archival pancreatic (n = 35) and ampullary (n = 6) adenocarcinomas, resected for cure, and their relationship to overall survival. Patients were treated with 5-fluorouracil and irradiation either pre- (n = 21) or postoperatively (n = 15); 5 patients received surgery alone. Using specific monoclonal antibodies, cytoplasmic bcl-2 and nuclear p53 proteins were detected in 22 of 40 (55%) and 20 of 37 (54%) tumors, respectively. No relationship was found between bcl-2 and p53 expression. Neither bcl-2 nor p53 correlated with histological response to preoperative chemoradiation. Lymph node involvement predicted poor overall survival (P = 0.02). A trend toward improved survival was seen in well-differentiated (P = 0.08) tumors and in those with increased bcl-2 expression (P = 0.06). p53 expression was not related to clinical outcome. In a multivariate analysis, nodal status was the single most important predictor of overall survival. Of note, the combined variable of bcl-2 expression and histological grade was a stronger prognostic variable than nodal status alone. Unlike nodal status, these features can potentially be evaluated in preoperative biopsy specimens.

Prognostic factors in resectable pancreatic cancer: p53 and bcl-2.

The p53 tumor suppressor gene and the Bcl-2 proto-oncogene regulate cell cycle progression and apoptosis. We evaluated the expression of these molecular markers with standard pathologic prognostic variables in patients who received multimodality therapy for resectable adenocarcinoma of the pancreas to study the effect of p53 and Bcl-2 on survival duration. Immunohistochemical staining of archival material was performed to determine levels of expression of p53 and Bcl-2 proteins in 70 patients with adenocarcinoma of pancreatic origin. All patients underwent a potentially curative pancreaticoduodenectomy and standardized pathologic analysis of resected specimens. Potential pathologic and molecular prognostic variables were assessed for their effect on survival duration. Nuclear staining for p53 was observed in 33 (47%) of 70 specimens. Immunostaining for Bcl-2 was observed in 23 specimens (33%). A trend toward improved survival duration was seen in patients whose tumors stained positive for either p53 or Bcl-2. Negative staining for both markers predicted short survival (P = 0.01). By univariate and multivariate analyses, no single pathologic factor was associated with survival duration. Immunohistochemical staging using both p53 and Bcl-2 significantly predicted survival duration by univariate and multivariate analysis; patients whose tumors stained positively for p53 and/or overexpressed Bcl-2 had a significantly longer survival than those whose tumors stained negative for both proteins.

Loci for efficient detection of microsatellite instability in hereditary non-polyposis colorectal cancer.

Most hereditary non-polyposis colorectal cancer (HNPCC) is due to germline mutations in DNA mismatch repair genes. Tumors arising as a result of these mutations display instability in microsatellites, which are short tandem repeats of DNA that are distributed throughout the genome. Although a subset of sporadic colorectal carcinomas also have microsatellite instability (MSI), the phenotype is a useful screening test in identifying patients with HNPCC caused by mutations in mismatch repair (MMR) genes. Studies have shown that some microsatellite markers are more efficient than others in identifying tumors with MSI. Furthermore, the frequency of instability can be assessed by categorizing patients into high (MSI-H, >/= 30-40% positive markers), low (MSI-L), and microsatellite stable (MSS) groups. Using a panel of 28 microsatellite markers, tumor and normal DNA from 10 HNPCC patients was used to identify the five most efficient markers for detecting MSI (BAT26, D2S123, FGA, D18S35, and TP53-DI). Each of the five markers detected MSI in 80-100% of the cases examined. We then expanded the sample size to 17 tumors from HNPCC patients. Each case had evidence for a mutation in either hMSH2 or hMLH1. We compared the efficiency of our panel of five best markers with another panel of five markers (BAT25, BAT26, D2S123, D17S250, and D5S346) identified as being efficient markers for detection of MSI at a recent NCI workshop. Our five selected markers were more efficient (85% vs. 79%) in detecting MSI. However, using either panel, 100% of the cases fell into the MSI-H category and the probability of misclassifying an MSI-H case as MSI-L is very low (0.002-0.008). We also examined four cases meeting the Amsterdam criteria for HNPCC, but with no evidence for mutation in either the hMSH2 or hMLH1 gene. With our panel, three were classified as MSI-H, while only two were classified as such with the NCI reference panel. The probability of misclassifying an MSI-L case as an MSI-H, using a panel of five markers is high (0.263).