Outcome according to KRAS-, NRAS- and BRAF-mutation as well as KRAS mutation variants: pooled analysis of five randomized trials in metastatic colorectal cancer by the AIO colorectal cancer study group.

BACKGROUND: To explore the impact of KRAS, NRAS and BRAF mutations as well as KRAS mutation variants in patients with metastatic colorectal cancer (mCRC) receiving first-line therapy. PATIENTS AND METHODS: A total of 1239 patients from five randomized trials (FIRE-1, FIRE-3, AIOKRK0207, AIOKRK0604, RO91) were included into the analysis. Outcome was evaluated by the Kaplan-Meier method, log-rank tests and Cox models. RESULTS: In 664 tumors, no mutation was detected, 462 tumors were diagnosed with KRAS-, 39 patients with NRAS- and 74 patients with BRAF-mutation. Mutations in KRAS were associated with inferior progression-free survival (PFS) and overall survival (OS) [multivariate hazard ratio (HR) for PFS: 1.20 (1.02-1.42), P = 0.03; multivariate HR for OS: 1.41 (1.17-1.70), P < 0.001]. BRAF mutation was also associated with inferior PFS [multivariate HR: 2.19 (1.59-3.02), P < 0.001] and OS [multivariate HR: 2.99 (2.10-4.25), P < 0.001]. Among specific KRAS mutation variants, the KRAS G12C-variant (n = 28) correlated with inferior OS compared with unmutated tumors [multivariate HR 2.26 (1.25-4.1), P = 0.001]. A similar trend for OS was seen in the KRAS G13D-variant [n = 71, multivariate HR 1.46 (0.96-2.22), P = 0.10]. More frequent KRAS exon 2 variants like G12D [n = 152, multivariate HR 1.17 (0.86-1.6), P = 0.81] and G12V [n = 92, multivariate HR 1.27 (0.87-1.86), P = 0.57] did not have significant impact on OS. CONCLUSION: Mutations in KRAS and BRAF were associated with inferior PFS and OS of mCRC patients compared with patients with non-mutated tumors. KRAS exon 2 mutation variants were associated with heterogeneous outcome compared with unmutated tumors with KRAS G12C and G13D (trend) being associated with rather poor survival.

Prognostic value of microsatellite instability and p53 expression in metastatic colorectal cancer treated with oxaliplatin and fluoropyrimidine-based chemotherapy.

PURPOSE: The aim of this study was to evaluate the prognostic value of MSI-H and p53 overexpression in metastatic colorectal cancer (mCRC) treated with oxaliplatin and fluoropyrimidine-based first line chemotherapy. METHODS: Tumour samples were retrospectively obtained from 229 patients from a prospective randomised phase III trial of the AIO colorectal study group, comparing CAPOX and FUFOX in mCRC. Immunohistochemistry of p53 and MMR proteins as well as microsatellite analysis were performed. RESULTS: The incidence of MSI-H and p53 overexpression was 7.9 % and 65.4 %, respectively. MSI-H status was not correlated with ORR, PFS and OS. We observed a trend to lower DCR for MSI-H tumours (65 % vs. 85 %, p = 0.055). p53 overexpression was not correlated with DCR, ORR and PFS. The median OS of patients with tumors with p53 overexpression was significantly longer compared to tumors withhout p53 overexpression (19.6 vs. 15.8 months; p = 0.05). The post-progression survival (PPS) of p53-positive patients undergoing 2nd and/or 3rd line chemotherapy with irinotecan and/or cetuximab was significantly longer compared to p53-negative patients. CONCLUSION: MSI-H tumours tend to have lower disease control rates when treated with an oxaliplatin/fluoropyrmidin combination. mCRC patients with p53 overexpression undergoing an irinotecan containing second- or third-line chemotherapy after oxaliplatin failure have a significantly longer post-progression survival compared to patients without p53 overexpression. To validate the clinical impact of p53 in patients with mCRC treated with irinotecan- and/or cetuximab further studies are needed.

Prognostic factors for 60-day mortality in first-line treatment of metastatic colorectal cancer (mCRC): individual patient analysis of four randomised, controlled trials by the AIO colorectal cancer study group.

BACKGROUND: The 60 day mortality is an established parameter for chemotherapy-related safety in randomised trials for metastatic colorectal cancer (mCRC). Prognostic factors associated with 60-day mortality would be helpful to identify high-risk patients in advance. PATIENTS AND METHODS: Individual baseline patient data from four randomised, controlled trials from the Arbeitsgemeinschaft Internistische Onkologie (AIO) study group were retrospectively analysed. Chemotherapy consisted of fluoropyrimidine (5-FU/capecitabine), irinotecan, oxaliplatin with or without bevacizumab or cetuximab. Prognostic factors were identified by univariate and multivariate logistic regression models in two cohorts: one limited to ECOG PS 0 and 1 and one including ECOG PS 2 patients. RESULTS: A total of 1377 patients were evaluated. The analysis of ECOG PS 0, 1 and 2 patients consisted of 898 patients where a total of 33 deaths within the first 60 days of treatment (3.7%) occurred. In multivariate analysis, 60-day mortality was significantly associated with ECOG PS 2 and high leucocyte count (white blood cell, WBC). Odds ratio was 6.28 for WBC and 12.92 for ECOG PS 2. Exclusion of ECOG PS 2 patients but inclusion of one trial limited to ECOG PS 0 and 1 patients resulted in 1302 assessable patients and 44 early deaths (3.4%). In both cohorts, around 50% of deaths were disease related. WBC was confirmed as a significant risk factor for early death (OR 7.60). A combined score using ECOG PS 2 and WBC ≥8.000/µl is able to identify high-risk patients with a sensitivity of 18% and specificity of 98%. CONCLUSIONS: In this large retrospective analysis of individual patient data, around 50% of early deaths were disease related. Elevated WBC was found strongly associated with increased 60-day mortality in first-line treatment of mCRC. The proposed AIO-60-Day-Mortality score serves as an additional trial exclusion criterion.

Cetuximab plus cisplatin-5-fluorouracil versus cisplatin-5-fluorouracil alone in first-line metastatic squamous cell carcinoma of the esophagus: a randomized phase II study of the Arbeitsgemeinschaft Internistische Onkologie.

BACKGROUND: This study assessed the activity of the mAb cetuximab in combination with cisplatin and 5-fluorouracil (5-FU) in advanced esophageal squamous cell carcinoma. PATIENTS AND METHODS: For a maximum of six 29-day cycles, patients received cisplatin 100 mg/m(2), day 1, plus 5-FU 1000 mg/m(2), days 1-5 (CF), either alone or in combination with cetuximab (CET-CF; 400 mg/m(2) initial dose followed by 250 mg/m(2) weekly thereafter). The primary end point was tumor response. Tumor material was obtained for analysis of KRAS mutation status. RESULTS: Sixty-two eligible patients were included, 32 receiving CET-CF and 30 CF. Cetuximab did not exacerbate grade 3/4 toxicity, except for rash (6% versus 0%) and diarrhea (16% versus 0%). The overall response rate according to RECIST criteria was 19% and 13% and the disease control rate 75% and 57% for the CET-CF and CF arms, respectively. With a median follow-up of 21.5 months, the median progression-free survival was 5.9 and 3.6 months and median overall survival 9.5 and 5.5 months for CET-CF and CF, respectively. No KRAS codon 12/13 tumor mutations were identified in 37 evaluated samples. CONCLUSION: Cetuximab can be safely combined with CF chemotherapy and may increase the efficacy of standard CF chemotherapy.

[Updated German S3 guidelines on esophageal cancer and supplements from a surgical perspective]. / Aktualisierte S3-Leitlinie zum Ösophaguskarzinom und Ergänzungen aus chirurgischer Sicht.

The updated German S3 guidelines recommend transthoracic subtotal esophagectomy with 2­field lymphadenectomy for surgical treatment of esophageal cancer in patients with squamous cell carcinoma and adenocarcinoma of the esophagogastric (AEG type I) junction of the middle and lower third. For AEG type III transhiatal extended total gastrectomy with distal esophageal resection is favored. Patients with AEG type II can be treated by both procedures under the prerequisite that an R0 resection can be achieved. A limited resection of the distal esophagus and the proximal stomach can only be considered in cT1 N0 M0 possibly cT2 AEG junction without an oncological risk constellation, i.e. grade G1/G2, intestinal type and no poorly cohesive carcinoma, because the rate of lymph node metastasis at the distal stomach is less than 2%. Minimally invasive procedures provide advantages compared to open esophagectomy due to the lower rate of postoperative total and especially pulmonary complications. This is true for hybrid esophagectomy (laparoscopy and thoracotomy) versus open access in cases of intrathoracic anastomoses and for total minimally invasive esophagectomy including robotic techniques versus open access in cervical esophagogastrostomy.

Vascularization of carcinomas of the esophagus and its correlation with tumor proliferation.

Vascularization and tumor cell proliferation were analyzed in 33 resected human squamous cell carcinomas of the esophagus using the endothelium-specific antibody BW 200 and the proliferation-associated antibody Ki-67. Vascular parameters (relative capillary volume, relative total vessel volume, vascular surface area, and vascular length) as well as the percentage of proliferating tumor cells (Ki-67 index) were evaluated on frozen sections by a morphometric method. Vascular parameters of the normal mucosa exceeded those of tumors significantly, by a factor of 1.4-2.3. The mean distance between tumor capillaries and the onset of necrosis was 92 +/- 34 microns. Global vascular density did not correlate with TNM stage, tumor diameter, or overall tumor proliferation (mean Ki-67 index, 35.1%; range, 14.2-64.1%). However, a significant negative correlation existed between the percentage of proliferating tumor cells per tumor cord and the intercapillary distance between capillaries located at the edges of these cords. This observation points to the fact that the esophageal cancers were composed of multiple tumor cords and that each of these cords possessed its own supply capillaries at the base of the cord. The sum of these "supply units" thus constitutes an esophageal cancer. The intercapillary distance may reflect the oxygenation status of tumor cells, which cannot be predicted on the basis of tumor staging or grading.

Hypermethylation of the p16INK4a promoter in colectomy specimens of patients with long-standing and extensive ulcerative colitis.

Functional inactivation of the p16INK4a gene has been reported to be involved in the development of a variety of human malignancies. Recent evidence shows that transcriptional silencing as a consequence of hypermethylation of CpG islands is the predominant mechanism of p16INK4a gene inactivation in sporadic colon cancer. This study sought to identify the significance of p16INK4a methylation in the colonic epithelium of patients with long-standing ulcerative colitis. A total of 89 tissue samples was retrieved from three colectomy specimens. A methylation-specific PCR assay was applied. The methylation status was compared with histological findings and the flow cytometrically determined DNA index. Hypermethylation of the p16INK4a promoter region was detected in 12.7% of samples that were negative for dysplasia. However, 70.0% of samples with dysplasia and all of the samples with carcinomatous lesions revealed hypermethylation. Hypermethylation of the p16INK4a gene promoter was detected already in 40% of specimens with lesions indefinite for dysplasia and in 13.7% of samples with exclusively diploid cell populations. These results suggest that hypermethylation of the p16INK4a promoter region is a frequent and early occurring event during the process of neoplastic progression in ulcerative colitis.

[New S3 guideline for esophageal cancer : Important surgical aspects]. / Die neue S3-Leitlinie zum Ösophaguskarzinom : Wichtige chirurgische Aspekte.

The current German S3 guideline represents the recommendations for the diagnosis and therapy of squamous cell carcinomas and adenocarcinomas of the esophagus based on evidence from the literature and interdisciplinary expert consensus. Esophagogastroscopy with biopsy, endosonography, and spiral CT scan of the neck, thorax, and abdomen are decisive in staging and the choice of therapy. For a curative approach, surgery, especially transthoracic esophagectomy and gastric pull-up, is the most important therapeutic option, except in the case of mucosal carcinomas or cervical squamous cell carcinomas. The significance of total minimally invasive esophageal resection or a hybrid technique is still uncertain. In category cT3 or resectable cT4 tumors, neoadjuvant radiochemotherapy should be performed in squamous cell carcinomas or adenocarcinomas. Alternatively, perioperative chemotherapy can be carried out in adenocarcinoma. Palliative resections should be avoided and replaced by interventional procedures for palliation.

Fluorouracil plus leucovorin as effective adjuvant chemotherapy in curatively resected stage III colon cancer: results of the trial adjCCA-01.

PURPOSE: Adjuvant postoperative treatment with fluorouracil (5-FU) and levamisole in curatively resected stage III colon cancer significantly reduces the risk of cancer recurrence and improves survival. Biochemical modulation of 5-FU with leucovorin has resulted in increased remission rates in metastatic colorectal cancer, thus reflecting an increased tumor-cell kill. The impact of 5-FU plus leucovorin on survival and tumor recurrence was analyzed in comparison with the effects of 5-FU plus levamisole in the prospective multicentric trial adjCCA-01. PATIENTS AND METHODS: Patients with a curatively resected International Union Against Cancer stage III colon cancer were stratified according to T, N, and G category and randomly assigned to receive one of the two adjuvant treatment schemes: 5-FU 400 mg/m(2) body-surface area intravenously in the first chemotherapy course, then 450 mg/m(2) x 5 days; 12 cycles, plus leucovorin 100 mg/m(2) (arm A), or 5-FU plus levamisole (Moertel scheme; arm B). RESULTS: Six hundred eighty (96.9%) of 702 patients enrolled onto this study were eligible. After a median follow-up time of 46.5 months, the 5-FU plus leucovorin combination significantly improved disease-free survival (P =.037) and significantly decreased overall mortality (P =.0089) in comparison with 5-FU plus levamisole. In a multivariate proportional hazards model, adjuvant chemotherapy emerged as a significant prognostic factor for survival (P =.0059) and disease-free survival (P =.03). Adjuvant treatment with 5-FU plus levamisole as well as with 5-FU plus leucovorin was generally well tolerated; only a minority of patients experienced grade 3 and 4 toxicities. CONCLUSION: After a curative resection of a stage III colon cancer, adjuvant treatment with 5-FU plus leucovorin is generally well tolerated and significantly more effective than 5-FU plus levamisole in reducing tumor relapse and improving survival.

Alteration of DNA ploidy status and cell proliferation induced by preoperative radiotherapy is a prognostic factor in rectal cancer.

To identify predictors of prognosis after preoperative radiotherapy, DNA ploidy and cell proliferation were investigated in 116 patients with rectal cancer. For flow cytometry, a nuclear suspension was prepared by pepsin digestion of paraffin samples of biopsies taken before preoperative radiotherapy (15 x 2 Gy) and also of the resected rectal tumors after radiotherapy. The median follow-up period was 6 years. The proportion of tumor necrosis was evaluated in histological sections before and after irradiation. There was a significant decrease (74 to 48%) in aneuploid tumors after radiation. Of 86 patients with aneuploid biopsies, 28 revealed no reduction in the proportion of aneuploid tumor cells [group AN(=/increase)], and 58 showed a reduction (mean 48.9%) or complete elimination of aneuploid tumor cells [group AN(decrease/psi)]. The incidence of local or distal failure was significantly reduced in the group AN(decrease/psi) (7.8%/20%), compared with the group AN (=/increase) (27%/54%) and the group of constant diploid tumors (n = 22; 13.6%/31.8 %; P = 0.034). There was a trend of decreased recurrence rate in diploid tumors with a reduced fraction of cells in S-phase after radiotherapy. Survival was significantly increased in group AN(decrease/psi) (P < 0.0001). In a multivariate regression analysis, variables of independent prognostic significance were increased proportion of necrosis after irradiation and DNA ploidy group and the postoperative tumor stage. These results suggest that alterations in tumor DNA ploidy and cell proliferation induced by preoperative radiotherapy might help to identify patients likely to benefit from preoperative radiation in rectal cancer.

17-Iodine-123 iodoheptadecanoic acid for metabolic liver studies in humans.

(17-123I)-Iodoheptadecanoic acid ([123I]HA) was used for dynamic planar scintigraphy of the liver in normal individuals (control I), in patients without liver disease but with elevated serum cholesterol and/or triglycerides (control II), and in patient groups with alcohol-induced fatty liver (PG I), fatty liver not due to alcohol (PG II), alcohol-induced liver cirrhosis (PG III), or liver cirrhosis of the posthepatitic type (PG IV). Tracer uptake and elimination time were assayed in different liver regions; mean elimination time was expressed for total liver. In control I, tracer uptake was homogeneous, and mean elimination time was 20.7 +/- 5.3 min without significant local variations. In control II, tracer uptake was reduced but homogeneous and mean elimination time was 59.4 +/- 35.8 min with some local variations. In PG I, uptake was reduced and inhomogeneous and elimination time was the same as in control I, irrespective of cholesterol and triglyceride values. In PG II, uptake was the same as in PG I but mean elimination time was 48 +/- 8.1 min with some local variations. In PG III, uptake was extremely reduced and spotty and elimination time correlated with the severity of disease from 19 to 881 min in different liver regions.

Comparative genomic hybridization analysis of chromosomal alterations in patients with long-standing ulcerative colitis.

Patients with ulcerative colitis (UC) are prone to develop colorectal cancer which is related to the duration and extent of the disease. One of the earliest events in tumor progression is the development of aneuploidy. Aneuploidy is correlated with the grade of dysplasia which serves as a common but not always reproducible marker for the prediction of UC associated formation of cancer. We analyzed 48 biopsy samples from 5 patients with long-standing ulcerative colitis by comparative genomic hybridization (CGH). The majority of these samples represented premalignant stages which are not well characterized at the molecular level as yet. We compared biopsy samples from different colon locations in regard to chromosomal alterations, dysplasia status and DNA index. Besides chromosomal changes occurring only in certain patients in restricted areas of the colon we also detected amplifications and deletions which were common in all persons throughout the colon. The stage of dysplasia seems to have no influence on the number and appearance of chromosomal changes. Amplifications in 2, 3, 6, 9, 11, 12 and 15 were found in almost all cases. In dysplastic samples chromosomal regions 3, 6 and 11 revealed gains of DNA. Deletions were detected within 8q, 15, 18q, 20p and 22q. The affected chromosomal regions may contain yet unknown oncogenes or tumor suppressor genes participating in UC associated carcinogenesis. The conspicuous regions found in the CGH experiments allow the selective and detailed characterization at a molecular level.

Growth inhibition of human gastrointestinal cancer cells by cyclosporin A.

We have studied the ability of cyclosporin A (CsA) to inhibit the growth of human AGS gastric and HT29 colon carcinoma cells in vitro. Using continuous drug exposure in growth assays of cultured tumour cells we found that CsA produced a dose-dependent growth inhibition in gastric and colon cancer cells with a half-maximal effect at 5 microM and 6 microM CsA respectively. The growth inhibition of CsA was reversible in AGS cells, when the tumour cells were incubated in normal growth medium following CsA treatment. Trypan blue dye exclusion in AGS cells indicated a cytostatic rather than a cytotoxic effect in the concentration range used. Coincubation of CsA-treated cells with 10-400 U/ml interleukin-2 (IL-2) could not abrogate this growth inhibition, suggesting an IL-2 independent mechanism of action. Flow-cytometric analysis did not reveal a phase arrest of the gastric cancer cells within the cell cycle. We conclude from our experiments that CsA cytostatically and reversibly inhibits the growth of human gastric cancer cells in a dose-dependent manner. In contrast to its mechanism of action in lymphocytes, this direct antiproliferative effect of CsA seems not to be mediated by an IL-2-dependent pathway or a cell-cycle-phase arrest of the tumour cells.

Protein kinase C and adenylate cyclase as targets for growth inhibition of human gastric cancer cells.

In the human gastric adenocarcinoma cell line AGS the effects of the protein-kinase-C-activating phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA), the protein kinase C inhibitor staurosporine, the adenylate-cyclase activating agent forskolin, and the permeable dibutyryl-adenosine 3',5'-monophosphate (Bt2cAMP) on the proliferation were assessed. Cell counting followed 5 days of incubation. Prolonged activation of protein kinase C by TPA, inhibition of protein kinase C by staurosporine, activation of adenylate cyclase by forskolin or a direct increase of the intracellular cAMP level all result in a dose-dependent growth inhibition of AGS gastric tumour cells. Half-maximal inhibition was achieved at 100 pM for TPA, 1 nM for staurosporine, 20 microM for forskolin, and 600 microM for Bt2cAMP. It is concluded that protein kinase C and adenylate cyclase play a fundamental role in the growth of AGS gastric cancer cells. Interference with these enzymes involved in the signal transduction of growth regulation in tumour cells may represent a target in the development of new antiproliferative principles.

The prognostic significance of DNA ploidy in adenocarcinomas of the esophagogastric junction.

DNA aneuploidy, as determined by flow cytometry, was detected in 36 out of 59 adenocarcinomas of the esophagogastric junction (61.0%). DNA aneuploidy was more frequent in tumors with infiltrative growth pattern and in high pT categories. No correlation was found with pN category, grading and Laurén's classification. In contrast to clinicopathological parameters, DNA ploidy has no impact on patients survival in univariate survival analysis.

Differential expression of mucins and trefoil peptides in native epithelium, Barrett's metaplasia and squamous cell carcinoma of the oesophagus.

BACKGROUND AND AIMS: In humans, trefoil peptides (TFF peptides) and some mucins have been reported to be expressed in a cell-specific manner at mucosal surfaces of normal gastrointestinal tissues. Neoplastic conditions cause characteristic changes of these expression patterns. To study such patterns in Barrett's metaplasia and squamous cell carcinoma of the oesophagus (SCC), the distribution of MUC1, MUC2, MUC5AC and the three TFF peptides (TFF1, TFF2 and TFF3) was investigated. METHODS: In 40 archival samples of SCC and in 21 samples of Barret's metaplasia, expression of the three mucins and two TFF peptides (TFF1 and TFF2) was assessed by specific antibodies. Reverse transcriptase/polymerase chain reaction amplification (RT-PCR) was performed on frozen tissue samples from the 11 biopsies of SCC for the three TFF peptides. RESULTS: Immunohistochemical tests for MUC2 and TFF2 were negative both in samples of Barret's metaplasia and in SCC. MUC1 expression was detected in 57.5% of the tumour samples, while TFF1 and MUC5AC were found in 10% and 7.5% of the cases respectively. In Barrett's metaplasia MUC1 was detected in 90.5% of the cases and TFF1 and MUC5AC in all of them. RT-PCR analysis revealed a more complex pattern: TFF1 and TFF3 expressed the corresponding mRNA in all samples investigated; the third member, TFF2, was active in 45.5% of the carcinoma biopsies and not in the corresponding native tissue. CONCLUSIONS: This finding in oesophageal carcinoma contrasts with the situation found in normal and neoplastic stomach epithelium where TFF1 and TFF2 are found co-expressed and TFF3 remains silent. Interestingly, MUC1 is expressed in a significant proportion of SCC. Both in Barett's metaplasia and in SCC the expression of MUC5AC mirrors the TFF1 synthesis in intensity and spatial distribution.

Isolation of Corynebacterium xerosis from clinical specimens: infection and colonization.

Eleven patients were colonized or infected with diphtheroids identified as Corynebacterium xerosis. All the patients were compromised hosts by nature of their underlying disease and/or therapy. Two patients developed bacteremia following colonization of the respiratory tract with C. xerosis. Other patients were colonized at various sites, which included the respiratory tract, abdominal and thoracic wounds, amputated limb, and arterial-venous shunt. Distinctive features for the identification of C. xerosis include negative reactions for hemolysis, urease, and motility, and positive reactions for catalase, glucose, sucrose and nitrate reduction. Antimicrobial susceptibility tests were performed by the disk diffusion method. In many instances the organisms were resistant to the antimicrobial regimens received by the patients. This was most frequent for nafcillin, gentamicin, kanamycin, clindamycin, and chloramphenicol. On the other hand, the organisms were highly susceptible to penicillin, ampicillin, cephalothin and carbenicillin.