Publications by authors named "Gisela Keller"

62 Publications

The immunologic tumor microenvironment in endometrioid endometrial cancer in the morphomolecular context: mutual correlations and prognostic impact depending on molecular alterations.

Cancer Immunol Immunother 2020 Dec 19. Epub 2020 Dec 19.

Institute of Pathology, Technical University Munich, Trogerstrasse 18, 81675, Munich, Germany.

Objective: POLE-mutant, microsatellite-instable (MSI), p53-mutant and non-specific molecular profile (NSMP) are TCGA-defined molecular subgroups of endometrial cancer (EC). Hypothesizing that morphology and tumor immunology might differ depending on molecular background concerning composition and prognostic impact, we aimed to comprehensively interconnect morphologic, immunologic and molecular data.

Methods: TCGA-defined molecular groups were determined by immunohistochemistry and sequencing in n = 142 endometrioid EC. WHO-defined histopathological grading was performed. The immunologic microenvironment (iTME) was characterised by the quantification of intraepithelial and stromal populations of tumor-infiltrating lymphocytes (TIL: overall T-cells; T-Killer cells; regulatory T-cells (Treg)). Immunologic parameters were correlated with WHO-grading, TCGA-subgroups and prognosis.

Results: High density TIL were significantly more frequent in high-grade (G3) compared to low-grade (G1/2) EC in the whole cohort and in the subgroup of POLE-wildtype-/microsatellite-stable-EC. MSI was associated with high-level TIL-infiltration when taking into account the type of mismatch repair defect (MLH1/PMS2; MSH2/MSH6). Prognostic impact of biomarkers depended on molecular subgroups: In p53-mutant EC, Treg were independently prognostic, in NSMP, the unique independently prognostic biomarker was WHO-grading.

Conclusions: EC morphology and immunology differ depending on genetics. Our study delineated two molecularly distinct subgroups of immunogenic EC characterized by high-density TIL-infiltration: MSI EC and high-grade POLE-wildtype/microsatellite-stable-EC. Prognostic impact of TIL-populations relied on TCGA-subgroups indicating specific roles for TIL depending on molecular background. In NSMP, histopathological grading was the only prognostic biomarker demonstrating the relevance of WHO-grading in an era of molecular subtyping.
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http://dx.doi.org/10.1007/s00262-020-02813-3DOI Listing
December 2020

Impact of Tumor Localization and Molecular Subtypes on the Prognostic and Predictive Significance of p53 Expression in Gastric Cancer.

Cancers (Basel) 2020 Jun 25;12(6). Epub 2020 Jun 25.

Institute of Pathology, TUM School of Medicine, Technical University of Munich, 81675 Munich, Germany.

We investigated the prognostic and predictive impact of p53 expression for gastric cancer (GC) patients treated without or with preoperative chemotherapy (CTx) and its relationship with specific molecular GC subtypes. Specimens from 694 GC patients (562 surgical resection specimens without or after CTx, 132 biopsies before CTx) were analyzed by p53 immunohistochemistry. High (H) and low (L) microsatellite instability (MSI) and Epstein-Barr virus positivity were determined previously. Our results show that aberrant p53 expression was a negative prognostic factor in uni- and multivariable analysis in the resection specimens cohort (each < 0.01). Subgroup analysis showed the strongest prognostic effect for patients with distally located tumors or no CTx treatment. In the biopsy cohort before CTx, p53 did not predict response or survival. p53 expression was significantly different among the molecular subtypes in surgical resection and bioptic specimens with strong association of altered p53 with MSI-L. Patients with MSI-H and aberrant p53 showed the worst survival in the biopsy cohort. In conclusion, the prognostic impact of p53 in GC differs according to tumor localization and CTx. Altered p53 is characteristic for MSI-L, and the p53 status in biopsies before CTx delineates MSI-H subtypes with inverse prognostic impact.
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http://dx.doi.org/10.3390/cancers12061689DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7352381PMC
June 2020

Prognostic implication of molecular subtypes and response to neoadjuvant chemotherapy in 760 gastric carcinomas: role of Epstein-Barr virus infection and high- and low-microsatellite instability.

J Pathol Clin Res 2019 10 17;5(4):227-239. Epub 2019 Jun 17.

Institute of Pathology, Technical University of Munich, Munich, Germany.

Epstein-Barr virus positivity (EBV(+)) and high-microsatellite instability (MSI-H) have been identified as molecular subgroups in gastric carcinoma. The aim of our study was to determine the prognostic and predictive relevance of these subgroups in the context of platinum/5-fluorouracil (5-FU) based preoperative chemotherapy (CTx). Additionally, we investigated the clinical relevance of the low-MSI (MSI-L) phenotype. We analysed 760 adenocarcinomas of the stomach or the gastro-oesophageal junction encompassing 143 biopsies before CTx and 617 resected tumours (291 without and 326 after CTx). EBV was determined by PCR and in situ hybridisation for selected cases. MSI was analysed by PCR using five microsatellite markers and classified as MSI-H and MSI-L. Frequencies of EBV(+), MSI-H and MSI-L in the biopsies before CTx were 4.2, 10.5 and 4.9% respectively. EBV(+) or MSI-H did not correlate with response, but MSI-L was associated with better response (p = 0.011). In the resected tumours, frequencies of EBV(+), MSI-H and MSI-L were 3.9, 9.6 and 4.5% respectively. Overall survival (OS) was significantly different in the non-CTx group (p = 0.014). Patients with EBV(+) tumours showed the best OS, followed by MSI-H. MSI-L was significantly associated with worse OS (hazard ratio [HR], 2.21; 95% confidence interval [CI], 1.21-4.04, p = 0.01). In the resected tumours after CTx, MSI-H was also associated with increased OS (HR, 0.54; 95% CI, 0.26-1.09, p = 0.085). In multivariable analysis, molecular classification was an independent prognostic factor in the completely resected (R0) non-CTx group (p = 0.035). In conclusion, MSI-H and EBV(+) are not predictive of response to neoadjuvant platinum/5-FU based CTx, but they are indicative of a good prognosis. In particular, MSI-H indicates a favourable prognosis irrespective of treatment with CTx. MSI-L predicts good response to CTx and its negative prognostic effect for patients treated with surgery alone suggests that MSI-L might help to identify patients with potentially high-benefit from preoperative CTx.
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http://dx.doi.org/10.1002/cjp2.137DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6817827PMC
October 2019

Genetic and immunological biomarkers predict metastatic disease recurrence in stage III colon cancer.

BMC Cancer 2018 Oct 19;18(1):998. Epub 2018 Oct 19.

Department of Surgery, Klinikum rechts der Isar, TUM, Ismaninger Str. 22, 81675, Munich, Germany.

Background: Even though the post-operative outcome varies greatly among patients with nodal positive colon cancer (UICC stage III), personalized prediction of systemic disease recurrence is currently insufficient. We investigated in a retrospective setting whether genetic and immunological biomarkers can be applied for stratification of distant metastasis occurrence risk.

Methods: Eighty four patients with complete resection (R0) of stage III colon cancer from two clinical centres were analysed for genetic biomarkers: microsatellite instability, oncogenic mutations in KRAS exon2 and BRAF exon15, expression of osteopontin and the metastasis-associated genes SASH1 and MACC1. Tumor-infiltrating CD3 and CD8 positive T-cells were quantified by immunocytochemistry. Results were correlated with outcome and response to 5-FU based adjuvant chemotherapy, using Cox's proportional hazard models and integrative two-step cluster analysis.

Results: Distant metastasis risk was significantly correlated with oncogenic KRAS mutations (p = 0.015), expression of SASH1 (p = 0.016), and the density of CD8-positive T-cells (p = 0.007) in Kaplan-Meier analysis. Upon multivariate Cox-regression analysis, KRAS mutation (p = 0.008) and density of CD8-positive TILs (p = 0.009) were retained as prognostic parameters for metachronous distant metastasis. Integrative two-step cluster analysis was used to combine all genetic markers, allowing stratification of patient subgroups. Post-operative distant metastasis risk ranged from 31% (low-risk) to 41% (intermediate), and 57% (high-risk) (p = 0.032). Increased expression of osteopontin (p = 0.019) and low density of CD8-positive T-cells (p = 0.043) were significantly associated with unfavourable response to 5-FU.

Conclusions: Integrative biomarker analysis allows stratification of stage III colon cancer patients for the risk of metastatic disease recurrence and may indicate response to 5-FU. Thus, biomarker analysis might facilitate the use of adjuvant therapy for high risk patients.
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http://dx.doi.org/10.1186/s12885-018-4940-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6194664PMC
October 2018

Evidence for PTGER4, PSCA, and MBOAT7 as risk genes for gastric cancer on the genome and transcriptome level.

Cancer Med 2018 10 6;7(10):5057-5065. Epub 2018 Sep 6.

Institute of Human Genetics, University of Bonn, Bonn, Germany.

Genetic associations between variants on chromosome 5p13 and 8q24 and gastric cancer (GC) have been previously reported in the Asian population. We aimed to replicate these findings and to characterize the associations at the genome and transcriptome level. We performed a fine-mapping association study in 1926 GC patients and 2012 controls of European descent using high dense SNP marker sets on both chromosomal regions. Next, we performed expression quantitative trait locus (eQTL) analyses using gastric transcriptome data from 143 individuals focusing on the GC associated variants. On chromosome 5p13 the strongest association was observed at rs6872282 (P = 2.53 × 10 ) and on chromosome 8q24 at rs2585176 (P = 1.09 × 10 ). On chromosome 5p13 we found cis-eQTL effects with an upregulation of PTGER4 expression in GC risk allele carrier (P = 9.27 × 10 ). On chromosome 8q24 we observed cis-eQTL effects with an upregulation of PSCA expression in GC risk allele carrier (P = 2.17 × 10 ). In addition, we found trans-eQTL effects for the same variants on 8q24 with a downregulation of MBOAT7 expression in GC risk allele carrier (P = 3.11 × 10 ). In summary, we confirmed and refined the previously reported GC associations at both chromosomal regions. Our data point to shared etiological factors between Asians and Europeans. Furthermore, our data imply an upregulated expression of PTGER4 and PSCA as well as a downregulated expression of MBOAT7 in gastric tissue as risk-conferring GC pathomechanisms.
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http://dx.doi.org/10.1002/cam4.1719DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6198243PMC
October 2018

A microsatellite based multiplex PCR method for the detection of chromosomal instability in gastric cancer.

Sci Rep 2018 08 22;8(1):12551. Epub 2018 Aug 22.

Department of Pathology, Technical University of Munich, Trogerstr. 18, 81675, Munich, Germany.

Chromosomal instability (CIN) is a hallmark of distinct subclasses of tumours with potential clinical relevance. The aim of our study was to establish a time and cost effective method for the determination of CIN in gastric carcinomas (GC). We developed a microsatellite based multiplex PCR assay for the detection of allelic imbalances (AI) using experimentally defined marker specific threshold values for AI. The assay was tested in 90 formalin-fixed paraffin-embedded GC and results were compared in a subset of 30 carcinomas with the Affymetrix OncoScan assay, which detects copy number variations on genome wide level. The ratios of alterations detected by the two methods demonstrated a significant correlation (r = 0.88). Based on the results of the OncoScan assay, tumours were classified in CIN-High and CIN-Low and a threshold of the AI ratio determined with the PCR assay was defined. Accordingly, 20 of the 90 GC (22%) were CIN-Low and 70 (78%) CIN-High. A significant association of CIN-High was found with intestinal type tumours and proximal tumour localization. In conclusion, we established a PCR based method to categorize AI as surrogate for CIN, which is easy to perform and useful for the clarification of the clinical relevance of CIN in large GC cohorts.
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http://dx.doi.org/10.1038/s41598-018-30971-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6105665PMC
August 2018

Comprehensive analysis of the promoter region in 480 patients with colorectal cancer and 1150 controls reveals new variants including one with a heritable constitutional epimutation.

J Med Genet 2018 04 22;55(4):240-248. Epub 2018 Feb 22.

Medizinische Klinik und Poliklinik IV, Klinikum der Universität München, Munich, Bavaria, Germany.

Background: Germline defects in , , and predisposing for Lynch syndrome (LS) are mainly based on sequence changes, whereas a constitutional epimutation of (CEM) is exceptionally rare. This abnormal promoter methylation is not hereditary when arising de novo, whereas a stably heritable and variant-induced CEM was described for one single allele. We searched for promoter variants causing a germline or somatic methylation induction or transcriptional repression.

Methods: We analysed the promoter sequence in five different patient groups with colorectal cancer (CRC) (n=480) composed of patients with i) CEM (n=16), ii) unsolved loss of MLH1 expression in CRC (n=37), iii) CpG-island methylator-phenotype CRC (n=102), iv) patients with LS (n=83) and v) MLH1-proficient CRC (n=242) as controls. 1150 patients with non-LS tumours also served as controls to correctly judge the results.

Results: We detected 10 rare promoter variants. One novel, complex variant c.-63_-58delins18 is present in a patient with CRC with CEM and his sister, both showing a complete allele-specific promoter methylation and transcriptional silencing. The other nine promoter variants detected in 17 individuals were not associated with methylation. For four of these, a normal, biallelic expression was found in the patients' cDNA.

Conclusion: We report the second promoter variant stably inducing a hereditary CEM. Concerning the classification of promoter variants, we discuss contradictory results from the literature for two variants, describe classification discrepancies between existing rules for five variants, suggest the (re-)classification of five promoter variants to (likely) benign and regard four variants as functionally unclear.
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http://dx.doi.org/10.1136/jmedgenet-2017-104744DOI Listing
April 2018

Loss of MSH2 and MSH6 due to heterozygous germline defects in MSH3 and MSH6.

Fam Cancer 2017 Oct;16(4):491-500

Medizinische Klinik und Poliklinik IV, Campus Innenstadt, Klinikum der Universität München, Ziemssenstr. 1, 80336, Munich, Germany.

Lynch Syndrome (LS) is the most common dominantly inherited colorectal cancer (CRC) predisposition and is caused by a heterozygous germline defect in one of the DNA mismatch repair (MMR) genes MLH1, MSH2, MSH6, or PMS2. High microsatellite instability (MSI-H) and loss of MMR protein expression in tumours reflecting a defective MMR are indicators for LS, as well as a positive family history of early onset CRC. MSH2 and MSH6 form a major functional heterodimer, and MSH3 is an alternative binding partner for MSH2. So far, the role of germline MSH3 variants remains unclear, as to our knowledge heterozygous truncating variants are not regarded causative for LS, but were detected in patients with CRC, and recently biallelic MSH3 defects have been identified in two patients with adenomatous polyposis. By gene screening we investigated the role of MSH3 in 11 LS patients with truncating MSH6 germline variants and an unexplained MSH2 protein loss in their corresponding MSI-H tumours. We report the first two LS patients harbouring heterozygous germline variants c.1035del and c.2732T>G in MSH3 coincidentally with truncating variants in MSH6. In the patient with truncating germline variants in MSH3 and MSH6, two additional somatic second hits in both genes abrogate all binding partners for the MSH2 protein which might subsequently be degraded. The clinical relevance of MSH3 germline variants is currently under re-evaluation, and heterozygous MSH3 defects alone do not seem to induce a LS phenotype, but might aggravate the MSH6 phenotype in affected family members.
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http://dx.doi.org/10.1007/s10689-017-9975-zDOI Listing
October 2017

Colorectal mixed adenoneuroendocrine carcinomas and neuroendocrine carcinomas are genetically closely related to colorectal adenocarcinomas.

Mod Pathol 2017 04 6;30(4):610-619. Epub 2017 Jan 6.

Institute of Pathology, Technical University of Munich, Munich, Germany.

Colorectal mixed adenoneuroendocrine carcinomas are rare and clinically aggressive neoplasms with considerable morphological heterogeneity. Data on their genomic characteristics and molecular associations to either conventional colorectal adenocarcinomas or poorly differentiated neuroendocrine neoplasms is still scarce, hampering optimized patient treatment and care. Tissue from 19 colorectal mixed adenoneuroendocrine carcinomas and eight colorectal poorly differentiated neuroendocrine neoplasms (neuroendocrine carcinomas) was microdissected and subjected to next-generation sequencing using a colorectal adenocarcinoma-specific panel comprising 196 amplicons covering 32 genes linked to colorectal adenocarcinoma, and poorly differentiated neuroendocrine neoplasm tumorigenesis. Mixed adenoneuroendocrine carcinomas were also examined for microsatellite instability and MLH-1 promoter methylation status. In three mixed adenoneuroendocrine carcinomas, exocrine and endocrine components were analyzed separately. Genetic testing of colorectal mixed adenoneuroendocrine carcinomas identified 43 somatic mutations clustering in 13/32 genes. Sixteen (84%) tumors harbored at least one somatic mutation, two tumors (11%) displayed high microsatellite instability. Compared with colorectal adenocarcinomas, mixed adenoneuroendocrine carcinomas were more frequently BRAF (37%; P=0.006), and less frequently KRAS (21%; P=0.043) and APC (16%; P=0.001) mutated. Point mutations in neuroendocrine neoplasm-related genes like RB1 or RET were not detected, but one tumor harbored a heterozygous RB1 deletion. Separately analyzed adenocarcinoma and neuroendocrine carcinoma components revealed a shared mutational trunk of driver genes involved in colorectal adenocarcinoma carcinogenesis. Colorectal neuroendocrine carcinomas were similar in their mutation profile to colorectal adenocarcinomas, but compared with mixed adenoneuroendocrine carcinomas, had a higher rate of APC mutations (P=0.027). Our data indicate that colorectal mixed adenoneuroendocrine carcinomas and neuroendocrine carcinomas are genetically closely related to colorectal adenocarcinomas, suggesting that the cells giving rise to these tumors primarily have an intestinal coinage. The identification of BRAF mutations and the frequently present KRAS wild-type status principally render some mixed adenoneuroendocrine carcinomas eligible to targeted treatment strategies used for colorectal adenocarcinomas.
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http://dx.doi.org/10.1038/modpathol.2016.220DOI Listing
April 2017

Clinical Significance of NOTCH1 and NOTCH2 Expression in Gastric Carcinomas: An Immunohistochemical Study.

Front Oncol 2015 22;5:94. Epub 2015 Apr 22.

Institute of Pathology, Technische Universität München , Munich , Germany.

Background: NOTCH signaling can exert oncogenic or tumor suppressive functions and can contribute to chemotherapy resistance in cancer. In this study, we aimed to clarify the clinicopathological significance and the prognostic and predictive value of NOTCH1 and NOTCH2 expression in gastric cancer (GC).

Methods: NOTCH1 and NOTCH2 expression was determined immunohistochemically in 142 primarily resected GCs using tissue microarrays and in 84 pretherapeutic biopsies from patients treated by neoadjuvant chemotherapy. The results were correlated with survival, response to therapy, and clinico-pathological features.

Results: Primarily resected patients with NOTCH1-negative tumors demonstrated worse survival. High NOTCH1 expression was associated with early-stage tumors and with significantly increased survival in this subgroup. Higher NOTCH2 expression was associated with early-stage and intestinal-type tumors and with better survival in the subgroup of intestinal-type tumors. In pretherapeutic biopsies, higher NOTCH1 and NOTCH2 expression was more frequent in non-responding patients, but these differences were statistically not significant.

Conclusion: Our findings suggested that, in particular, NOTCH1 expression indicated good prognosis in GC. The close relationship of high NOTCH1 and NOTCH2 expression with early tumor stages may indicate a tumor-suppressive role of NOTCH signaling in GC. The role of NOTCH1 and NOTCH2 in neoadjuvantly treated GC is limited.
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http://dx.doi.org/10.3389/fonc.2015.00094DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4406075PMC
May 2015

Biallelic MUTYH mutations can mimic Lynch syndrome.

Eur J Hum Genet 2014 Nov 12;22(11):1334-7. Epub 2014 Feb 12.

1] Medizinische Klinik und Poliklinik IV, Campus Innenstadt, Klinikum der Universität München, Munich, Germany [2] MGZ - Medizinisch Genetisches Zentrum, Munich, Germany.

The hallmarks of Lynch syndrome (LS) include a positive family history of colorectal cancer (CRC), germline mutations in the DNA mismatch repair (MMR) genes, tumours with high microsatellite instability (MSI-H) and loss of MMR protein expression. However, in ∼10-15% of clinically suspected LS cases, MMR mutation analyses cannot explain MSI-H and abnormal immunohistochemistry (IHC) results. The highly variable phenotype of MUTYH-associated polyposis (MAP) can overlap with the LS phenotype, but is inherited recessively. We analysed the MUTYH gene in 85 'unresolved' patients with tumours showing IHC MMR-deficiency without detectable germline mutation. Biallelic p.(Tyr179Cys) MUTYH germline mutations were found in one patient (frequency 1.18%) with CRC, urothelial carcinoma and a sebaceous gland carcinoma. LS was suspected due to a positive family history of CRC and because of MSI-H and MSH2-MSH6 deficiency on IHC in the sebaceous gland carcinoma. Sequencing of this tumour revealed two somatic MSH2 mutations, thus explaining MSI-H and IHC results, and mimicking LS-like histopathology. This is the first report of two somatic MSH2 mutations leading to an MSI-H tumour lacking MSH2-MSH6 protein expression in a patient with MAP. In addition to typical transversion mutations in KRAS and APC, MAP can also induce tumourigenesis via the MSI-pathway.
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http://dx.doi.org/10.1038/ejhg.2014.15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4200426PMC
November 2014

A retrospective comparative exploratory study on two methylentetrahydrofolate reductase (MTHFR) polymorphisms in esophagogastric cancer: the A1298C MTHFR polymorphism is an independent prognostic factor only in neoadjuvantly treated gastric cancer patients.

BMC Cancer 2014 Feb 3;14:58. Epub 2014 Feb 3.

Department of Surgery, University Hospital of Heidelberg, Im Neuenheimer Feld 110, Heidelberg 69120, Germany.

Background: Methylentetrahydrofolate reductase (MTHFR) plays a major role in folate metabolism and consequently could be an important factor for the efficacy of a treatment with 5-fluorouracil. Our aim was to evaluate the prognostic and predictive value of two well characterized constitutional MTHFR gene polymorphisms for primarily resected and neoadjuvantly treated esophagogastric adenocarcinomas.

Methods: 569 patients from two centers were analyzed (gastric cancer: 218, carcinoma of the esophagogastric junction (AEG II, III): 208 and esophagus (AEG I): 143). 369 patients received neoadjuvant chemotherapy followed by surgery, 200 patients were resected without preoperative treatment. The MTHFR C677T and A1298C polymorphisms were determined in DNA from peripheral blood lymphozytes. Associations with prognosis, response and clinicopathological factors were analyzed retrospectively within a prospective database (chi-square, log-rank, cox regression).

Results: Only the MTHFR A1298C polymorphisms had prognostic relevance in neoadjuvantly treated patients but it was not a predictor for response to neoadjuvant chemotherapy. The AC genotype of the MTHFR A1298C polymorphisms was significantly associated with worse outcome (p = 0.02, HR 1.47 (1.06-2.04). If neoadjuvantly treated patients were analyzed based on their tumor localization, the AC genotype of the MTHFR A1298C polymorphisms was a significant negative prognostic factor in patients with gastric cancer according to UICC 6th edition (gastric cancer including AEG type II, III: HR 2.0, 95% CI 1.3-2.0, p = 0.001) and 7th edition (gastric cancer without AEG II, III: HR 2.8, 95% CI 1.5-5.7, p = 0.003), not for AEG I. For both definitions of gastric cancer the AC genotype was confirmed as an independent negative prognostic factor in cox regression analysis. In primarily resected patients neither the MTHFR A1298C nor the MTHFR C677T polymorphisms had prognostic impact.

Conclusions: The MTHFR A1298C polymorphisms was an independent prognostic factor in patients with neoadjuvantly treated gastric adenocarcinomas (according to both UICC 6th or 7th definitions for gastric cancer) but not in AEG I nor in primarily resected patients, which confirms the impact of this enzyme on chemotherapy associated outcome.
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http://dx.doi.org/10.1186/1471-2407-14-58DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3922603PMC
February 2014

Independent validation of a prognostic genomic signature (ColoPrint) for patients with stage II colon cancer.

Ann Surg 2013 Jun;257(6):1053-8

Department of Surgery, Klinikum rechts der Isar, Technische Universität München, Munich, Germany.

Objectives: The aim of this study was to independently validate a genomic signature developed both to assess recurrence risk in stage II patients and to assist in treatment decisions.

Background: Adjuvant therapy is recommended for high-risk patients with stage II colon cancer, but better tools to assess the patients' prognosis accurately are still required.

Methods: Previously, an 18-gene signature had been developed and validated on an independent cohort, using full genome microarrays. In this study, the gene signature was translated and validated as a robust diagnostic test (ColoPrint), using customized 8-pack arrays. In addition, clinical validation of the diagnostic ColoPrint assay was performed on 135 patients who underwent curative resection (R0) for colon cancer stage II in Munich. Fresh-frozen tissue, microsatellite instability status, clinical parameters, and follow-up data for all patients were available. The diagnostic ColoPrint readout was determined blindly from the clinical data.

Results: ColoPrint identified most stage II patients (73.3%) as at low risk. The 5-year distant-metastasis free survival was 94.9% for low-risk patients and 80.6% for high-risk patients. In multivariable analysis, ColoPrint was the only significant parameter to predict the development of distant metastasis with a hazard ratio of 4.28 (95% confidence interval, 1.36-13.50; P = 0.013). Clinical risk parameters from the American Society of Clinical Oncology (ASCO) recommendation did not add power to the ColoPrint classification. Technical validation of ColoPrint confirmed stability and reproducibility of the diagnostic platform.

Conclusions: ColoPrint is able to predict the development of distant metastasis of patients with stage II colon cancer and facilitates the identification of patients who may be safely managed without chemotherapy.
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http://dx.doi.org/10.1097/SLA.0b013e31827c1180DOI Listing
June 2013

Epidermal growth factor receptor, phosphatidylinositol-3-kinase catalytic subunit/PTEN, and KRAS/NRAS/BRAF in primary resected esophageal adenocarcinomas: loss of PTEN is associated with worse clinical outcome.

Hum Pathol 2013 May 14;44(5):829-36. Epub 2012 Nov 14.

Teilgemeinschaftspraxis Molekularpathologie Südbayern, D-81675 Munich, Germany.

Alterations of the epidermal growth factor receptor (EGFR) can be observed in a significant subset of esophageal adenocarcinomas (EACs), and targeted therapy against EGFR may become an interesting approach for the treatment of these tumors. Mutations of KRAS, NRAS, BRAF, and phosphatidylinositol-3-kinase catalytic subunit (PIK3CA) and deregulation of PTEN expression influence the responsiveness against anti-EGFR therapy in colorectal carcinomas. We investigated the prevalence of these events in a collection of 117 primary resected EACs, correlated the findings with EGFR expression and amplification, and determined their clinicopathologic impact. KRAS mutations were detected in 4 (3%) of 117 tumors (3× G12D and 1 G12V mutation). One tumor had a PIK3CA E545K mutation. Neither NRAS nor BRAF mutations were detected. Sixteen (14%) of 117 cases were negative for PTEN expression, determined by immunohistochemistry. Loss of PTEN was observed predominantly in advanced tumor stages (P = .004). There was no association between PTEN and EGFR status. Loss of PTEN was associated with shorter overall and disease-free survival (P < .001 each) and also an independent prognostic factor in multivariate analysis (P = .015). EGFR status had no prognostic impact in this case collection. In summary, loss of PTEN can be detected in a significant subset of EAC and is associated with an aggressive phenotype. Therefore, PTEN may be useful as a prognostic biomarker. In contrast, mutations of RAS/RAF/PIK3CA appear only very rarely, if at all, in EAC. A possible predictive role of PTEN in anti-EGFR treatment warrants further investigations, whereas determination of RAS/RAF/PIK3CA mutations may only have a minor impact in this context.
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http://dx.doi.org/10.1016/j.humpath.2012.08.005DOI Listing
May 2013

Expression profiling of stem cell-related genes in neoadjuvant-treated gastric cancer: a NOTCH2, GSK3B and β-catenin gene signature predicts survival.

PLoS One 2012 10;7(9):e44566. Epub 2012 Sep 10.

Institute of Pathology, Technische Universität München, München, Germany.

Cancer stem cell (CSC) based gene expression signatures are associated with prognosis in various tumour types and CSCs are suggested to be particularly drug resistant. The aim of our study was first, to determine the prognostic significance of CSC-related gene expression in residual tumour cells of neoadjuvant-treated gastric cancer (GC) patients. Second, we wished to examine, whether expression alterations between pre- and post-therapeutic tumour samples exist, consistent with an enrichment of drug resistant tumour cells. The expression of 44 genes was analysed in 63 formalin-fixed, paraffin embedded tumour specimens with partial tumour regression (10-50% residual tumour) after neoadjuvant chemotherapy by quantitative real time PCR low-density arrays. A signature of combined GSK3B(high), β-catenin (CTNNB1)(high) and NOTCH2(low) expression was strongly correlated with better patient survival (p<0.001). A prognostic relevance of these genes was also found analysing publically available gene expression data. The expression of 9 genes was compared between pre-therapeutic biopsies and post-therapeutic resected specimens. A significant post-therapeutic increase in NOTCH2, LGR5 and POU5F1 expression was found in tumours with different tumour regression grades. No significant alterations were observed for GSK3B and CTNNB1. Immunohistochemical analysis demonstrated a chemotherapy-associated increase in the intensity of NOTCH2 staining, but not in the percentage of NOTCH2. Taken together, the GSK3B, CTNNB1 and NOTCH2 expression signature is a novel, promising prognostic parameter for GC. The results of the differential expression analysis indicate a prominent role for NOTCH2 and chemotherapy resistance in GC, which seems to be related to an effect of the drugs on NOTCH2 expression rather than to an enrichment of NOTCH2 expressing tumour cells.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0044566PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3438181PMC
March 2013

Association of amphiregulin with the cetuximab sensitivity of gastric cancer cell lines.

Int J Oncol 2012 Aug 14;41(2):733-44. Epub 2012 May 14.

Institute of Pathology, Technische Universität München, Klinikum rechts der Isar, Munich, Germany.

The therapeutic activity of the epidermal growth factor receptor (EGFR)-directed monoclonal antibody cetuximab in gastric cancer is currently being investigated in clinical studies. Reliable biomarkers for the identification of patients who are likely to benefit from this treatment are not available. In this study, we assessed the activity of cetuximab in five gastric cancer cell lines (AGS, AZ521, Hs746T, LMSU and MKN1). The viability of two of these cell lines, AZ521 and MKN1, was significantly reduced by cetuximab treatment. High expression and secretion levels of the EGFR-binding ligand, amphiregulin (AREG), were associated with cetuximab responsiveness. MET activation and mutations in Kirsten-Ras gene (KRAS) were associated with cetuximab resistance. By introducing a hierarchy between these markers, we established a model that facilitated the correct classification of all five gastric cancer cell lines as cetuximab responsive or non-responsive. The highest priority was allocated to activating KRAS mutations, followed by MET activation and finally by the levels of secreted AREG. In order to validate these results, we used three additional human gastric cancer cell lines (KATOIII, MKN28 and MKN45). In conclusion, we propose that our model allows the response of gastric cancer cell lines to cetuximab treatment to be predicted.
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http://dx.doi.org/10.3892/ijo.2012.1479DOI Listing
August 2012

The impact of cyclin D1 mRNA isoforms, morphology and p53 in mantle cell lymphoma: p53 alterations and blastoid morphology are strong predictors of a high proliferation index.

Haematologica 2012 Sep 7;97(9):1422-30. Epub 2012 Feb 7.

Institute of Pathology, University Hospital Tübingen, Liebermeisterstrasse 8, D-72076 Tübingen, Germany.

Background: Mantle cell lymphoma is a clinically heterogeneous disease characterized by overexpression of cyclin D1 protein. Blastoid morphology, high proliferation, and secondary genetic aberrations are markers of aggressive behavior. Expression profiling of mantle cell lymphoma revealed that predominance of the 3'UTR-deficient, short cyclin D1 mRNA isoform was associated with high cyclin D1 levels, a high "proliferation signature" and poor prognosis.

Design And Methods: Sixty-two cases of mantle cell lymphoma were analyzed for cyclin D1 mRNA isoforms and total cyclin D1 levels by real-time reverse transcriptase polymerase chain reaction, and TP53 alterations were assessed by immunohistochemistry and molecular analysis. Results were correlated with proliferation index and clinical outcome.

Results: Predominance of the short cyclin D1 mRNA was found in 14 (23%) samples, including four with complete loss of the standard transcript. TP53 alterations were found in 15 (24%) cases. Predominance of 3'UTR-deficient mRNA was significantly associated with high cyclin D1 mRNA levels (P=0.009) and more commonly found in blastoid mantle cell lymphoma (5/11, P=0.060) and cases with a proliferation index of >20% (P=0.026). Both blastoid morphology (11/11, P<0.001) and TP53 alterations (15/15, P<0.001) were significantly correlated with a high proliferation index. A proliferation index of 10% was determined to be a significant threshold for survival in multivariate analysis (P=0.01).

Conclusions: TP53 alterations are strongly associated with a high proliferation index and aggressive behavior in mantle cell lymphoma. Predominance of the 3'UTR-deficient transcript correlates with higher cyclin D1 levels and may be a secondary contributing factor to high proliferation, but failed to reach prognostic significance in this study.
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http://dx.doi.org/10.3324/haematol.2011.055715DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3436245PMC
September 2012

Relevance of MET activation and genetic alterations of KRAS and E-cadherin for cetuximab sensitivity of gastric cancer cell lines.

J Cancer Res Clin Oncol 2012 May;138(5):843-58

Institut für Allgemeine Pathologie und Pathologische Anatomie, Technische Universität München, Klinikum rechts der Isar, Trogerstr. 18, 81675, Munich, Germany.

Purpose: The therapeutic activity of the epidermal growth factor receptor (EGFR)-directed monoclonal antibody cetuximab in gastric cancer is currently being investigated. Reliable biomarkers for the identification of patients who are likely to benefit from the treatment are not available. The aim of the study was to examine the drug sensitivity of five gastric cancer cell lines towards cetuximab as a single agent and to establish predictive markers for chemosensitivity in this cell culture model. The effect of a combination of cetuximab with chemotherapy was compared between a sensitive and a nonsensitive cell line.

Methods: EGFR expression, activation and localisation, the presence and subcellular localisation of the cell adhesion molecule E-cadherin as well as MET activation were examined by Western blot analysis, flow cytometry and immunofluorescence staining. Cells were treated with varying concentrations of cetuximab and cisplatin and 5-fluorouracil in tumour-relevant concentrations. The biological endpoint was cell viability, which was measured by XTT cell proliferation assay. Response to treatment was evaluated using statistical methods.

Results: We assessed the activity of cetuximab in five gastric cancer cell lines (AGS, KATOIII, MKN1, MKN28 and MKN45). The viability of two cell lines, MKN1 and MKN28, was significantly reduced by cetuximab treatment. High EGFR expression and low levels of receptor activation were associated with cetuximab responsiveness. MET activation as well as mutations of KRAS and CDH1 (gene encoding E-cadherin) was associated with cetuximab resistance.

Conclusion: These data indicate that our examinations may be clinically relevant, and the candidate markers should therefore be tested in clinical studies.
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http://dx.doi.org/10.1007/s00432-011-1128-4DOI Listing
May 2012

TFAP2E-DKK4 and chemoresistance in colorectal cancer.

N Engl J Med 2012 Jan;366(1):44-53

Department of Medicine II, Universitätsmedizin Mannheim, Ruprecht-Karls-Universität Heidelberg, Mannheim, Germany.

Background: Chemotherapy for advanced colorectal cancer leads to improved survival; however, predictors of response to systemic treatment are not available. Genomic and epigenetic alterations of the gene encoding transcription factor AP-2 epsilon (TFAP2E) are common in human cancers. The gene encoding dickkopf homolog 4 protein (DKK4) is a potential downstream target of TFAP2E and has been implicated in chemotherapy resistance. We aimed to further evaluate the role of TFAP2E and DKK4 as predictors of the response of colorectal cancer to chemotherapy.

Methods: We analyzed the expression, methylation, and function of TFAP2E in colorectal-cancer cell lines in vitro and in patients with colorectal cancer. We examined an initial cohort of 74 patients, followed by four cohorts of patients (total, 220) undergoing chemotherapy or chemoradiation.

Results: TFAP2E was hypermethylated in 38 of 74 patients (51%) in the initial cohort. Hypermethylation was associated with decreased expression of TFAP2E in primary and metastatic colorectal-cancer specimens and cell lines. Colorectal-cancer cell lines overexpressing DKK4 showed increased chemoresistance to fluorouracil but not irinotecan or oxaliplatin. In the four other patient cohorts, TFAP2E hypermethylation was significantly associated with nonresponse to chemotherapy (P<0.001). Conversely, the probability of response among patients with hypomethylation was approximately six times that in the entire population (overall estimated risk ratio, 5.74; 95% confidence interval, 3.36 to 9.79). Epigenetic alterations of TFAP2E were independent of mutations in key regulatory cancer genes, microsatellite instability, and other genes that affect fluorouracil metabolism.

Conclusions: TFAP2E hypermethylation is associated with clinical nonresponsiveness to chemotherapy in colorectal cancer. Functional assays confirm that TFAP2E-dependent resistance is mediated through DKK4. In patients who have colorectal cancer with TFAP2E hypermethylation, targeting of DKK4 may be an option to overcome TFAP2E-mediated drug resistance. (Funded by Deutsche Forschungsgemeinschaft and others.).
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http://dx.doi.org/10.1056/NEJMoa1009473DOI Listing
January 2012

Biomarker analysis of cetuximab plus oxaliplatin/leucovorin/5-fluorouracil in first-line metastatic gastric and oesophago-gastric junction cancer: results from a phase II trial of the Arbeitsgemeinschaft Internistische Onkologie (AIO).

BMC Cancer 2011 Dec 7;11:509. Epub 2011 Dec 7.

Institut für Allgemeine Pathologie und Pathologische Anatomie, Technische Universität München, Trogerstraße 18, 81675 München, Germany.

Background: The activity of the epidermal growth factor receptor (EGFR)-directed monoclonal antibody cetuximab combined with oxaliplatin/leucovorin/5-fluorouracil (FUFOX) was assessed in first-line metastatic gastric and oesophago-gastric junction (OGJ) cancer in a prospective phase II study showing a promising objective tumour response rate of 65% and a low mutation frequency of KRAS (3%). The aim of the correlative tumour tissue studies was to investigate the relationship between EGFR gene copy numbers, activation of the EGFR pathway, expression and mutation of E-cadherin, V600E BRAF mutation and clinical outcome of patients with gastric and OGJ cancer treated with cetuximab combined with FUFOX.

Methods: Patients included in this correlative study (n = 39) were a subset of patients from the clinical phase II study. The association between EGFR gene copy number, activation of the EGFR pathway, abundance and mutation of E-cadherin which plays an important role in these disorders, BRAF mutation and clinical outcome of patients was studied. EGFR gene copy number was assessed by FISH. Expression of the phosphorylated forms of EGFR and its downstream effectors Akt and MAPK, in addition to E-cadherin was analysed by immunohistochemistry. The frequency of mutant V600E BRAF was evaluated by allele-specific PCR and the mutation profile of the E-cadherin gene CDH1 was examined by DHPLC followed by direct sequence analysis. Correlations with overall survival (OS), time to progression (TTP) and overall response rate (ORR) were assessed.

Results: Our study showed a significant association between increased EGFR gene copy number (≥ 4.0) and OS in gastric and OGJ cancer, indicating the possibility that patients may be selected for treatment on a genetic basis. Furthermore, a significant correlation was shown between activated EGFR and shorter TTP and ORR, but not between activated EGFR and OS. No V600E BRAF mutations were identified. On the other hand, an interesting trend between high E-cadherin expression levels and better OS was observed and two CDH1 exon 9 missense mutations (A408V and D402H) were detected.

Conclusion: Our finding that increased EGFR gene copy numbers, activated EGFR and the E-cadherin status are potentially interesting biomarkers needs to be confirmed in larger randomized clinical trials.

Trial Registration: Multicentre clinical study with the European Clinical Trials Database number 2004-004024-12.
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http://dx.doi.org/10.1186/1471-2407-11-509DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3252322PMC
December 2011

DNA methyltransferase 1 as a predictive biomarker and potential therapeutic target for chemotherapy in gastric cancer.

Eur J Cancer 2011 Aug 31;47(12):1817-25. Epub 2011 Mar 31.

Institute of Pathology, Technische Universität München, Trogerstr. 18, 81675 München, Germany.

Purpose: DNA methylation contributes to carcinogenesis by mediating transcriptional regulation and chromatin remodelling, which may influence the effect of DNA-damaging drugs. We examined the prognostic and predictive impact of DNA methyltransferase (DNMT) 1 and 3b expression in gastric carcinomas (GC) treated by neoadjuvant chemotherapy. In vitro, DNMT1 expression and chemosensitivity were investigated for a functional relationship and the DNMT inhibitor decitabine (DAC) was tested as an alternative treatment option.

Patients And Methods: DNMT1/3b expression was analysed immunohistochemically in 127 pretherapeutic biopsies of neoadjuvant (platinum/5-fluorouracil)-treated GC patients and correlated with response and overall survival (OS). Short hairpin RNA technology was used to knockdown DNMT1 in the GC cell line, AGS. The chemosensitivity of GC cell lines to DAC alone and to DAC in combination with cisplatin was analysed by XTT or colony formation assays.

Results: High DNMT1 and DNMT3b expression was found in 105/127 (83%) and 79/127 (62%) carcinomas, respectively. Patients with low DNMT1 expression demonstrated a significantly better histopathological/clinical response (P=0.03/P=0.008) and OS (P(log-rank)=0.001). In vitro, knockdown of DNMT1 caused an increased chemosensitivity towards cisplatin. Combined treatment with cisplatin and DAC showed a synergistic effect leading to increased cytotoxicity in the cisplatin-resistant cell line AGS.

Conclusion: Low DNMT1 expression defines a subgroup of GC patients with better outcomes following platinum/5FU-based neoadjuvant chemotherapy. In vitro data support a functional relationship between DNMT1 and cisplatin sensitivity. Besides its potential use as a predictive biomarker, DNMT1 may represent a promising target for alternative therapeutic strategies for a subset of GC patients.
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http://dx.doi.org/10.1016/j.ejca.2011.02.024DOI Listing
August 2011

DNA repair gene and MTHFR gene polymorphisms as prognostic markers in locally advanced adenocarcinoma of the esophagus or stomach treated with cisplatin and 5-fluorouracil-based neoadjuvant chemotherapy.

Ann Surg Oncol 2011 Sep 24;18(9):2688-98. Epub 2011 Feb 24.

Department of Surgery, University Hospital Heidelberg, Heidelberg, Germany.

Background: DNA repair plays an important role in chemoresistance to platinum-based therapy, and therefore polymorphisms in the genes may modulate therapeutic response. We assessed 12 polymorphisms in 7 DNA repair genes and 2 polymorphisms in the MTHFR gene for association with disease response and prognosis.

Methods: A total of 258 patients included in the study had adenocarcinoma of the esophagus (n = 114) or gastric cancer (n = 144), at stage cT3/4 and cM0, and had been treated with platinum-based neoadjuvant polychemotherapy. The patients were genotyped for polymorphisms in the XPC, XPD, XPG, APEX, XRCC1, NBS1, XRCC3, and MTHFR genes by the allelic discrimination method and the data correlated with various clinical parameters.

Results: None of the investigated polymorphisms was associated with histopathological response. XRCC3 polymorphisms rs861539 (P = 0.02) and rs861530 (P = 0.05) showed association with clinical response in gastric cancer. The variants in XRCC3 (rs861539, P = 0.05; rs1799794, P = 0.03) and MTHFR (rs1801131, P = 0.02) were associated with survival in esophageal and gastric cancer, respectively. In R0 resected patients, XRCC3 variants (rs861539, P = 0.04; rs861530, P = 0.02) in esophageal cancer, and XRCC3 (rs1799794, P = 0.02) and MTHFR (rs1801131, P = 0.005) in gastric cancer predicted survival. Cox regression revealed ypT category (P = 0.001) and lymphatic vessel invasion (P = 0.03) to be independent prognostic factors for esophageal cancer, and histopathological response (P = 0.01), MTHFR variant (rs1801131, P = 0.002), and ypN category (P = 0.02) to be prognostic factors for gastric cancer.

Conclusion: In gastric cancer patients, MTHFR variant (rs1801131) could serve as a potential prognostic marker. In esophageal cancer patients, none of the polymorphisms studied had conclusive results in multivariate analysis, although XRCC3 variant (rs861539) showed an effect on survival in Kaplan-Meier univariate analysis.
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http://dx.doi.org/10.1245/s10434-011-1601-yDOI Listing
September 2011

Genetic aberrations in primary esophageal melanomas: molecular analysis of c-KIT, PDGFR, KRAS, NRAS and BRAF in a series of 10 cases.

Mod Pathol 2011 Apr 3;24(4):495-501. Epub 2010 Dec 3.

Institute of Pathology, Technische Universität München, München, Germany.

We present a series of 10 primary esophageal melanomas of Caucasian patients characterized clinicopathologically and on the molecular level. Mutation analysis for c-Kit (exons 9, 11, 13 and 17), PDGFR (exons 12, 14 and 18), NRAS and KRAS were determined using PCR and direct sequencing. Analysis of the V600E mutation of BRAF was performed using mutation-specific PCR. Expression of c-Kit and PDGFR-A was additionally determined using immunohistochemistry. One tumor harbored a missense mutation in the c-Kit (p.F504L) and in the KRAS gene (p.G12S). A different c-Kit mutation (c.1507_1508 ins TTGCCT) was detected in another case. A third case had a V600E BRAF mutation. Using immunohistochemistry, c-Kit expression could be detected in all cases. The two cases with c-Kit mutations showed high c-Kit expression. None of the tumors showed a PDGFR mutation or expression or a NRAS mutation. We conclude that molecular analysis can identify targets for a specific therapy such as tyrosin kinase inhibitors as additional treatment option in these highly malignant tumors.
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http://dx.doi.org/10.1038/modpathol.2010.220DOI Listing
April 2011

A multicenter study to validate the reproducibility of MSI testing with a panel of 5 quasimonomorphic mononucleotide repeats.

Diagn Mol Pathol 2010 Dec;19(4):236-42

Department of A.C.A.D.E.M., University of Trieste, Trieste, Italy.

Microsatellite instability (MSI) testing in clinics is becoming increasingly widespread; therefore, there is an urgent need for methodology standardization and the availability of quality control. This study is aimed to assess the interlaboratory reproducibility of MSI testing in archive samples by using a panel of 5 recently introduced, mononucleotide repeats (MNR). The quality control involved 8 European institutions. Participants were supplied with DNA extracted from 15 archive colon carcinoma samples and from the corresponding normal tissues. Every group was asked to assess the MSI status of the samples by using the BAT25, BAT26, NR21, NR24, and NR27 mononucleotide markers. Four institutions repeated the analysis using the NCI reference panel to confirm the results obtained with the MNR markers. The overall concordance among institutions for MSI analyses at single locus level was 97.7% when using the MNR panel and 95.0% with the NCI one. The laboratories obtained a full agreement in scoring the MSI status of each patient sample, both using the mononucleotide and the NCI marker sets. With the NCI marker set, however, concordance was lowered to 85.7% when considering the MSI-Low phenotype. Concordance between the 2 panels in scoring the MSI status of each sample was complete if no discrimination was made between MSI-Stable and MSI-L, whereas it dropped to 76.7% if MSI-L was considered. In conclusion, the use of the MNR panel seems to be a robust approach that yields a very high level of reproducibility. The results obtained with the 5 MNR are diagnostically consistent with those obtained by the use of the NCI markers, except for the MSI-Low phenotype.
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http://dx.doi.org/10.1097/PDM.0b013e3181db67afDOI Listing
December 2010

Expression of class I histone deacetylases (HDAC1 and HDAC2) in oesophageal adenocarcinomas: an immunohistochemical study.

J Clin Pathol 2010 Nov 5;63(11):994-8. Epub 2010 Oct 5.

Institute of Pathology, Technische Universität München, Munich, Germany.

Background: Histone deacetylases (HDACs) are enzymes which play a central role in post-translational histone and non-histone protein modification. Deregulation of HDACs has been detected in various human malignancies and may also influence response to chemotherapy.

Aims: To investigate the expression of class I histone deacetylase (HDAC) isoforms 1 and 2 in oesophageal adenocarcinomas.

Methods: 132 primary resected tumours and 48 tumours treated by chemotherapy were analysed. Expression of HDAC1 and HDAC2 was determined by immunohistochemistry, applied on a tissue microarray and on pretherapeutic biopsies, and correlated with pathological features and prognosis.

Results: There was negative or low expression of HDAC1 in 54% of tumours, moderate expression in 41% and high expression in 5%. HDAC2 expression was negative or low in 30% of tumours, moderate in 47% and high in 21%. In primary resected tumours, high HDAC2 levels were associated with lymphatic tumour spread and lower tumour differentiation grade. HDAC1 levels were not associated with pT, pN category or tumour differentiation grade. For neoadjuvant treated tumours, there was only a trend for an association with high pretherapeutic HDAC2 expression and tumour regression after chemotherapy. Pretherapeutic HDAC1 levels were not associated with regression after chemotherapy. Survival analysis failed to show any prognostic impact of HDAC1 or HDAC2 expression.

Conclusions: High HDAC2 expression is associated with aggressive tumour behaviour in oesophageal adenocarcinomas. No significant prognostic value could be found with respect to overall survival or an association with response to conventional chemotherapy for HDAC expression. Immunohistochemical determination of HDACs may be useful for prediction of response to specific HDAC inhibitors.
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http://dx.doi.org/10.1136/jcp.2010.080952DOI Listing
November 2010

Histone deacetylase (HDAC) 1 and 2 expression and chemotherapy in gastric cancer.

Ann Surg Oncol 2010 Dec 29;17(12):3336-43. Epub 2010 Jun 29.

Institute of Pathology, Technische Universität München, Munich, Germany.

Background: Histone deacetylases (HDACs) modulate chromatin and may influence the effect of DNA-damaging drugs. We investigated HDAC1 and -2 expression in gastric carcinomas (GCs) for an association of patient outcome with conventional neoadjuvant chemotherapy. In vitro, HDAC inhibitors were evaluated as alternative treatment options.

Methods: HDAC1/2 expression was analyzed immunohistochemically in 127 pretherapeutic biopsy samples of neoadjuvant (platinum/5-fluorouracil) chemotherapy-treated GC patients and correlated with response and overall survival (OS). Chemosensitivity of four GC cell lines to cisplatin and the HDAC inhibitors suberoylanilide hydroxamic acid (SAHA) and valproic acid was determined by XTT assays. Efficiencies of combined drug schedules were analyzed.

Results: High expression of HDAC1/2 was found in 69 (54%) of 127 and 108 (85%) of 127 carcinomas, respectively, and was not associated with response or OS. In patients whose disease responded to therapy, high HDAC1 expression was associated with worse OS (P = 0.005). In cell lines, sequential treatment with SAHA and cisplatin showed synergistic effects irrespective of the initial cisplatin sensitivity.

Conclusions: HDAC1 and -2 expression is not suitable to predict response or survival for neoadjuvant-treated GC patients, but HDAC1 expression may be used for risk stratification in patients whose disease responds to therapy. Sequential treatment with SAHA and cisplatin may represent an alternative treatment option for GC patients.
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http://dx.doi.org/10.1245/s10434-010-1182-1DOI Listing
December 2010

Association of the VEGF 936C>T polymorphism with FDG uptake, clinical, histopathological, and metabolic response in patients with adenocarcinomas of the esophagogastric junction.

Mol Imaging Biol 2011 Feb;13(1):178-86

National Center of Tumor Diseases, University of Heidelberg, Heidelberg, Germany.

Purpose: The MUNICON trial confirmed prospectively the usefulness of early response evaluation by 2-deoxy-2-[F-18]fluoro-D-glucose-positron emission tomography (FDG-PET) . Metabolic responders (R) showed initially a higher FDG uptake compared with nonresponders (p = 0.018). An association of the vascular endothelial growth factor (VEGF) 936C>T polymorphism and FDG uptake was reported for breast cancer. Therefore, we investigated the VEGF 936C>T polymorphism for an association with response and survival.

Procedures: The study was based on 110 patients included in the MUNCON trial (103 male, seven female; 75 AEG I, 35 AEG II, event-free survival (EFS) median 21.1 ± 4.6 months). Response was significantly associated with EFS. The VEGF 936C>T polymorphism was determined by PCR and restriction fragment length polymorphism analysis. For analysis, the T-variants were combined.

Results: One hundred two patients were evaluable. Seventy-two patients showed the CC, 24 the CT, and six the TT genotype. Median EFS was 29.3 months for CC and 11.7 months for CT/TT (p = 0.04). No association of the genotypes (CC or CT/TT) with the SUV or response was found. Multivariate analysis revealed histopathological regression (p = 0.003) and genotype (p = 0.04) as independent prognostic factors. A combination of genotype and PET response (Gen-PET) defines three prognostic groups early in the course of treatment (p = 0.002). Cox regression analysis including clinical and histopathological response and Gen-PET reveals Gen-PET as independent prognostic factor (p = 0.003).

Conclusion: The VEGF 936C>T polymorphism is a prognostic factor in patients undergoing neoadjuvant chemotherapy, although it is not associated with FDG uptake and response. The combination of metabolic response and VEGF 936C>T polymorphism defines three different prognostic groups. These findings need to be confirmed prospectively. This study has been registered in the European Clinical Trials Database as trial 2007-003356-11.
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http://dx.doi.org/10.1007/s11307-010-0330-0DOI Listing
February 2011

CyclinD1 and interleukin-1 receptor antagonist polymorphisms are associated with prognosis in neoadjuvant-treated gastric carcinoma.

Eur J Cancer 2009 Dec 12;45(18):3326-35. Epub 2009 Oct 12.

Institute of Pathology, Technische Universität München, Trogerstr. 18, 81675 München, Germany.

Purpose: We evaluated DNA polymorphisms in genes related to DNA repair, cell-cycle control and tumour microenvironment to determine possible associations with response and survival in neoadjuvant-treated gastric cancer patients.

Patients And Methods: One hundred and seventy eight patients who received platinum/5FU-based chemotherapy were genotyped for 10 polymorphisms in nine genes (ERCC1: Asn118Asn, C > T; ERCC1: 8092C > A; TP53: Arg72Pro, G < C; cyclinD1: Pro241Pro, G > A; STK15: Phe31Ile, A > T; VEGF: 936C > T; TNF-alpha: -308G > A; interleukin-1b (IL-1B): -511C >T; IL-1 receptor antagonist (IL-1RN): variable tandem repeat; IL-8: -251T>A). Genotypes were correlated with histopathological and clinical response and overall (OS) and progression-free survival (PFS).

Results: Only the cyclinD1 genotypes were associated with clinical response (P(x)(2)=0.044). Significantly worse survival rates were noted in patients homozygous for the G-allele as compared to patients with the AG or AA genotypes of the cyclinD1 polymorphism (OS: P(log-rank) = 0.024; PFS: P(log-rank)=0.007) and in patients homozygous for the short allele compared to all other genotypes at the IL-1RN polymorphic locus (OS: P(log-rank) = 0.026; PFS: P(log-rank) = 0.013). The combination of both unfavourable genotypes demonstrated strong prognostic relevance (OS: P(log-rank) = 0.006; PFS: P(log-rank) = 0.001). Multivariate analysis for OS in the group of completely resected patients (n = 139) revealed statistical significance for ypM (P < 0.001), histopathological response (P < 0.001) and the combined cyclinD1/IL-1RN genotypes (P = 0.043).

Conclusion: The cyclinD1 and IL-1RN polymorphisms were associated with survival. The combination of specific cyclinD1 and IL-1RN genotypes showed a particular prognostic relevance and should be considered an independent prognostic marker for neoadjuvant-treated gastric cancer patients.
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http://dx.doi.org/10.1016/j.ejca.2009.09.021DOI Listing
December 2009

Novel multiple, monoallelic KRAS mutations at codon 12 and 13.

Int J Cancer 2009 Dec;125(11):2744-5

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http://dx.doi.org/10.1002/ijc.24635DOI Listing
December 2009

Germline mutation of the E-cadherin gene in three sibling cases with advanced gastric cancer: clinical consequences for the other family members.

Eur J Gastroenterol Hepatol 2010 Mar;22(3):306-10

Fourth Department of Internal Medicine, Klinikum Wels-Grieskirchen, Grieskirchnerstrasse 42, Wels, Austria.

Background And Aims: Germline mutations in the E-cadherin (CDH1) gene have been found in families with hereditary diffuse gastric cancer (HDGC). These families are characterized by a highly penetrant susceptibility to diffuse gastric cancer with an autosomal dominant pattern of inheritance. We describe the clinical presentation of three sibling cases with advanced gastric cancer, the way of confirming the suspicion of underlying HDGC and the clinical management of the other healthy family members.

Methods: Screening for CDH1 germline mutation was carried out by denaturing high-performance liquid chromatography and automated DNA sequencing. The clinical suspicion of HDGC has been confirmed by identifying a frameshift mutation in exon 9 (1302_1303insA, 1306_1307delTT) of the E-cadherin gene.

Results: Eight of nine tested family members were positive for the CDH1 germline mutation. Prophylactic laparoscopic gastrectomies were performed in five mutation carriers. After pathological examination, we could identify intramucosal malignant signet-ring cell carcinoma in all resected stomachs.

Conclusion: This report underlines that prophylactic gastrectomy remains the only option to eliminate the high risk for gastric cancer in CDH1 mutation carriers.
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http://dx.doi.org/10.1097/MEG.0b013e32832bab9aDOI Listing
March 2010