PAMM Laboratory for Pathology

Eindhoven, Netherlands

PAMM Laboratory for Pathology

Eindhoven, Netherlands

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Farina-Sarasqueta A.,PAMM Laboratory for Pathology | Farina-Sarasqueta A.,Fontys University of Applied Sciences | van Lijnschoten G.,PAMM Laboratory for Pathology | Moerland E.,PAMM Laboratory for Pathology | And 5 more authors.
Annals of Oncology | Year: 2010

Background: Molecular markers in colon cancer are needed for a more accurate classification and personalized treatment. We determined the effects on clinical outcome of the BRAF mutation, microsatellite instability (MSI) and KRAS mutations in stage II and stage III colon carcinoma. Patients and methods: Stage II colon carcinoma patients (n = 106) treated with surgery only and 258 stage III patients all adjuvantly treated with 5-fluorouracil chemotherapy were included. KRAS mutations in codons 12 and 13, V600E BRAF mutation and MSI status were determined. Results: Older patients (P < 0.001), right-sided (P = 0.018), better differentiated (P = 0.003) and MSI tumors (P < 0.001) were significantly more frequent in stage II than stage III. In both groups, there was a positive association between mutated BRAF and MSI (P = 0.001) and BRAF mutation and right-sided tumors (P = 0.001). Mutations in BRAF and KRAS were mutually exclusive. In a multivariate survival analysis with pooled stage II and stage III data, BRAF mutation was an independent prognostic factor for overall survival (OS) and cancer-specific survival [hazards ratio (HR) = 0.45, 95% confidence interval (CI) 0.25-0.8 for OS and HR = 0.47, 95% CI 0.22-0.99]. KRAS mutation conferred a poorer disease-free survival (HR = 0.6, 95% CI 0.38-0.97). Conclusions: The V600E BRAF mutation confers a worse prognosis to stage II and stage III colon cancer patients independently of disease stage and therapy. © The Author 2010. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved.


Van Dam P.M.E.L.,Maxima Medisch Centrum | Alexander S.M.,Maxima Medisch Centrum | Degreef E.,PAMM Laboratory for Pathology | Salemans J.M.J.I.,Maxima Medisch Centrum | Roumen R.M.H.,Maxima Medisch Centrum
BMJ Case Reports | Year: 2013

A 43-year-old woman was admitted to the gastroenterology department with colicky pain in the upper abdomen. Four years earlier, she had undergone a laparoscopic cholecystectomy because of cholecystitis. She recognised her current complaints from that previous episode. An endoscopic retrograde cholangiopancreatography showed a cavity with a diameter of 2 cm which contained multiple concrements near the liver hilus. An elective surgical exploration was performed. Near the clip of the previous cholecystectomy a bulging of the biliary tract with its own duct was visualised and resected. Histological examination of this "neo" gallbladder showed that the bulging was consistent with the formation of a reservoir secondary to bile leakage, probably caused by a small peroperative lesion of the common bile duct during the previous cholecystectomy. In conclusion, our patient presented with colicky pain caused by concrements inside a 'neo' gallbladder. Copyright 2013 BMJ Publishing Group. All rights reserved.


Sarasqueta A.F.,PAMM Laboratory for Pathology | Sarasqueta A.F.,Fontys University of Applied Sciences | Zeestraten E.C.M.,Leiden University | Van Wezel T.,Leiden University | And 11 more authors.
Cellular Oncology | Year: 2011

Background PIK3CA mutations in the helical domain (in exon 9) and in the kinase domain (exon 20) cause tumor formation by different means. We aimed to determine the effects of each of these mutations on survival of colon carcinoma patients. Methods A large cohort of 685 colon carcinoma patients was tested for PIK3CA mutations in exons 9 and 20 by single nucleotide primer extension (N=428) or by real time PCR (N=257). Results PIK3CA mutation rate was 13%. 66 of 83 (79.5%) were in exon 9 and 17 of 83 (20.5%) in exon 20. In survival analysis, PIK3CA mutations in exon 9 and 20 had different effects on patient outcome. The PIK3CA exon 20 mutation conferred a poorer disease free survival compared to patients with wild type alleles and exon 9 mutations (Log rank p=0.04 and p=0.03 respectively) and cancer specific survival (Log rank p=0.03 and p=0.056 respectively) in stage III patients. In stage I and II this negative effect on outcome was not seen. Conclusions PIK3CA mutation in exon 20 is a negative prognostic factor in stage III colon cancer patients. Moreover, this negative effect is not present in stage I and II patients. © International Society for Cellular Oncology 2011.


Farina-Sarasqueta A.,PAMM Laboratory for Pathology | Farina-Sarasqueta A.,Fontys University of Applied Sciences | Gosens M.J.E.M.,Fontys University of Applied Sciences | Moerland E.,PAMM Laboratory for Pathology | And 7 more authors.
Cellular Oncology | Year: 2011

Aim Although the predictive and prognostic value of thymidylate synthase (TS) expression and gene polymorphism in colon cancer has been widely studied, the results are inconclusive probably because of methodological differences. With this study, we aimed to elucidate the role of TS gene polymorphisms genotyping in therapy response in stage III colon carcinoma patients treated with 5-FU adjuvant chemotherapy. Patients and methods 251 patients diagnosed with stage III colon carcinoma treated with surgery followed by 5-FU based adjuvant therapy were selected. The variable number of tandem repeats (VNTR) and the single nucleotide polymorphism (SNP) in the 5'untranslated region of the TS gene were genotyped. Results There was a positive association between tumor T stage and the VNTR genotypes (p=0.05). In both univariate and multivariate survival analysis no effects of the studied polymorphisms on survival were found. However, there was an association between both polymorphisms and age. Among patients younger than 60 years, the patients homozygous for 2R seemed to have a better overall survival, whereas among the patients older than 67 this longer survival was seen by the carriers of other genotypes. Conclusion We conclude that the TS VNTR and SNP do not predict response to 5-FU therapy in patients with stage III colon carcinoma. However, age appears to modify the effects of TS polymorphisms on survival. © International Society for Cellular Oncology 2011.


Faria Sarasqueta A.,PAMM Laboratory for Pathology | Faria Sarasqueta A.,Fontys University of Applied Sciences | Van Lijnschoten G.,PAMM Laboratory for Pathology | Lemmens V.E.P.P.,Comprehensive Cancer Center South | And 2 more authors.
Molecular Diagnosis and Therapy | Year: 2011

Purpose: Adjuvant chemotherapy improves survival in stage III colon cancer patients. However, a subgroup of patients still develops recurrent disease at some point in time, partly because of the ineffectiveness of the chemotherapy. Predictive markers of response are therefore crucial. Our aim was to study the predictive value of functional polymorphisms in genes involved in the metabolism of oxaliplatin and in DNA repair in stage III colon cancer patients. Materials and Methods: Normal DNA was isolated from 98 patients diagnosed with stage III colon carcinoma. Single nucleotide polymorphisms (SNPs) in three genes (the excision repair cross-complementing genes ERCC1 [19007T>C] and ERCC2 [2251A>C], and the glutathione S-transferase pi 1 gene [GSTP1 313A>G]) were tested by PCR followed by digestion with restriction enzymes or by direct sequencing. These genes and SNPs were selected on the basis of their reported associations with oxaliplatin response in colorectal cancer. Results: The genotype frequencies were in Hardy-Weinberg equilibrium. GSTP1 and ERCC2 polymorphisms were significantly associated with sex. The AA genotype of GSTP1 313A>G was more frequent in men than in women (59% vs 30%, p= 0.02), and the CC genotype of ERCC2 2251A>C was significantly more frequent in women than in men (24%vs 6%, p = 0.02). In univariate and multivariate survival analysis, none of the tested polymorphisms seemed to influence disease-free survival. The GSTP1 AA genotype had different effects on survival between men and women; homozygous A men had significantly worse cancerspecific survival and overall survival than women with the same genotype (log rank p = 0.029 and p = 0.015, respectively). Conclusion: None of the tested polymorphisms is likely to be a reliable marker of response to oxaliplatin therapy. The GSTP1 313A>G homozygous A genotype may have a prognostic value in male patients. © 2011 Adis Data Information BV. All rights reserved.


Sarasqueta A.F.,PAMM Laboratory for Pathology | Sarasqueta A.F.,Fontys University of Applied Sciences | Moerland E.,PAMM Laboratory for Pathology | De Bruyne H.,PAMM Laboratory for Pathology | And 4 more authors.
Journal of Molecular Diagnostics | Year: 2011

Although direct sequencing is the gold standard for KRAS mutation detection in routine diagnostics, it remains laborious, time consuming, and not very sensitive. Our objective was to evaluate SNaPshot and the KRAS StripAssay as alternatives to sequencing for KRAS mutation detection in daily practice. KRAS exon 2-specific PCR followed by sequencing or by a SNaPshot reaction was performed. For the StripAssay, a mutant-enriched PCR was followed by hybridization to KRAS-specific probes bound to a nitrocellulose strip. To test sensitivities, dilution series of mutated DNA in wild-type DNA were made. Additionally, direct sequencing and SNaPshot were evaluated in 296 colon cancer samples. Detection limits of direct sequencing, SNaPshot, and StripAssay were 20%, 10%, and 1% tumor cells, respectively. Direct sequencing and SNaPshot can detect all 12 mutations in KRAS codons 12 and 13, whereas the StripAssay detects 10 of the most frequent ones. Workload and time to results are comparable for SNaPshot and direct sequencing. SNaPshot is flexible and easy to multiplex. The StripAssay is less time consuming for daily laboratory practice. SNaPshot is more flexible and slightly more sensitive than direct sequencing. The clinical evaluation showed comparable performances between direct sequencing and SNaPshot. The StripAssay is rapid and an extremely sensitive assay that could be considered when few tumor cells are available. However, found mutants should be confirmed to avoid risk of false positives. Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.


PubMed | PAMM Laboratory for Pathology
Type: Journal Article | Journal: Analytical cellular pathology (Amsterdam) | Year: 2010

Although the predictive and prognostic value of thymidylate synthase (TS) expression and gene polymorphism in colon cancer has been widely studied, the results are inconclusive probably because of methodological differences. With this study, we aimed to elucidate the role of TS gene polymorphisms genotyping in therapy response in stage III colon carcinoma patients treated with 5-FU adjuvant chemotherapy.251 patients diagnosed with stage III colon carcinoma treated with surgery followed by 5-FU based adjuvant therapy were selected. The variable number of tandem repeats (VNTR) and the single nucleotide polymorphism (SNP) in the 5-untranslated region of the TS gene were genotyped.There was a positive association between tumor T stage and the VNTR genotypes (p=0.05).In both univariate and multivariate survival analysis no effects of the studied polymorphisms on survival were found. However, there was an association between both polymorphisms and age. Among patients younger than 60 years, the patients homozygous for 2R seemed to have a better overall survival, whereas among the patients older than 67 this longer survival was seen by the carriers of other genotypes.We conclude that the TS VNTR and SNP do not predict response to 5-FU therapy in patients with stage III colon carcinoma. However, age appears to modify the effects of TS polymorphisms on survival.


PubMed | PAMM Laboratory for Pathology
Type: Journal Article | Journal: Cellular oncology (Dordrecht) | Year: 2011

PIK3CA mutations in the helical domain (in exon 9) and in the kinase domain (exon 20) cause tumor formation by different means. We aimed to determine the effects of each of these mutations on survival of colon carcinoma patients.A large cohort of 685 colon carcinoma patients was tested for PIK3CA mutations in exons 9 and 20 by single nucleotide primer extension (N=428) or by real time PCR (N=257).PIK3CA mutation rate was 13%. 66 of 83 (79.5%) were in exon 9 and 17 of 83 (20.5%) in exon 20. In survival analysis, PIK3CA mutations in exon 9 and 20 had different effects on patient outcome. The PIK3CA exon 20 mutation conferred a poorer disease free survival compared to patients with wild type alleles and exon 9 mutations (Log rank p=0.04 and p=0.03 respectively) and cancer specific survival (Log rank p=0.03 and p=0.056 respectively) in stage III patients. In stage I and II this negative effect on outcome was not seen.PIK3CA mutation in exon 20 is a negative prognostic factor in stage III colon cancer patients. Moreover, this negative effect is not present in stage I and II patients.


PubMed | PAMM Laboratory for Pathology
Type: Journal Article | Journal: Molecular diagnosis & therapy | Year: 2011

Adjuvant chemotherapy improves survival in stage III colon cancer patients. However, a subgroup of patients still develops recurrent disease at some point in time, partly because of the ineffectiveness of the chemotherapy. Predictive markers of response are therefore crucial. Our aim was to study the predictive value of functional polymorphisms in genes involved in the metabolism of oxaliplatin and in DNA repair in stage III colon cancer patients.Normal DNA was isolated from 98 patients diagnosed with stage III colon carcinoma. Single nucleotide polymorphisms (SNPs) in three genes (the excision repair cross-complementing genes ERCC1 [19007T>C] and ERCC2 [2251A>C], and the glutathione S-transferase pi 1 gene [GSTP1 313A>G]) were tested by PCR followed by digestion with restriction enzymes or by direct sequencing. These genes and SNPs were selected on the basis of their reported associations with oxaliplatin response in colorectal cancer.The genotype frequencies were in Hardy-Weinberg equilibrium. GSTP1 and ERCC2 polymorphisms were significantly associated with sex. The AA genotype of GSTP1 313A>G was more frequent in men than in women (59% vs 30%, p=0.02), and the CC genotype of ERCC2 2251A>C was significantly more frequent in women than in men (24% vs 6%, p=0.02). In univariate and multivariate survival analysis, none of the tested polymorphisms seemed to influence disease-free survival. The GSTP1 AA genotype had different effects on survival between men and women; homozygous A men had significantly worse cancer-specific survival and overall survival than women with the same genotype (log rank p=0.029 and p=0.015, respectively).None of the tested polymorphisms is likely to be a reliable marker of response to oxaliplatin therapy. The GSTP1 313A>G homozygous A genotype may have a prognostic value in male patients.


PubMed | PAMM Laboratory for Pathology
Type: Evaluation Studies | Journal: The Journal of molecular diagnostics : JMD | Year: 2011

Although direct sequencing is the gold standard for KRAS mutation detection in routine diagnostics, it remains laborious, time consuming, and not very sensitive. Our objective was to evaluate SNaPshot and the KRAS StripAssay as alternatives to sequencing for KRAS mutation detection in daily practice. KRAS exon 2-specific PCR followed by sequencing or by a SNaPshot reaction was performed. For the StripAssay, a mutant-enriched PCR was followed by hybridization to KRAS-specific probes bound to a nitrocellulose strip. To test sensitivities, dilution series of mutated DNA in wild-type DNA were made. Additionally, direct sequencing and SNaPshot were evaluated in 296 colon cancer samples. Detection limits of direct sequencing, SNaPshot, and StripAssay were 20%, 10%, and 1% tumor cells, respectively. Direct sequencing and SNaPshot can detect all 12 mutations in KRAS codons 12 and 13, whereas the StripAssay detects 10 of the most frequent ones. Workload and time to results are comparable for SNaPshot and direct sequencing. SNaPshot is flexible and easy to multiplex. The StripAssay is less time consuming for daily laboratory practice. SNaPshot is more flexible and slightly more sensitive than direct sequencing. The clinical evaluation showed comparable performances between direct sequencing and SNaPshot. The StripAssay is rapid and an extremely sensitive assay that could be considered when few tumor cells are available. However, found mutants should be confirmed to avoid risk of false positives.

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