[show abstract][hide abstract] ABSTRACT: Background:Vulvar cancer is the fourth most common gynaecological malignancy, with an annual incidence of 2 out of 100 000 women. Although most cases of early stage vulvar cancer have a good prognosis, recurrence and rapid tumour progression can occur. We investigated the prevalence of spindle cell morphology in vulvar cancer and its association with survival.Methods:This retrospective cohort study included 108 patients with primary vulvar squamous cell carcinoma who were treated at the Leiden University Medical Center during 2000-2009. Paraffin-embedded tissue was examined for the presence of spindle cell morphology. Survival and histology data were compared between cases with spindle and without spindle cell morphology.Results:Twenty-two (20%) tumours showed spindle cells infiltrating the stromal tissue. All spindle cell tumours were human papillomavirus (HPV) negative. Spindle cell morphology was strongly associated with poor prognosis and with a high risk of lymph node involvement at the time of diagnosis (relative risk 2.26 (95% CI 1.47-3.47)). Five-year disease-specific survival was lower in patients with vs without spindle cell morphology (45.2% vs 79.7%, respectively; P=0.00057).Conclusion:Vulvar spindle cell morphology occurs frequently and seems to develop through the non-HPV pathway. It is associated with a worse prognosis than conventional vulvar squamous cell carcinoma.British Journal of Cancer advance online publication, 24 September 2013; doi:10.1038/bjc.2013.563 www.bjcancer.com.
British Journal of Cancer 09/2013; · 5.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: Treatment choices for cervical cancer are primarily based on clinical FIGO stage and the post-operative evaluation of prognostic parameters including tumor diameter, parametrial and lymph node involvement, vaso-invasion, infiltration depth, and histological type. The aim of this study was to evaluate genomic changes in bulky cervical tumors and their relation to clinical parameters, using single nucleotide polymorphism (SNP)-analysis. Flow-sorted tumor cells and patient-matched normal cells were extracted from 81 bulky cervical tumors. DNA-index (DI) measurement and whole genome SNP-analysis were performed. Data were analyzed to detect copy number alterations (CNA) and allelic balance state: balanced, imbalanced or pure LOH, and their relation to clinical parameters. The DI varied from 0.92–2.56. Pure LOH was found in $40% of samples on chromosome-arms 3p, 4p, 6p, 6q, and 11q, CN gains in .20% on 1q, 3q, 5p, 8q, and 20q, and losses on 2q, 3p, 4p, 11q, and 13q. Over 40% showed gain on 3q. The only significant differences were found between histological types (squamous, adeno and adenosquamous) in the lesser allele intensity ratio (LAIR) (p = 0.035) and in the CNA analysis (p = 0.011). More losses were found on chromosome-arm 2q (FDR = 0.004) in squamous tumors and more gains on 7p, 7q, and 9p in adenosquamous tumors (FDR = 0.006, FDR = 0.004, and FDR = 0.029). Whole genome analysis of bulky cervical cancer shows widespread changes in allelic balance and CN. The overall genetic changes and CNA on specific chromosome-arms differed between histological types. No relation was found with the clinical parameters that currently dictate treatment choice. Copyright: ß 2013 van den Tillaart et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: D. Ruano Neto received funding from grant 93518025 of the Netherlands Genomic Initiative (NGI). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist.
[show abstract][hide abstract] ABSTRACT: The switch/sucrose non-fermentable (SWI/SNF) subunit ARID1A (AT-rich interactive domain 1A gene) has been recently postulated as a novel tumor suppressor of gynecologic cancer and one of the driver genes in endometrial carcinogenesis. However, specific relationships with established molecular alterations in endometrioid endometrial cancer (EEC) are currently unknown. We analyzed the expression of ARID1A in 146 endometrial cancers (130 EECs and 16 non-EECs) in relation to alterations in the PI3K-Akt pathway (PTEN expression/KRAS/PIK3CA mutations), TP53 status (TP53 immunohistochemistry) and microsatellite instability. To discriminate between microsatellite instability due to somatic MLH1 promoter hypermethylation or germline mutations in one of the mismatch repair genes (Lynch syndrome), we included a 'Lynch syndrome set'. This set included 21 cases with confirmed germline mutations and 15 cases that were suspected to have a germline mutation. Loss of ARID1A expression was exclusively found in EECs in 31% (40/130) of the EEC cases. No loss of expression of the other subunits of the SWI/SNF complex, SMARCD3 and SMARCB1, was detected. Alterations in the PI3K-Akt pathway were more frequent when ARID1A expression was lost. Loss of ARID1A and mutant-like TP53 expression was nearly mutually exclusive (P=0.0004). In contrast to Lynch-associated tumors, a strong association between ARID1A loss and sporadic microsatellite instability was found. Only five cases (14%) of the 'Lynch syndrome set' as compared with 24 cases (75%, P<0.0001) of the sporadic microsatellite-unstable tumors showed loss of ARID1A. These observations suggest that ARID1A is a causative gene, instead of a target gene, of microsatellite instability by having a role in epigenetic silencing of the MLH1 gene in endometrial cancer.Modern Pathology advance online publication, 24 May 2013; doi:10.1038/modpathol.2013.96.
[show abstract][hide abstract] ABSTRACT: Background:Multiparameter flow cytometry is a robust and reliable method for determining tumour DNA content applicable to formalin-fixed paraffin-embedded (FFPE) tissue. This study examined the clinical and pathological associations of DNA content in primary breast cancer using an improved multiparametric technique.Methods:The FFPE tissue from 201 primary breast cancers was examined and the cancers categorised according to their DNA content using multiparametric flow cytometry incorporating differential labelling of stromal and tumour cell populations. Mathematical modelling software (ModFit 3.2.1) was used to calculate the DNA index (DI) and percentage S-phase fraction (SPF%) for each tumour. Independent associations with clinical and pathological parameters were sought using backward stepwise Binary Logistic Regression (BLR) and Cox's Regression (CR) analysis.Results:Tumours were grouped into four categories based on the DI of the tumour cell population. Low DI tumours (DI=0.76-1.14) associated with progesterone receptor-positive status (P=0.012, BLR), intermediate DI (DI=1.18-1.79) associated with p53 mutant tumours (P=0.001, BLR), high DI (DI1.80) tumours with human epidermal growth factor receptor 2 (HER2)-positive status (P=0.004, BLR) and 'multiploid tumours' (two or more tumour DNA peaks) did not show any significant associations. Tumours with high SPF% (10%) independently associated with poor overall survival (P=0.027, CR).Conclusion:Multiparametric flow analysis of FFPE tissue can accurately assess tumour DNA content. Tumour sub-populations associated with biomarkers of prognosis or likely response to therapy. The alterations in DNA content present the potential for greater understanding of the mechanisms underlying clinically significant biomarker changes in primary breast cancer.
British Journal of Cancer 03/2013; 108(4):873-80. · 5.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: Treatment choices for cervical cancer are primarily based on clinical FIGO stage and the post-operative evaluation of prognostic parameters including tumor diameter, parametrial and lymph node involvement, vaso-invasion, infiltration depth, and histological type. The aim of this study was to evaluate genomic changes in bulky cervical tumors and their relation to clinical parameters, using single nucleotide polymorphism (SNP)-analysis. Flow-sorted tumor cells and patient-matched normal cells were extracted from 81 bulky cervical tumors. DNA-index (DI) measurement and whole genome SNP-analysis were performed. Data were analyzed to detect copy number alterations (CNA) and allelic balance state: balanced, imbalanced or pure LOH, and their relation to clinical parameters. The DI varied from 0.92-2.56. Pure LOH was found in ≥40% of samples on chromosome-arms 3p, 4p, 6p, 6q, and 11q, CN gains in >20% on 1q, 3q, 5p, 8q, and 20q, and losses on 2q, 3p, 4p, 11q, and 13q. Over 40% showed gain on 3q. The only significant differences were found between histological types (squamous, adeno and adenosquamous) in the lesser allele intensity ratio (LAIR) (p = 0.035) and in the CNA analysis (p = 0.011). More losses were found on chromosome-arm 2q (FDR = 0.004) in squamous tumors and more gains on 7p, 7q, and 9p in adenosquamous tumors (FDR = 0.006, FDR = 0.004, and FDR = 0.029). Whole genome analysis of bulky cervical cancer shows widespread changes in allelic balance and CN. The overall genetic changes and CNA on specific chromosome-arms differed between histological types. No relation was found with the clinical parameters that currently dictate treatment choice.
PLoS ONE 01/2013; 8(7):e67414. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: To investigate if analysis of genetic alterations in the main pathways involved in endometrioid type carcinogenesis (PI3K-AKT, Wnt/β-catenin, P53-activation and MSI) improves the current risk assessment based on clinicopathological factors.
Formalin fixed paraffin embedded (FFPE) primary tumor samples of 65 patients with FIGO-stage I endometrioid type endometrial cancer (EEC) were selected from the randomized PORTEC-2 trial. Tumors were stained by immunohistochemistry for P53, PTEN and β-catenin. Tumor DNA was isolated for sequence analysis of TP53 (exons 4 to 8), hotspot mutation analysis of KRAS (exon 1) and PI3K (exon 9 and 20) and microsatellite-instability (MSI) analysis including MLH1 promotor-methylation status. Univariate and multivariate analyses for disease-free survival (DFS) using Cox regression models were performed.
P53 status (HR 6.7, 95%CI 1.75-26.0, p=0.006) and MSI were the strongest single genetic prognostic factors for decreased DFS, while high PI3K-AKT pathway activation showed a trend and β-catenin was not prognostic. The combination of multiple activated pathways was the most powerful prognostic factor for decreased DFS (HR 5.0; 95%CI 1.59-15.6 p=0.006). Multiple pathway activation, found in 8% of patients, was strongly associated with aggressive clinical course. In contrast, 40% of patients had no alterations in the investigated pathways and had a very low risk of disease progression.
Activation of multiple oncogenic pathways in EEC was the most powerful prognostic factor for decreased DFS, resulting in an individual risk assessment superior to the current approach based on clinicopathological factors.
[show abstract][hide abstract] ABSTRACT: The CXC chemokine receptor (CXCR)7 is involved in tumour development and metastases formation. The aim of the present study was to determine protein expression of CXCR7, its putative co-receptors epidermal growth factor receptor (EGFR) and CXCR4, its predominant ligand CXCL12, their co-dependency and their association with survival in cervical cancer patients.
CXC chemokine receptor 7, EGFR, CXCR4 and CXCL12 expression were determined immunohistochemically in 103 paraffin-embedded, cervical cancers. Subsequently, associations with patient characteristics were assessed and survival analyses were performed.
CXC chemokine receptor 7 was expressed by 43% of tumour specimens, in a large majority of cases together with either EGFR or CXCR4 (double positive), or both (triple positive). The CXCR7 expression was associated with tumour size (P=0.013), lymph node metastasis (P=0.001) and EGFR expression (P=0.009). CXC chemokine receptor 7 was independently associated with disease-free survival (hazard ratio (HR)=4.3, 95% confidence intervals (CI) 1.7-11.0, P=0.002), and strongly associated with disease-specific survival (HR=3.9, 95% CI 1.5-10.2, P=0.005).
CXC chemokine receptor 7 expression predicts poor disease-free and disease-specific survival in cervical cancer patients, and might be a promising new therapeutic marker. In a large majority of cases, CXCR7 is co-expressed with CXCR4 and/or EGFR, supporting the hypothesis that these receptors assist in CXCR7 signal transduction.
British Journal of Cancer 04/2012; 106(9):1520-5. · 5.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: Cancer-associated fibroblasts (CAFs) have been recognized as important contributors to cancer development and progression. However, opposing evidence has been published whether CAFs, in addition to epigenetic, also undergo somatic genetic alterations and whether these changes contribute to carcinogenesis and tumour progression.
We combined multiparameter DNA flow cytometry, flow-sorting and 6K SNP-arrays to study DNA aneuploidy, % S-phase, loss of heterozygosity (LOH) and copy number alterations (CNAs) in cervical cancer-associated stromal cell fractions (n = 57) from formalin-fixed, paraffin-embedded (FFPE) samples. Tissue sections were examined for the presence of CAFs. Microsatellite analysis was used to confirm LOH findings.
Smooth muscle actin and vimentin immunohistochemistry verified the presence of CAFs in all cases tested. However, we found no evidence for DNA aneuploidy, somatic genetic alterations in the vimentin-positive stromal cell fractions of any samples, while high frequencies of DNA content abnormalities (43/57) and substantial numbers of CNAs and LOH were identified in the keratin-positive epithelial cell fractions. LOH hot-spots on chromosomes 3p, 4p and 6p found were confirmed by microsatellite analysis.
From our study we conclude that stromal cell fractions from cervical carcinomas are DNA diploid, have a genotype undistinguishable from patient-matched normal tissue and are genetically stable. Using flow cytometry and SNP-arrays, stromal genetic changes do not seem to play a role during cervical carcinogenesis and progression. In addition, the stromal cell fraction of cervical carcinomas can be used as reference allowing large retrospective studies of archival FFPE tissues for which no normal reference tissue is available.
[show abstract][hide abstract] ABSTRACT: This unit contains a detailed protocol for the simultaneous flow cytometric measurement of tumor cells, stromal cells, and DNA content of formalin-fixed, paraffin-embedded (FFPE) tissues. The vimentin-positive stromal cell fraction can be used as an internal reference for DNA content assessments. This allows clear detection of keratin-positive tumor cells with a DNA index lower than 1.0 and of keratin-positive tumor cells with a DNA close to 1.0 in overall DNA aneuploid samples, thus improved DNA ploidy assessment in FFPE carcinomas. Furthermore, the protocol is useful for studying molecular genetic alterations and intratumor heterogeneity in archival FFPE samples. Keratin-positive tumor cell fractions can be flow-sorted for further molecular genetic analysis, while DNA from the sorted vimentin-positive stromal cells can serve as a reference when normal tissue of the patient is not available.
Current protocols in cytometry / editorial board, J. Paul Robinson, managing editor ... [et al.] 01/2011; Chapter 7:Unit 7.37.
[show abstract][hide abstract] ABSTRACT: Chromosomal aberrations are a common characteristic of cancer and are associated with copy number abnormalities and loss of heterozygosity (LOH). Tumor heterogeneity, low tumor cell percentage, and lack of knowledge of the DNA content impair the identification of these alterations especially in aneuploid tumors. To accurately detect allelic changes in carcinomas, we combined flow-sorting and single nucleotide polymorphism arrays. Cells derived from archival cervical and colon cancers were flow-sorted based on differential vimentin and keratin expression and DNA content and analyzed on single nucleotide polymorphism arrays. A new algorithm, the lesser allele intensity ratio, was used to generate a molecular measure of chromosomal aberrations for each case. Flow-sorting significantly improved the detection of copy number abnormalities; 31.8% showed an increase in amplitude and 23.2% were missed in the unsorted fraction, whereas 15.9% were detected but interpreted differently. Integration of the DNA index in the analysis enabled the identification of the allelic state of chromosomal aberrations, such as LOH ([A]), copy-neutral LOH ([AA]), balanced amplifications ([AABB]), and allelic imbalances ([AAB] or [AAAB], etc.). Chromosomal segments were sharply defined. Fluorescence in situ hybridization copy numbers, as well as the high similarity between the DNA index and the allelic state index, which is the average of the allelic states across the genome, validated the method. This new approach provides an individual molecular measure of chromosomal aberrations and will likely have repercussions for preoperative molecular staging, classification, and prognostic profiling of tumors, particularly for heterogeneous aneuploid tumors, and allows the study of the underlying molecular genetic mechanisms and clonal evolution of tumor subpopulations.
Cancer Research 01/2009; 68(24):10333-40. · 8.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: In cervical cancer, an important mechanism by which tumour cells escape immune surveillance is loss of HLA class I, enabling tumours to evade recognition and lysis by cytotoxic T lymphocytes. Some tumours, however, escape from immune surveillance without accumulating defects in antigen presentation. We hypothesized that tumours with no or partial loss of HLA class I develop alternative mechanisms to prevent immune elimination. To investigate this hypothesis, genome-wide expression profiling using Illumina arrays was performed on cervical squamous cell carcinomas showing overall loss of HLA class I, partial, and normal HLA class I protein expression. Statistical analyses revealed no significant differences in gene expression between tumours with partial (n = 11) and normal HLA class I expression (n = 10). Comparison of tumours with normal/partial HLA class I expression (n = 21) with those with overall loss of HLA class I expression (n = 11) identified 150 differentially expressed genes. Most of these genes were involved in the defence response (n = 27) and, in particular, inflammatory and acute phase responses. Especially SerpinA1 and SerpinA3 were found to be up-regulated in HLA-positive tumours (3.6- and 8.2-fold, respectively), and this was confirmed by real-time PCR and immunohistochemistry. In a group of 117 tumours, high SerpinA1 and SerpinA3 expression in association with normal/partial HLA expression correlated significantly with poor overall survival (p = 0.035 and p = 0.05, respectively). Thus, HLA-positive tumours are characterized by higher expression of genes associated with an inflammatory profile. In addition, expression of the acute phase proteins SerpinA1 and SerpinA3 in HLA-positive tumours is associated with worse prognosis.
The Journal of Pathology 07/2008; 215(3):222-30. · 7.59 Impact Factor
[show abstract][hide abstract] ABSTRACT: Genetic instability is known to drive colorectal carcinogenesis. Generally, a distinction is made between two types of genetic instability: chromosomal instability (CIN) and microsatellite instability (MIN or MSI). Most CIN tumours are aneuploid, whereas MSI tumours are considered near-diploid. However, for MUTYH-associated polyposis (MAP) the genetic instability involved in the carcinogenesis remains unclear, as near-diploid adenomas, aneuploid adenomas and near-diploid carcinomas have been reported. Remarkably, our analysis of 26 MAP carcinomas, using SNP arrays and flow sorting, showed that these tumours are often near-diploid (52%) and mainly contain chromosomal regions of copy-neutral loss of heterozygosity (LOH) (71%). This is in contrast to sporadic colon cancer, where physical loss is the main characteristic. The percentage of chromosomal gains (24%) is comparable to sporadic colorectal cancers with CIN. Furthermore, we verified our scoring of copy-neutral LOH versus physical loss in MAP carcinomas by two methods: fluorescence in situ hybridization, and LOH analysis using polymorphic markers on carcinoma fractions purified by flow sorting. The results presented in this study suggest that copy-neutral LOH is an important mechanism in the tumorigenesis of MAP.
The Journal of Pathology 04/2008; 216(1):25-31. · 7.59 Impact Factor
[show abstract][hide abstract] ABSTRACT: Infection with oncogenic human papillomavirus (HPV) is considered to be the major etiologic event for cervical cancer. Tumor necrosis factor alpha (TNFalpha), a proinflammatory cytokine, may be involved in orchestrating an antitumor immune response against human papillomavirus expressing cervical cancer cells. Hence, loss of TNFalpha could be advantageous for tumor cells to escape immune clearance. The aim of our study was to investigate TNFalpha gene expression and epigenetic characteristics associated with the loss of TNFalpha expression in cervical cancer. To this end, we examined TNFalpha expression, loss of heterozygosity (LOH) at 6p21.3, the locus of TNFalpha, mutational status of the TNFalpha locus, loss of the TNFalpha promoter variant 2 allele and CpG hypermethylation of the TNFalpha promoter. RNA in situ hybridization showed absence of TNFalpha expression in 45% of 63 tumors. LOH occurred in 57% of the tumors and was not concordant with absence of TNFalpha mRNA. No mutations in the TNFalpha gene were identified in 15 cases deficient in TNFalpha expression exhibiting LOH. Furthermore, lack of TNFalpha expression did not correlate with promoter methylation. In conclusion, TNFalpha mRNA expression is absent in nearly half of the cervical tumors analyzed. Neither promoter methylation nor genetic causes for lack of expression were evident.
[show abstract][hide abstract] ABSTRACT: Transporter associated with antigen processing (TAP) loss causes human leukocyte antigen (HLA) class I downregulation which is frequently found in cervical carcinomas and their precursors. HLA class I molecules activate T-cells by antigen presentation and are therefore essential for immunological surveillance. To add to the hitherto limited knowledge of molecular mechanisms underlying TAP loss, we investigated TAP expression, loss of heterozygosity (LOH) and possible TAP mutations.
Twenty-three cervical carcinomas and adjacent precursor lesions were stained with HLA-A-, HLA-B/C-, beta2 -microglobulin-, TAP1- and TAP2- antibodies. In order to separate tumour and non-tumour cells, cervical carcinoma samples were sorted by flow-cytometry and were subsequently analysed for LOH with 3 markers in the TAP region on chromosome 6p21.3. Mutation analysis of the complete TAP1 gene was performed.
Aberrant TAP1 expression was detected in 10/23 cervical carcinoma lesions and in 5/10 adjacent cervical intraepithelial neoplasia (CIN) lesions. All the lesions with low TAP expression also had reduced HLA class I expression. LOH was found in 7 out of 10 lesions with TAP loss. Mutation analysis detected no aberrations, but identified a polymorphism in the 5'-untranslated region (UTR) of the TAP1 gene in two lesions.
This study shows that defective TAP expression in cervical carcinoma is often associated with LOH in the TAP region but not with mutations in the TAP1 gene.
[show abstract][hide abstract] ABSTRACT: Loss at chromosome 6p21.3, the human leukocyte antigen (HLA) region, is the main cause of HLA downregulation, occurring in the majority of invasive cervical carcinomas. To identify the stage of tumor development at which HLA class I aberrations occur, we selected 12 patients with cervical carcinoma and adjacent cervical intraepithelial neoplasia (CIN). We investigated HLA class I and beta2-microglobulin expression by immunohistochemistry in tumor and adjacent CIN. Loss of heterozygosity (LOH) was studied using microsatellite markers covering the HLA region. Fluorescent in situ hybridization with HLA class I probes was performed to investigate the mechanism of HLA loss. Immunohistochemistry showed absent or weak HLA class I expression in 11/12 cases. In 10 of these 11 cases, downregulation occurred in both tumor and CIN. Only in one case did the concomitant CIN lesion show normal expression. In 9/12 cases, LOH was present for at least one marker in both tumor and CIN, 1 case showed only LOH in the CIN lesion, and 1 case showed retention of heterozygosity for all markers in both tumor and CIN. We conclude that HLA class I aberrations occur early and frequently in cervical carcinogenesis. This might allow premalignant CIN lesions to escape immune surveillance and progress to invasive cancer.
Human Immunology 12/2005; 66(11):1167-73. · 2.30 Impact Factor
[show abstract][hide abstract] ABSTRACT: The accuracy of DNA ploidy measurements of paraffin-embedded tissues is limited by the lack of resolution and the inability to identify the DNA diploid population unequivocally in bimodal DNA histograms. A multi-parameter DNA flow cytometric method has been developed that enables the simultaneous detection of neoplastic and stromal cells in samples from dewaxed 50 microm sections or 2 mm diameter punches of archival tissue blocks. The method combines heat pretreatment in sodium citrate buffer and subsequent enzymatic dissociation with a collagenase/dispase mixture. Cells were simultaneously stained for keratin (FITC), vimentin (R-PE), and DNA (PI) before flow cytometric analysis. The method was applied to 12 paraffin-embedded cervical carcinomas and four colorectal carcinomas. In all cervical cancers, distinct keratin-positive and vimentin-positive cell populations were observed. While the exclusive vimentin-positive cell fractions always yielded unimodal DNA content distributions, bimodal distributions were observed for the keratin-positive cell fractions in nine cervical carcinomas, whereas one cervical carcinoma showed three distinct G0G1 populations. Coefficients of variation of the G0G1 peaks ranged from 1.70% to 4.79%. Average background, aggregate, and debris values were 14.7% (vimentin-positive fraction) and 33.8% (keratin-positive fraction). Flow sorting confirmed that the exclusively vimentin-positive cell fractions represent different normal stromal and infiltrate cells that can serve as an internal ploidy reference enabling discrimination between DNA hypo-diploid and DNA hyper-diploid tumour cell subpopulations. The neoplastic origin of the keratin-vimentin co-expressing cells from two cervical carcinomas was confirmed by genotyping of flow-sorted samples revealing loss of heterozygosity (LOH) of 6p. This improved method obviates the need for fresh/frozen tumour tissue for high-resolution DNA ploidy measurements and enables the isolation of highly purified tumour subpopulations for subsequent genotyping.
The Journal of Pathology 07/2005; 206(2):233-41. · 7.59 Impact Factor