Direct inhibition of STAT signaling by platinum drugs contributes to their anti-cancer activity.

Platinum-based chemotherapeutics are amongst the most powerful anti-cancer drugs. Although their exact mechanism of action is not well understood, it is thought to be mediated through covalent DNA binding. We investigated the effect of platinum-based chemotherapeutics on signaling through signal transducer and activator of transcription (STAT) proteins, which are involved in many oncogenic signaling pathways. We performed in vitro experiments in various cancer cell lines, investigating the effects of platinum chemotherapeutics on STAT phosphorylation and nuclear translocation, the expression of STAT-modulating proteins and downstream signaling pathways. Direct binding of platinum to STAT proteins was assessed using an AlphaScreen assay. Nuclear STAT3 expression was determined by immunohistochemistry and correlated with disease-free survival in retrospective cohorts of head and neck squamous cell carcinoma (HNSCC) patients treated with cisplatin-based chemoradiotherapy (n= 65) or with radiotherapy alone (n = 32). At clinically relevant concentrations, platinum compounds inhibited STAT phosphorylation, resulting in loss of constitutively activated STAT proteins in multiple distinct cancer cell lines. Platinum drugs specifically inhibited phospho-tyrosine binding to SH2 domains, thereby blocking STAT activation, and subsequently downregulating pro-survival- and anti-apoptotic- target genes. Importantly, we found that active STAT3 in tumors directly correlated with response to cisplatin-based chemoradiotherapy in HNSCC patients (p = 0.006). These findings provide insight into a novel, non-DNA-targeted mechanism of action of platinum drugs, and could be leveraged into the use of STAT expression as predictive biomarker for cisplatin chemotherapy and to potentiate other therapeutic strategies such as immunotherapy.

Variation in guideline adherence in non-Hodgkin's lymphoma care: impact of patient and hospital characteristics.

BACKGROUND: The objective of this observational study was to assess the influence of patient, tumor, professional and hospital related characteristics on hospital variation concerning guideline adherence in non-Hodgkin's lymphoma (NHL) care. METHODS: Validated, guideline-based quality indicators (QIs) were used as a tool to assess guideline adherence for NHL care. Multilevel logistic regression analyses were used to calculate variation between hospitals and to identify characteristics explaining this variation. Data for the QIs regarding diagnostics, therapy, follow-up and organization of care, together with patient, tumor and professional related characteristics were retrospectively collected from medical records; hospital characteristics were derived from questionnaires and publically available data. RESULTS: Data of 423 patients diagnosed with NHL between October 2010 and December 2011 were analyzed. Guideline adherence, as measured with the QIs, varied considerably between the 19 hospitals: >20 % variation was identified in all 20 QIs and high variation between the hospitals (>50 %) was seen in 12 QIs, most frequently in the treatment and follow-up domain. Hospital variation in NHL care was associated more than once with the characteristics age, extranodal involvement, multidisciplinary consultation, tumor type, tumor aggressiveness, LDH level, therapy used, hospital region and availability of a PET-scanner. CONCLUSION: Fifteen characteristics identified at the patient level and at the hospital level could partly explain hospital variation in guideline adherence for NHL care. Particularly age was an important determinant: elderly were less likely to receive care as measured in the QIs. The identification of determinants can be used to improve the quality of NHL care, for example, for standardizing multidisciplinary consultations in daily practice.

Improvement of hospital care for patients with non-Hodgkin's lymphoma: protocol for a cluster randomized controlled trial (PEARL study).

BACKGROUND: Malignant lymphomas constitute a diverse group of cancers of lymphocytes. One well-known disease is Hodgkin's lymphoma; the others are classified as non-Hodgkin's lymphoma (NHL). NHLs are the most common hematologic neoplasms in adults worldwide, and in 2012 over 170,000 new cases were estimated in the United States and Europe.In previous studies, several practice gaps in hospital care for patients with NHL have been identified. To decrease this variation in care, the present study aims to perform a problem analysis in which barriers to and facilitators for optimal NHL care will be identified and, based on these findings, to develop (tailored) improvement strategies. Subsequently, we will assess the effectiveness, feasibility and costs of the improvement strategies. METHODS/DESIGN: Barriers and facilitators will be explored using the literature, using interviews and questionnaires among physicians involved in NHL care, and patients diagnosed with NHL. The results will be used to develop a tailored improvement strategy. A cluster randomized controlled trial involving 19 Dutch hospitals will be conducted. Hospitals will be randomized to receive either an improvement strategy tailored to the barriers and facilitators found or, a standard strategy of audit and feedback.The effects of both strategies will be evaluated using previously developed quality indicators. Adherence to the indicators will be measured before and after the intervention period based on medical records from newly diagnosed NHL patients. To study the feasibility of both strategies, a process evaluation will be additionally performed. Data about exposure to the different elements of the strategies will be collected using questionnaires. Economic evaluation from a healthcare perspective will compare the two implementation strategies, where the costs of the implementation strategy and changes in healthcare consumption will be assessed. DISCUSSION: The presence of variation in the use of diagnostic tests, treatment, and follow-up between different physicians in different hospitals in the Netherlands is important for patients. To reduce the existing variation in care, implementation of tailored interventions to improve NHL care is necessary. TRIAL REGISTRATION: This trial is registered at ClinicalTrial.gov as the PEARL study, registration number NCT01562509.

Application of microfluidic technology to the BIOMED-2 protocol for detection of B-cell clonality.

The BIOMED-2 protocol is widely used for detecting clonality in lymphoproliferative disorders. The protocol requires multiple PCR reactions, which are analyzed by either capillary electrophoresis (GeneScan analysis) or heteroduplex PAGE analysis. We tested a microfluidic chip-based electrophoresis device (Agilent 2100 Bioanalyzer) for the analysis of B-cell clonality using PCR for the three framework subregions (FR) of the Ig heavy chain gene (IGH) and PCR for two rearrangements occurring in the Ig κ chain gene (IGK-VJ and IGK-DE). We analyzed 62 B-cell lymphomas (33 follicular and 29 nonfollicular) and 16 reactive lymph nodes. Chip-based electrophoresis was conclusive for monoclonality in 59/62 samples; for 20 samples, it was compared with GeneScan analysis. Concordant results were obtained in 45/55 IGH (FR1, FR2, and FR3) gene rearrangements, and in 34/37 IGK gene rearrangements. However, when the chip device was used to analyze selected IGK gene rearrangements (biallelic IGK rearrangements or IGK rearrangements in a polyclonal background), its performance was not completely accurate. We conclude, therefore, that this microfluidic chip-based electrophoresis device is reliable for testing cases with dominant PCR products but is less sensitive than GeneScan in detecting clonal peaks in a polyclonal background for IGH PCR, or with complex IGK rearrangement patterns.

Platinum-based drugs disrupt STAT6-mediated suppression of immune responses against cancer in humans and mice.

Tumor microenvironments feature immune inhibitory mechanisms that prevent T cells from generating effective antitumor immune responses. Therapeutic interventions aimed at disrupting these inhibitory mechanisms have been shown to enhance antitumor immunity, but they lack direct cytotoxic effects. Here, we investigated the effect of cytotoxic cancer chemotherapeutics on immune inhibitory pathways. We observed that exposure to platinum-based chemotherapeutics markedly reduced expression of the T cell inhibitory molecule programmed death receptor-ligand 2 (PD-L2) on both human DCs and human tumor cells. Downregulation of PD-L2 resulted in enhanced antigen-specific proliferation and Th1 cytokine secretion as well as enhanced recognition of tumor cells by T cells. Further analysis revealed that STAT6 controlled downregulation of PD-L2. Consistent with these data, patients with STAT6-expressing head and neck cancer displayed enhanced recurrence-free survival upon treatment with cisplatin-based chemoradiation compared with patients with STAT6-negative tumors, demonstrating the clinical relevance of platinum-induced STAT6 modulation. We therefore conclude that platinum-based anticancer drugs can enhance the immunostimulatory potential of DCs and decrease the immunosuppressive capability of tumor cells. This dual action of platinum compounds may extend their therapeutic application in cancer patients and provides a rationale for their use in combination with immunostimulatory compounds.

Mutation analysis of KRAS prior to targeted therapy in colorectal cancer: development and evaluation of quality by a European external quality assessment scheme.

In Europe, the use of anti-EGFR monoclonal antibodies is restricted to Kirsten RAS (KRAS) wild-type colorectal tumors. Information on the KRAS status of the patients tumor is thus key for clinical practice; however, there is little guidance or definition on which KRAS mutations to assess and how to assess them. To ensure the consistency and the quality of KRAS test results in Europe, an interlaboratory control network needs to be set up. This pilot study aimed to identify the variables that need to be assessed in a quality control scheme and to provide a first assessment in a selected set of laboratories. Fourteen different tumor cases were circulated between 13 laboratories by a central laboratory acting as the referent for the mutation status determination. This study illustrated that of 13 experienced laboratories that perform KRAS testing only ten correctly identified the KRAS in all 14 cases that were circulated. There was no harmonization in DNA isolation and KRAS mutation detection method between the laboratories. These results indicate that future standardization is needed in KRAS mutation detection methodology. An expansion of the European Society of Pathology KRAS program could identify areas of difficulty in KRAS testing and provide the basis for harmonization.

Preparing pathology for personalized medicine: possibilities for improvement of the pre-analytical phase.

With the introduction of new biological agents for cancer treatment enabling 'personalized medicine', treatment decisions based on the molecular features of the tumour are more common. Consequently, tissue evaluation in tumour pathology is becoming increasingly based on a combination of classical morphological and molecular analysis. The results of diagnostic tests rely not only on the quality of the method used but, to a large extent, also on the quality of specimens, which is dependent on the pre-analytical procedures and storage. With the introduction of predictive immunohistochemical and molecular tests in clinical pathology, improvement and standardization of pre-analytical procedures has become crucial. The aim of this review is to increase awareness with regard to tissue handling and for standardization of the pre-analytical phase of a diagnostic process. In addition, several processing steps in tissue handling that need to be improved in order to obtain the quality needed for modern molecular medicine will be discussed. Optimal, standardized procedures are crucial if a high standard of test results is to be achieved, which is what each patient deserves.

Protein profiling in pathology: analysis and evaluation of 239 frozen tissue biopsies for diagnosis of B-cell lymphomas.

PURPOSE: We determined the potential value of protein profiling of tissue samples by assessing how precise this approach enables discrimination of B-cell lymphoma from reactive lymph nodes, and how well the profiles can be used for lymphoma classification. EXPERIMENTAL DESIGN: Protein lysates from lymph nodes (n=239) from patients with the diagnosis of reactive hyperplasia (n=44), follicular lymphoma (n=63), diffuse large B-cell lymphoma (n=43), mantle cell lymphoma (n=47), and chronic lymphocytic leukemia/small lymphocytic B-cell lymphoma (n=42) were analysed by SELDI-TOF MS. Data analysis was performed by (i) classification and regression tree-based analysis and (ii) binary and polytomous logistic regression analysis. RESULTS: After internal validation by the leave-one-out principle, both the classification and regression tree and logistic regression classification correctly identified the majority of the malignant (87 and 96%, respectively) and benign cases (73 and 75%, respectively). Classification was less successful since approximately one-third of the cases of each group were misclassified according to the histological classification. However, an additional mantle cell lymphoma case that was misclassified as chronic lymphocytic leukemia/small lymphocytic B-cell lymphoma initially was identified based on the protein profile. CONCLUSIONS AND CLINICAL RELEVANCE: SELDI-TOF MS protein profiling allows for reliable identification of the majority of malignant lymphoma cases; however, further validation and testing robustness in a diagnostic setting is needed.

Biomarkers as disease definition: mantle cell lymphoma as an example.

The introduction in pathology practice of a new biomarker that is contributing to define a disease requires a series of investigations. Now that new biomarkers are being discovered continuously it is important to learn from successful examples of markers that are presently widely used. In this historical account the steps are described that have led to the use of immunohistochemical staining of tissue samples with an antibody against cyclinD1 to diagnose mantle cell lymphoma. Furthermore, a short outlook is given on the introduction of proteomics as a tool in the diagnosis of lymphoma and the potential route to be taken for introducing this technology into clinical practice.

Correlation of FCGR3A and EGFR germline polymorphisms with the efficacy of cetuximab in KRAS wild-type metastatic colorectal cancer.

BACKGROUND: Next to KRAS mutation status, additional predictive markers are needed for the response to cetuximab in patients with metastatic colorectal cancer (mCRC). Previous studies indicated that germline polymorphisms in specific genes may predict efficacy and toxicity of cetuximab in mCRC patients. METHODS: Germline DNA was isolated from 246 KRAS wild-type mCRC patients who were treated in the phase III CAIRO2 study with chemotherapy and bevacizumab alone or with the addition of cetuximab. Associations of epidermal growth factor (EGF) 61A>G, EGF receptor (EGFR) CA(14-22), cyclin D1 (CCND1) 932G>A, fragment-C gamma receptor (FCGR) 2A 535A>G and FCGR3A 818A>C polymorphisms with progression-free survival (PFS) and cetuximab-related skin toxicity were studied. RESULTS: In cetuximab-treated patients, the FCGR3A 818C-allele was associated with decreased PFS compared with the FCGR3A 818AA genotype (median PFS, 8.2 [95%CI, 6.7-10.3] versus 12.8 [95%CI, 10.3-14.7] months, respectively; HR, 1.57 [95%CI, 1.06-2.34]; P=.025). The EGFR20 genotype was associated with decreased PFS compared with the EGFR<20 genotype (median PFS, 7.6 [95%CI, 6.7-10.0] versus 12.4 [95%CI, 10.3-13.4] months, respectively; HR, 1.58 [95%CI, 1.06-2.35]; P=.024). The FCGR3A and EGFR polymorphisms were not associated with PFS in patients treated without cetuximab. None of the polymorphisms were associated with the incidence of grades 2-3 skin toxicity. CONCLUSION: EGFR and FCGR3A germline polymorphisms are associated with PFS in KRAS wild-type mCRC patients treated with cetuximab, bevacizumab and chemotherapy.

High sensitivity of both sequencing and real-time PCR analysis of KRAS mutations in colorectal cancer tissue.

The KRAS mutation status predicts the outcome of treatment with epidermal growth factor receptor targeted agents, and therefore the testing for KRAS mutations has become an important diagnostic procedure. To optimize the quality of this test, we compared the results of the two most commonly used KRAS mutation tests, cycle sequencing and a real-time PCR-based assay, in DNA extracted from formalin-fixed paraffin-embedded (FFPE) colorectal cancer samples of 511 patients. The results were interpreted in the context of the tumour cell percentage and the assay parameters. In 510 samples KRAS mutation status assessment was successful. A KRAS mutation was detected in 201 tumours (39.4%). Sequencing and the real-time PCR-based assay generated the same result in 486 samples (95.3%). The sequencing result was considered false positive in one (0.2%) and false negative in nine samples (1.8%). The assay result was considered false positive in six (1.2%) and false negative in seven samples (1.4%). Explanations for discrepant test results were a higher sensitivity of the assay in samples with a low tumour cell percentage, occurrence of mutations that are not covered by the assay and δ Ct values approximating the cut-off value of the assay. In conclusion, both sequencing and the real-time PCR-based assay are reliable tests for KRAS mutation analysis in FFPE colorectal cancer samples, with a sensitivity of 95.5% (95% confidence interval [CI] 91.7-97.9%) and 96.5% (95% CI 93.0-98.6%), respectively. The real-time PCR based assay is the method of choice in samples with a tumour cell percentage below 30%.

Lymph node retrieval in rectal cancer is dependent on many factors--the role of the tumor, the patient, the surgeon, the radiotherapist, and the pathologist.

Lymph node status is the strongest prognostic factor for survival in colorectal cancer. There are several guidelines concerning the minimum numbers of lymph nodes that need to be examined to make reliable staging possible, but there is no consensus in the available literature. In this study, we determine in patients with rectal cancer factors that relate to the number of lymph nodes found and the presence of lymph node metastasis. In addition, the number of examined lymph nodes was correlated with prognosis. A total of 1227 patients were selected from a multicenter prospective randomized trial investigating the value of neoadjuvant radiotherapy. The median number of examined lymph nodes in all patients was 7.0. The number of retrieved lymph nodes in patients with node metastasis was significantly higher than in node negative patients. After neoadjuvant radiotherapy fewer lymph nodes were retrieved (6.9 vs. 8.5; P<0.0001). Variations in lymph node yield between pathology laboratories and individual pathologists were striking. The following patient and tumor characteristics are associated with a significant lower lymph node retrieval: age over 60 years, overweight, small size, and low invasion depth of the tumor, poor differentiation grade, and absence of a lymphoid reaction. Node negative patients in whom seven or less lymph nodes were examined had a lower recurrence free interval than patients in whom at least 8 lymph nodes were examined (17.0% vs. 10.7%, P=0.016). We conclude that in pathology laboratories a median of at least 8 lymph nodes need to be examined in rectal cancer specimens, but that higher numbers are desirable and achievable in most cases, even after preoperative radiotherapy.

High-resolution genomic profiling of pediatric lymphoblastic lymphomas reveals subtle differences with pediatric acute lymphoblastic leukemias in the B-lineage.

Lymphoblastic lymphoma (LBL) is one of the most frequent occurring pediatric non-Hodgkin lymphomas. In the WHO classification scheme, pediatric LBL is considered to be the same disease entity as pediatric acute lymphoblastic leukemia (ALL). However, it is unclear whether the genetic basis of pediatric LBL development is similar to that of pediatric ALL. We performed genome-wide analyses of copy number aberrations in 12 T-LBL and 7 precursor B-cell LBL pediatric cases using high-resolution SNP-based array CGH. Similar to what previously has been found in T-ALL, T-LBL exhibited recurrent deletions of the CDKN2A locus, occurring in 92% of the cases. Additionally, we detected deletions of RB1 (16%), duplications of MYB (16%), and an amplification of ABL1 in one case. These results show that, similar to T-ALL, the genomic alterations in T-LBL predominantly target genes involved in cell cycle progression. The majority of precursor B-cell LBL was characterized by high-hyperdiploidy (71%), and showed high resemblance with high-hyperdiploid precursor B-cell ALL. Taken together, our data suggest that pediatric LBL and ALL exhibit similar genomic abnormalities within confined immunophenotypic and cytogenetic subgroups, but that the representations of these subgroups differs between the two entities.

Chemotherapy, bevacizumab, and cetuximab in metastatic colorectal cancer.

BACKGROUND: Fluoropyrimidine-based chemotherapy plus the anti-vascular endothelial growth factor (VEGF) antibody bevacizumab is standard first-line treatment for metastatic colorectal cancer. We studied the effect of adding the anti-epidermal growth factor receptor (EGFR) antibody cetuximab to a combination of capecitabine, oxaliplatin, and bevacizumab for metastatic colorectal cancer. METHODS: We randomly assigned 755 patients with previously untreated metastatic colorectal cancer to capecitabine, oxaliplatin, and bevacizumab (CB regimen, 378 patients) or the same regimen plus weekly cetuximab (CBC regimen, 377 patients). The primary end point was progression-free survival. The mutation status of the KRAS gene was evaluated as a predictor of outcome. RESULTS: The median progression-free survival was 10.7 months in the CB group and 9.4 in the CBC group (P=0.01). Quality-of-life scores were lower in the CBC group. The overall survival and response rates did not differ significantly in the two groups. Treated patients in the CBC group had more grade 3 or 4 adverse events, which were attributed to cetuximab-related adverse cutaneous effects. Patients treated with cetuximab who had tumors bearing a mutated KRAS gene had significantly decreased progression-free survival as compared with cetuximab-treated patients with wild-type-KRAS tumors or patients with mutated-KRAS tumors in the CB group. CONCLUSIONS: The addition of cetuximab to capecitabine, oxaliplatin, and bevacizumab resulted in significantly shorter progression-free survival and inferior quality of life. Mutation status of the KRAS gene was a predictor of outcome in the cetuximab group. (ClinicalTrials.gov number, NCT00208546.)

Hypermutation in mantle cell lymphoma does not indicate a clinical or biological subentity.

Mantle cell lymphoma is a prime example of a well-defined entity based on morphology, phenotype, genetics and also clinical features. Although most patients have an adverse clinical course, some have a better survival than others. The most consistently reported adverse prognostic parameter is a high mitotic rate. Recently, it has been shown that hypermutation in the immunoglobulin heavy-chain gene occurs in a subset of mantle cell lymphomas. It is, however, unclear whether the mutational status is stable over time within a given case, whether hypermutation might be influenced by therapy and how it is related to other relevant biological features of mantle cell lymphoma. In this study, we analyzed 23 typical mantle cell lymphoma cases with respect to mutational status and compared the results with clinicopathological and genetic data to determine whether the presence of mutation indicates a subentity with clinical or pathological relevance. We found somatic hypermutation in 26% of our cases and, interestingly, one case showed ongoing somatic hypermutation. In tumor cells of both mutated and unmutated cases, we found a preferential usage of V(H)3-21 (23%) and V(H)4-34 (19%). No significant correlations were found between mutation status and the other morphological and genetic features analyzed. In conclusion, our results provide additional evidence that mutation status in mantle cell lymphoma is better interpreted as a feature within the spectrum of disease that seems to have little clinical or pathological relevance.

Integrated genomic and expression profiling in mantle cell lymphoma: identification of gene-dosage regulated candidate genes.

Mantle cell lymphoma (MCL) is characterized by the t(11;14)(q13;q32) translocation and several other cytogenetic aberrations, including heterozygous loss of chromosomal arms 1p, 6q, 11q and 13q and/or gains of 3q and 8q. The common intervals of chromosomal imbalance have been narrowed down using array-comparative genomic hybridization (CGH). However, the chromosomal intervals still contain many genes potentially involved in MCL pathogeny. Combined analysis of tiling-resolution array-CGH with gene expression profiling on 11 MCL tumours enabled the identification of genomic alterations and their corresponding gene expression profiles. Only subsets of genes located within given cytogenetic anomaly-intervals showed a concomitant change in mRNA expression level. The genes that showed consistent correlation between DNA copy number and RNA expression levels are likely to be important in MCL pathology. Besides several 'anonymous genes', we also identified various fully annotated genes, whose gene products are involved in cyclic adenosine monophosphate-regulated pathways (PRKACB), DNA damage repair, maintenance of chromosome stability and prevention of rereplication (ATM, ERCC5, FBXO5), energy metabolism (such as genes that are involved in the synthesis of proteins encoded by the mitochondrial genome) and signal transduction (ARHGAP29). Deregulation of these gene products may interfere with the signalling pathways that are involved in MCL tumour development and maintenance.

Gastrointestinal zygomycosis due to Rhizopus microsporus var. rhizopodiformis as a manifestation of chronic granulomatous disease.

A case of gastrointestinal zygomycosis in a 10-month-old boy with chronic granulomatous disease (CGD) is presented. Zygomycetes are an uncommon cause of fungal disease in CGD patients and gastrointestinal zygomycosis has not been previously described in individuals with CGD. To improve outcome, a timely and correct diagnosis is of utmost importance.

Protein profiling of B-cell lymphomas using tissue biopsies: A potential tool for small samples in pathology.

Non-Hodgkin's lymphoma comprises many related but distinct diseases and diagnosis and classification is complex. Protein profiling of lymphoma biopsies may be of potential value for use in this lymphoma classification and the discovery of novel markers. In this study, we have optimized a method for SELDI-TOF MS based protein profiling of frozen tissue sections, without dissection of tumour cells. First we have compared chip surfaces and lysis buffers. Also, we have determined the minimal input using laser dissection microscopy. Subsequently, we have analyzed and compared protein profiles of diffuse large B-cell lymphoma (n=8), follicular lymphoma (n=8) and mantle cell lymphoma (n=8). Benign, reactive lymph nodes (n=14) were used as a reference group.CM10 chip surface in combination with urea lysis buffer and an input of approximately 50,000 lymphocytes allowed the detection of many differential peaks. Identification of the diffuse large B-cell lymphoma cases was reliably made in the supervised classification. Unsupervised clustering showed segregation into a benign/indolent cluster predominantly formed by benign, reactive lymph nodes and follicular lymphoma cases and into a more aggressive cluster formed by diffuse large B-cell lymphoma and mantle cell lymphoma cases. In conclusion, our protocol enables protein profiling of protein lysates derived from small histological samples and the subsequent detection of many differentially expressed proteins, without the need of tumour cell dissection. These results support further evaluation of protein profiling of small lymphoma biopsies as an additional tool in pathology.

Pitfalls in TCR gene clonality testing: teaching cases.

Clonality testing in T-lymphoproliferations has technically become relatively easy to perform in routine laboratories using standardized multiplex polymerase chain reaction protocols for T-cell receptor (TCR) gene analysis as developed by the BIOMED-2 Concerted Action BMH4-CT98-3936. Expertise with clonality diagnostics and knowledge about the biology of TCR gene recombination are essential for correct interpretation of TCR clonality data. Several immunobiological and technical pitfalls that should be taken into account to avoid misinterpretation of data are addressed in this report. Furthermore, we discuss the need to integrate the molecular data with those from immunohistology, and preferably also flow cytometric immunophenotyping, for appropriate interpretation. Such an interactive, multidisciplinary diagnostic model guarantees integration of available data to reach the most reliable diagnosis.

Promoter methylation of PARG1, a novel candidate tumor suppressor gene in mantle-cell lymphomas.

BACKGROUND AND OBJECTIVES: Mantle cell lymphoma (MCL), a mature B-cell neoplasm, is genetically characterized by the translocation t(11;14)(q13;q32). However, secondary alterations are required for malignant transformation. The identification of inactivated tumor suppressor genes contributing to the development of MCL may lead to further elucidation of the biology of this disease and help to identify novel targets for therapy. DESIGN AND METHODS: Whole genome microarray-based gene expression profiling on treated versus untreated MCL cell lines was used to identify genes induced by 5-aza-2'-deoxycytidine. The degree of promoter methylation and transcriptional silencing of selected genes was then proven in MCL cell lines and primary cases by methylation-specific polymerase chain reaction (PCR) techniques, real-time PCR and gene expression profiling. RESULTS: After 5-aza-2'-deoxycytidine treatment, we identified more than 1000 upregulated genes, 16 of which were upregulated > or =3-fold. Most of them were not known to be silenced by methylation in MCL. A low expression of ING1, RUNX3 and BNIP3L was observed in three of the five the MCL cell lines. In addition, the expression of PARG1, which is located in the frequently deleted region 1p22.1, was substantially reduced and displayed at least partial promoter methylation in all investigated MCL cell lines as well as in 31 primary MCL cases. INTERPRETATION AND CONCLUSIONS: In summary, we identified interesting novel candidate genes that probably contribute to the progression of MCL and suggest that PARG1 is a strong candidate tumor suppressor gene in MCL.