Botond Timár

Microsatellite instability and hMLH1 promoter hypermethylation in Richter's transformation of chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is an indolent B cell non-Hodgkin lymphoma (NHL) that may transform into diffuse large B cell lymphoma (DLBL). This transformation is referred to as Richter’s syndrome or transformation. To analyze whether microsatellite instability (MSI) and DNA mismatch repair defects are associated with Richter’s transformation, we have performed microsatellite analysis, mutational analysis of hMLH1 and hMSH2 genes and methylation status analysis of CpG island of the hMLH1 promoter on serial biopsy specimens from 19 patients with CLL. Ten cases of CLL showed no histologic alteration in the second biopsy, and nine cases of CLL underwent morphologic transformation to DLBL in the second biopsy. Using eight microsatellite loci, high level of MSI was associated with Richter’s transformation in four cases of CLL, but none of the CLLs displayed this level of MSI without transformation. Mutations of the hMLH1 or hMSH2 genes were not detected in any of the lymphoma samples. In five cases of Richter’s transformation the hMLH1 promoter was hypermethylated in both CLL and DLBL samples. Hypermethylation of the hMLH1 promoter associated with high-level of MSI in four cases, and low-level of MSI in one case. These results suggest that in certain cases of Richter’s transformation the DNA mismatch-repair defect-initiated genetic instability may play a role in tumor progression.

Relationship between the mutational status of VH genes and pathogenesis of diffuse large B-cell lymphoma in Richter's syndrome.

Patients with chronic lymphocytic leukemia (CLL) may develop diffuse large B-cell lymphoma (DLBL), also known as Richters syndrome. Mutational status of immunoglobulin (Ig) heavy-chain variable region (VH) genes have prognostic impact in CLL. Patients with mutated VH genes have a stable disease, whereas patients with unmutated VH gene have more aggressive disease. The mutational status of CLLs that transform to DLBL is unknown. To reveal whether Richters syndrome occurs in CLLs with mutated or unmutated VH genes, we have performed mutational analysis on serial specimens from eight patients. CLL and DLBL tumorclones were identical in five cases and they were different in three cases. Six CLLs expressed unmutated and two cases expressed mutated VH genes. In five of the six unmutated CLLs, the DLBL clones evolved from CLL tumorclones and the VH genes expressed by DLBLs were also unmutated. In one unmutated and two mutated CLLs, the DLBLs expressed mutated VH genes, but in these three cases the DLBL tumorclones developed as independent secondary neoplasm. These results suggest that Richters syndrome may develop in both mutated or unmutated CLLs, but clonal transformation of CLL to DLBL occur only in the unmutated subgroup of CLL.

ROR1 expression is not a unique marker of CLL.

Recent studies have identified receptor tyrosine kinase‐like orphan receptor 1 (ROR1) on the surface of chronic lymphoid leukaemia (CLL) cells. In order to determine whether ROR1 expression is a suitable surrogate marker for the diagnosis of CLL we analysed the mRNA level of ROR1 in different types of non‐Hodgkin lymphomas (NHL), and detected elevated levels of ROR1 compared to control peripheral mononuclear cells in several entities (CLL ≥ mantle cell lymphoma (MCL) > marginal zone lymphoma (MZL) >> diffuse large B‐cell lymphoma > follicular lymphoma). ROR1 protein was expressed intensely on the cell surface of lymphoma cells with leukaemic blood count detected by three colour immunofluorescence. Our results indicate that ROR1 expression is not limited to CLL cases, but it is more prevalent in NHLs, mainly in MCL where it is expressed intensely and MZL where it is expressed moderately, suggesting a general role of ROR1 in lymphoma genesis and/or maintenance. Copyright

PRDM1/BLIMP1 is commonly inactivated in anaplastic large T-cell lymphoma.

Anaplastic large cell lymphoma (ALCL) is a mature T-cell lymphoma that can present as a systemic or primary cutaneous disease. Systemic ALCL represents 2% to 5% of adult lymphoma but up to 30% of all pediatric cases. Two subtypes of systemic ALCL are currently recognized on the basis of the presence of a translocation involving the anaplastic lymphoma kinase ALK gene. Despite considerable progress, several questions remain open regarding the pathogenesis of both ALCL subtypes. To investigate the molecular pathogenesis and to assess the relationship between the ALK(+) and ALK(-) ALCL subtypes, we performed a genome-wide DNA profiling using high-density, single nucleotide polymorphism arrays on a series of 64 cases and 7 cell lines. The commonest lesions were losses at 17p13 and at 6q21, encompassing the TP53 and PRDM1 genes, respectively. The latter gene, coding for BLIMP1, was inactivated by multiple mechanisms, more frequently, but not exclusively, in ALK(-)ALCL. In vitro and in vivo experiments showed that that PRDM1 is a tumor suppressor gene in ALCL models, likely acting as an antiapoptotic agent. Losses of TP53 and/or PRDM1 were present in 52% of ALK(-)ALCL, and in 29% of all ALCL cases with a clinical implication.

Somatic hypermutation of IGVH genes and aberrant somatic hypermutation in follicular lymphoma without BCL-2 gene rearrangement and expression

The findings of this study suggest that follicular lymphoma with typical morphological and immunophenotypic features may develop even without the involvement of the BCL-2 gene. See related perspective article on page 1773. Background Follicular lymphoma is characterized by the t(14;18) translocation resulting in constitutive expression of BCL-2 protein; however approximately 10–15% of follicular lymphomas do not express BCL-2 protein, and a small fraction of these cases does not exhibit translocation of the BCL-2 gene either. It is highly debated whether cases of follicular lymphoma without BCL-2 gene rearrangement and expression represent a separate lymphoma entity with distinct biological characteristics, different from the BCL-2-positive cases. Design and Methods To further characterize follicular lymphomas without BCL-2 gene rearrangement and expression, we analyzed and compared the mutational status of IGVH genes as well as other genes (C-MYC, PAX-5 and RHOH) frequently involved in the specific type of genomic instability called aberrant somatic hypermutation in 11 cases of BCL-2-negative and 7 cases of BCL-2-positive follicular lymphomas. We also determined the levels of expression of activation-induced cytidine deaminase in these cases. Results The analyzed cases were grade 2 and grade 3A follicular lymphomas. Our findings demonstrate that follicular lymphomas without BCL-2 gene rearrangement and expression are associated with ongoing somatic hypermutation of the IGVH genes, low activity of aberrant somatic hypermutation and elevated activation-induced cytidine deaminase expression. These results were in concordance with the results found in the cases of BCL-2-positive follicular lymphoma. Conclusions Although, BCL-2 protein overexpression is considered to be a critical pathogenic event in the development of follicular lymphoma, our findings suggest that follicular lymphomas with the same morphology, immunophenotype, mutational pattern and activation-induced cytidine deaminase expression may develop without the involvement of BCL-2 gene. The present data support the hypothesis that BCL-2-positive and BCL-2-negative follicular lymphomas (grades 1-3A) represent a homogenous group with different initial but several common additional molecular pathways.

Activity and complexes of mTOR in diffuse large B-cell lymphomas—a tissue microarray study

Diffuse large B-cell lymphoma is a heterogeneous group of diseases with different responses to therapy. Targeting mTOR (mammalian target of rapamycin) offers a new approach to improve the treatment. mTOR inhibitors are being developed and are in clinical use in mantle cell lymphoma therapy and clinical trials are ongoing in other high-grade lymphomas as well. However, there is limited data about mTOR activity and the expression of its different complexes in diffuse large B-cell lymphomas. Tissue microarray blocks were constructed from paraffin-embedded biopsy specimens. More than 700 immunohistochemical stainings (mTOR signaling-related proteins and phosphoproteins, markers for lymphoma classification) were evaluated from 68 diffuse large B-cell lymphoma biopsies from conventionally treated and followed patients. Approximately 30% of cases were characterized as germinal center-derived diffuse large B-cell lymphomas, which showed virtually no mTOR activity, as determined by phospho-ribosomal S6 expression, the most sensitive marker of mTOR activity. In about 80% of non-germinal center-derived diffuse large B-cell lymphoma cases, positivity of mTOR-related phosphoproteins was observed, denoting mTOR activity. Moreover, Rictor (a characteristic protein of the mTOR complex2) was overexpressed in 43% of all diffuse large B-cell lymphomas and in 63% of mTOR-active non-germinal center diffuse large B-cell lymphoma samples. Rictor overexpression with mTOR activity indicated significantly worse survival for patients than mTOR inactivity or mTOR activity with low Rictor expression. These results suggest that mTOR activity is characteristic in most non-germinal center-derived diffuse large B-cell lymphomas with potentially variable mTOR-inhibitor sensitivity. Taken together, mTOR inhibitors may be useful in addition to regular therapy in diffuse large B-cell lymphomas, however, patient and inhibitor selection criteria must be carefully considered.

Prolonged survival using anti-CD20 combined chemotherapy in primary prostatic intravascular large B-cell lymphoma.

Here we report a case of a 73-year-old man with primary intravascular large B-cell lymphoma localized to the prostate. Total prostatectomy was performed due to a benign adenoma suggested by ultrasonography. The diagnosis of IVLBL was obtained incidentally from the prostatectomy specimen. Eight months after the initial R-CHOP chemotherapy a relapse was detected in the left inguinal lymph node, where histologic examination revealed common diffuse large B-cell lymphoma with minimal intravascular component. The second complete remission was achieved by R-IEV therapy. Five months later a second relapse occurred and the patient died in the widespread disease and pneumonia. Primary prostate IVLBL is extremely uncommon; to date only four cases have been described. This is a well documented case, where we also confirmed that the initial primary IVLBL and the secondary lymph node involvement are clonally related. Successful treatment depends on early diagnosis of IVLBL, aggressive chemotherapy and the fact that IVLBL should be considered as a generalized disease in spite of negative staging results.

Foetal craniopharyngioma diagnosed by prenatal ultrasonography and confirmed by histopathological examination

Craniopharyngioma is an intracranial tumour, which usually develops in children and young adults and rarely occurs in intrauterine life.

Protocol for qRT-PCR analysis from formalin fixed paraffin embedded tissue sections from diffuse large b-cell lymphoma: Validation of the six-gene predictor score

As a part of an international study on the molecular analysis of Diffuse Large B-cell Lymphoma (DLBCL), a robust protocol for gene expression analysis from RNA extraction to qRT-PCR using Formalin Fixed Paraffin Embedded tissues was developed. Here a study was conducted to define a strategy to validate the previously reported 6-gene (LMO2, BCL6, FN1, CCND2, SCYA3 and BCL2) model as predictor of prognosis in DLBCL. To avoid variation, all samples were tested in a single centre and single platform. This study comprised 8 countries (Brazil, Chile, Hungary, India, Philippines, S. Korea, Thailand and Turkey). Using the Kaplan-Meier and log rank test on patients (n=162) and two mortality risk groups (with those above and below the mean representing high and low risk groups) confirmed that the 6-gene predictor score correlates significantly with overall survival (OS, p<0.01) but not with event free survival (EFS, p=0.18). Adding the International Prognostic Index (IPI) shows that the 6-gene predictor score correlates significantly with high IPI scores for OS (p<0.05), whereas those with low IPI scores show a trend not reaching significance (p=0.08). This study defined an effective and economical qRT-PCR strategy and validated the 6-gene score as a predictor of OS in an international setting.

Quantitative assessment of JAK2 V617F and CALR mutations in Philadelphia negative myeloproliferative neoplasms

BACKGROUND Philadelphia negative myeloproliferative neoplasms (MPNs) are characterized by frequent mutations of driver genes including JAK2, CALR and MPL. While the influence of JAK2 V617F mutant allele burden on the clinical phenotype of MPN patients is well-described, the impact of CALR mutant allele burden on clinical features needs further investigation. PATIENTS AND METHODS Quantitative assessment of JAK2 and CALR mutations was performed on diagnostic DNA samples from 425 essential thrombocythemia (ET) and 227 primary myelofibrosis patients using real-time quantitative PCR and fragment length analysis. Characterization of CALR mutations and detection of MPL mutations were performed by Sanger sequencing. RESULTS Twelve novel CALR mutations have been identified. ET patients with CALRmut load exceeding the median value exhibited lower hemoglobin values (12.0 vs. 13.6 g/dL), higher LDH levels (510 vs. 351 IU/L) and higher rate of myelofibrotic transformation (19% vs. 5%). The CALRmut load was higher among ET patients presenting with splenomegaly compared to those without splenomegaly (50.0% vs. 43.5%). CONCLUSION Our study confirms the clinical significance of driver mutational status and JAK2mut load in MPNs; in addition, unravels a novel clinical association between high CALRmut load and a more proliferative phenotype in ET.