Brigitte Maes

Identification of novel fusion partners of ALK, the anaplastic lymphoma kinase, in anaplastic large‐cell lymphoma and inflammatory myofibroblastic tumor

ALK‐positive anaplastic large‐cell lymphoma (ALCL) has been recognized as a distinct type of lymphoma in the heterogeneous group of T/Null‐ALCL. While most of the ALK‐positive ALCL (ALKomas) are characterized by the presence of the NPM‐ALK fusion protein, the product of the t(2;5)(p23;q35), 10–20% of ALKomas contain variant ALK fusions, including ATIC‐ALK, TFG‐ALK, CLTC‐ALK (previously designated CLTCL‐ALK), TMP3‐ALK, and MSN‐ALK. TMP3‐ALK and TMP4‐ALK fusions also have been detected in inflammatory myofibroblastic tumors (IMTs), making clear that aberrations of the ALK gene are not associated exclusively with the pathogenesis of ALK‐positive ALCL. Here we report results of molecular studies on two lymphoma cases and one IMT case with variant rearrangements of ALK. Our study led to the detection of the CLTC‐ALK fusion in an ALCL case and to the identification of two novel fusion partners of ALK: ALO17 (KIAA1618), a gene with unknown function, which was fused to ALK in an ALCL case with a t(2;17)(p23;q25), and CARS, encoding the cysteinyl‐tRNA synthetase, which was fused to ALK in an IMT case with a t(2;11;2)(p23;p15;q31). These results confirm the recurrent involvement of ALK in IMT and further demonstrate the diversity of ALK fusion partners, with the ability to homodimerize as a common characteristic.

Checklist for optimization and validation of real-time PCR assays

Real‐time polymerase chain reaction (PCR) is a frequently used technique in molecular diagnostics. To date, practical guidelines for the complete process of optimization and validation of commercial and in‐house developed molecular diagnostic methods are scare. Therefore, we propose a practical guiding principle for the optimization and validation of real‐time PCR assays. Based on literature, existing guidelines, and personal experience, we created a checklist that can be used in different steps of the development and validation process of commercial and in‐house developed real‐time PCR assays. Furthermore, determination of target values and reproducibility of internal quality controls are included, which allows a statistical follow‐up of the performance of the assay. Recently, we used this checklist for the development of various qualitative and quantitative assays for microbiological and hematological applications, for which accreditation according to ISO 15189:2007 was obtained. In our experience, the use of the proposed guidelines leads to a more efficient and standardized optimization and validation. Ultimately, this results in reliable and robust molecular diagnostics. The proposed checklist is independent of environment, equipment, and specific applications and can be used in other laboratories. A worldwide consensus on this kind of checklist should be aimed at. J. Clin. Lab. Anal. 23:145–151, 2009.

Marginal zone cell lymphoma--an update on recent advances.

Marginal zone cell lymphoma—an update on recent advances

Additional Prognostic Value of Bone Marrow Histology in Patients Subclassified According to the International Prognostic Scoring System for Myelodysplastic Syndromes

PURPOSE The most recent and powerful prognostic instrument established for myelodysplastic syndromes (MDS) is the International Prognostic Scoring System (IPSS), which is primarily based on medullary blast cell count, number of cytopenias, and cytogenetics. Although this prognostic system has substantial predictive power in MDS, further refinement is necessary, especially as far as lower-risk patients are concerned. Histologic parameters, which have long proved to be associated with outcome, are promising candidates to improve the prognostic accuracy of the IPSS. Therefore, we assessed the additional predictive power of the presence of abnormally localized immature precursors (ALIPs) and CD34 immunoreactivity in bone marrow (BM) biopsies of MDS patients. PATIENTS AND METHODS Cytogenetic, morphologic, and clinical data of 184 MDS patients, all from a single institution, were collected, with special emphasis on the determinants of the IPSS score. BM biopsies of 173 patients were analyzed for the presence of ALIP, and CD34 immunoreactivity was assessable in 119 patients. Forty-nine patients received intensive therapy. RESULTS The presence of ALIP and CD34 immunoreactivity significantly improved the prognostic value of the IPSS, with respect to overall as well as leukemia-free survival, in particular within the lower-risk categories. In contrast to the IPSS, both histologic parameters also were predictive of outcome within the group of intensively treated MDS patients. CONCLUSION Our data confirm the importance of histopathologic evaluation in MDS and indicate that determining the presence of ALIP and an increase in CD34 immunostaining in addition to the IPSS score could lead to an improved prognostic subcategorization of MDS patients.

The product of the t(ll;18), an API2-MLT fusion, is an almost exclusive finding in marginal zone cell lymphoma of extranodal MALT-type

BACKGROUND Extranodal marginal zone cell lymphoma (MZCL) of MALT-type share similar features with nodal and splenic MZCL regarding morphology and immunophenotype. At the genetic level, recent cytogenetic studies have shown that t(11;18) is a recurring abnormality in extranodal MALT-type MZCL but has hitherto never been reported in nodal or splenic MZCL. The aim of the present study was to determine the prevalence of t(11;18) in a large series of nodal, splenic and extranodal MALT-type MZCL, using a sensitive real-time RT-PCR method. MATERIALS AND METHODS Ninety-three MZCL cases were divided on clinical grounds into 61 extranodal MALT-type, 19 splenic and 12 nodal MZCL. One case that presented with a massive splenomegaly but for which also gastro-intestinal localisations were found, was left unclassified. A real-time RT-PCR method for the detection of the API2-MLT fusion resulting from t(11;18) was performed on RNA extracted from frozen tissue sections. RESULTS The API2-MLT fusion was detected in 12 cases, which were all extranodal MALT-type lymphomas of the stomach, except for one case. The remaining positive case was the unclassified case, for which the translocation was detected in the spleen and in hilar lymph node tissue. CONCLUSIONS While similarities between MZCL from different anatomic sites have lend us to propose that all MZCL have a common normal counterpart, the almost exclusive detection of t(11;18) in gastric MALT-type lymphoma favours its recognition as a separate lymphoma entity. The absence of the translocation in nodal and splenic MZCL challenges the idea of these lymphomas being secondary to MALT-type lymphomas of the gut. The unclassified case illustrates the inadequate approaches available at present to identify and define the various MZCL.

Evaluation of B cell lymphoid infiltrates in bone marrow biopsies by morphology, immunohistochemistry, and molecular analysis

Aims—Morphological criteria to distinguish between reactive and neoplastic B cell lymphocytoid infiltrates in trephines have been defined but are not always reliable. Polymerase chain reaction (PCR) analysis of the CDR3 region of the immunoglobulin heavy chain (IgH) gene which, by demonstrating monoclonality, can provide additional arguments in favour of lymphoid malignancy is now frequently used for the detection and follow up of B cell lymphoma (NHL). The aim of this study was to investigate the usefulness of morphological findings in bone marrow biopsies in comparison with data obtained by PCR analysis. Methods—Eighty nine bone marrow biopsies displaying lymphoid infiltrates were evaluated by morphology and immunohistochemistry as well as by CDR3-PCR using consensus framework 3 (FRW3) and JH primers. Results—The presence of a clonal B cell proliferation was demonstrated by PCR analysis in 45 biopsies, including 21 samples considered to be positive, 17 to be suspicious, and seven to be negative by morphology. In the remaining 44 trephines we found no evidence of clonality, although 12 of these trephines were thought to be positive by morphology. Conclusions—These results, revealing an incomplete correlation between CDR3-PCR data and immunomorphological findings, indicate that molecular analysis may be more sensitive and specific in general. However, false negative PCR results do occur, which emphasises the necessity to combine both diagnostic tools in the evaluation of lymphoid infiltrates.

Application of the International Prognostic Scoring System for myelodysplastic syndromes

BACKGROUND In March 1997 an international workshop introduced a new International Prognostic Scoring System (IPSS) for MDS. The goal of the present study was to apply the IPSS to a large group of MDS patients from one centre and to compare it to the FAB-classification. PATIENTS One hundred eighty-four MDS patients were included on the basis of similar criteria as used by the workshop but some of them (30) received AML-type therapy. RESULTS The IPSS separated our patients into distinctive prognostic subgroups (P = 0.0001). Median survival was respectively 6.5, 2.6, 1.3 and 0.75 years for the low-risk (22% of patients), the intermediate-1-risk (INT-1) (46%), the intermediate-2-risk (INT-2) (25%) and the high-risk group (7%). The IPSS also discriminated within each of the FAB-categories: RA patients (58 patients) were present in low-risk, INT-1-risk and INT-2-risk subgroups, RARS patients (23) were separated into low-risk and INT-1-risk subgroups. RAEB patients (53) were distributed predominantly between INT-1-risk and INT-2-risk groups, RAEB-t patients (23) between INT-2-risk and high-risk subgroups. CMML patients (27) were present in the low-risk, the INT-1-risk and the INT-2-risk group. CONCLUSIONS Our results confirm the effectiveness of the IPSS in predicting clinical outcome in MDS patients and indicate that it is an improved method compared to the FAB-classification.

Among diffuse large B-cell lymphomas, T-cell-rich/histiocyte-rich BCL and CD30+ anaplastic B-cell subtypes exhibit distinct clinical features

BACKGROUND The EORTC clinical trial 20901, activated in 1990, was designed to treat non-Hodgkins lymphomas (NHL) of intermediate/high-grade malignancy according to the Working Formulation. Established in 1994, the R.E.A.L. Classification on NHL has now replaced all former classifications. PATIENTS AND METHODS We reanalysed all cases (n = 273) documented by material available for review according to the R.E.A.L. Classification. In addition, we subdivided cases recognised as diffuse large B-cell lymphoma (DLBCL) into three morphologically distinct categories, namely, large cleaved DLBCL (LC-DLBCL), T-cell-rich/histiocyte-rich B-cell lymphoma (T-cell-rich/histiocyte-rich BCL) and CD30+ DLBCL with anaplastic cell features (CD30+ DLBCL). Finally, T/NULL anaplastic large-cell lymphoma (ALCL) cases were subdivided into ALK+ and ALK- lymphomas. Review was performed independently by two pathologists from two different centres. RESULTS DLBCL (61%), T/NULL ALCL (15%) and mantle-cell lymphoma (MCL, 50%) were the main NHL categories represented in the study. Fifty-seven of one hundred sixty DLBCL cases were further subclassified as LC-DLBCL (33 cases), T-cell-rich/histiocyte-rich BCL (13 cases) or CD30+ DLBCL (11 cases). The remaining cases were indicated as unspecified DLBCL. A clinico-pathological correlation confirmed the findings of previous studies suggesting that MCL, DLBCL and ALCL represent distinct entities with MCL being characterised by a short survival, in contrast with the longer survival and less frequent progression typical of ALK+ compared to ALK- ALCL. Within DLBCL, T-cell-rich/histiocyte-rich BCL showed distinctive features at presentation whereas CD30+ DLBCL showed a trend towards a more favourable prognosis, that might be comparable to that of ALK+ ALCL. CONCLUSIONS Our data further support the usefulness of the R.E.A.L. Classification and illustrate the feasibility of DLBCL subtyping. Moreover, our results demonstrate the distinct clinical characteristics of T-cell-rich/histiocyte-rich BCL and CD30+ DLBCL with anaplastic cell features suggesting that they may represent clinico-pathologic entities.

Guidelines for subtyping small B-cell lymphomas in bone marrow biopsies

Abstract In this review, we summarise the patterns of bone marrow involvement by small B-cell lymphomas. Both our own experience and the literature reports on the subject show that each subtype of lymphoma can be recognised from a distinct combination of a suggestive growth pattern and a particular cytological composition. A predominantly paratrabecular infiltrate composed of centrocytes is characteristic of follicle centre cell lymphoma. In mantle cell lymphoma, prominent intertrabecular nodules, each consisting of a monotonous proliferation of small to intermediate-sized lymphoid cells with an irregular nucleus, are the most frequent finding. Marginal zone cell lymphoma displays similar intertrabecular nodules, but the infiltrates are rather loose and polymorphic, whereas the lymphoid cells exhibit monocytoid features. Diffuse infiltrates composed of small lymphocytes with clumped chromatin, of plasma cells with Dutcher bodies and of mast cells are observed in most cases of lymphoplasmacytoid lymphoma/immunocytoma. Although chronic lymphocytic leukaemia / small lymphocytic lymphoma can present with a comparable pattern of bone marrow involvement, an interstitial infiltrate of small lymphoid cells is usually observed. A comparable interstitial pattern also prevails in hairy cell leukaemia. This lymphoma subtype, however, can be readily identified by the abundant clear cytoplasm of the neoplastic cells, erythrocyte extravasation and associated abnormalities in the haematopoietic series.

Chromosomal gains and losses are uncommon in hairy cell leukemia: a study based on comparative genomic hybridization and interphase fluorescence in situ hybridization

In contrast to other subtypes of lymphoproliferative malignancies, the genetic mechanisms underlying the pathogenesis of hairy cell leukemia (HCL) are unknown. We studied densely infiltrated splenic tissue of 14 cases of HCL for the presence of chromosomal gains and losses by comparative genomic hybridization (CGH). Chromosomal imbalances were detected in only four of the 14 cases. Chromosomal gains involved the regions 5q13-q31 (two cases) and 1p32-p36.2 (one case). A loss of the region 11q14-q22 was found in one additional patient. The imbalances affecting the regions 5q and 11q were confirmed by interphase fluorescence in situ hybridization (FISH) using PAC clone 144G9 (5q31) and YAC clones 755B11 (11q22.3-q23.1) and 801E11 (11q22.3-q23.1 spanning the ATM gene) and occurred in 61% to 75% of analyzed nuclei. The latter DNA probes and probes hybridizing to chromosomal regions, which are frequently deleted in other subtypes of non-Hodgkin lymphomas (NHL), namely 9p21/ P16(INK4A), 13q14/D13S25, and 17p13/P53 were subsequently applied to all 14 cases of HCL, but no additional abnormalities were found. We conclude that overrepresentation of chromosome 5 represents a recurrent aberration in HCL and that the commonly overrepresented region resides in 5q13-q31. Chromosomal imbalances including deletions of the tumor suppressor gene loci 9p21/P16(INK4A), 13q14/D13S25, and 17p13/P53 rarely occur in HCL in contrast to some other subtypes of B-cell NHL. The pathogenetic role of 11q/ATM alterations in HCL remains to be determined.