Chris De Wolf-Peeters

Keratin immunohistochemistry in normal human liver. Cytokeratin pattern of hepatocytes, bile ducts and acinar gradient

A panel of 2 polyclonal and 7 monoclonal antibodies directed against cytokeratins was tested on cryostat and paraffin sections of 14 normal human liver biopsies using an immunoperoxidase procedure. The staining characteristics of hepatocytes and bile ducts are reported. On cryostat sections, monoclonal antibodies directed against individual cytokeratins no8 and no18 stained both bile ducts and hepatocytes, whereas monoclonals anti-cytokeratin no7 and no19 exclusively stained bile ducts. The potential use of these 4 monoclonal antibodies in liver histopathology is briefly discussed. Monoclonal antibody anti-type II cytokeratins and the polyclonal rabbit anti-human keratin stained only bile ducts on both cryostat and paraffin sections. Using monoclonal antibody CAM 5.2 on paraffin sections, both bile ducts and parenchyma were positive. An acinar gradient was apparent in that zone 1 hepatocytes were more intensely stained. Moreover, a rim of hepatocytes around terminal hepatic venules and adjacent to subhepatic veins showed more intense staining. The same gradient could be seen in some paraffin sections stained with the monoclonals anti-cytokeratin no18 and KL1, and the rabbit polyclonal anti-keratin “wide spectrum screening”. The gradient is interpreted as reflecting quantitative differences in keratin content between hepatocytes. Polyclonal rabbit anti-human keratin is proposed as the most reliable antibody for identification of bile ducts in paraffin sections. The usefulness of reliable bile duct staining in several pathological conditions is emphasized.

S-phase kinase-associated protein 2 expression in non-Hodgkin's lymphoma inversely correlates with p27 expression and defines cells in S phase.

The protein expression of the cyclin-dependent kinase inhibitor p27 is often deregulated in human tumors. In lymphomas the inactivation of p27 is achieved through either increased degradation(1) or sequestration via D cyclins,(2) and p27 protein levels have been shown to have a prognostic significance.(1,3) Recently, S-phase kinase-associated protein 2 (Skp2) has been proved to mediate p27 degradation in normal cells(4-7) and to have oncogenetic properties.(8,9) In this study, B-, T-, and myeloid hematopoietic cell lines and a well-characterized panel of human lymphomas (n = 244) were studied for the expression of Skp2. In human lymphomas, the expression of Skp2 strongly related to the grade of malignancy, being low in indolent tumors and very high in aggressive lymphomas. Moreover, the percentages of Skp2- and S-phase-positive cells, as measured by DNA content or BrdU labeling, strictly matched and closely parallel that of Ki-67 and cyclin A. An inverse correlation between Skp2 and p27 was found in the majority of lymphoma subtypes. Nonetheless, most mantle cell lymphomas and a subset of diffuse large cell lymphomas failed to show this correlation, suggesting that alternative pathway(s) for the regulation of p27 might exist. The detection of Skp2 protein either by flow cytometry or by immunohistochemistry represents a simple method to precisely assess the S phase of lymphomas. The potential diagnostic and prognostic value of Skp2 is discussed.

Further characterization of morphologically defined typical and atypical CLL: a clinical, immunophenotypic, cytogenetic and prognostic study on 390 cases

We analysed a group of 390 patients, diagnosed with chronic lymphocytic leukaemia (CLL). Cases were subclassified as morphologically typical and atypical CLL according to the criteria of the FAB proposal. Typical CLL cases were mostly diagnosed at a low‐risk stage (Binet A/Rai 0), required no immediate treatment and expected a long survival; atypical CLL cases mostly presented at a more advanced risk stage (Binet B/Rai I–II), usually required immediate treatment and their survival was shorter. Moreover, clinical staging was of prognostic significance in typical but not in atypical cases.  In typical CLL, del(11q) was the most common chromosomal abnormality (21%) whereas in atypical CLL trisomy 12 was found in about 65% of the cases documented with an abnormal karyotype. Although chromosomal abnormalities were associated with a poor survival in typical CLL, they are of no prognostic significance in atypical CLL.  Based on these data, we conclude that subtyping CLL by morphology enables the identification of two groups of cases, each characterized by a specific clinical presentation, different cytogenetic abnormalities and prognostic parameters. We speculate that these two groups may represent two related, but different, diseases with different prognostic parameters and a different survival.

Translocations targeting CCND2, CCND3, and MYCN do occur in t(11;14)-negative mantle cell lymphomas

The genetics of t(11;14)(q13;q32)/cyclin D1-negative mantle cell lymphoma (MCL) is poorly understood. We report here 8 MCL cases lacking t(11;14) or variant CCND1 rearrangement that showed expression of cyclin D1 (2 cases), D2 (2 cases), and D3 (3 cases). One case was cyclin D negative. Cytogenetics and fluorescence in situ hybridization detected t(2;12)(p11;p13)/IGK-CCND2 in one of the cyclin D2-positive cases and t(6;14)(p21;q32)/IGH-CCND3 in one of the cyclin D3-positive cases. Moreover, we identified a novel cryptic t(2;14)(p24;q32) targeting MYCN in 2 blastoid MCLs: one negative for cyclin D and one expressing cyclin D3. Interestingly, both cases showed expression of cyclin E. Notably, all 3 blastoid MCLs showed a monoallelic deletion of RB1 associated with a lack of expression of RB1 protein and monoallelic loss of p16. In sum-mary, this study confirms frequent aberrant expression of cyclin D2 and D3 in t(11;14)-negative MCLs and shows a t(11;14)-independent expression of cy-clin D1 in 25% of present cases. Novel findings include cyclin E expression in 2 t(11;14)-negative MCLs characterized by a cryptic t(2;14)(p24;q32) and identification of MYCN as a new lymphoma oncogene associated with a blastoid MCL. Clinically important is a predisposition of t(11;14)-negative MCLs to the central nervous system involvement.

Expression of the endothelin-B receptor in pigment cell lesions of the skin

Abstract. Endothelins (ETs) exert several functions in human melanocytes, including proliferation, dendrite formation, and melanin synthesis. Among the ET receptors, the non-selective endothelin-B (ETB) receptor is the major receptor in melanocytes and malignant melanoma (MM) cells. In spite of the important role of ETs and their receptors in the growth and differentiation of melanocytes, the distribution and expression levels of ETB receptors in tissue sections of benign and malignant pigment cell lesions is still unknown. We combined immunohistochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR) to study ETB receptor expression in benign and malignant pigment cell lesions and in normal skin. Immunohistochemistry on paraffin-embedded tissue sections of 159 cases revealed a significant increase in intensity of ETB receptor expression from common nevi over dysplastic nevi and primary MM to metastatic MM. Quantitative PCR using real-time detection on 75 samples confirmed the immunohistochemical results. These data add the ETB receptor to the growing list of tumor progression markers in MM and suggest that ETs play a role in the progression of MM in the skin.

Fluorescence in situ hybridization study of chromosome 7 aberrations in hepatosplenic T-cell lymphoma: Isochromosome 7q as a common abnormality accumulating in forms with features of cytologic progression

Hepatosplenic γδ T‐cell lymphoma (HSγδTCL) is a rare and aggressive subtype of peripheral T‐cell lymphoma that has been associated cytogenetically with the isochromosome 7q [i(7)(q10)]. The incidence of this aberration and its relevance to pathogenesis of HSγδTCL is still unknown. We investigated the status of chromosome 7 in 12 HSTCL cases, including nine with a typical γδ phenotype, one with a so‐called T‐cell receptor (TCR)–silent phenotype, and two with the variant αβ phenotype. We analyzed available fresh and archival material using a dual‐color interphase fluorescence in situ hybridization (FISH) approach with 7p and 7q probes. A significant population of cells with predominance of 7q signals was detected in 10 cases (eight γδ, one αβ, and one TCR silent), and two lymphomas did not show clonal 7p/7q signal imbalances. In four of 10 cases with chromosome 7 aberrations, a hybridization pattern indicative of the presence of one chromosome 7 and one i(7)(q10) was found. In four other cases, the configuration of signals (2x7p/3x7q) suggested the presence of the i(7)(q10) and additional structural aberrations involving the second chromosome 7. In two cases, including one αβ phenotypic variant, a variety of FISH patterns equivalent to two to five copies of i(7)(q10) or numerical and structural aberrations of second chromosome 7 has been detected. These findings support cytogenetic data pointing to a characteristic association of i(7)(q10) with HSTCL, irrespective of the immunophenotype of malignant cells. An increased number of 7q signals was found in three cases with cytologic features of progression, indicating a tendency of HSTCL to multiply the i(7)(q10) chromosome during evolution.

Plasmacytoid T cells: a cell population normally present in the reactive lymph node. An immunohistochemical and electronmicroscopic study.

Plasmacytoid T cells (PTCs) are medium-sized cells characterized by abundant rough endoplasmic reticulum. They occur in the thymic-dependent area in human lymph nodes. PTCs are hardly identified in routinely stained sections. We studied their occurrence in 100 reactive lymph nodes with the use of monoclonal antibodies MB2, MT1, LN1, LN2, reactive on paraffin-embedded tissue, and with electron microscopy in nine selected cases. PTCs strongly reactive with MT1 and LN2 were found in 87 of 100 lymph nodes. They were observed in clusters, loose aggregates, and as singular cells. An association between PTCs, postcapillary venules, small T lymphocytes, and interdigitating reticulum cells (IDRCs) was found. Our results indicate that PTCs are normally present in the human lymph node. Their immunophenotype suggests a relationship with a monocyte/macrophage lineage, but does not rule out a T cell origin. If the various distribution patterns represent the morphologic substrate of functional stages of PTCs it can be assumed that PTCs play a role in T cell-mediated immune response.

JAK2 rearrangements, including the novel SEC31A-JAK2 fusion, are recurrent in classical Hodgkin lymphoma

The genetics of classical Hodgkin lymphoma (cHL) is poorly understood. The finding of a JAK2-involving t(4;9)(q21;p24) in 1 case of cHL prompted us to characterize this translocation on a molecular level and to determine the prevalence of JAK2 rearrangements in cHL. We showed that the t(4;9)(q21;p24) leads to a novel SEC31A-JAK2 fusion. Screening of 131 cHL cases identified 1 additional case with SEC31A-JAK2 and 2 additional cases with rearrangements involving JAK2. We demonstrated that SEC31A-JAK2 is oncogenic in vitro and acts as a constitutively activated tyrosine kinase that is sensitive to JAK inhibitors. In vivo, SEC31A-JAK2 was found to induce a T-lymphoblastic lymphoma or myeloid phenotype in a murine bone marrow transplantation model. Altogether, we identified SEC31A-JAK2 as a chromosomal aberration characteristic for cHL and provide evidence that JAK2 rearrangements occur in a minority of cHL cases. Given the proven oncogenic potential of this novel fusion, our studies provide new insights into the pathogenesis of cHL and indicate that in at least some cases, constitutive activation of the JAK/STAT pathway is caused by JAK2 rearrangements. The finding that SEC31A-JAK2 responds to JAK inhibitors indicates that patients with cHL and JAK2 rearrangements may benefit from targeted therapies.

Large Cleaved and Immunoblastic Lymphoma May Represent Two Distinct Clinicopathologic Entities Within the Group of Diffuse Large B-Cell Lymphomas

PURPOSE The reliability of immunohistochemistry for subdividing diffuse large B-cell lymphomas (DLBCL) into germinal center B-cell-like (GCB) and non-GCB prognostic subgroups is debated. In this study we evaluated the prognostic significance of such subgrouping on a series of 153 DLBCL patients. Furthermore, we investigated whether both subgroups could comprise clinicopathologic entities recognized by their morphology and characterized by a distinct phenotype, specific genetic abnormalities, and clinical characteristics. PATIENTS AND METHODS All samples from patients were reviewed and morphologically subdivided into large cleaved, immunoblastic, and not otherwise specified DLBCL. GCB and non-GCB immunohistochemical profiles were established. The presence of chromosomal translocations involving BCL2, BCL6, and MYC and/or rearrangements of these genes was investigated. RESULTS Subdividing DLBCL with either a GCB or non-GCB immunophenotypic profile was not of prognostic significance. Nevertheless, CD10 expression was a predictor of favorable outcome, whereas high bcl-2 expression and BCL6 rearrangement were adverse predictors of disease-free survival. Interestingly, large cleaved DLBCL was clearly associated with a GCB immunophenotypic profile, CD10 expression, BCL2 rearrangement, age younger than 60 years, and low to low/intermediate International Prognostic Index risk, but was not of prognostic significance. In contrast, immunoblastic morphology was associated with a non-GCB profile and was a significant predictor of unfavorable DFS. CONCLUSION Subdividing DLBCL into subgroups based on their immunohistochemical profile was not of prognostic significance. Nevertheless, it allowed the additional characterization of two lymphoma subgroups previously recognized in the Working Formulation. Both correspond to two distinct clinicopathologic entities within the DLBCL.

ALK-positive large B-cell lymphomas with cryptic SEC31A-ALK and NPM1-ALK fusions

We report 2 ALK-positive large B-cell lymphoma cases showing granular cytoplasmic and cytoplasmic/nuclear ALK immunostaining in which cryptic ALK rearrangements were identified by fluorescent in situ hybridization and molecular analysis. In the first case, the ALK-involving t(2;3)(p23;q27) masked the cryptic SEC31A-ALK fusion generated by an insertion of the 5′ end of SEC31A (4q21) upstream of the 3′ end of ALK. This rearrangement was associated with loss of the 5′ end of ALK and duplication of SEC31A-ALK on der(20). In the second case with complex rearrangements of both chromosomes 2, a submicroscopic NPM1-ALK fusion created by insertion of the 3′ end of ALK into the NPM1 locus was evidenced. Further studies of SEC31A-ALK showed that this variant fusion transforms IL3-dependent Ba/F3 cells to growth factor independence, and that the ALK inhibitor TAE-684 reduces cell proliferation and kinase activity of SEC31A-ALK and its downstream effectors ERK1/2, AKT, STAT3 and STAT5.