Cathepsin-k as a diagnostic marker in the identification of micro-granulomas in Crohn's disease.

Crohn's disease is a chronic inflammatory bowel disease, whose aetiology and pathogenesis are still unknown. The occurrence of epithelioid granulomas is one characteristic feature of the disease since these lesions are found in the bowel wall in 50-87% of colectomy specimens. Although granulomas are not pathognomonic, their identification is considered a relevant element for diagnosis. Cathepsin-k, a papain-like cysteine protease, is involved in bone remodelling, and has been widely used as a immunohistochemical marker for the in situ detection of osteoclasts. Interestingly, the expression of this potent protease is also significantly increased in stimulated tissue macrophages, epithelioid cells and granulomas, but is not expressed in resident tissue macrophages. In the present study, we evaluated Cathepsin-k expression as a diagnostic tool in the identification of small granulomas in Crohn's disease. Formalin-fixed and paraffin-embedded samples of 10 cases of Crohn's disease were collected from surgical ileo-colic resections followed by comparison of Cathepsin-k and CD68 immunoreactivity. Granulomas were identified in 4 of 10 cases examined in haematoxylin & eosin preparations. Cathepsin-k enabled the identification of small granulomas (with a diameter between 100 and 200 microm) in 6 of 10 cases, mainly localized within the submucosa and muscular layers. When compared to CD68, Cathepsin-k immunoreactivity was generally absent or only weakly expressed in resting tissue macrophages, thus allowing better identification of activated epithelioid cells. Based on these results, Cathepsin-k appears to be a reliable tool for the precise and rapid identification of small epithelioid granulomas in Crohn's disease.

Fluorescent cytogenetics of renal cell neoplasms.

Renal cell neoplasms are a heterogeneous group of tumours in terms of pathological features and prognostic behaviour. The genetics of these tumours may aid in correct diagnosis and accurate assessment of prognosis. In ambiguous cases it may be necessary to utilise new markers that are capable of further discerning renal cell neoplasms. Fluorescence in situ hybridization (FISH) on formalin-fixed, paraffin-embedded tissue is an increasingly useful technique in the detection of many diagnostic chromosomal abnormalities, among which chromosomes 1, 2, 3p, 6, 7, 10, 17 and Y are the most common. The addition of FISH to histological evaluation improves the diagnostic accuracy of core biopsies from renal masses, which may have an important impact in clinical management of many cases due to newer therapeutic approaches, including cryo- or radiofrequency ablation, nephron-sparing surgeries and target therapies.

Expression of transmembrane protein tyrosine phosphatase gamma (PTPgamma) in normal and neoplastic human tissues.

AIMS: To establish the conditions for protein tyrosine phosphatase gamma (PTPgamma) detection in paraffin tissues using two antibodies raised against its NH(2)- (anti-P4) and COOH-termini (gammaTL1); to analyse its expression in normal tissues and to perform an initial screening of neoplastic tissues. METHODS AND RESULTS: Membranous and/or cytoplasmic PTPgamma expression was detected in the majority of epithelial cell types and in endocrine cells, with the highest expression in adrenal medulla, endocrine cells of the gastrointestinal tract and pancreatic islets. Both antibodies stained the thyroid follicular epithelium, but only anti-P4 antibody stained the colloid matrix, suggesting shedding/secretion of the PTPgamma extracellular domain. Marked loss of PTPgamma immunoreactivity was detected in subsets of ovarian (21%), breast (56%) and lung (80%) neoplasms. Conversely, cytoplasmic positivity was found in 37% of lymphomas, mainly of high-grade histotypes, while normal lymphocytes were negative. Brain tissue showed PTPgamma expression in a few neuronal and glial elements and PTPgamma was overexpressed in the majority of high-grade astrocytomas. CONCLUSIONS: We have analysed PTPgamma expression in archival paraffin-embedded tissues for the first time, demonstrating particularly high expression in endocrine cells and both down- and up-regulation in neoplasia, the latter possibly reflecting the undifferentiated state of the neoplastic cells, suggesting a complex role for this phosphatase.

Independent prognostic value of fascin immunoreactivity in stage III-IV colonic adenocarcinoma.

Fascin, an actin-bundling protein involved in cell motility, has been shown to be upregulated in several types of carcinomas. In this study, we investigated the expression of fascin in 228 advanced colonic adenocarcinoma patients with a long follow-up. Fascin expression was compared with several clinicopathologic parameters and survival. Overall, fascin immunoreactivity was detected in 162 (71%) tumours with a prevalence for right-sided tumours (P<0.001). Fascin correlated significantly with sex, tumour grade and stage, mucinous differentiation, number of metastatic lymph nodes, extranodal tumour extension, and the occurrence of distant metastases. Patients with fascin-expressing tumours experienced a shorter disease-free and overall survival in comparison with those with negative tumours, and fascin immunoreactivity emerged as an independent prognostic factor in the multivariate analysis. Moreover, patients with the same tumour stages could be stratified in different risk categories for relapse and progression according to fascin expression. Our findings suggest that fascin is a useful prognostic marker for colonic adenocarcinomas.

ZAP-70 expression, as detected by immunohistochemistry on bone marrow biopsies from early-phase CLL patients, is a strong adverse prognostic factor.

Zeta-associated protein-70 (ZAP-70), mostly assessed by flow-cytometry (FC), recently emerged as reliable prognostic factor in chronic lymphocytic leukaemia (CLL) at presentation. We evaluated ZAP-70 expression in 156 CLL patients by immunohistochemistry (IHC) on formalin-fixed bone marrow (BM) biopsies at diagnosis. At presentation, 117 patients (75%) were with Binet stage A, 27 (17%) stage B and 12 (8%) stage C. Median follow-up was 61 months (range 6-242). ZAP-70 was expressed in neoplastic lymphocytes of 69 patients (44%). Concordance between ZAP-70 by IHC and ZAP-70 by FC, immunoglobulin heavy chain variable genes (IGHV) mutational status and CD38 expression was found in 41/46 (89%), 41/49 (80%) and in 60/88 (68%) tested cases, respectively. ZAP-70 expression significantly correlated with advanced Binet stage (B-C), diffuse BM infiltration, increased lactate dehydrogenase (LDH) and beta2-microglobulin serum levels and lymphocyte doubling time <12 months. ZAP-70 positivity was significantly related to poorer time to progression (median 16 months vs 158 of ZAP-70-negative cases) (P<0.0001) and overall survival (median 106 months vs not reached) (P=0.0002); this correlation was confirmed at multivariate analysis. ZAP-70 expression correlated with poorer outcome also when evaluated only in the 117 stage A patients. In conclusion, immunohistological detection of ZAP-70 on formalin-fixed BM biopsies at diagnosis appears a useful methodological approach to identify patients with poor prognosis in CLL.

[The Haemolymphopathology Italian Group (H.I.G.): an essential resource for the new technical and organization problems troubling modern haemolymphopathology diagnostics]. / II Gruppo Italiano di Emolinfopatologia (G.I.E.): una esigenza per le nuove e complesse problematiche tecnico-organizzative della moderna diagnostica emolinfopatologica.

Recently, many progresses have been recorded in the molecular and histogenetic characterization of the haematopoietic and lymphoid tumours, resulting in important classifying changes. As a consequence, the exact definition of lymphoma subtype requires an integration between traditional morphologic "expertise" and several bio-functional data obtained from advanced and complex ancillary techniques (immunohistochemistry, molecular biology and cytogenetics). At the same time, the data provided by gene expression profiling studies are going to deeply modify the therapies in haematological cancers. These studies are expected to allow the achievement of single-patient-tailored genic therapy; for this reason it is necessary to get biological samples of good quality. Indeed, while these progresses contribute to highlight the pathologist's diagnostic role, they should make us reflect on the state of the art of the Italian haemolymphopathology diagnostics and on its ability to cope up with the new challanges. The aim of this article is to outline a realistic picture of the present condition, and to explain the reasons for setting up, inside SIAPEC-IAP, the Haemolymphopathology Italian Group (H.I.G.). The purpose of H.I.G. will be twofold: first of all, scheduling of a series of projects so as to the haemolymphopathological diagnostic standardization; secondly, building a national network among all the pathologists involved in this exciting and complex field of the anatomic pathology.

Insights into the multistep transformation process of lymphomas: IgH-associated translocations and tumor suppressor gene mutations in clonally related composite Hodgkin's and non-Hodgkin's lymphomas.

Clonally related composite lymphomas of Hodgkin's lymphoma (HL) and Non-Hodgkin's lymphoma (NHL) represent models to study the multistep transformation process in tumorigenesis and the development of two distinct tumors from a shared precursor. We analyzed six such lymphomas for transforming events. The HLs were combined in two cases with follicular lymphoma (FL), and in one case each with B-cell chronic lymphocytic leukemia, splenic marginal zone lymphoma, mantle cell lymphoma (MCL) and diffuse large B-cell lymphoma (DLBCL). In the HL/FL and HL/MCL combinations, BCL2/IGH and CCND1/IGH translocations, respectively, were detected in both the HL and NHL. No mutations were found in the tumor suppressor genes FAS, NFKBIA and ATM. The HL/DLBCL case harbored clonal replacement mutations of the TP53 gene on both alleles exclusively in the DLBCL. In conclusion, we present the first examples of molecularly verified IgH-associated translocations in HL, which also show that BCL2/IGH or CCND1/IGH translocations can represent early steps in the pathogenesis of composite HL/FL or HL/MCL. The restriction of the TP53 mutations to the DLBCL in the HL/DLBCL case exemplifies a late transforming event that presumably happened in the germinal center and affected the fate of a common lymphoma precursor cell towards development of a DLBCL.

Primary mediastinal large B-cell lymphoma (PMLBCL): long-term results from a retrospective multicentre Italian experience in 138 patients treated with CHOP or MACOP-B/VACOP-B.

The optimal treatment of primary mediastinal large B-cell lymphoma (PMLBCL) is still undefined. In the absence of randomised studies, we retrospectively analysed: (a) the effectiveness of two chemotherapy regimens (CHOP vs MACOP-B/VACOP-B) in complete remission (CR) achievement and event-free survival (EFS) and (b) the role of mediastinal involved-field radiotherapy (IF-RT) as consolidation. From 1982 to 1999, 138 consecutive patients affected by PMLBCL were treated in 13 Italian institutions with CHOP (43) or MACOP-B/VACOP-B (95). The two groups of patients were similar as regard to age, gender, presence of bulky mediastinal mass, pleural effusion, stage and international prognostic indexes category of risk. Overall, 75.5% of patients in CR received IF-RT as consolidation. Complete remission was 51.1% in the CHOP group and 80% in MACOP-B/VACOP-B (P<0.001). Relapse occurred in 22.7% of CHOP- and in 9.2% of MACOP-B/VACOP-B-treated patients (n.s.). Event-free patients were 39.5% in CHOP and 75.7% in the MACOP-B/VACOP-B group (P<0.001). The addition of IF-RT as consolidation improved the outcome, irrespectively of the type of chemotherapy (P=0.04). At a multivariate analysis, achievement of CR (P<0.0001) and type of CT (MACOP-B/VACOP-B) retained the significance for OS (P=0.008) and EFS (P=0.03). In our experience, MACOP-B/VACOP-B appears to positively influence OS and EFS in patients affected by PMLBCL, as compared to CHOP. Consolidation IF-RT on mediastinum further improves the outcome of CR patients.

Genetic alterations in primary mediastinal B-cell lymphoma: an update.

Primary mediastinal B-cell lymphoma (PMBL) is a distinct clinical entity among non-Hodgkin's lymphoma. The malignancy has received little attention from a standpoint of basic research due in part to its rarity. However, based on recent studies consistent trends are beginning to emerge regarding the molecular and chromosomal alterations commonly observed in this disease. By both CGH and AP-PCR, genetic gains involving chromosomes 2, 5, 7, 9p, 12, and Xq are among the most frequently observed events. From a molecular standpoint, alterations in the c-myc, p16(INK4) and p53 genes have been observed in up to 30% of cases. This information along with the well-established histological, immunological, and clinical features should convince the few remaining disbelievers that PMBL is a distinct pathological entity among non-Hodgkin's lymphomas.

Alteration of chromosome arm 6p is characteristic of primary mediastinal B-cell lymphoma, as identified by genome-wide allelotyping.

Five cases of primary mediastinal B-cell lymphoma (PMBL) each have been studied with 375 microsatellite markers from all 22 autosomes. Of the 151 genomic alterations among the 1,875 assays, only five were allelic losses. The remainder of the microsatellite alterations consisted of 114 allelic imbalances and 32 instabilities. Microsatellite alterations were found in all cases on chromosomal arms 6p and 9p. These allelic imbalances most likely are indicative of genetic amplification, a finding agreeing well with those of studies using either comparative genomic hybridization or arbitrarily primed polymerase chain reaction, in which amplification of chromosome arm 9p in PMBL has been found. The allelic imbalances on chromosome arm 6p always included marker D6S276 located at 6p21.3-p22.3, where the MHC class I genes reside. These allelic imbalances may be reflective of alterations in the expression of the MHC gene products, characteristic of PMBL. Allelic anomalies close to the MYB gene locus on 6q were detected in two cases and prompted the analysis of MYB rearrangements in a series of 30 lymphomas. One rearrangement was detected in one of 18 cases of PMBL and in none of 10 diffuse, large B-cell lymphomas and two T-cell lymphomas. Our genome-wide microsatellite analysis provides independent confirmation that PMBL is characterized by infrequent chromosomal losses and by frequent genetic alterations involving chromosomal arm 9p. For the first time, chromosomal arm 6p has been identified as a highly frequent target of genetic alterations in this tumor type. Finally, MYB may also be involved occasionally in PMBL pathogenesis.

Low expression of p27 and low proliferation index do not correlate in hairy cell leukaemia.

The molecular basis accounting for the peculiar clinical and biological features of hairy cell leukaemia (HCL) is currently unknown. Deregulation of cell cycle genes plays a significant role in oncogenesis and there is considerable evidence suggesting that Cdk inhibitors (Ckis) function as tumour suppressors. We and others have recently demonstrated low expression of Cki p27 in very aggressive neoplasms and high-grade lymphomas. To investigate whether HCL cases express normal p27 protein, as in other low-grade lymphomas with a low proliferation index, 58 cases of HCL were characterized using a sensitive biotin-streptavidin-immunoperoxidase technique and specific antibodies against p27. All HCL cases showed either no or very weak reactivity, in contrast to other types of low-grade B-cell lymphoma [22 cases of chronic lymphocytic leukaemia (CLL), 12 cases of gastric marginal B-cell lymphoma (MALT), 16 cases of follicular lymphomas and two cases of splenic marginal zone lymphomas]. To investigate the possible mechanism(s) accounting for the low p27 expression observed in hairy cells, multiple approaches were used. According to these molecular studies, low levels of p2 7 are not as a result of (1) increased ubiquitin-mediated degradation, (2) decreased levels of p27 transcription or (3) p27 somatic mutations and/or allelic loss. These findings suggest that low p27 protein expression in HCL may be achieved through post-transcriptional regulation. Finally, our data demonstrate that p27 expression in HCL does not correlate with either cell cycle progression or proliferation index, suggesting that low levels of p27 in hairy cells may be associated with their unique stage of B-cell differentiation and/or the activation of as yet unknown pathways.

Molecular features of primary mediastinal B-cell lymphoma: involvement of p16INK4A, p53 and c-myc.

Primary mediastinal B-cell lymphoma (PMBL) shows chromosome 9p anomalies in 50% of cases. Based on reports that p16INK4A gene, located on this chromosomal arm, is frequently altered in aggressive lymphomas, we analysed for alterations of this gene in 27 cases of PMBL, which were part of a series of 32 PMBL cases that have been characterized for alterations in c-myc, p53, N-ras, bcl-1, bcl-2, bcl-6 and for Epstein-Barr virus (EBV) infection. Four cases showed p16INK4A gene anomalies, including three with promoter methylation and one homozygous deletion. Eight PMBLs showed c-myc rearrangements. Three additional cases showed sequence variations in the c-myc P2 promoter, two of which consisted of the same germline variation involving a novel polymorphic XhoI site. Four tumours contained p53 gene mutations and three had clonal EBV infection. One case had a bcl-6 rearrangement. In conclusion, our study shows that p16INK4, c-myc and p53 alterations occur in 15%, 25% and 13% of PMBLs, respectively. EBV monoclonality was found in 9% of cases, whereas no abnormality was detected in bcl-1, bcl-2 and N-ras. Thus, none of the common genetic aberrations seen in other types of non-Hodgkin's lymphomas appears to be stringently involved in the pathogenesis of this unique lymphoma type.

Nonrandom chromosomal imbalances in primary mediastinal B-cell lymphoma detected by arbitrarily primed PCR fingerprinting.

We used arbitrarily primed polymerase chain reaction (AP-PCR) fingerprinting to identify chromosomal imbalances in six primary mediastinal B-cell lymphomas (PMBLs). Seventy-four chromosomal imbalances were detected, consisting of 49 sequence gains and 25 losses. Amplifications on chromosome X were seen in five cases, four of which involved the same chromosomal locus. Nonrandom gains at the same locus were also identified on chromosomes 2 and 7 in four cases and on chromosomes 5, 9, and 12 in three cases. Five PMBLs were also analyzed by comparative genomic hybridization (CGH), which found chromosome arm 9p amplification as the only nonrandom imbalance. Our data demonstrate that chromosomal amplifications outnumber losses in PMBL. These mainly involve chromosomes 9 and X and may reflect more complex phenomena, such as translocations or other chromosomal rearrangements, as AP-PCR found coexistent gains and losses on these chromosomes. Comparison between AP-PCR and CGH suggests that anomalies affecting the same chromosomal regions may occur at much higher frequencies than expected by CGH, suggesting that genomic amplifications are usually confined to DNA segments smaller than the megabase long segments required for detection in CGH. Modest increases in genetic material may be as effective as higher-level amplifications when affecting sites where a proto-oncogene resides.

Correlation of poly(ADP-ribose)polymerase and p53 expression levels in high-grade lymphomas.

The steady-state levels of mRNA for the poly(ADP-ribose)polymerase (PARP), c-myc, p53, and histone H3 genes were investigated in 31 high-grade B-cell lymphomas by northern blot analysis. The panel included 15 nodal large B-cell lymphomas, nine mediastinal large B-cell lymphomas, and seven sporadic Burkitt's lymphomas. The PARP mRNA level was significantly higher in lymphomas than in control tissues and corresponded with the amount of PARP protein, as assessed by immunoblot analysis in six samples. The level of PARP mRNA was positively correlated with that of p53 mRNA. No correlation was found between the mRNA expression levels of PARP and histone H3, suggesting that PARP expression levels are independent of the proliferation rate of neoplastic cells. In this setting, the strong correlation between PARP and p53 suggests that the high expression of PARP may be associated with ongoing DNA damage in high-grade lymphomas.

ALK+ lymphoma: clinico-pathological findings and outcome.

A distinct pathologic entity (ALK+ lymphoma) that is characterized by expression of the anaplastic lymphoma kinase (ALK) protein has recently emerged within the heterogeneous group of CD30(+) anaplastic large-cell lymphomas. Information on clinical findings and treatment outcome of ALK+ lymphoma is still limited, and no data are available concerning the value of the International Prognostic Index when applied to this homogeneous disease entity. To clarify these issues, a recently developed monoclonal antibody ALKc (directed against the cytoplasmic portion of ALK) was used to detect expression of the ALK protein in paraffin-embedded biopsies from 96 primary, systemic T/null anaplastic large-cell lymphomas, and the ALK staining pattern was correlated with morphological features, clinical findings, risk factors (as defined by the International Prognostic Index), and outcome in 78 patients (53 ALK+ and 25 ALK-). Strong cytoplasmic and/or nuclear ALK positivity was detected in 58 of 96 ALCL cases (60.4%), and it was associated with a morphological spectrum (common type, 82.7%; giant cell, 3.5%; lymphohistiocytic, 8. 6%; and small cell, 5.2%) that reflected the ratio of large anaplastic elements (usually showing cytoplasmic and nuclear ALK positivity) to small neoplastic cells (usually characterized by nucleus-restricted ALK expression). Clinically, ALK+ lymphoma mostly occurred in children and young adults (mean age, 22.01 +/- 10.87 years) with a male predominance (male/female [M/F] ratio, 3.0) that was particularly striking in the second-third decades of life (M/F ratio, 6.5) and usually presented as an aggressive, stage III-IV disease, frequently associated with systemic symptoms (75%) and extranodal involvement (60%), especially skin (21%), bone (17%), and soft tissues (17%). As compared with ALK+ lymphoma, ALK- cases occurred in older individuals (mean age, 43.33 +/- 16.15 years) and showed a lower M/F ratio (0.9) as well as lower incidence of stage III-IV disease and extranodal involvement at presentation. Overall survival of ALK+ lymphoma was far better than that of ALK- anaplastic large-cell lymphoma (71% +/- 6% v 15% +/- 11%, respectively). However, within the good prognostic category of ALK+ lymphoma, survival was 94% +/- 5% for the low/low intermediate risk group (age-adjusted International Prognostic Index, 0 to 1) and 41% +/- 12% for the high/high intermediate risk group (age-adjusted International Prognostic Index, >/=2). Multivariate analysis identified ALK expression and the International Prognostic Index as independent variables that were able to predict survival among T/null primary, systemic anaplastic large-cell lymphoma. Thus, we suggest that such parameters should be taken into consideration for the design of future clinical trials.

CD138/syndecan-1: a useful immunohistochemical marker of normal and neoplastic plasma cells on routine trephine bone marrow biopsies.

Detection of abnormal numbers and/or distribution of bone marrow (BM) plasma cells (PCs) on trephine biopsies can be important in the differential diagnosis of multiple myeloma (MM) and other PC disorders. A variety of immunohistochemical markers can potentially improve the specificity and sensitivity of PC detection on routine histological sections obtained from trephine BM biopsies, but most of them are not completely satisfactory. In this study, we investigated whether the antibody CD138/B-B4, which is an optimal marker for PC detection on BM aspirates by flow cytometry, can be used successfully for the identification of PCs also on formalin-fixed, decalcified biopsies. A series of samples including normal BM [12], MM [65], monoclonal gammopathies of undetermined significance [44], and B-cell lymphoma of various types [94], including B-cell precursor lymphoblastic leukemia [9], lymphoplasmacytoid [17], immunoblastic [14], lymphocytic/CLL [23], hairy cell leukemia [4], large B-cell [8], mantle-cell [3], marginal zone [6] and follicular [10] lymphomas, have been investigated for CD138 expression using a sensitive immunohistochemical technique. Within the BM microenvironment, CD138 was characterized by excellent sensitivity and specificity. Virtually all normal and neoplastic PCs expressed clear-cut membrane CD138 immunostaining, whereas all other cell types did not. All cases of MM, including plasmablastic and leukemic cases, showed strong immunoreactivity. Conversely, all B-cell lymphomas, including all cases characterized by secretive features, lymphoplasmacytoid, and immunoblastic lymphomas, were completely negative. These results demonstrate that CD138 is a highly sensitive and specific marker that is useful for the rapid and precise localization of normal and neoplastic PCs on routine BM sections. In addition, because of its clear-cut cell membrane localization, CD138 can be used successfully in double-marker immunostaining reactions to evaluate precisely nuclear prognostic markers such as Ki67 and p53 in MMs.