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Aims: Peripheral T cell lymphoma not otherwise specified (PTCL/NOS) is the commonest PTCL subtype. Recently, proliferation pathways have been found to be commonly altered in PTCL/NOS. CDKN1B/p27, a critical regulator of cell cycle and proliferation, has been suggested to be involved in T cell lymphomagenesis. This study aimed to evaluate the possible occurrence of CDKN1B/p27 aberrations in PTCL/NOS., Methods: CDKN1B/p27 expression at RNA and protein level by DNA and tissue microarrays, in 28 and 98 cases, respectively, was studied. Additionally, direct sequencing of CDKN1B in 81 PTCL/NOS was performed., Results: CDKN1B mRNA was similarly expressed in PTCL/NOS and normal T lymphocytes. In addition, structural abnormalities were not found; these included mutations and deletions in any exons, exon-intron junctions or regulatory regions. Furthermore, physiological expression of p27 in neoplastic cells was demonstrated by immunohistochemistry; this was mutually exclusive with Ki-67, as expected when the system is intact. Consistently, the expression of other molecules that are functionally related to CDKN1B/p27 in controlling cell cycle (including CCNE1) did not appear to be affected at either the mRNA or protein level. Finally, it was found that p27 expression was not significantly related with overall survival., Conclusion: CDKN1B/p27 aberrations seem to be uncommon in PTCL/NOS pathogenesis.

Peripheral T cell lymphomas (PTCL) account for about 12% of lymphoid tumours worldwide. Almost half show such morphological and molecular variability as to hamper any further classification, and to justify their inclusion in a waste-basket category termed "not otherwise specified (NOS)". The latter term is used for neoplasms with aggressive presentation, poor response to therapy and dismal prognosis. In contrast to B cell lymphomas, PTCL have been the subject of only a limited number of studies to elucidate their pathobiology and identify novel pharmacological approaches. Herewith, the authors revise the most recent contributions on the subject based on the experience they have gained in the extensive application of microarray technologies. PTCL/NOS are characterised by erratic expression of T cell associated antigens, including CD4 and CD52, which have recently been proposed as targets for ad hoc immunotherapies. PTCL/NOS also show variable Ki-67 marking, with rates >80% heralding a worse prognosis. Gene expression profiling studies have revealed that PTCL/NOS derive from activated T lymphocytes, more often of the CD4+ type, and bear a signature composed of 155 genes and related products that play a pivotal role in cell signalling transduction, proliferation, apoptosis and matrix remodelling. This observation seems to pave the way for the use of innovative drugs such as tyrosine kinase and histone deacetylase inhibitors whose efficacy has been proven in PTCL primary cell cultures. Gene expression profiling also allows better distinction of PTCL/NOS from angioimmunoblastic T cell lymphoma, the latter being characterised by follicular T helper lymphocyte derivation and CXCL13, PD1 and vascular endothelial growth factor expression.

Background and Aims: Chronic lymphocytic leukaemia (CLL) is a frequent non-Hodgkin lymphoma characterised by a heterogeneous clinical course. Assessment of cell cycle phase kinetics might be important for prediction of clinical behaviour and prognosis., Methods: Distribution of neoplastic cells in CLL within the cell cycle was evaluated by determining the labelling indices (LI, i.e. percentage of positive cells) of markers specific for late G1-phase (cyclin E), S-phase (cyclin A), and G2/M-phase (cyclin B1), and Mcm2, a novel marker of proliferative potential, in a large cohort of patients (n = 79) using tissue microarray (TMA) technology. Utilising a combination of these markers, an algorithm was developed--subtracting the combined LIs of cyclin E, cyclin A and cyclin B1 from the LI of Mcm2--to determine the percentage of tumour cells residing in early G1-phase, which is probably a critical state for the malignant potential of CLL., Results: 27.11% of cells had acquired proliferative potential as indicated by expression of Mcm2. Only a small number of cells were found to be in late G1-phase (7.16%), S-phase (3.31%) or G2/M-phase (0.98%), while 15.66% of cells were considered to be in early G1-phase., Conclusion: Cell cycle phase distribution can easily be assessed by immunohistochemistry in routinely processed paraffin-embedded specimens. A large number of neoplastic cells in CLL have proliferative potential, with a significant sub-population residing in early G1-phase. Estimates of these cells may identify cases likely to exhibit a more aggressive biological behaviour and adverse clinical course.

Background: The use of tissue microarrays (TMAs) is now a generally accepted method for the investigation of solid tumours. However, little is known about the applicability of the TMA technique for analysis of patients with acute leukaemia. A bone marrow (BM)-TMA analysis with 15 different immunohistochemical markers was performed. The TMA was validated by comparison with the corresponding full tissue sections., Materials and Methods: A BM-TMA comprising 148 cases of acute leukaemia, including 115 acute myeloid leukaemia (AML) and 33 acute lymphoblastic leukaemia (ALL) cases, was constructed. Expression of CD3, CD10, CD15, CD20, CD34, CD61, CD68, CD79a, CD99, CD117, CD138, myeloperoxidase, haemoglobin A1, glycophorin and terminal deoxynucleotidyl transferase was immunohistochemically analysed. 50 cases of the TMA were directly compared with the corresponding full tissue section to validate the results., Results: Morphologically and immunohistochemically, 6 (4%) of 148 cases and 765 (11%) cores of 6912 individual analyses were not evaluable. A direct comparison of TMA cases with conventional full sections showed a concordance of the results of 100%., Conclusions: The small size of bone-marrow biopsies and the presence of bony trabeculae do not preclude construction and analysis of acute leukaemia TMAs. Acute leukaemia cases on TMA displayed the characteristic phenotypic profiles expected in different AML and ALL subtypes. Therefore, the TMA technique is also a promising method for high-throughput analysis of combined marker expression and clinicopathological correlations in patients with leukaemia.

Background/aims: Paediatric primary follicular lymphoma of the testis (PPFLT) is exceptional: the few reported cases seem to lack BCL-2 gene rearrangement and/or protein expression. The aim of this study was to characterise a PPFLT arising in a 4 year old boy., Methods: This case was characterised using conventional histological analysis, immunohistochemistry, and a polymerase chain reaction based method for the detection of immunoglobulin V(H) chain rearrangements., Results: The neoplasm was staged I(E)/A; left orchiectomy and chemotherapy were performed, producing complete remission. Histology showed a predominantly follicular lymphoid infiltrate mainly composed of centroblast-like cells. The phenotype was CD20(+), CD79a(+), CD10(+), bcl-6(+), B cell specific activating protein(+), kappa light chain(+), CD30(-/+), interferon regulating factor 4(-/+), c-myc(-/+), lambda light chain(-), CD3(-), bcl-2(-), p53(-), cytokeratin(-), and placental alkaline phosphatase(-). Lymphomatous elements were found within a CD21(+) follicular dendritic cell network and 70% were positive for Ki-67/MIB-1. Molecular analysis revealed monoclonal immunoglobulin heavy chain gene rearrangement and BCL-6 mutations, in the absence of BCL-2 major breakpoint and BCL-2 minor cluster region rearrangements, p53 mutations, and death associated protein kinase gene hypermethylation., Conclusions: These findings suggest a different pathogenesis of PPTFL compared with adult follicular lymphoma and might explain its favourable course in spite of aggressive histology.

Despite its well known histological and clinical features, Hodgkin's lymphoma (HL) has recently been the object of intense research activity, leading to a better understanding of its phenotype, molecular characteristics, histogenesis, and possible mechanisms of lymphomagenesis. There is complete consensus on the B cell derivation of the tumour in most cases, and on the relevance of Epstein-Barr virus infection and defective cytokinesis in at least a proportion of patients. The REAL/WHO classification recognises a basic distinction between lymphocyte predominance HL (LP-HL) and classic HL (CHL), reflecting the differences in clinical presentation and behaviour, morphology, phenotype, and molecular features. CHL has been classified into four subtypes: lymphocyte rich, nodular sclerosing, with mixed cellularity, and lymphocyte depleted. The borders between CHL and anaplastic large cell lymphoma have become sharper, whereas those between LP-HL and T cell rich B cell lymphoma remain ill defined. Treatments adjusted to the pathobiological characteristics of the tumour in at risk patients have been proposed and are on the way to being applied.

Serum cryoglobulins are found in a wide spectrum of disorders but are often transient and without clinical implications. Monoclonal cryoglobulins are usually associated with haematological disorders, whereas mixed cryoglobulins are found in many infectious and systemic disorders. So called essential mixed cryoglobulinaemia shows a striking association with hepatitis C virus (HCV) infection (> 90%). It is a systemic vasculitis (leucocytoclastic vasculitis) with cutaneous and multiple visceral organ involvement. Chronic HCV infection can lead to a constellation of autoimmune and neoplastic disorders. In this review, the aetiology, diagnosis, disease heterogeneity, and treatment of cryoglobulinaemia are discussed.

Aims: To investigate the expression of retinoblastoma protein (pRb) in invasive breast tumours and compare its expression with the major biopathological prognostic indicators to identify more aggressive subgroups., Material: Archival paraffin embedded tissues from 153 consecutive primary breast carcinomas., Methods: pRb, Ki-67, and oestrogen receptor/progesterone receptor proteins were identified by immunohistochemistry and score values were recorded by image cytometric analysis; p53 and EGFr expression was also evaluated., Results: pRb scores correlated strongly with proliferation activity as determined by Ki-67 staining. Positive relations were also observed between pRb scores, tumour size, nuclear and histological grade, and oestrogen receptor/progesterone receptor content, while abnormal p53 accumulation was not associated with pRb expression. Among the high proliferating carcinomas it was possible to identify 13 cases with loss of pRb expression., Conclusions: pRb expression paralleled proliferative activity in the majority of breast carcinomas examined, suggesting that in these cases the protein behaves normally in regulating the cell cycle. Conversely in cases with loss of pRb immunostaining, the combined expression of specific highly aggressive factors (EGFr and p53 expression, oestrogen receptor/progesterone receptor negative status, and high K67) seems to characterise a more aggressive phenotype showing growth advantage and cellular "progression" rather than significant nodal involvement.

Aim: To assess a newly developed immunohistochemical detection system, the EnVision++., Methods: A large series of differently processed normal and pathological samples and 53 relevant monoclonal antibodies were chosen. A chessboard titration assay was used to compare the results provided by the EnVision++ system with those of the APAAP, CSA, LSAB, SABC, and ChemMate methods, when applied either manually or in a TechMate 500 immunostainer., Results: With the vast majority of the antibodies, EnVision++ allowed two- to fivefold higher dilutions than the APAAP, LSAB, SABC, and ChemMate techniques, the staining intensity and percentage of expected positive cells being the same. With some critical antibodies (such as the anti-CD5), it turned out to be superior in that it achieved consistently reproducible results with differently fixed or overfixed samples. Only the CSA method, which includes tyramide based enhancement, allowed the same dilutions as the EnVision++ system, and in one instance (with the anti-cyclin D1 antibody) represented the gold standard., Conclusions: The EnVision++ is an easy to use system, which avoids the possibility of disturbing endogenous biotin and lowers the cost per test by increasing the dilutions of the primary antibodies. Being a two step procedure, it reduces both the assay time and the workload.