Prolegomenon for chronic lymphocytic leukaemia.

Chronic lymphocytic leukaemia (CLL) is a unique lymphoproliferative disorder that scarcely occurs under the age of 40; thereafter the incidence of CLL increases exponentially with age. CLL is characterized by progressive expansion of malignant CD5+ME+ B-cell clone accompanied by a myriad of cellular and humoral immune defects. Each of them might be linked to different clinically manifested complications such as increasing rate of infections, autoimmune disorders and disturbed immune surveillance against tumour cells. We assume that CLL occurs as a consequence of age-dependent, genetically related functional restrictions of the thymic microenvironment in supporting common lymphoid progenitor cells (CD5+ME+CD4-CD8-) to differentiate into mature T-cell and B-cell descendants. In conjunction with genetic abnormalities developing in B-cell progenitors, presumably expressing P glycoprotein (Pgp+), we postulate that developmentally altered T-cell descendants, along with quantitative imbalance among CD4+, their subsets and CD8+ lymphocytes in the peripheral blood, play an important additional role in facilitating the malignant B-cell clone emergence and in modulating the CLL clinical evolution. Namely, imbalance of any of T-cell-mediated cell interactive homeostatic mechanisms accompanied by imbalance in the production of various cytokines might in CLL influence leukaemic B-cell growth by deregulating inducer (c-myc and p53) and/or suppressor (bcl-2 and mutant p53) oncogenes responsible for the promotion or suppression of B-cell mitogenesis that may in turn further contribute to their impaired differentiation and/or differentiation arrest. In conclusion, CLL might be interpreted as a primary immunodeficiency syndrome developing in elderly population due to gradually evolving restriction of genetically controlled programs in the thymic microenvironment responsible for irregular maturation of common lymphoid progenitor cells that constitutively express CD5 antigen and ME receptor into T-cell and B-cell descendants.

Molecular insight into the diagnosis of lymphoma.

Malignant lymphoma may be very difficult to diagnose with routine histopathological methods because they may recapitulate benign architecture or contain benign infiltrates. The best method of diagnosis is to establish the clonal profile of the lymphocyte population, since a monoclonal proliferation is highly suggestive of neoplasia. By means of a PCR (polymerase chain reaction) method it is possible to detect the immunoglobulin heavy chain (IgH) gene rearrangement and therefore establish the lymphocyte clonality. PCR with primers complementary to relatively conserved regions called frameworks (FR1-FR3) laying among hyper variable regions (CDR1-CDR3) of IgH gene unable us to detect monoclonal versus polyclonal B-cell population. The length of the PCR product with these primers is unique if we deal with a monoclonal population. On the contrary, a polyclonal population gives PCR products of a different size. In this retrospective study we used a semi-nested PCR to analyse 37 paraffin-embedded specimens. All of them had been evaluated previously by pathohistological and immunophenotypic criteria. A number of polyclonal (PBL and tonsils from healthy donors) and monoclonal cells (PBL from CLL patients, Raji cell line) were analyzed as controls. Clonality was successfully determined in all specimens. Our results support the concept that molecular techniques such as PCR provide a helpful approach for detection of monoclonal immunoglobulin rearrangements in malignant lymphoma. This is especially true for border cases, but always in the combination with other pathohistological methods.

Monoclonality in Helicobacter pylori-positive gastric biopsies: an early detection of mucosa-associated lymphoid tissue lymphoma.

Mucosa-associated lymphoid tissue (MALT) is not present in healthy gastric mucosa, but it can develop in sites of long-persisting inflammation and is connected with the development of MALT lymphoma. A monoclonal lymphocyte population is one of the characteristics of such lymphomas. In this study we analyzed gastric biopsies (formalin fixed and paraffin embedded or frozen) in 93 patients with dyspepsia accompanied by Helicobacter pylori infection. We applied PCR and single-cell immunocytochemistry to detect the clonality of the gastric B-cell population. Immunocytochemistry performed on 33 frozen biopsies showed two samples with monoclonal pattern. PCR analysis of immunoglobulin heavy-chain (IgH) gene rearrangements revealed two monoclonal populations out of 161 biopsies from 60 patients. We conclude that PCR analysis was the most sensitive method, which gave us insight into the nature of the earliest stage of MALT lymphoma in gastric biopsies.

Nested polymerase chain reaction for detection of hepatitis C virus RNA in blood derivatives.

Testing for the presence of hepatitis C virus (HCV) in blood derivatives used in clinical medicine is of enormous importance to ensure the production of safe preparations. So far, no system has been developed for the isolation and detection of hepatitis C virus from blood derivatives. The recently introduced commercial kit for the detection of hepatitis C virus is designed for the isolation and detection of virus from blood serum. A reliable and reproducible method for the isolation of hepatitis C virus RNA, subsequent reverse transcription and nested polymerase chain reaction (PCR) from blood derivatives is described. Of 17 batches of factor VIII, gamma-globulin and anti tetanus, cytomegalovirus and Varicella-zoster immunoglobulin concentrates, respectively (14 negative for anti HCV and 3 of unknown anti HCV status) five were found positive in RNA-PCR.

Immunohistochemical detection of TGF-alpha, EGF-R, c-erbB-2, c-H-ras, c-myc, estrogen and progesterone in benign and malignant human breast lesions: a concomitant expression.

The expression of transforming growth factor-alpha (TGF-alpha), epidermal growth factor receptor (EGF-R) and oncogenes c-erbB-2, c-H-ras, c-myc, as well as estrogen (ER) and progesterone (PR) receptors were studied immunohistochemically in the tissue of 21 benign and 58 malignant human breast lesions. Twenty nine (50%) of 58 carcinomas were positive for EGF-R and c-erbB-2 product, 55 (94.8%) for c-myc product, 9 (15.5%) for c-H-ras product and 17 (29%) for TGF-alpha. Eighteen of 58 (31%) carcinomas were estrogen receptor positive and 22 (38%) were positive for progesterone receptor. No correlation was found between expression of each investigated parameter and the clinical stage or degree of histological differentiation of the carcinomas. However, a significant positive correlation was observed between lymph node involvement and c-erbB-2 and EGF-R/c-erbB-2 positive tumors. A strong correlation was also observed between high levels of EGF-R and low levels of estrogen receptor. In 15 of 17 cases we found simultaneous expression of EGF-R and TGF-alpha. We also found interesting patterns in concomitant expression of the investigated parameters suggesting a possible cascade of events that occur in breast cancer cells.

Functional differences of T cells in B-chronic lymphocytic leukemia.

B cell chronic lymphocytic leukemia (B-CLL) is a disease characterized by an accumulation of monoclonal lymphocytes of B cell origin. Although the neoplastic process involves the B lymphocyte compartment, phenotypic and functional defects within the T lymphocyte population implicate their possible role in the pathogenesis of the disease. We analyzed the functional and morphological integrity of T lymphocytes from the peripheral blood of 64 patients with B-CLL. The activation of B-CLL T cells after PHA stimulation was determined by measuring [3H]-thymidine incorporation, assessing cell numbers in parallel cultures, and by monitoring the lymphocyte subsets during 9 days of cultivation. Our results indicate the presence of three functionally different populations of T cells in the peripheral blood of B-CLL patients. We present evidence for an increased proliferative potential of T lymphocytes from a group of patients with B-CLL.

Defects of natural killer cell activity in children with untreated acute lymphocytic leukemia.

Natural killer (NK) activity against cells of the K-562 line was significantly depressed in 12 of 18 children (66%) with untreated acute lymphocytic leukemia (ALL). No suppression of allogeneic NK activity was observed with sera of the patients, regardless of the level of NK depression. The ability of peripheral blood lymphocytes (PBLs) to suppress allogeneic NK activity was tested in two ALL patients - one with no detectable NK activity, and one with high NK activity. No NK-suppressive activity was found with PBLs of the areactive patient; PBLs of the reactive patients exhibited some suppressive activity, but only at a particular suppressor-to-effector cell ratio. Leukemic blasts were resistant to killing by autologous NK cells stimulated by IFN, as well as to killing by allogeneic, IFN-stimulated PBLs. Leukemic blasts of an ALL patient inhibited lysis of K-562 cells in an 18-h, but not in a 4-h NK assay. The inhibition could partly be reversed by pretreatment of ALL cells with alpha interferon, suggesting that the blasts might inhibit the lysis of K-562 targets in a competitive manner. Disturbed function and/or regulation of NK cells may influence attempts at NK cell activation by lymphokines.