Biomedical subjects
Publications and source records attributed to Terry J Hamblin.
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Chronic lymphocytic leukemia is a frightening diagnosis which we believe is mistakenly applied to patients who will live their whole lives without symptoms referable to this diagnosis and never require treatment for it. Changes in the threshold of lymphocytosis required for the diagnosis have led to many more people being inappropriately diagnosed. In addition, modern prognostic factors have enabled us to predict with a fair degree of accuracy which patients presenting with a lymphocytosis will have a benign course and which will require treatment. We propose a new category of benign monoclonal lymphocytosis be recognized, better to reflect reality.
Autoimmune complications are common in chronic lymphocytic leukemia (CLL), occurring in up to a quarter of all patients during the course of the illness. By far the most common manifestation is autoimmune hemolytic anemia (AIHA), followed by immune thrombocytopenia (ITP). It is not true to say that autoimmunity is confined to the formed elements of the blood since conditions such as paraneoplastic pemphigus and acquired angioedema do occur in CLL, but nonhematologic autoimmunity is very rare indeed. The pathogenesis of autoimmunity in CLL is unknown. It may be related to the ability of the CLL cells to act as antigen-presenting cells (APCs), and to process antigen (particularly the Rh protein) so as to reveal cryptic peptides that are seen as foreign by helper T cells. It is likely that a failure of regulatory T-cell function is also involved. Autoimmune episodes may be triggered by treatment, particularly with purine analogues. Such episodes are often severe and may be fatal. Treatment of CLL-associated autoimmunity follows conventional protocols, but non-response to primary treatments is not uncommon. Promising results have been obtained with cyclosporine and rituximab.
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How to treat anemia in the myelodysplastic syndrome (MDS) is controversial. Many health care systems refuse to fund erythropoietin on grounds of health economics. A new paper on the effect of anemia in MDS on cardiac remodeling has suggested that a higher treatment threshold level for hemoglobin should be introduced. This would have an impact of the calculation of risk/benefits.
It has become clear that the heterogeneity of chronic lymphocytic leukaemia (CLL) is not a continuous spectrum, but is bipolar. Originally distinguished by the mutational status of the immunoglobulin variable region genes, the two poles are perhaps better identified by the expression of ZAP-70, a signalling molecule normally utilised by T cells rather than B cells, but anomalously expressed in the more aggressive subtype of CLL. Assaying ZAP-70 expression has become progressively simplified so that a directly stained flow cytometric test is currently being evaluated, and a version of this should shortly be available to routine laboratories. In addition, the understanding of the nature of CLL has been advanced rapidly and this should lead to new, better targeted therapies, which in contrast to the current armoury, will work better for the more malignant variants of CLL than for the more benign. In particular, ZAP-70 is especially attractive because its aberrant expression in tumour cells from the more aggressive forms of CLL requires the chaperoning action of activated heat-shock protein 90, which may be specifically inhibited.
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BACKGROUND: Chronic lymphocytic leukaemia (CLL) is a heterogeneous disease; many patients never need treatment, whereas some have poor outcomes. New treatments, which can induce complete remissions, allow patients with poor outlook to be treated while they are still asymptomatic. Whether or not the IgVH gene is mutated is the best predictor of clinical outcome, but this assay is unsuited to the routine laboratory. The gene coding for ZAP-70, a tyrosine kinase protein normally expressed in T and NK cells, has been shown by gene-expression profiling to be differentially expressed between patients with mutated and unmutated IgVH genes. We assessed whether ZAP-70 could be used as a prognostic marker in CLL. METHODS: We developed a flow cytometry assay for ZAP-70 protein expression and investigated its concordance with ZAP-70 mRNA expression, IgVH gene mutational status, and clinical outcome in 167 patients with CLL. FINDINGS: We showed high concordance between ZAP-70 protein expression and IgVH gene mutations. 108 patients (65%) had mutated IgVH genes and were ZAP-70 negative; 46 (28%) had unmutated IgVH genes and were ZAP-70 positive. Findings were discordant in 13 patients: six (4%) had mutated IgVH genes but were ZAP-70 positive, and seven (4%) had unmutated IgVH genes and were ZAP-70 negative. Expression of mRNA showed 97% concordance with ZAP-70 protein. Median survival was 24.4 years (95% CI 15.1-33.8) in ZAP-70 negative patients and 9.3 years (7.0-11.5) in those who were ZAP-70 positive (hazard ratio 5.5, 2.8-.8). INTERPRETATION: ZAP-70 protein, which can be measured by flow cytometry in the general laboratory, is a reliable prognostic marker in CLL, equivalent to that of IgVH gene mutational status.
The presence or absence of somatic mutations in the expressed immunoglobulin heavy chain variable regions (IgVH) of chronic lymphocytic leukemia (CLL) cells provides prognostic information. Patients whose leukemic cells express unmutated IgVH regions (Ig-unmutated CLL) often have progressive disease, whereas patients whose leukemic cells express mutated IgVH regions (Ig-mutated CLL) more often have an indolent disease. Given the difficulty in performing IgVH sequencing in a routine diagnostic laboratory, this prognostic distinction is currently unavailable to most patients. Pilot gene expression profiling studies in patients with CLL identified genes that were differentially expressed between the Ig-unmutated and Ig-mutated CLL subtypes. Here, we have profiled an expanded cohort of 107 patients and show that ZAP-70 is the gene that best distinguishes the CLL subtypes. Ig-unmutated CLL expressed ZAP-70 5.54-fold more highly than Ig-mutated CLL (P < 10(-21)). ZAP-70 expression correctly predicted IgVH mutation status in 93% of patients. ZAP-70 expression and IgVH mutation status were comparable in their ability to predict time to treatment requirement following diagnosis. In 7 patients, ZAP-70 expression and IgVH mutation status were discordant: 4 Ig-mutated CLLs had high ZAP-70 expression and 3 Ig-unmutated CLLs had low ZAP-70 expression. Among these ZAP-70 "outliers," those with Ig-mutated CLL had clinical features that are uncharacteristic of this CLL subtype: 2 required early treatment and 2 used a mutated VH3-21 gene, an IgVH gene that has been associated with progressive disease. We developed reverse transcriptase-polymerase chain reaction and immunohistochemical assays for ZAP-70 expression that can be applied clinically and would yield important prognostic information for patients with CLL.
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The B-cell receptor (BCR) for antigen is composed of surface immunoglobulin (sIg), which provides antigen specificity, and a noncovalently associated signaling unit, the CD79a/b heterodimer. Defects in CD79 can influence both BCR expression and signaling and may explain why cells from certain malignancies, such as B-chronic lymphocytic leukemia (B-CLL), often express diminished and inactive BCR. Recently, an alternative transcript of CD79b (DeltaCD79b) has been reported that is up-regulated in B-CLL and may explain this diminished BCR expression. Here we assess the expression of DeltaCD79b in B-CLL and other lymphoid malignancies and investigate its function. High relative expression of DeltaCD79b was confirmed in most cases of B-CLL and found in 6 of 6 cases of splenic lymphomas with villous lymphocytes (SLVLs) and hairy cell leukemia. In a range of Burkitt lymphoma cell lines, expression of DeltaCD79b was relatively low but correlated inversely with the ability of the BCR to signal apoptosis when cross-linked by antibody (Ab). Interestingly, when Ramos-EHRB cells, which express low DeltaCD79b, were transfected with this transcript, they were transformed from being sensitive to anti-Fcmu-induced apoptosis to being highly resistant. Although DeltaCD79b was expressed as protein, its overexpression did not reduce the level of cell surface BCR. Finally, we showed that the inhibitory activity of DeltaCD79b depended on an intact leader sequence to ensure endoplasmic reticulum (ER) trafficking and a functional signaling immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic tail. These results point to DeltaCD79b being a powerful modulator of BCR signaling that may play an important role in normal and malignant B cells.
This study evaluates the prognostic significance of genetic abnormalities (detected at or shortly after presentation), clinical stage, lymphocyte morphology, CD38 expression, and IGVH gene status in 205 patients with chronic lymphocytic leukemia (B-CLL). Deletion of chromosome 11q23, absence of a deletion of chromosome 13q14, atypical lymphocyte morphology, and more than 30% CD38 expression are significantly associated with the presence of unmutated IGVH genes. Advanced stage, male sex, atypical morphology, more than 30% CD38 expression, trisomy 12, deletion of chromosome 11q23, loss or mutation of the p53 gene, and unmutated IGVH genes are all poor prognostic factors in a univariate analysis. However, only 98% or more homology of IGVH genes to the germline sequence, loss or mutation of the p53 gene, and clinical stage retain prognostic significance in a multivariate analysis. The median survival of patients with mutated IGVH genes, unmutated IGVH genes, and loss or mutation of the p53 gene regardless of IGVH gene status is 310, 119, and 47 months, respectively. These data should facilitate the design of new trials for the management of patients presenting with advanced disease or poor prognosis early stage disease.
There exists a wide spectrum of IgM-secreting B-cell tumors with different clinical behavior. Knowledge of the V(H) gene status can reveal their origin and clonal history. For Waldenstrom macroglobulinemia (WM), a distinct subtype of lymphoplasmacytic lymphoma, early data on limited sequences showed evidence for somatic mutation. A recent report of one case demonstrated intraclonal mutational activity occurring after transformation, a characteristic of germinal center lymphomas. To extend the investigation, we have analyzed 7 cases of WM. V(H) genes were somatically mutated with no evidence of intraclonal variation in all cases. In contrast to IgM-secreting multiple myeloma, there was no evidence for isotype switch transcripts in any of the cases. These data support the concept that typical WM is derived from a B cell that has undergone somatic mutation prior to transformation, at a point where isotype switch events have not been initiated. (Blood. 2002;100:1505-1507)
Most patients with follicular lymphoma (FL) have somatically mutated V genes with intraclonal variation, consistent with location in the germinal center site. Using our own and published sequences, we have investigated the frequency of potential N-glycosylation sites introduced into functional V(H) genes as a consequence of somatic mutation. FL cells were compared with normal memory B cells or plasma cells matched for similar levels of mutation. Strikingly, novel sites were detected in 55 of 70 (79%) patients with FL, compared to 7 of 75 (9%) in the normal B-cell population (P <.001). Diffuse large B-cell lymphoma (DLCL) showed an intermediate frequency (13 of 32 [41%] patients). Myeloma and the mutated subset of chronic lymphocytic leukemia showed frequencies similar to those of normal cells in 5 of 64 (8%) patients and 5 of 40 (13%) patients, respectively. In 3 of 3 random patients with FL, immunoglobulin was expressed as recombinant single-chain Fv in Pichia pastoris, and glycosylation was demonstrated. These findings indicate that N-glycosylation of the variable region may be common in FL and in a subset of DLCL. Most novel sites are located in the complementarity-determining regions. V(H) sequences of nonfunctional V(H) genes contained few sites, arguing for positive selection in FL. One possibility is that the added carbohydrate in the variable region contributes to interaction with elements in the germinal center environment. This common feature of FL may be critical for tumor behavior.
Although the presence or absence of somatic mutations in the immunoglobulin variable region (IgV(H)) genes in chronic lymphocytic leukemia (B-CLL) identifies subtypes with very different prognoses, the assay is technically complex and unavailable to most laboratories. CD38 expression has been suggested as a surrogate marker for the 2 subtypes. IgV(H) mutations and CD38 expression in 145 patients with B-CLL with a long follow-up were compared. The 2 assays gave discordant results in 41 patients (28.3%). Multivariate analysis demonstrated that Binet stage, IgV(H) mutations and CD38 were independent prognostic indicators. Median survival time in patients whose cells had unmutated IgV(H) genes and expressed CD38 was 8 years; in those with mutated IgV(H) genes not expressing CD38, it was 26 years. For those with discordant results, median survival time was 15 years. Thus, although CD38 expression does not identify the same 2 subsets as IgV(H) mutations in CLL, it is an independent risk factor that can be used with IgV(H) mutations and clinical stage to select patients with B-CLL with the worst prognoses. Using cryopreserved cells taken at intervals during the course of the disease, however, changes of CD38 expression over time were demonstrated in 10 of 41 patients. Causes of the variation of CD38 expression require further study. Additional prospective studies are required for comparing CD38 expression with other prognostic factors and for taking sequential measurements during the course of the disease.
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This update of early stage B-cell chronic lymphocytic leukemia (B-CLL) embraces current information on the diagnosis, biology, and intervention required to more fully develop algorithms for management of this disease. Emphasis on early stage is based on the rapid advancement in our understanding of the disease parameters and our increasing ability to predict for a given early stage patient whether there is a need for more aggressive management. In Section I, Dr. Terry Hamblin addresses the nature of the disease, accurate diagnostic procedures, evidence for an early "preclinical" phase, the use of newer prognostic features to distinguish who will be likely to progress or not, and whether it is best to watch or treat early stage disease. In Section II, Dr. Neil Kay and colleagues address the biologic aspects of the disease and how they may relate to disease progression. Review of the newer insights into gene expression, recurring genetic defects, role of cytokines/autocrine pathways, and the interaction of the CLL B cell with the microenvironment are emphasized. The relationship of these events to both trigger disease progression and as opportunities for future therapeutic intervention even in early stage disease is also considered. In Section III, Dr. John Byrd and colleagues review the historical and now current approaches to management of the previously untreated progressive B-CLL patient. They discuss what decision tree could be used in the initial decision to treat a given patient. The use of single agents versus newer combination approaches such as chemoimmunotherapy are discussed here. In addition, the place of marrow transplant and some of the newer antibodies available for treatment of B-CLL are considered. Finally, a challenge to utilize our growing knowledge of the biology of B-CLL in the early stage B-CLL is proffered.