Early Chronic Lymphocytic Leukemia Research Articles

Identification of protamine 1 as a novel cancer‐testis antigen in early chronic lymphocytic leukaemia

Early chronic lymphocytic leukaemia (CLL) is an ideal disease for immunotherapy. We previously showed that SEMG 1 is a cancer-testis (CT) antigen in CLL. In this study, SEMG 1 was applied as the bait in a yeast two-hybrid system of a testicular cDNA library. Seven clones were isolated and Protamine (Prm) 1 was identified as a novel CT antigen in early CLL. PRM1 transcripts were detected in 11/41 (26.8%) patients. Prm 1 protein was also expressed but heterogeneously within individual patients. Of the 11 patients expressing Prm 1, four expressed Zap 70 protein and seven did not. These results, therefore, indicate that Prm 1 could potentially be a suitable target for the design of tumour vaccine for patients with early CLL, including for those with poor risk CLL. High titres of Prm 1 IgG antibodies could be detected in 20 of these 41 CLL patients but not in any of the 20 healthy donors (P = 0.0001), suggesting the presence of Prm 1-reactive immune responses within the immune repertoire of patients with early CLL. Further work is warranted, especially in approaches to upregulate Prm 1 expression, and to determine the role of Prm 1 as an immunotherapeutic target for early CLL.

SEMG-1 expression in early stage chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is an indolent disease. It is currently recommended that patients with CLL stages 0 and I follow a watchful waiting strategy. These patients are, therefore, a suitable group for testing immunotherapeutic approaches to avoid problems of immunosuppression as a result of disease progression and chemotherapy. In this study, we investigated the expression of SEMG-1 in early CLL to determine the suitability of SEMG-1 as a target for further development of tumor vaccines for early CLL. A combination of reverse transcriptase (RT)-polymerase chain reaction (PCR) and immunocytochemistry was used to evaluate the expression of SEMG-1 in early CLL. The results were correlated with Zap 70 expression. Recombinant SEMG-1 protein was used in an enzyme-linked immunosorbent assay (ELISA) to determine the presence of SEMG-1 antibodies (Ab) in serum from these patients. The SEMG-1 gene was expressed in 19/41 (46%) patients with early CLL. Gene expression was associated with protein synthesis in CLL cells. Protein expression, however, was heterogeneous within individual patients. Only transcripts encoding the SEMG-1(50) variant and not SEMG-1(43) were detected. SEMG-1(50) was expressed irrespective of the Zap 70 status. High-titer SEMG-1 IgG but not IgM Ab were detected in some of these patients, suggesting that SEMG-1-reactive immune responses are intact within the immune repertoire of early CLL patients. SEMG-1 is expressed in nearly half of patients with early CLL and may be a target for further investigations into its use for immunotherapy of early CLL.

Monoclonal B-Lymphocytosis: Analysis of the Incidence, Demographics, Nature and Subclassification in 414 Patients

The 2008 Guidelines for Chronic Lymphocytic Leukemia require a B-cell count ≥5.0×109/L (Hallek et al, Blood , 111, 5446, 2008). Clonal B-cell populations below <5.0×109/L, and in the absence of adenopathy, organomegaly or cytopenia, are defined as Monoclonal B-Lymphocytosis (MBL) (Marti et al., Br. J. Haematol., 2005). We analysed the frequency and nature of small B-cell clonal populations identified in a large, non-hospital based pathology laboratory servicing both metropolitan and regional areas of New South Wales, Australia. There were 414 patients identified from 2000 to 2005 with an incidental finding of a B-cell clone with total B-lymphocytes (defined as CD19+ cells) <5.0×109/L fulfilling the criteria for MBL. There were 212 males (51%) and 202 females (49%) with a mean age of 69.7 years (range 14–97 years). Patients could be clearly divided into two major groups: 322 (77.7%) with a typical chronic lymphocytic leukaemia (CLL) phenotype (MBL[cll]) and 92 (22.3%) with a ‘non-CLL’ phenotype (MBL[nhl]). Analysis of MBL[cll] showed a near equal gender distribution of 168 (52.2%) males and 154 (47.8%) females with a mean age of 70.6 years (range 41–94 years). No patient had a CLL clone aged younger than 40 years, and for each subsequent age decade there were 4% (40–49 yrs), 13% (50–59yrs), 23% (60–69yrs), 40% (70–79yrs), 18% (80–89yrs) and 2% (>90yrs) of the patients with a CLL clone, mirroring the age distribution of CLL. The mean clonal level (CD19/CD5+) was 2.36×109/L (range 0.01–5.21×109/L), the mean B-cell count (CD19+) 2.50×109/L (range 0.02–4.97 ×109/L) and the mean absolute lymphocyte count (ALC) was 4.71 ×109/L (range 0.4–10.5×109/L. Over half (52%) of these patients fulfilled NCI-1988/1996 criteria for the diagnosis of CLL (with ALC ≥5.0×109/L). The ALC was within the laboratory reference range (1.0–4.0 ×109/L) in only 22% of patients. Follow-up analysis on a representative cohort of 146 patients showed progression to a lymphocyte count >30×109/L in 15 patients over a mean of 4.03 (range 1–8) years, and including 3 patients with clonal populations originally measuring <1.5×109/L. Analysis of the 92 patients with a ‘non-CLL’ phenotype (MBL[nhl]) showed 44 (47.8%) males and 48 (52.2%) females, with a mean age of 66.7 years (range 14–97 years). The mean clonal level in this group was 1.27×109/L. The level of the clonal population with MBL[nhl] was <2.0×109/L in 77% and <3.0×109/L in 92% of patients. There were 66 patients (71.7%) with MBL[nhl] with a phenotype of “lymphoma not otherwise unspecified” (i.e. pan-B cell+, monoclonal surface immunoglobulin++, CD5−, CD10−, CD103−) and the remainder had ‘specific’ diagnosis possible on review of the peripheral blood morphology and phenotype as follows: 'Mantle Cell Lymphoma'-like (CD5+, CD23+) - 10 (10.9%), Splenic Lymphoma with Villous Lymphocytes - 7 (7.6%), 'Follicular Lymphoma' (CD10+) - 4 (4.3%), Hairy Cell Leukaemia (CD103+) - 4 (4.3%), Hairy Cell Leukemia Variant - 1 (1%). MBL is not uncommon in the immunophenotyping laboratory and can be conveniently divided into two dominant groups, the majority with a phenotype typical of CLL (MBL[cll]), while there are a range of non-CLL phenotype identified (MBL[nhl]), some with a ‘specific’ diagnosis. In 322 patients with an MBL[cll] clone below the defined cut-off point for CLL of B-cells <5.0×109/L, the absolute B-cell count is a linear continuous variable. This data suggests that MBL[cll] is not a precursor state or predisposition but rather the same biological process as early CLL (separated by an arbitrary definitional cut-off) from which some will progress but many will remain stable.

Semg 1 Expression in Early Chronic Lymphocytic Leukemia

SEMG 1 is major protein of semen coagulum shown to inhibit human sperm capacitation. It plays an important role in sperm clotting and is normally degraded into smaller fragments by prostate-specific antigen. The gene encoding SEMG 1 has been localized to the long arm of chromosome 20, a region of chromosome 20 that is frequently deleted in myeloproliferative diseases and myelodysplastic syndrome. We previously found SEMG 1 to be a Cancer-Testis (CT) antigen that was immunogenic in the cancer-bearing patients, supporting its potential as a target for tumor immunotherapy. CLL patients are generally immunosuppressed even before any therapy is given. The immunosuppression increases as the disease progresses and may prevent successful immunotherapy unless the therapeutic approach is used to patients with early stage disease (Stage 0 or I), even prior to use of any immunosuppressive chemotherapy. However, most CT antigens are expressed in low frequency in early stage cancer. Accordingly, we set out to determine the expression frequency, variant and pattern of expression of SEMG 1 in patients with early CLL.Using a pair of sequence-specific primers in RT-PCR on total RNA derived from patients with early CLL, we found that SEMG 1 transcripts could be detected in 19/41 (46%) patients. The high frequency at which SEMG 1 is expressed in early CLL is in contrast to the generally low frequency of expression of other CT antigens in early malignancies. SEMG 1 expression not only occurred at the transcript level but also protein level, as determined by immunocytochemistry using SEMG 1 MoAbs. These results indicate that SEMG 1 could potentially be a suitable target for the design of tumor vaccine that could be applicable to a large proportion of patients with early CLL. Of the 19 patients expressing SEMG 1, eight expressed Zap 70 protein and 11 did not (p = 0.4; N.S.). Therefore, any SEMG 1-based tumor vaccine could be applicable even to patients with early poor risk CLL (as determined by Zap 70 expression). However, SEMG 1 expression, as determined by immunocytochemistry, within individual CLL patients was heterogeneous, suggesting that tumor vaccine targeting SEMG 1 may have to be administered with therapeutic agents that upregulate SEMG 1 expression to overcome the potential problem of tumor antigen heterogeneity that could prevent the success of the tumor vaccine.Since SEMG 1 exists in two variants, we next determined which SEMG 1 variant was expressed in CLL cells. Using a primer pairs in PCR to amplify across the 180 bp transcript that is deleted in SEMG 143, we showed that in all cases of SEMG 1-positive CLL specimens, only the non-truncated transcripts were detected, indicating the expression of SEMG 150 variant in CLL.Finally, applying diluted patient serum to an ELISA system, we found that high titers SEMG 1 IgG antibodies could be detected in six of these 41 patients but not in any of the 20 healthy donors. Leukemia cells from three of these patients expressed SEMG 1. These results, therefore, suggest that SEMG 1-reactive lymphocytes are present in the immune repertoire of patients with early CLL, irrespective of whether the leukemia cells express SEMG 1, and further support the feasibility of applying a SEMG 1-based tumor vaccine to these patients.In conclusion, SEMG 150 is expressed by the leukemia cells in a high proportion of patients with early CLL, irrespective of the Zap 70 expression status. SEMG 1 may be an immunological target for immunotherapy of nearly half of early CLL patients, especially when SEMG 1-reactive lymphocytes are present in the immune repertoire of these patients.

Pattern of Chromosomal Aberrations and IgVH Mutation Status in Patients with Monoclonal B-Cell Lymphocytosis (MBL)

The separation between chronic lymphocytic leukemia (CLL) and monoclonal B-cell lymphocytosis (MBL) with CLL-phenotype by the presence of more than 5,000/μl lymphocytes in peripheral blood has been debated due to the rather continuous spectrum of clinical and biological features of both entities. Furthermore, this separation is influenced by non-malignant cells since it is based on the total lymphocyte count and not only on the count of cells with CLL-phenotype. To broaden the insights into the genetic background of MBL at the boarder to CLL we analyzed 188 cases with less than 5,000/μl CLL-phenotype cells for chromosomal aberrations by fluorescence in situ hybridization (FISH) using a standard panel of probes as well as for their IgVH mutation status. In addition, in 116 of these cases conventional chromosome analysis has been performed. Patient characteristic were (median/range): age, 66.0/27.5–86.6 years; WBC count, 10,600/1,700–22,660/μl; cells with CLL-phenotype 26/4-94% and 3,081/264–4,998/μl, respectively. The proportion of aberrant/analyzed cases for the respective chromosome aberrations as determined by FISH were: 11q-, 10/182 (5.5%); +12, 39/183 (21.3%); 6q-, 2/182 (1.1%); 13q-, 88/185 (47.6%); 17p-, 3/183 (1.6%); and t(11;14), 8/183 (4.4%). In 57/185 (30.8%) cases no aberration was found by FISH. A comparison to a previous series of 518 CLL cases revealed similar frequencies for +12, 6q-, and t(11;14) but higher frequencies in CLL for 11q-, 5.5 vs. 13.1%, p=0.004; 13q-, 47.6 vs. 57.8%, p=0.020; and 17p-, 1.6 vs. 8.1%, p=0.001. Within the present series, a relation between the respective chromosome aberration and the count of cells with CLL-phenotype was found for 6q- vs. no 6q-, 1,214 vs. 2,910/μl, p=0.079; 13q- vs. no 13q-, 3,285 vs. 2,486/μl, p<0.001; t(11;14) vs. no t(11;14), 2,025 vs. 2,920/μl, p=0.087 but not for 11q-, +12, and 17p- aberrations. IgVH mutation status has been analyzed in 172 cases and was found unmutated (IgVHunmut) in 44 (25.6%) und mutated (IgVHmut) in 111 (64.5%). No clonal IgVH rearrangement was detectable in 17 (9.9%). A relation to the count of cells with CLL-phenotype was found: IgVHmut vs. IgVHunmut, 3,071 vs. 2,629/μl, p=0.052. Conventional chromosome analysis has been performed in addition in 116 of these cases resulting in 111 (95.7%) evaluable cases. In general, results obtained by FISH were confirmed: 11q-, n=3 (1.8%); +12, n=21 (21.3%); 6q- 1 (0.9%); 13q-, n=39 (35.1%), and t(11;14), n=5 (4.5%). Additional aberrations were found and included other trisomies (n=13), other balanced translocations/inversions (n=13), 14q- (n=8), loss of sex chromosome (n=3), t(14;18) (n=1), and 2q- (n=1). Thus, a total of 39 chromosome aberrations have been found in 111 cases as assessed by conventional chromosome analysis which could not be detected by FISH using a standard panel of probes. In 30 cases (27%) a normal karyotype was found. These data indicate that the genetic background of MBL is highly related to the one of classical CLL as defined today although 11q-, 13q- and 17p- occur more frequently in CLL. Chromosome aberrations are present in clearly more than half of the cases and an unmutated IgVH status can be detected in a quarter of cases. Similar to CLL, conventional chromosome analysis reveals a substantial number of aberrations not detectable by FISH using a standard panel of probes. It is suggested to further analyze the genetic spectrum of MBL and early CLL cases and its clinical impact and to challenge based on these data the rather arbitrarily defined cut-off values like 5,000/μl lymphocytes.

Inherited predisposition to chronic lymphocytic leukemia

Inherited susceptibility to chronic lymphocytic leukemia (CLL) has been recognized for decades. Approximately 10% of individuals with CLL report a family history of CLL or a related lymphoproliferative disorder, and genetic predisposition is the best understood risk factor for CLL. Studies of familial CLL have suggested that the disease features are largely similar to sporadic CLL, although recent data suggest that familial CLL may more commonly show somatic hypermutation of the immunoglobulin heavy-chain variable region, suggesting a more indolent disease course. Monoclonal B-cell lymphocytosis (MBL) has been identified recently as a likely precursor to CLL; it is found in the general population with increasing age and enriched in unaffected relatives of individuals with familial CLL. Studies of MBL as well as mouse models of CLL may lead to better understanding of early CLL pathogenesis that is relevant to familial predisposition. To date, the identification of genes that predispose to familial CLL has been slow, primarily due to the relatively few families available for study, the small size of those families and disease causation most likely by multiple genes that each confer smaller risks. In the coming years, the application of systematic genomics approaches to familial CLL should, hopefully, lead to the identification of novel loci involved in the disease.

Serum thrombopoietin compared with ZAP-70 and immunoglobulin heavy-chain gene mutation status as a predictor of time to first treatment in early chronic lymphocytic leukemia

In an effort to confirm previous reports we analyzed clinico-biological implications of increased serum levels of thrombopoietin (TPO) in a series of 71 previously untreated Binet stage A B-cell chronic lymphocytic leukemia (CLL) patients. Serum levels of TPO did not correlate with peripheral blood lymphocytosis (p = 0.928), Rai substages (p = 0.516), platelet count (p = 0.572), hemoglobin level (p = 0.228), LDH (p = 0.144) and β2-microglobulin (p = 0.520). The same applied when correlation with ZAP-70 (p = 0.562), CD38 (p = 0.258) or mutational status of IgVH (p = 0.0794) were sought. The risk of disease-progression according to known and putative prognostic parameters was also evaluated as time to first treatment (TFT). The univariate Cox proportional hazard model demonstrated that the absence of IgVH mutational status (p = 0.0005) and ZAP-70-positivity (p = 0.02) were associated with a shorter TFT. In contrast, Kaplan-Meier estimates of TFT, plotted after setting as cut-off the median value for TPO (i.e., 46 pg/mL), failed to demonstrate any statistical difference between two groups (p = 0.342). Looking for cellular source of TPO we investigated the presence of TPO at gene expression level in 60 B-CLL patients belonging to an independent series. Here we provide evidence for the presence of a low TPO gene expression transcript in B-CLL cells. In conclusion, our results indicate that in early B-cell CLL circulating level of TPO does not provide a useful insight into the complex interrelationship of prognostic variables.

Prospective Evaluation of Prognostic Parameters in Early Stage Chronic Lymphocytic Leukemia (CLL): Results of the CLL1-Protocol of the German CLL Study Group (GCLLSG).

Background: Early stage CLL is a heterogeneous disease with a prognosis ranging from an overall survival of a few months to two decades. Prognostic markers helping to predict the individual course of CLL are highly warranted. The CLL1 trial was initiated to investigate all major prognostic parameters used at the time of its initiation for prediction of the course of CLL.Patients: From 1997 to 2004 877 Binet A pts (median age 60 yrs) were enrolled. The median follow up was 45 months (mo). Risk stratification was possible for 788 pts. Pts with high risk (HR) were defined by elevated thymidine kinase (TK) or beta-2-microglobulin (beta-2-MG)) and either short lymphocyte doubling time (LDT, <12 months)) or diffuse bone marrow infiltration (BMI). 99 pts were not eligible due to trial violation. HR pts were randomized to W&W vs. immediate therapy with fludarabine. Treated pts (n=104) were excluded from this analysis. Analyses on PFS and OS on 585 pts are presented. Progression was defined by slightly modified NCI-WG criteria.Results: 114 pts (19.5%) were stratified to HR, 471 pts (80.5%) to low risk (LR). At enrolment there was no significant difference with regard to age, performance state, comorbidity. Significantly more male pts were assigned to HR (p=0.03). Beside parameters for stratification the cohorts differed in leukocyte/lymphocyte count (p<0.001), cervical and inguinal lymphadenopathy (LN) (p=0.001 resp. 0.005) and splenomegaly (p=0.02). B symptoms, hemoglobin, platelets, hepatomegaly and axillary LN were similar distributed in both groups. The median PFS for all was 57.3 mo. The median PFS for HR was significantly shorter than for LR (88 vs.18 mo; p<0,001). Although the median OS was not reached for both, HR had significantly shorter OS time (p<0,001). Pts had a significantly shorter PFS (25 vs. 88 mo; p<0.001) if they had TK>7 U/L, beta-2-MG>3.5 mg/L (13 vs. 75 mo; p<0.001) or LDT <12 mo (20 vs. 75 mo; p<0.001). For these parameters OS was also significantly shorter for HR, although the median OS was not reached. Pts with diffuse BMI had shorter PFS (49 vs. 75 mo; p=0.003), but OS was not different (p=0.2). Furthermore lymphocyte counts >30 G/L (PFS 17 vs. 88 mo; p<0.001) or non-smouldering CLL (PFS 46 mo vs. n.r.; p<0.001) predicted shorter PFS/OS. Male pts had shorter PFS (49 mo vs. n.r.; p=0.001), but not OS (p=0.08). OS was significantly shorter for older pts than 55 (p=0.014) or 65 yrs (p<0.001), while PFS was not different. A multivariate Cox analysis revealed that TK, LDT, beta-2-MG, absolute lymphocyte count, sex and age were independent variables for PFS, while LDT, beta-2-MG, lymphocytes and age were also independent for OS.Conclusion: This prospective trial defined clinical and biological factors (TK, LDT, beta-2-MG, absolute lymphocyte count, age, sex) which help to predict progression in early CLL. Our model for risk stratification reliably separated between HR and LR. Due to the weak impact of BMI we do not further recommend the use of BM biopsy for assessment of prognosis in CLL.

Increased expression of angiopoietin-2 characterizes early B-cell chronic lymphocytic leukemia with poor prognosis

We measured the angiopoietin-2 (Ang-2) expression in early chronic lymphocytic leukemia (CLL) patients, pointing our attention on the association with immunoglobulin (IgV H) mutational status, CD38 expression and clinical outcome. Our results indicate that Ang-2 expression is heterogeneous among Binet stage A CLL patients. CLL patients can be divided into two subgroups (Ang-2 positive and Ang-2 negative CLL) with 30% of them displaying Ang-2 RNA levels above the cut off. A shorter progression-free survival was observed in Ang-2 positive CLL subset ( p = 0.032). Abnormal Ang-2 expression was also associated with unmutated IgV H genes ( p < 0.0001) and increased bone marrow angiogenesis ( p = 0.028), suggesting a role of Ang-2 in disease-progression of early CLL patients.

Using Smudge Cells on Routine Blood Smears to Predict Clinical Outcome in Chronic Lymphocytic Leukemia: A Universally Available Prognostic Test

Recently developed prognostic tests in early Rai and Binet stage chronic lymphocytic leukemia (CLL) require considerable technologic expertise and are not available worldwide. Smudge cells are CLL cells ruptured during smear preparation. We hypothesized that smudge cell formation is inversely correlated with expression of vimentin, a cytoskeletal protein and prognostic marker, and that the percentage of smudge cells would predict prognosis in CLL. We reviewed the blood smears of 75 patients with previously untreated early and intermediate-stage CLL (Rai stage 0-II) who were seen at the Mayo Clinic in Rochester, Minn, between September 1989 and December 2000. A total of 200 lymphocytes and smudge cells were counted on each slide and the results expressed as a percentage of the total lymphocytes (intact and smudged). The median percentage of smudge cells was 27% (range, 4%-72%). The percentage of smudge cells inversely correlated with vimentin expression (r=-0.57; P=.007). The median percentage of smudge cells was higher in patients with the mutated immunoglobulin heavy chain gene than in those with the unmutated immunoglobulin heavy chain gene (31% vs 13%; P=.02). Patients with less than 30% smudge cells had a median time from diagnosis to initial treatment of 72.7 months, whereas the median time from diagnosis to initial treatment in patients with 30% or more smudge cells was not reached (P=.001). The percentage of smudge cells as a continuous variable correlated with overall survival (P=.04). The estimation of smudge cells on a blood smear could be a universally available prognostic test in early-stage CLL.

Lymphocytes from patients with early stage of B-cell chronic lymphocytic leukaemia and long survival synthesize decorin

mRNA/cDNA gene expression of both small leucine-rich proteoglycans decorin and biglycan was evaluated by PCR real time in lymphocytes collected from patients with chronic lymphocytic leukaemia (CLL) at different stages of disease and from healthy controls. Lymphocytes obtained from healthy controls showed no or very low levels of mRNA expression of both decorin and biglycan. Biglycan expression was very low in CLL patients, values being close to those of controls. On the contrary, decorin mRNA was clearly expressed in patients with early B-cell CLL, while a low expression was found in advanced clinical stages. Furthermore, a significant higher decorin expression was found in patients with non-progressive CLL type in comparison with patients with aggressive type of the disease. Decorin expression resulted especially high in the low-progressive low-risk patients. The synthesis of decorin was also assessed by Western blot analysis. The peculiar occurrence of decorin in the non-aggressive type of CLL is consistent with its suggested anti-oncogenic role. Intracellular Bcl-2 level does not correlate with decorin mRNA transcription, suggesting that a Bcl-2 independent anti-cancer mechanism may occur. The measurement of galactosamine-containing proteoglycans concentration in plasma confirmed decorin expression results, with significant differences between CLL patients and controls. Significant changes were also seen between groups of patients of Rai stage 0 with recent diagnosis (less than 5 years, from analysis), (low amount of decorin) and less recent diagnosis (more than 5 years), (high amount of decorin).

Serum levels of syndecan-1 in B-cell chronic lymphocytic leukemia: Correlation with the extent of angiogenesis and disease-progression risk in early disease

Syndecan-1 is a transmembrane proteoglycan generally not expressed in mature B-cell neoplasias like chronic lymphocytic leukemia (CLL). Moreover, information dealing with the evaluation of soluble syndecan-1 in CLL are lacking. We measured syndecan-1 concentrations in serum drawn at the time of diagnosis from 67 B-cell CLL patients (Binet stage A, 46; stage B, 7; stage C, 14). For this purpose a syndecan-1 enzyme-linked immunosorbent assay (ELISA, Diaclone, Besancon, France) was used. Detectable levels of syndecan-1 were found in all patients, although serum concentrations were significantly lower in CLL patients in comparison to age- and sex-matched controls (P = 0.02; Mann-Whitney test). No correlation was found with Binet clinical stages (P = 0.796), β2-microglobulin (P = 0.923), hemoglobin level (P = 0.605), platelet count (P = 0.992) and lymphocyte doubling time (P = 0.709). Only an association with absolute peripheral blood lymphocytosis (PBL) (P = 0.01) and LDH (P = 0.05) could be detected. Serum levels of syndecan-1 did not parallel those of several angiogenic cytokines such as vascular endothelial growth factor (VEGF) (P = 0.963), basic fibroblastic growth factor (FGF-2) (P = 0.216), angiogenin (P = 0.478), metalloproteinase-9 (MMP-9) (P = 0.125) as well as bone marrow (BM) microvessel density (P = 0.110). The same applied with adhesion molecules such as CD54 (P = 0.233), CD108 (P = 0.799), CD44 (P = 0.816) and CD31 (P = 0.508). Interestingly, the inverse correlation (r = −0.4967; P = 0.03) between serum concentrations of syndecan-1 and plasma levels of stromal derived growth factor-1 (SDF-1) is in keeping with the different function, respectively, pro- and anti-apoptotic, of these molecules. In 46 Binet stage A patients, serum levels of syndecan-1 were further evaluated as a dichotomous variable with respect to progression-free survival (PFS), an end-point surrogate for overall survival in early B-cell CLL. The best separation of curves was seen with a cut-off point at the median value of syndecan-1 (i.e. 36.5 pg/ml). Median PFS was not reached in the patient group with low syndecan-1, compared to a median of 34 months observed in the remaining patients (P = 0.018; HR = 0.208; 95% CI = 0.115 – 0.816). At the multivariate analysis performed including variables significant in the univariate analysis [i.e. PBL (P = 0.03) and syndecan-1 (P = 0.01)], only syndecan-1 retained a trend of significance (P = 0.08).Despite the pro-angiogenic activity of syndecan-1 which mediates FGF-2 binding and activity, no correlation with either angiogenic cytokines or the extent of BM angiogenesis was found in CLL. The inverse correlation with plasma levels of SDF-1 suggests an involvement in the processes leading to apoptosis. Finally, our results highlight the involvement of syndecan-1 in the mechanisms of disease-progression of early CLL.

Serum insulin‐like growth factor is not elevated in patients with early B‐cell chronic lymphocytic leukemia but is still a prognostic factor for disease progression

Insulin-like growth factor 1 (IGF-1) is an important growth and anti-apoptotic factor for the cancer cells in several malignancies and in multiple myeloma recent studies support the hypothesis of a role for IGF-1 in disease progression; however, clinico-biological relevance of IGF-1 was never studied in B-cell chronic lymphocytic leukemia (CLL). Using a quantitative sandwich immunoassay technique (ELISA) (Quantikine, Human IGF-1 and IGFBP-3, R&D Systems), we measured the concentration of IGF-1 and its major binding protein IGF-binding protein 3 (IGFBP-3) in serum drawn at the time of diagnosis from 77 Binet stage A CLL patients. Either IGF-1 or IGFBP-3 were significantly decreased compared with healthy age- and sex-matched controls (P < 0.0001 for both; Mann-Whitney test). Serum levels of IGF-1 and IGFBP-3 paralleled each other (P = 0.002); in contrast, no significant correlation was found between serum levels of IGF-1 and clinico-hematological variables including age (P = 0.253), sex (P = 0.270), Rai clinical substages (P = 0.140), lactate dehydrogenase (P = 0.956), beta2-microglobulin (P = 0.368), lymphocyte count (P = 0.703) and lymphocyte doubling time (LDT, P = 0.233). When correlation were attempted with circulating levels of angiogenic cytokines such as vascular endothelial growth factor (P = 0.971), basic fibroblastic growth factor (P = 0.695), angiogenin (P = 0.282) or adhesion molecules such as vascular cell adhesion molecule-1 (P = 0.318), intercellular adhesion molecule-1 (P = 0.883) and platelet endothelial cell adhesion molecule-1 (P = 0.772) similar results were found. Serum levels of IGF-1 were further evaluated as a dichotomous variable with respect to progression-free survival (PFS), an endpoint surrogate for overall survival in early B-cell CLL. The best separation of curves was seen with the cutoff point at the 75th percentile of IGF-1 levels (i.e., 93 pg/mL). Median PFS was 63 months in the patient group with low IGF-1, compared with a median PFS of 40 months in the remaining patients (P = 0.03). In the multivariate analysis performed including variables significant at univariate analysis [i.e. Rai substage (P = 0.002); LDT (P = 0.004), IGF-1 (P = 0.01)], only Rai substage retained prognostic significance (P = 0.006). However, after removing from analysis LDT (only six of 77 had an LDT < 12 months), either IGF-1 or Rai substage entered the model at a significant level (P = 0.03 and P = 0.01, respectively). IGF-1 did not correlate with markers of tumor burden or clinical status in CLL thus suggesting that levels of this cytokine do not reflect the intrinsic malignancy of disease. Results of the present study highlight, however, its involvement in mechanisms of disease progression in early CLL.

Serum Free Light Chain (FLC) Measurement Can Aid Capillary Zone Electrophoresis in Detecting Subtle FLC-Producing M Proteins

We hypothesized that using a free light chain (FLC) assay as an adjunct to capillary zone electrophoresis (CZE) could improve detection of lymphoplasmacytic processes. We prospectively studied 1,003 consecutive serum samples submitted for routine protein electrophoresis and/or immunofixation electrophoresis by CZE and FLC. Samples from patients previously characterized as having M proteins were excluded. Protein electrophoresis was read by a pathologist unaware of the FLC results. Sixteen cases revealed an abnormal free kappa/lambda ratio in which CZE did not demonstrate an M protein. Nine cases of B-lymphocyte or plasma cell proliferative processes were detected by an abnormal free kappa/lambda ratio in which CZE did not demonstrate an M protein. Cases with low free kappa/lambda ratios included 1 chronic lymphocytic leukemia (CLL), 1 IgM lambda with aplastic anemia, and 1 lambda light chain myeloma. Cases with high free kappa/lambda ratios included 2 CLL, 1 lymphocytosis (possibly early CLL), 1 kappa light chain myeloma, 1 atypical lymphoma with neuropathy, and 1 nonsecretory myeloma. Addition of the free kappa/lambda ratio to CZE increases the yield of lymphocyte and plasma cell proliferative processes detected by 56%.

The prognostic value of CD38 expression in chronic lymphocytic leukaemia patients studied prospectively at diagnosis: a single institute experience

The purpose of this study was to assess in chronic lymphocytic leukaemia (CLL) patients the prevalence and clinical impact of CD38 expression, evaluated prospectively at disease presentation, and to verify whether this parameter changes over time. In 242 consecutive and untreated CLL patients, the percentage of CD38+ cases, according to the 7%, 20% and 30% cut-off points, was 21%, 17% and 14%, respectively. Using the 7% threshold, CD38 positivity correlated with male sex, intermediate and high-risk (Rai I-IV) disease, lower Hb and platelet levels, and higher lymphocyte count. Furthermore, patients with a CD38 expression>or=7% showed a significantly lower 3-year probability of treatment-free survival (TFS) than CD38- patients (P<0.0001). At multivariate analysis, CD38 expression remained significantly associated to TFS, together with stage, lymphocyte count and morphology. Also, in the 146 patients with stage 0 CLL a CD38 expression>or=7% identified a subgroup of patients with a significantly lower 3-year probability of TFS (P=0.0005). Furthermore, this parameter did not change in 30 of 31 (97%) re-evaluated patients. In conclusion, this study indicates that, when tested at diagnosis and on fresh material, a CD38 expression>or=7% is an important parameter for the identification of early CLL patients with more aggressive disease and that its expression remains stable over time.

A gender-based score system predicts the clinical outcome of patients with early B-cell chronic lymphocytic leukemia

To facilitate the development of a prognostic model for early B-cell chronic lymphocytic leukemia (CLL), the Gruppo Italiano Malattie EMatologiche Maligne dell'Adulto (GIMEMA) proposes its multi-institutional effort as a working model. In total, 1138 newly diagnosed Binet stage A patients managed over the last 10 years outside the setting of clinical trials according to a "wait and see" policy form the basis of the present study aimed at investigating prognostic variables affecting disease progression, a surrogate endpoint for overall survival. A 3-stage risk system, simply obtained by summing the variables that proved significant in the multivariate analysis (i.e. short lymphocyte doubling time, advanced Rai substage, high peripheral blood lymphocytosis), is proposed. Clear-cut differences in the 10 year progression-free survival (PFS) were observed among patients scoring 0 (low risk), 1 (intermediate risk), 2-3 (high risk): 67.8, 41.0 and 24.8%, respectively (P<0.0001). The results of the Medical Research Council (MRC) suggesting a better clinical outcome for females prompted us to verify such a gender-related difference within our prognostic categories. Because changes in PFS only reflected gender for patients scoring 0 (P=0.04), the following prognostic subgroups are proposed: (1) females scoring 0; (2) males scoring 0; (3) patients scoring 1-3 whatever gender (10 year PFS: 76.2, 61.4 and 37.8%; P<0.00001). Our long-term database provides an adequate patient sample to generate a generalized risk stratification model based on clinical data. The indolent clinical outcome of women with early CLL is also supported by the higher frequency of the immunoglobulin heavy-chain variable (IgVH) mutational status and lower proportion of 17p and 11q deletions found in such a patient subset in the MRC CLL4 trial.

Serum Insulinlike Growth Factor Is Not Elevated in Patients with Early B-Cell Chronic Lymphocytic Leukemia but Is Still a Prognostic Factor for Disease-Progression.

Insulin like growth factor 1 (IGF-1) is an important growth and antiapoptotic factor for the cancer cells in several malignancies and in multiple myeloma recent studies support the hypotesis of a role for IGF-1 in disease progression. Clinico-biological relevance of IGF-1 was never studied in B-cell chronic lymphocytic leukemia (CLL). Using a quantitative sandwich immunoassay technique (ELISA)(QUANTIKINE®, Human IGF-1 and IGFBP-3, R & D Systems), we measured the concentration of IGF-1 and its major binding protein IGF-binding protein 3 (IGFBP-3) in serum drawn at the time of diagnosis from 77 Binet stage A CLL patients. Either IGF-1 or IGFBP-3 were significantly decreased compared to healthy age- and sex-matched controls (P<0.0001 for both; Mann-Whitney test).Serum levels of IGF-1 and IGFBP-3 paralleled each other (P=0.002); in contrast, no significant correlation was found between serum levels of IGF-1 and clinico-hematological variables including age (P=0.253), sex (P=0.270), Rai clinical substages (P=0.140), LDH (P=0.956), β2-microglobulin (P=0.368), lymphocyte count (P=0.703) and lymphocyte doubling time [LDT](P=0.233). When correlation were attempted with circulating levels of angiogenic cytokines such as vascular endothelial growth factor (VEGF)(P=0.971), basic fibroblastic growth factor (FGF-2)(P=0.695), angiogenin (P=0.282) or adhesion molecules such as vascular cell adhesion molecule-1 [VCAM-1] (P=0.318), intercellular adhesion molecule-1 [ICAM-1] (P=0.883) and platelet endothelial cell adhesion-1 [PECAM-1] (P=0.772) similar results were found. Serum levels of IGF-1 were further evaluated as a dichotomous variable with respect to progression-free survival (PFS), an endpoint surrogate for overall survival in early B-cell CLL. The best separation of curves was seen with the cutoff point at the 75th percentile of IGF-1 levels (i.e., 93 pg/ml). Median PFS was 63 months in the patient group with low IGF-1, compared to a median PFS of 40 months in the remaining patients (P=0.002; HR, 0.311, 95% C.I, 0.085–0.630). In the multivariate analysis performed including variables significant at univariate analysis [i.e., Rai substage (P=0.002); LDT (P=0.004), IGF-1 (P=0.01)], only Rai substage retained prognostic significance (P=0.006). However, after removing from analysis LDT (only 6 out of 77 had an LDT< 12 months), either IGF-1 or Rai substage entered the model at a significant level ((P=0.03 and P=0.01, respectively).In conclusion, IGF-1 did not correlate with markers of tumor burden or clinical status in CLL thus suggesting that levels of this cytokine do not reflect the intrinsic malignancy of disease. Results of the present study highlight, however, its involvement in mechanisms of disease-progression in early CLL.

Prognostic Value of ZAP-70 Expression Detected by Immunohistochemistry on Bone Marrow Biopsies in Early Phase Chronic Lymphocytic Leukaemia.

New biological prognostic factors have been developed over the last years with the aim of predicting at presentation the heterogeneous clinical course of B chronic lymphocytic leukaemia (B-CLL) and of planning a risk-adapted treatment strategy. Among them ZAP-70 expression on leukemic cells as evaluated by molecular analysis or flow cytometry, initially proposed as surrogate of IgVH mutational status, appears a strong prognostic marker in B-CLL. However the optimal methodological approach to immunophenotipical demonstration of ZAP-70 expression as still matter of debate. We evaluated the cytoplasmic expression of ZAP-70 protein in leukemic cells by immunohistochemical method on bone marrow trephine biopsies taken at diagnosis of 84 patients with B-CLL. We used a mouse anti-human monoclonal antibody to ZAP-70 (clone 2F3.2, 1/200, Upstate, Lake Placid NY) with a polymeric labelling two-step method (Dako cytomation EnVision+, HRP). The results were correlated with age, sex, Binet stage, pattern of bone marrow infiltration, survival and clinical outcome. They were 54 males (64%), aged 34 to 80 years (median 62.5). At presentation 69 (82%) were Binet stage A, 9 (11%) stage B and 6 (7%) stage C. Among the 60 survivors, the median follow-up period from diagnosis was 78.5 months (range 22 – 236 months) Thirty-nine cases (46%) of B-CLL patients had cytoplasmic expression of ZAP-70. This group of patients presented higher percentage of advanced Binet stage (B–C) (p= 0.001). The ZAP-70 positivity was significantly related to inferior OS (55% vs 90% at 7 years)(HR 4.67 CI 1.95–11.14) and PFS (15% vs 57% at 7 years) (HR 5.52 CI 2.57–11.86), confirmed in multivariate analysis. ZAP-70 expression was correlated to poorer outcome also when we evaluated only the 69 stage A patients and the 56 cases with non-diffuse bone marrow infiltration, whereas in patients with diffuse pattern ZAP-70 expression had no prognostic significance. In conclusion, immunohistological detection of ZAP-70 on bone marrow samples at diagnosis appears a useful methodological approach to identify patients with different prognosis in B-CLL.

Serum angiogenin is not elevated in patients with early B-cell chronic lymphocytic leukemia but is prognostic factor for disease progression.

The association between angiogenin and cancer progression and poor outcome in solid tumors has been documented, but its significance in leukemias has not been evaluated. Using an ELISA technique (Quantikine Human Angiogenin Immunoassay; R&D Systems), we measured serum angiogenin levels in 77 previously untreated Binet stage A B-cell chronic lymphocytic leukemia (CLL) patients. No difference in angiogenin serum levels could be found between patients (median: 295 ng/mL; range: 74-1700) and 15 age- and sex-matched healthy controls (median: 264 ng/mL; range: 29-1835) (P = NS; Mann-Whitney test). Increased angiogenin serum level was associated with higher LDH (P = 0.03) and beta2-m (P = 0.007) concentrations. However, angiogenin did not reflect the extent of bone marrow (BM) angiogenesis as evaluated by microvessel area (P = 0.611), circulating levels of vascular endothelial growth factor (VEGF) (P = 0.873) and basic fibroblastic growth factor (FGF-2) (P = 0.421). When the 25 patients with available data were stratified into the four major cytogenetic categories (normal karyotype, 13q as a sole aberration, 12q trisomy, 11q or 17p deletion) and aberrations were compared with angiogenin serum levels, no correlation was found (P = 0.651; Kruskall-Wallis test). A cut-off of angiogenin serum level corresponding to median (i.e. 330 ng/mL) or higher identified later upstaging and longer progression-free survival (PFS). The 5-yr PFS was 51.5% for patients with angiogenin levels lower than median and 85% for patients with higher values [P = 0.03; hazard ratio (HR) = 2.86; 95% CI: 1.08-6.72]. Although in multivariate analysis only Rai substages (P = 0.00001) and peripheral blood lymphocytosis (P = 0.009) retained their prognostic significance, angiogenin could be incorporated into the Rai substages thus leading to the identification of the following risk categories: (i) stage 0 (angionenin >330 ng/mL); (ii) stage 0 (angiogenin <330 ng/mL) + stage I-II (angiogenin >330 ng/mL); and (iii) stage I-II (angiogenin <330 ng/mL). The 40-month PFS were as follows: 85%, 65%, 25% (chi(2) for trend = 6.33; d.f. = 1; P = 0.01). In conclusion, serum angiogenin levels although not increased in comparison with healthy controls, may predict clinical outcome of patients with early CLL and help to refine Rai's stratification.

Clinicoprognostic implications of increased serum levels of vascular endothelial growth factor and basic fibroblastic growth factor in early B-cell chronic lymphocytic leukaemia.

To assess the relative merit of increased serum levels of vascular endothelial growth factor and basic fibroblastic growth factor in predicting the risk of disease progression of patients with early B-cell chronic lymphocytic leukaemia we analyzed 81 Binet stage A patients whose sera were taken at the time of diagnosis and evaluated for the presence of vascular endothelial growth factor and basic fibroblast growth factor using an enzyme-linked immunosorbent assay. Serum levels of vascular endothelial growth factor positively correlated with Rai sub-stages (P=0.03), peripheral blood lymphocytosis (P=0.03), bone marrow histology (P=0.04) and β2-microglobulin (β2-m) (P=0.006). When dealing with basic fibroblast growth factor only a correlation with Rai sub-stages (P=0.02) could be found. Different cut-offs set on the basis of a stratification in quartiles, failed to demonstrate any correlation between serum levels of basic fibroblast growth factor and disease progression. In contrast, patients with increased serum levels of vascular endothelial growth factor (above median value, 203 pg ml−1) had a three times increased risk of disease progression, although, in multivariate analysis only Rai sub-stages (P=0.0001) and lymphocyte doubling time (P=0.002) retained their prognostic significance. Low levels of vascular endothelial growth factor were indicative of good clinical outcome in the subgroup of patients with either low (P=0.02) or high (P=0.03) β2-m concentration. Finally, the highest prognostic power was obtained when serum vascular endothelial growth factor and β2-m were examined in combination. Median of progression-free survival of patients who had both serum vascular endothelial growth factor and β2-m higher than median value was only 13 months, in contrast median progression-free survival of patients with one marker increased (i.e. above the 50th percentile) was 40 months. Patients with both markers below the median experienced the best clinical outcome (median progression-free survival not reached at 40 months). In conclusion, serum levels of either vascular endothelial growth factor or basic fibroblast growth factor are high in patients with early chronic lymphocytic leukaemia, however, only vascular endothelial growth factor predicts behaviour of disease and helps to refine the prognosis of stage A patients.British Journal of Cancer (2002) 86, 31–35. DOI: 10.1038/sj/bjc/6600022 www.bjcancer.com© 2002 The Cancer Research Campaign