Clinical significance of LAIR1 (CD305) as assessed by flow cytometry in a prospective series of patients with chronic lymphocytic leukemia.

Most patients affected by chronic lymphocytic leukemia are diagnosed by flow cytometry. Several immunophenotypic markers have been identified as significant and independent prognostic variables, especially from retrospective cohorts. However, while attractive because their detection is inexpensive and feasible in most laboratories, only few have been validated by independent series. The expression of leukocyte-associated immunoglobulin-like receptor-1 (also known as LAIR1, LAIR-1 or CD305), an inhibitor of B-cell receptor-mediated signaling, has been reported to be lacking in high-risk chronic lymphocytic leukemia. However, its correlation with biological variables and its prognostic significance remain unknown. We investigated 311 consecutive patients, prospectively enrolled since 2007. Methods for studying patients were standardized and included clinical assessment, immunophenotype, fluorescence in situ hybridization, and status of immunoglobulin heavy chain variable region genes. Overall, 22.1% of patients had Binet stage B or C disease, 38.5% had unmutated immunoglobulin genes, 15.1% had high-risk cytogenetic abnormalities, 23.4% were CD38(+), 37.8% CD49d(+), and 59.8% LAIR1(+). Expression of LAIR1 was inversely related to that of CD38 (P=0.0005), but was not associated with CD49d expression (P=0.96). A significantly lower expression of LAIR1 was observed in patients with Binet stage B or C disease (P=0.023), and in the presence of high-risk cytogenetic abnormalities (P=0.048) or unmutated immunoglobulin heavy chain variable region genes (P<0.0001). At univariate analysis LAIR1(+) was significantly associated with longer time to first treatment (P=0.0002). This favorable effect of LAIR1(+) was confirmed by multivariate analysis (hazard ratio=2.1, P=0.03 for LAIR1). Our results indicate that LAIR1 expression is a reliable and inexpensive marker capable of independently predicting time to first treatment in newly diagnosed unselected patients with chronic lymphocytic leukemia.

Variant Philadelphia translocations: molecular-cytogenetic characterization and prognostic influence on frontline imatinib therapy, a GIMEMA Working Party on CML analysis.

Variant Philadelphia (Ph) chromosome translocations have been reported in 5%-10% of patients with newly diagnosed chronic myeloid leukemia (CML). Variant translocations may involve one or more chromosomes in addition to 9 and 22, and can be generated by 2 different mechanisms, 1-step and 2-step rearrangements, as revealed by fluorescence in situ hybridization. The prognostic significance of the occurrence of variant translocations has been discussed in previous studies. The European LeukemiaNet recommendations do not provide a "warning" for patients with variant translocations, but there is limited information about their outcome after therapy with tyrosine kinase inhibitors. To identify the role of variant translocations in early chronic phase (CP) CML patients treated with imatinib mesylate, we performed an analysis in a large series of 559 patients enrolled in 3 prospective imatinib trials of the Gruppo Italiano Malattie EMatologiche dell'Adulto (GIMEMA) Working Party on CML. Variant translocations occurred in 30 patients (5%). Our data show that the presence of variant translocations has no impact on the cytogenetic and molecular response or on outcome, regardless of the involvement of different mechanisms, the number of involved chromosomes, or the presence of deletions. Therefore, we suggest that patients with variant translocations do not constitute a "warning" category in the imatinib era. This study is registered at www.clinicaltrials.gov as NCT00514488 and NCT00510926.

B-cell receptor configuration and adverse cytogenetics are associated with autoimmune hemolytic anemia in chronic lymphocytic leukemia.

The development of autoimmune hemolytic anemia (AIHA) in patients with chronic lymphocytic leukemia (CLL) is associated with specific biological features. The occurrence of AIHA was hereby investigated in a retrospective series of 585 CLL patients with available immunoglobulin heavy chain variable (IGHV) gene status. AIHA occurred in 73 patients and was significantly associated with an IGHV unmutated (UM) status (P < 0.0001) and unfavorable [del(17)(p13) and del(11)(q23)] cytogenetic lesions (P < 0.0001). Stereotyped HCDR3 sequences were identified in 29.6% of cases and were similarly represented among patients developing or not AIHA; notably, subset #3 was associated with a significantly higher risk of AIHA than the other patients (P = 0.004). Multivariate analysis showed that UM IGHV, del(17)(p13) and del(11)(q23), but not stereotyped subset #3, were the strongest independent variables associated with AIHA. Based on these findings, we generated a biological risk score for AIHA development according to the presence of none (low risk), one (intermediated risk), or two (high risk) of the independent risk factors. Overall, our data indicate that UM IGHV status and/or unfavorable cytogenetic lesions are associated with the risk of developing secondary AIHA in CLL patients and suggest a possible role of specific stereotyped B-cell receptor subsets in a proportion of cases.

Increased apoptosis in circulating lymphocyte cultures of anti-RNA polymerase III positive patients with systemic sclerosis.

The sera of 39 patients (38 women and 1 man), 16 with limited and 23 with diffuse clinical form of systemic sclerosis (SSc), were tested for anti-centromere (ACA), anti-topoisomerase I (ATA) and anti-RNA polymerase III (ARA) antibodies. The presence of apoptotic cells in cultures of circulating lymphocytes was investigated using the TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling) technique. ACAs were present in 16 (41%), ATA in 15 (38%) and ARA in 8 (21%) cases. The mean frequency of apoptotic lymphocytes was statistically higher in the ARA positive patients with respect to that in the control population (P < 0.001), in ACA (P < 0.001) and in the ATA (P < 0.001) groups. Moreover, apoptosis was distributed homogenously in ACA and ATA positive subjects, but not in the ARA patients. Our results show that there is an increase in apoptosis in the lymphocytes of ARA positive SSc patients.

Unstabilized DNA breaks in lymphocytes of patients with different subsets of systemic sclerosis.

The clastogenic effects on DNA, proven by the presence of micronuclei (MN) and the protective cellular mechanisms normally used to stabilize DNA breaks were investigated in three subsets of patients with systemic sclerosis (SSc). The frequency of MN found in cultures of peripheral lymphocytes in patients with anticentromere and antitopoisomerase I antibodies was significantly higher than that in the control group. The group with anticentromere antibody showed a significantly higher frequency of MN than did the subjects with antitopoisomerase antibody (4.22% versus 2.34%, P < 0.001). Patients with anti-RNA polymerase III, instead, had a low prevalence of typical micronucleated cells (0.98%), not significantly different from that of the healthy controls (0.82%). Moreover, when MN was characterized for the presence or absence of DNA fragments with free 3'-OH ends by digoxigenin-dUTP (DIG-dUTP) using terminal deoxynucleotidil transferase, its frequency was found to be increased in the groups with anticentromere and antitopoisomerase I antibodies with respect to that in the controls. The increase was significantly higher in the lymphocytes of the patients with anticentromere than in those with antitopoisomerase I antibody (35% versus 20.08%, P < 0.001). Nonetheless, the prevalence of unstable DNA fragments in patients with anti-RNA polymerase III antibody was low (2.05%) and not significantly different from that of the control group (1.18%). Our results indicate that there is a clastogenic effect on DNA and an interference in the protective cellular mechanisms normally stabilizing DNA breaks only in some subsets of SSc patients.

Genetics and Autosomal Dominant Polycystic Kidney Disease Progression.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary kidney disease, accounting for 10% of European patients on renal replacement therapy. In the previous years, many approaches to slow the progression of ADPKD were studied and many clinical trials published. In addition to having diagnostic role, the description of the genotype is even important to predict the progression of the disease and contributes, combined with several other factors, to a more precise patients classification. With the availability of disease-modifying drugs, "fast progression factors" are needed to early recognize those patients who would likely progress, before cyst growth reaches a critical value. ERA-EDTA working group on inherited kidney disorders included a series of recommendations resulting in a hierarchical decision algorithm to select patients who are most likely to benefit from the treatment. Beyond diagnosis, we will also discuss the important role of genetics in ADPKD progression and management.

Unstabilized DNA breaks in lymphocytes of patients with systemic sclerosis.

The clastogenic effects on DNA, proven by the presence of micronuclei (MN), and the protective cellular mechanisms normally used to stabilize DNA breaks were investigated in patients with systemic sclerosis (SSc). The frequency of micronucleated cells found in cultures of peripheral lymphocytes in patients was significantly higher than in the control group. The patient group with anti-centromere antibodies showed a significantly higher frequency of micronucleated cells than that observed in the patients with anti-topoisomerase I antibodies (4.22% versus 2.34%, p < 0.001). Moreover, we attempted to characterize MN for the presence or absence of DNA fragments with free 3'-OH ends by digoxigenin-dUTP (DIG-dUTP) using terminal deoxynucleotidil transferase. It was found that the frequency of MN containing DNA fragments with 3'-OH free ends (unstable fragments) increased in SSc patients compared to that observed in the control group. Moreover, this increase was significantly higher in lymphocytes of the patients with anti-centromere antibodies than in those with anti-topoisomerase I antibodies (35% versus 20.08%, p < 0.001). Our results indicate that in SSc patients there is an interference in the protective cellular mechanisms, normally stabilizing DNA breaks.