Clinical utility of molecular and flow cytometric markers in chronic lymphocytic leukaemia

Background: Chronic lymphocytic leukaemia (CLL) is a clinically heterogeneous disease. While immunoglobulin variable region heavy chain (IgVH) mutational status remains the ‘gold standard’ in molecular prognostication, a range of additional markers is increasingly being used in clinical trials. As awareness of trial data increases, requests to determine these prognostic markers for new CLL patients are becoming more prevalent in Australia. Aim: To explore the clinical utility of currently available prognostic markers for CLL in an Australian cohort. Methods: IgVH mutational status and gene usage was determined and compared with other reported immunophenotypic markers, cytogenetics and clinical outcome as defined by treatment‐free survival (TFS), lymphocyte doubling time and clinical stage in a cohort of 65 CLL patients. Results: An unmutated IgVH gene, high expression of CD38, ZAP‐70, CD25, CD49d, CD54 or low expression of CD49c was associated with shorter TFS indicating an adverse clinical prognosis in our cohort. High expression of each of CD38, ZAP‐70, CD49d and CD54 was significantly associated with an unmutated IgVH gene; however, associations were not absolute. IgVH and CD25 expression retained their significance in multivariate analysis. Concordant CD25^high/IgVH unmutated CLL patients had the shortest median TFS interval (40 months) in our cohort. Conclusions: Molecular and immunophenotypic markers remain useful as adjuncts to clinical prognostication; however, as single parameters they are unable to dictate the timing of therapeutic intervention. The combined use of CD25 and IgVH mutational status may be clinically relevant to CLL prognostication while also providing insight into the biological pathways involved in disease progression.     

MDM2 promotor polymorphism and disease characteristics in chronic lymphocytic leukemia: results of an individual patient data-based meta-analysis

A number of single nucleotide polymorphisms have been associated with disease predisposition in chronic lymphocytic leukemia. A single nucleotide polymorphism in the MDM2 promotor region, MDM2SNP309, was shown to soothe the p53 pathway. In the current study, we aimed to clarify the effect of the MDM2SNP309 on chronic lymphocytic leukemia characteristics and outcome. We performed a meta-analysis of data from 2598 individual patients from 10 different cohorts. Patients’ data and genetic analysis for MDM2SNP309 genotype, immunoglobulin heavy chain variable region mutation status and fluorescence in situ hybridization results were collected. There were no differences in overall survival based on the polymorphism (log rank test, stratified by study cohort; P=0.76; GG genotype: cohort-adjusted median overall survival of 151 months; TG: 153 months; TT: 149 months). In a multivariable Cox proportional hazards regression analysis, advanced age, male sex and unmutated immunoglobulin heavy chain variable region genes were associated with inferior survival, but not the MDM2 genotype. The MDM2SNP309 is unlikely to influence disease characteristics and prognosis in chronic lymphocytic leukemia. Studies investigating the impact of individual single nucleotide polymorphisms on prognosis are often controversial. This may be due to selection bias and small sample size. A meta-analysis based on individual patient data provides a reasonable strategy for prognostic factor analyses in the case of small individual studies. Individual patient data-based meta-analysis can, therefore, be a powerful tool to assess genetic risk factors in the absence of large studies.     

Haplotype structure and selection of the MDM2 oncogene in humans

The MDM2 protein is an ubiquitin ligase that plays a critical role in regulating the levels and activity of the p53 protein, which is a central tumor suppressor. A SNP in the human MDM2 gene (SNP309 T/G) occurs at frequencies dependent on demographic history and has been shown to have important differential effects on the activity of the MDM2 and p53 proteins and to associate with altered risk for the development of several cancers. In this report, the haplotype structure of the MDM2 gene is determined by using 14 different SNPs across the gene from three different population samples: Caucasians, African Americans, and the Ashkenazi Jewish ethnic group. The results presented in this report indicate that there is a substantially reduced variability of the deleterious SNP309 G allele haplotype in all three populations studied, whereas multiple common T allele haplotypes were found in all three populations. This observation, coupled with the relatively high frequency of the G allele haplotype in both and Caucasian and Ashkenazi Jewish population data sets, suggests that this haplotype could have undergone a recent positive selection sweep. An entropy-based selection test is presented that explicitly takes into account the correlations between different SNPs, and the analysis of MDM2 reveals a significant departure from the standard assumptions of selective neutrality.     

Single-nucleotide polymorphisms p53 G72C and Mdm2 T309G in patients with psoriasis, psoriatic arthritis, and SAPHO syndrome

Psoriasis (Ps), psoriatic arthritis (PsA), and SAPHO syndrome are diseases of unknown etiology that share common clinical features; however, family studies support the hypothesis of a genetic background for each of these diseases. To study the two common single-nucleotide polymorphisms (SNP) in the murine-double-minute-2-(Mdm2) and p53 genes in patients with Ps, PsA, and SAPHO syndrome. Genomic DNA was obtained from 187 patients with Ps, 50 with PsA, and 36 with SAPHO as well as 478 healthy controls. Mdm2-gene SNP T309G and p53-gene SNP G72C genotypes were determined by the polymerase chain reaction. Genotype and allele frequencies were analyzed with χ²-tests. Among the patients with Ps and PsA, no differences in allele or genotype frequencies of the p53-gene SNP G72C and Mdm2-gene SNP T309G were detected. However, in the SAPHO patients group, the frequencies of the Mdm2 SNP309 G allele and the genotype SNP 309 GG were significantly increased compared with the controls (G allele: 51.4 vs. 38.7%, P = 0.034; genotype GG: 36.1 vs. 14.2%, P = 0.002). In addition, the frequencies of the p53 SNP72 C allele and the genotype SNP 72 CC were also increased in the SAPHO patients cohort (C allele: 36.1 vs. 25.6%, P = 0.05; genotype CC: 16.7 vs. 6.3%, P = 0.05). SAPHO syndrome may be linked to an imbalance between MDM2 and p53 regulation with a “weak” p53-response associated with the Mdm2 SNP 309 G allele. In contrast, the p53 network does not seem to play a major role in pathogenesis of Ps or PsA.     

Association between MDM2-SNP309 and hepatocellular carcinoma in Taiwanese population

AIM: To investigate the risk association and compare the onset age of hepatocellular carcinoma (HCC)patients in Taiwan with different genotypes of MDM2-SNP309.METHODS: We analyzed MDM2-SNP309 genotypes from 58 patients with HCC and 138 cancer-free healthy controls consecutively. Genotyping of MDM2-SNP309 was conducted by restriction fragment length polymorphism assay.RESULTS: The proportion of homozygous MDM2-SNP309 genotype (G/G) in cases and cancer-free healthy controls was similar (17.2% vs 16.7%). Multivariate analysis showed that the risk of G/G genotype of MDM2-SNP309 vs wild-type T/T genotype in patients with HCC was not significant (OR = 1.265, 95%CI = 0.074-21.77) after adjustment for sex, hepatitis B or C virus infection, age, and cardiovascular disease/diabetes. Nevertheless, there was a trend that GG genotype of MDM2-SNP309 might increase the risk in HCC patients infected with hepatitis virus (OR = 2.568,95% CI = 0.054-121.69). Besides, the homozygous MDM2-SNP309 genotype did not exhibit a significantly earlier age of onset for HCC.CONCLUSION: Current data suggest that the association between MDM2-SNP309 GG genotype and HCC is not significant, while the risk may be enhanced in patients infected by hepatitis virus in Taiwan.     

Current evidence on the relationship between murine double minute 2 T309G polymorphism and esophageal cancer susceptibility

The relationship between murine double minute 2 (MDM2) T309G polymorphism and esophageal cancer risk has been discussed with discrepant results. The aim of our study is to investigate the systematic association between the potentially functional MDM2 T309G polymorphism and esophageal cancer risk. Eligible studies were included through searching the databases of PubMed, EMBASE, and Chinese National Knowledge Infrastructure (up to April 2014). The crude odds ratio (OR) and 95% confidence interval (CI) were used to estimate the strength of the association. Six published case–control studies, including 1899 cases and 3016 controls, were identified. Overall, our study suggested that MDM2 T309G polymorphism was significantly associated with increased risk of esophageal cancer (TT vs. GG: OR = 0.77, 95% CI = 0.65–0.90, P = 0.002; T vs. G: OR = 0.88, 95% CI = 0.81–0.96, P = 0.002). In subgroup analyses stratified by source of controls, ethnicity, and quality score assessment, respectively, similar results were obtained (TT vs. GG: OR = 0.65, 95% CI = 0.48–0.89, P = 0.007 for hospital‐based studies; T vs. G: OR = 0.90, 95% CI = 0.81–0.99, P = 0.04 for population‐based studies; and T vs. G: OR = 0.85, 95% CI = 0.78–0.93, P = 0.004 for Asians). The results of Begg's test and Egger's test did not suggest publication bias in the studies. Therefore, the MDM2 T309G polymorphism may be significantly associated with increased esophageal cancer risk, especially among Asians.     

The G‐Allele of the PSMA6−8C>G polymorphism is associated with poor outcome in multiple myeloma independently of circulating proteasome serum levels

Introduction: The proteasome system plays a crucial role in several malignant disorders, especially in multiple myeloma (MM). The G‐allele of a single nucleotide polymorphism (SNP) ?8C>G in the gene PSMA6, one of seven α‐subunit genes of the 20S proteasome, was associated with myocardial infarction. Moreover, PSMA6 mRNA expression in human B‐cell lines depended on genotypes. We investigated a potential role of this novel SNP in patients with MM. Methods: PSMA6 genotypes of 116 patients with MM were associated with survival. Circulating proteasome levels (CPL) dependent on ?8C>G genotypes of 70 newly diagnosed patients were studied using an anti‐20S proteasome enzyme‐linked immunoabsorbant assay (ELISA). Results: Genotype distribution (69 CC, 44 CG, 3 GG) was compatible with Hardy–Weinberg equilibrium. Kaplan–Meier curves revealed a significant association of PSMA6?8C>G with 5‐yr survival (P = 0.014). Median survival time was 43 months for the GG genotype and 50 months for the CG genotype. It was not reached within follow‐up by the CC genotype (CC 5‐yr survival rate 61.2%). Following hazard ratio (HR) for overall survival was calculated: G‐allele vs. CC genotype: 2.038, 95% CI 1.14–3.65, P = 0.017. In multivariate analysis the G‐allele was an independent prognostic factor (HR 2.1, P = 0.014). CPL were not significantly different between genotypes [mean CPL: CC 284.9 ng/mL vs. 303.3 ng/mL G‐allele carriers (P = 0.709)]. Conclusions: These results suggest the G‐allele of the PSMA6?8C>G polymorphism as a possible survival prognosticator.     

Significant change in ZAP‐70 expression during the course of chronic lymphocytic leukemia

Introduction: It is widely accepted that expression of ZAP‐70 in chronic lymphocytic leukemia (CLL) remains stable in time. However, data supporting this notion are surprisingly scarce. Therefore, we assessed expression of ZAP‐70 in serial samples taken during the course of the disease. Patients and Methods: We studied 44 patients with CLL diagnosed according to NCI‐WG criteria (34 men, 10 women, median age 62, range, 36–81). A total of 104 samples were examined; all patients had at least two measurements. Median interval between the first and the second sample was 13 months (range, 2–36). ZAP‐70 expression was detected by flow cytometry using phycoerythrin‐conjugated monoclonal antibody clone 1E7.2 and negative isotype control. Twenty percent of positive cells were considered as the threshold of positivity. Results: Significant change in ZAP‐70 expression (i.e. from positivity to negativity and vice versa) was detected in 15/44 patients (34%). Interestingly, 7/8 patients whose ZAP‐70 expression converted to positivity had unmutated IgVH genes. In addition, the conversion was accompanied by clinical progression or relapse in all but one patient. On the other hand, 5/7 patients with loss of ZAP‐70 had stable clinical course. One patient became ZAP‐70‐negative during treatment with prednisone for autoimmune hemolytic anemia. Conclusions: In contrast to commonly accepted opinion, significant change in ZAP‐70 expression in time was detected in a substantial proportion of our patients with CLL. While the conversion to ZAP‐70 negativity was found predominantly in patients with stable disease, change to positivity was typical in patients with unmutated IgVH genes at the time of progression or relapse. Based on our pilot results, repeated assessment of ZAP‐70 expression might be especially useful at the time of progression or relapse in patients who were initially ZAP‐70‐negative.     

Comparison between oral and intravenous fludarabine plus cyclophosphamide regime as front-line therapy in patients affected by chronic lymphocytic leukaemia: influence of biological parameters on the clinical outcome

The fludarabine plus cyclophosphamide (FC) regimen was reported to be superior to chlorambucil or fludarabine alone in terms of complete response (CR), overall response (OR) and progression-free survival (PFS) in previously untreated patients with chronic lymphocytic leukaemia (CLL). In the present study, we compared the efficacy and toxicity of FC administered through oral and intravenous route in 65 untreated patients affected by advanced CLL. No statistical differences were noticed between the two routes of administration in terms of OR, PFS, time to re-treatment (TTR) and overall survival (OS) of analysed patients. We also assessed the influence on the clinical outcome of the mutation status of the immunoglobulin variable region heavy chain (IgVH) gene, of the cytogenetic abnormalities and of the expression of ZAP70 and CD38 in patients' primary samples. Among the 58 evaluable patients, 31 (53%) achieved a CR and 18 (31%) a partial response. The median PFS was 35?months, median TTR was 42?months and median OS was not reached after 45?months (range, 1?C161). A significantly lower OR rate was noticed in patients with high-risk cytogenetic abnormalities (del 17p, del 11q). In this study, high-risk cytogenetic abnormalities and unmutated IgVH genes were independent predictors of TTR. These results underline the importance of biological stratifications in front-line treatment of CLL patients. We confirm that FC is an effective regimen with mild toxicities; it could be recommended for patients with low-risk biological parameters who represent, in our experience, about 30% of the total.     

Comprehensive biomarker and genomic analysis identifies p53 status as the major determinant of response to MDM2 inhibitors in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is the most common leukemia in the Western world and remains incurable with conventional therapies. Patients with relapsed or resistant CLL have a significantly shortened lifespan. MDM2 inhibitors have been developed and may have significant potential in the treatment of CLL. Clinical development of these compounds would be aided through knowledge of molecular predictors of activity. To understand determinants of sensitivity or resistance to MDM2 inhibitor therapy in CLL, we comprehensively analyzed a large cohort of CLL patient-derived samples for response to MDM2 inhibition and correlated these responses with clinically important biomarkers. Furthermore, we employed high-density single nucleotide polymorphism (SNP) arrays to analyze genomewide changes of copy number and allele status, including that of p53. The results of these studies conclusively demonstrate that p53 status is the major determinant of response to MDM2 inhibitors in CLL. Additional defects in the p53 regulatory cascade do not appear operational in this leukemia. Further, we identify a novel subgroup of patients with CLL with early progressive disease that appears particularly sensitive to MDM2 inhibitor treatment. These data provide definitive evidence for target-specific and predictive activity and a rationale to proceed with this potentially important class of compounds in the treatment of CLL.     

MDM2 SNP309 and TP53 Arg72Pro interact to alter therapy-related acute myeloid leukemia susceptibility

The p53 tumor suppressor directs the cellular response to many mechanistically distinct DNA-damaging agents and is selected against during the pathogenesis of therapy-related acute myeloid leukemia (t-AML). We hypothesized that constitutional genetic variation in the p53 pathway would affect t-AML risk. Therefore, we tested associations between patients with t-AML (n = 171) and 2 common functional p53-pathway variants, the MDM2 SNP309 and the TP53 codon 72 polymorphism. Although neither polymorphism alone influenced the risk of t-AML, an interactive effect was detected such that MDM2 TT TP53 Arg/Arg double homozygotes, and individuals carrying both a MDM2 G allele and a TP53 Pro allele, were at increased risk of t-AML (P value for interaction is .009). This interactive effect was observed in patients previously treated with chemotherapy but not in patients treated with radiotherapy, and in patients with loss of chromosomes 5 and/or 7, acquired abnormalities associated with prior exposure to alkylator chemotherapy. In addition, there was a trend toward shorter latency to t-AML in MDM2 GG versus TT homozygotes in females but not in males, and in younger but not older patients. These data indicate that the MDM2 and TP53 variants interact to modulate responses to genotoxic therapy and are determinants of risk for t-AML.     

The effect of IgVH mutational status on the induction of apoptosis by rituximab in patients with heavily pretreated B-cell chronic lymphocytic leukemia: evidence from a clinical phase I/II trial

We investigated tumor cell apoptosis in vivo in 14 heavily pretreated patients with B-cell chronic lymphocytic leukemia undergoing rituximab monotherapy. Apoptosis induction was more pronounced in patients with mutated IgVH genes than in those with unmutated IgVH genes, independently of the levels of expression of CD20, CD38, and ZAP-70 and of the presence of 17p deletion. Our results suggest an association between IgVH gene mutational status and rituximab-induced apoptosis.     

Prognostic relevance of the FAB morphological criteria in chronic lymphocytic leukemia: correlations with IgVH gene mutational status and other prognostic markers

Morphological examination is the routine first step in the diagnosis of hematological malignancies, including chronic lymphocytic leukemia (CLL). Atypical cell morphology according to the FAB criteria is known to herald disease progression. Several years ago, it was proposed that FAB morphology at diagnosis had a considerable prognostic impact. However, this proposal has not been widely adopted in practice. Thus we questioned the prognostic value of the morphological examination, which was performed retrospectively in 88 patients out of our 110 institutional registry patients (70 males and 40 females, median age 57 yrs) with CLL at diagnosis. We related the results to the more modern prognostic markers. Atypical FAB morphology was shown to correlate with IgVH gene mutation status, trisomy of chromosome 12 and deletion of 17p detected either by conventional G-banding or by fluorescence in situ hybridization (FISH) analysis. The correlation of FAB morphology with CD38 antigen expression or with the histopathological pattern of bone marrow infiltration was not significant. Overall survival (OS) data were available for 84 morphologically examined patients. The patients with atypical morphology (64 patients) had a significantly shorter OS (103 months) than the 20 patients presenting with typical CLL morphology (237 months; p=0.03). Only the mutation status of IgVH genes correlated more closely with OS (p=0.002). Of note, there was no leukemia-related death within "unmutated" cases who had typical FAB morphology (p=0.14), and vice versa, the mutation status had a significant prognostic impact within the morphologically atypical cases (p=0.01). Thus FAB morphology and the mutation status may yield complementary prognostic information. OS was affected both by the presence of cytogenetic aberrations (p=0.03) - most adversely by deletions of 17p and 11q, and by CD38 expression (p=0.003). We conclude that careful examination of peripheral blood smears according to FAB is a simple, cheap and valuable tool in the first-line assessment of prognosis of CLL patients and should not be overlooked even in 3rd millennium when more sophisticated prognostic markers are at hand. This ought to be confirmed in larger prospective studies with multivariate analysis of data.     

FCRL2 mRNA expression is inversely associated with clinical progression in chronic lymphocytic leukemia

Fc receptor‐like 2 (FCRL2) is highly expressed on B‐cell chronic lymphocytic leukemia (B‐CLL) cells and could possibly influence disease pathogenesis. Therefore, we investigated FCRL2 mRNA expression in a large cohort with 152 CLL patients in order to assess its role in risk prediction in B‐CLL. FCRL2 mRNA expression was found to be expressed at considerably higher levels in peripheral blood mononuclear cells (PBMC) of B‐CLL patients compared to controls (range 1.35‐ to 210‐fold upregulation; P < 0.0001) and cells of other hematological diseases. Patients with high FCRL2 expression (according to ROC‐analysis) had a significantly longer treatment‐free survival (TFS) and overall survival (OS) than patients with low FCRL2 expression (median TFS: 119 vs. 34 months, P < 0.0001; median OS: 321 months vs. not reached, P = 0.009). Univariate comparisons found that FCRL2 expression was weakly associated with IGHV mutation status (P = 0.05), CD38 status (P < 0.0001) and ZAP‐70 status (P < 0.0001). Furthermore, we show that the combination of FCRL2 with ZAP70‐, CD38‐ or IGHV‐status could further significantly refine the prognostic information provided by either of the factors alone in TFS and OS. In multivariate analysis low FCRL2 expression was a significant independent prognostic factor (HR 2.4; P = 0.005). Here we demonstrate that the level of FCRL2 expression is correlated with prognosis in B‐CLL.     

Is MDM2 SNP309 Variation a Risk Factor for Head and Neck Carcinoma?: An Updated Meta-Analysis Based on 11,552 Individuals

Murine double minute-2 (MDM2) is a negative regulator of P53, and its T309G polymorphism has been suggested as a risk factor for a variety of cancers. Increasing evidence has shown the association of MDM2 T309G polymorphism with head and neck carcinoma (HNC) risk. However, the results are inconsistent. Thus, we performed a meta-analysis to elucidate the association.The meta-analysis retrieved studies published up to August 2015, and essential information was extracted for analysis. Separate analyses on ethnicity, source of controls, sample size, detection method, and cancer types were also conducted. Odds ratios (ORs) and their 95% confidence intervals (CIs) were used to estimate the association.Pooled data from 16 case–control studies including 4625 cases and 6927 controls failed to indicate a significant association. However, in the subgroup analysis of sample sizes, an increased risk was observed in the largest sample size group (>1000) under a recessive model (OR = 1.52; 95% CI = 1.08–2.13). Increased risks were also found in the nasopharyngeal cancer in the subgroup analysis of cancer types (GG vs TT: OR = 2.07; 95% CI = 1.38–3.12; dominant model: OR = 1.48; 95% CI = 1.13–1.93; recessive model: OR = 1.76; 95% CI = 1.17–2.65).The results suggest that homozygote GG alleles of MDM2 SNP309 may be a low-penetrant risk factor for HNC, and G allele may confer nasopharyngeal cancer susceptibility.     

The absolute percent deviation of IGHV mutation rather than a 98% cut‐off predicts survival of chronic lymphocytic leukaemia patients treated with fludarabine,cyclophosphamide and rituximab

The degree of somatic hypermutation, determined as percent deviation of immunoglobulin heavy chain gene variable region sequence from the germline (IGHV%), is an important prognostic factor in chronic lymphocytic leukaemia (CLL). Currently, a cut‐off of 2% deviation or 98% sequence identity to germline in IGHV sequence is routinely used to dichotomize CLL patients into mutated and unmutated groups. Because dissimilar IGHV% cut‐offs of 1–5% were identified in different studies, we wondered whether no cut‐off should be applied and IGHV% treated as a continuous variable. We analysed the significance of IGHV% in 203 CLL patients enrolled on the original frontline fludarabine, cyclophosphamide and rituximab (FCR) trial with a median of 10 years follow‐up. Using the Cox Proportional Hazard model, IGHV% was identified as a continuous variable that is significantly associated with progression‐free (PFS) and overall survival (OS) (P < 0·001). Furthermore, we validated this finding in 323 patients treated with FCR off‐protocol and in the total cohort (n = 535). Multivariate analysis revealed a continuous trend. Higher IGHV% levels were incrementally associated with favorable PFS and OS in both FCR‐treated cohorts (P < 0·001, both cohorts). Taken together, our data suggest that IGHV% is a continuous variable in CLL patients treated with FCR.