Rai Stage Research Articles

KRAS, NRAS and BRAF Mutations Are Highly Enriched in TRI12 Chronic Lymphocytic Leukemia and Are Associated to Shorter Time to First Treatment

BackgroundExtensive sequencing data highlighted the recurrence of mutations resulting in constitutive MAP kinase signaling in chronic lymphocytic leukemia (CLL), and proposed these mutations as late drivers of CLL progression (X.S. Puente, Nature, 2015; D.A. Landau, Nature, 2015). Although some preliminary studies suggested KRAS, NRAS and BRAF mutations as possibly associated with trisomy 12 (TRI12) and/or unmutated (UM) IGHV genes (C.D. Herling, Blood, 2016; K. Takahashi, Blood, 2018), dedicated analysis of these aspects is still missing.AimTo correlate the occurrence of KRAS, NRAS and BRAF mutations with specific clinico-biological features in CLL.MethodsThe study included 534 primary CLL purposely enriched with 300 TRI12 cases: 190 cases with TRI12 as sole chromosomal aberration (TRI12only) and 110 cases harboring TRI12 plus other chromosomal aberrations (TRI12plus). The cohort was also enriched in cases with NOTCH1 aberrations (n=214) to evaluate their possible role in RAS-RAF mutations incidence.Amplicon-based targeted next-generation sequencing assay was performed using Miseq Illumina on DNA from purified CLL samples. Analyzed amplicons mapped in the hotspot regions of KRAS, NRAS (exons 2, 3 and 4) and BRAF (exons 11 and 15) genes. Variant allele frequency (VAF) was obtained by Miseq report. CLL cases were characterized for IGHV mutational status, main cytogenetic abnormalities, NOTCH1 and TP53 aberrations. Time to first treatment (TTFT) data were correlated with molecular findings.ResultsWe found 90 missense point mutations (Fig.1) in 64 CLL cases, with prevalence of KRAS (44 mutations in 38 patients), followed by BRAF (31 mutations in 24 patients) and NRAS (15 mutations in 13 patients). Nearly all mutations have been previously associated with gain-of-function phenotype and increased RAS/ERK downstream signaling. The co-occurrence of 2 mutated genes (mainly KRAS and BRAF) were observed in 11 patients. Mutations were mainly subclonal (median VAF 6.15%, range 1.3%-61.6%) with only one third of mutations (27/90) above 15% VAF.A high association between the presence of KRAS/NRAS/BRAF mutations and UM IGHV and the presence of TRI12 was observed. Overall, 87.3% of KRAS/NRAS/BRAF mutated cases had a UM IGHV (p<0.0001) and 79.7% were TRI12 CLL (p<0.0001) (Fig.2). Moreover, in the context of the UM IGHV CLL group (332 cases), TRI12 CLL had a 4-fold higher KRAS/NRAS/BRAF mutation frequency (46/182, 25.3%) than non-TRI12 (9/150, 6%; p<0.0001). Indeed, we found 29/182 (15.9%) KRAS, 9/182 (4.9%) NRAS and 17/182 (9.3%) BRAF mutated cases in the TRI12 group vs. 4/150 (2.7%) KRAS, 3/150 (2%) NRAS and 3/150 (2%) BRAF mutated cases in the non-TRI12 group. The KRAS/NRAS/BRAF mutation frequency was even higher when considering the TRI12only group in the context of UM IGHV (38/133, 28.6%). Conversely, CLL cases with del(13q) as sole chromosomal aberration (n=94) showed the lowest frequency of KRAS/NRAS/BRAF mutations (2/94, 2.1% in the whole cohort and 2/53, 3.8% in the context of UM IGHV cases).Furthermore, a higher prevalence of KRAS/NRAS/BRAF mutations was found in NOTCH1 wild type (wt) (22.1%) when compared to NOTCH1 mutated (11.2%) cases, in the context of the UM IGHV group (p=0.008), pointing to a mutual exclusivity of these mutations in the pathogenesis of disease.No other significant associations with clinical variables as RAI stage at diagnosis, presence of TP53 mutations or gender were observed.We finally evaluated if KRAS/NRAS/BRAF mutations had an impact on TTFT in the context of UM IGHV/TRI12only/NOTCH1wt, that showed the highest incidence of mutations (26/70, 37%). Both KRAS mutations alone (p=0.005) and KRAS/NRAS mutations (p=0.04) were associated with shorter TTFT (Fig.3).ConclusionsTaken together our data show that KRAS, NRAS and BRAF mutations are almost exclusively found in UM IGHV/TRI12/NOTCH1wt CLL and, in the context of this CLL group the presence of KRAS and NRAS mutations is associated with unfavorable prognosis.The high incidence of KRAS, NRAS and BRAF activating mutations in TRI12 CLL together with the observation of higher level of ERK phosphorylation (S. Decker, Blood, 2012) and higher sensitivity to MEK/ERK inhibitors (S. Dietrich, J Clin Invest, 2018) described in the TRI12, reinforce the evidence of an essential role for MEK/ERK signaling in TRI12 CLL, pointing to this pathway as the most appropriate therapeutic target. [Display omitted] DisclosuresZaja:Novartis: Honoraria, Research Funding; Janssen: Honoraria; Abbvie: Honoraria; Celgene: Honoraria, Research Funding; Amgen: Honoraria; Takeda: Honoraria; Sandoz: Honoraria.

Acalabrutinib in Patients with Relapsed/Refractory (R/R) and High-Risk, Treatment-Naive (TN) Chronic Lymphocytic Leukemia (CLL)

Background: Bruton tyrosine kinase (BTK) is a critical component of B-cell receptor signaling and a validated target for CLL. Acalabrutinib is a highly selective, potent, covalent BTK inhibitor, which has shown promising efficacy and safety in patients with CLL, including high-risk patients. We present preliminary efficacy, safety, and pharmacodynamic results from an ongoing single-center, open-label, phase 2 study of acalabrutinib monotherapy in patients with R/R and high-risk, TN CLL.Methods: Patients with R/R or high-risk (chromosome 17p deletion [del17p] or mutation in TP53 or NOTCH1) TN CLL/small lymphocytic lymphoma (SLL) who met International Workshop on Chronic Lymphocytic Leukemia (IWCLL) 2008 criteria for treatment and had an Eastern Cooperative Oncology Group performance status ≤2 were eligible. Patients who had prior BTK inhibitor therapy were excluded. Patients were randomized to receive oral acalabrutinib 100 mg twice daily (BID) or 200 mg daily (QD) until progressive disease or unacceptable toxicity. The primary endpoint was investigator-assessed overall response rate (ORR) by IWCLL 2008 criteria with modification for lymphocytosis. Secondary endpoints included safety and BTK occupancy. BTK occupancy was measured with a biotin-tagged analogue probe in peripheral blood cells at drug trough time points after 3 days of dosing and after 1, 6, and 12 mo of treatment. BTK occupancy in lymph node samples was measured at drug trough time points after 3 days of dosing.Results: Forty-six patients were enrolled and treated (100 mg BID, n=22; 200 mg QD, n=24). The median age was 64 years (range, 45-83), and 35% (16/46) were TN. Approximately 39% of patients (25% of TN) had bulky lymph nodes ≥5 cm, 37% (50% of TN) had Rai stage III-IV disease at baseline, 76% (88% of TN) had unmutatedIGHV, 21% (40% of TN) had del(17p), 21% (23% of TN) had TP53 mutation, and 47% (54% of TN) had NOTCH1 mutation. As of April 13, 2018, the median time on study for all treated patients was 20 mo (range 1-39), with 89% (41/46) remaining on acalabrutinib. Two patients (9%) in the BID group and 3 patients (13%) in the QD group discontinued treatment due to an adverse event (AE; n=1), progressive disease (n=1), and other reasons (n=3). The patient who discontinued due to progressive disease (BID group) achieved partial response at 2 mo and developed Richter transformation at 6 mo.The ORR was 90% (95% CI: 76, 97) for efficacy evaluable patients (N=39), defined per protocol as patients who had ≥ 6 mo of acalabrutinib (Table). ORR was 95% (75, 100) and 84% (60, 97) for the BID and QD group, respectively. For the intent-to-treat population (N=46), ORR was 80% (66, 91). Most AEs were grade 1/2 and did not require dose delays or modifications. The most common AEs (all grades; >25%) were headache (63%), contusion (50%), diarrhea (43%), upper respiratory tract infection (43%), arthralgia (33%), influenza-like illness (28%), maculo-papular rash (28%), myalgia (26%), and nausea (26%). Grade 3/4 AEs occurred in 33% (15/46) of patients (BID, 27% [6/22]; QD, 38% [9/24]), most commonly (>10%) infections (13%; urinary tract infection, lung infection, hepatitis B reactivation, which led to treatment discontinuation and fatal hepatic failure after 10 mo of treatment, and an invasive pulmonary aspergillosis at 2 mo in the setting of prolonged neutropenia and recent systemic corticosteroid use that led to treatment discontinuation) and neutropenia (11%). Approximately 33% (15/46) of patients (BID, 23% [5/22]; QD, 42% [10/24]) reported serious AEs (all grades), most commonly (>5%) lung infection (7%). No atrial fibrillation was reported, and one grade 1 atrial flutter occurred (BID).On day 4 of cycle 1, median trough BTK occupancy was significantly higher for the BID group versus the QD group in the peripheral blood (95% vs 87%; P<0.001) and in the lymph node (98% vs 90%, P<0.001). Median trough BTK occupancy in the peripheral blood was also higher for the BID group at 1, 6, and 12 mo (range, 98%-99% for BID vs 95%-97% for QD; P<0.05 at all time points).Conclusion: Acalabrutinib monotherapy produced high ORR in R/R and high-risk TN CLL, with an acceptable safety profile. The study was not designed to detect a statistically significant difference in clinical outcomes between the dosing groups. Near complete target coverage (>95%) was more rapidly achieved with 100 mg BID than 200 mg QD dosing in the lymph node and peripheral blood. [Display omitted] DisclosuresNierman:National Institutes of Health: Employment. Covey:Acerta Pharma: Employment; AstraZeneca: Equity Ownership. Hamdy:Acerta Pharma: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: various patents for ACP-196. Izumi:Acerta Pharma: Employment, Equity Ownership, Patents & Royalties: Acerta Pharma, various patents for ACP-196. Liu:Acerta Pharma: Employment. Patel:Acerta Pharma: Employment, Equity Ownership. Wiestner:Pharmacyclics LLC, an AbbVie Company: Research Funding.

Acalabrutinib in Treatment-Naive (TN) Chronic Lymphocytic Leukemia (CLL): Updated Results from the Phase 1/2 ACE-CL-001 Study

Acalabrutinib in Treatment-Naive (TN) Chronic Lymphocytic Leukemia (CLL): Updated Results from the Phase 1/2 ACE-CL-001 Study

Validation of Clinical Prognostic Models and Integration of Genetic Biomarkers of Drug Resistance in CLL Patients Treated with Ibrutinib

Introduction:We previously reported a prognostic scoring system in CLL using pre-treatment factors in patients treated with ibrutinib [Ahn et al, 2016 ASH Annual Meeting]. Here we present long-term follow-up results and validation of the prognostic models in a large independent cohort of patients. We also determine the incidence of resistance-conferring mutations in BTK and PLCG2 genes in different clinical risk groups.Methods and Patients:The discovery cohort comprised 84 CLL patients on a phase II study with either TP53 aberration (deletion 17p or TP53 mutation) or age ≥65 years (NCT01500733). The validation cohort comprised 607 patients pooled from four phase II and III studies for ibrutinib in treatment-naïve or relapsed/refractory CLL (NCT01105247; NCT01578707; NCT01722487; NCT01744691). All patients received single-agent ibrutinib 420mg once daily. We used Cox regression models to identify independent predictors of PFS, Kaplan-Meier method to estimate probabilities of PFS, log-rank test to compare PFS, and Cochran-Armitage trend test to compare the incidence of mutation among subgroups. We used R version 3.5.0 or SAS® version 9.3 for statistical analyses. For biomarker correlation, we tested cellular DNA or cell-free DNA collected from patients in the discovery cohort with the targeted sequencing of BTK and PLCG2 genes.Result:At a median follow-up of 5.2 years, 28 (33.3%) of 84 patients in the discovery cohort progressed or died. 52 (61.9%) patients had treatment-naïve CLL. Independent factors of PFS on univariate analysis were; TP53 aberration, prior treatment, and β-2 microglobulin (B2M) >4mg/L (P<0.05 for all tests). Unmutated IGHV and advanced Rai stage (III/IV) showed a trend toward inferior outcome without reaching statistical significance. Because higher levels of B2M were associated with relapsed/refractory CLL, we performed two multivariate Cox regression models to assess B2M and prior treatment status separately. Risk groups were determined by the presence of TP53 aberration, advanced Rai stage, and B2M >4mg/L for Model 1, and TP53 aberration, advanced Rai stage, and relapsed/refractory CLL for Model 2 (Table 1). The high-risk group had all three adverse risk factors; the intermediate-risk group had two risk factors; and the low-risk group, none or one. The median PFS of the high-risk group was 38.9 months for Model 1 and 38.4 months for Model 2, and was significantly shorter than those of intermediate and low-risk groups.In the validation cohort, 254 (41.8%) of 607 patients progressed or died at a median follow-up of 4.2 years. 167 (27.5%) patients had treatment-naïve CLL. Both models showed statistically significant differences in PFS by risk groups (Table 1). For the high-risk group, 4-year PFS was 30.2% in Model 1 and 30.5% in Model 2, which were inferior to those of intermediate (53.4 and 52.4%) and low-risk groups (68.7 and 73.7%). Model 1 classified 20% of patients and Model 2 classified 28% of patients to the high-risk group.BTK and PLCG2 mutations are common genetic drivers of ibrutinib resistance in CLL. To determine whether the incidence of these mutations correlates with prognostic risk groups, we performed targeted sequencing of BTK and PLCG2 of samples collected from patients in the discovery cohort. We used cell-free DNA for patients who received long-term ibrutinib (≥3 years) and had low circulating tumor burden, and cellular DNA, for samples collected within 3 years on ibrutinib or at progression. Of 84 patients, 69 (82.1%) were tested at least once, and 37 (44.0%) were tested at least twice. The frequency of testing was similar across the risk groups by two models (P>0.05). The cumulative incidences of mutations at 5 years in the low-, intermediate-, and high-risk groups were: 21.4%, 44.8% and 50%, respectively, by Model 1 (P=0.02); and 22.6%, 41.4% and 66.7%, respectively, by Model 2 (P=0.01).Conclusion:We developed and validated prognostic models to predict the risk of disease progression or death in CLL patients treated with ibrutinib. Risk groups classified by three commonly available pre-treatment factors showed statistically significant differences in PFS. The clinically-defined high-risk disease was linked to higher propensity to develop clonal evolution with BTK and/or PLCG2 mutations, which heralded ibrutinib resistance. DisclosuresAlbitar:Neogenomics Laboratories: Employment. Ma:Neogenomics Laboratories: Employment. Ipe:Pharmacyclics, an AbbVie Company: Employment, Other: Travel; AbbVie: Equity Ownership. Tsao:Pharmacyclics LLC, an AbbVie Company: Employment. Cheng:Pharmacyclics LLC, an AbbVie Company: Employment. Dean:CTI BioPharma Corp.: Employment, Equity Ownership; Pharmacyclics LLC, an AbbVie Company: Employment, Equity Ownership. Wiestner:Pharmacyclics LLC, an AbbVie Company: Research Funding.

A Phase Ib/II Study of Ibrutinib in Combination with Obinutuzumab-Gazyva As First-Line Treatment for Patients with Chronic Lymphocytic Leukemia > 65 Years Old or with Coexisting Conditions

A Phase Ib/II Study of Ibrutinib in Combination with Obinutuzumab-Gazyva As First-Line Treatment for Patients with Chronic Lymphocytic Leukemia > 65 Years Old or with Coexisting Conditions

Reduction of Tumor Burden Rather Than Off-Target Effects Drives Changes in T-Cell Number and Profile during Prolonged Ibrutinib Treatment in Relapsed or Refractory Chronic Lymphocytic Leukemia Patients

In this study, we analyzed the changes occurring in the peripheral blood mononuclear cells (PBMC) during long-term treatment with the Bruton's tyrosine kinase inhibitor ibrutinib in relapsed or refractory (R/R) chronic lymphocytic leukemia (CLL) patients (pts). PBMC populations were assessed by flow-cytometry including CLL cells, Natural Killer (NK) cells, T-cell memory subsets, helper subpopulations (Ths) and regulatory T cell (Tregs) subsets. Peripheral blood samples were collected before start of treatment and at week (wk) 4, 10, 16, 22 and at 8, 12, 24 and 36 months (mo) of treatment.Eleven pts were included in the study, 8 males and 3 females, with median age 73 years. Nine age- and sex-matched healthy individuals were included as controls. Comparisons between pts and controls were done with the non-parametric Mann-Whitney U test; between follow-up time points and baseline in pts by Wilcoxon rank test for paired comparisons. Simple regression analyses and the Pearson´s rank test were used to estimate the relationship between different cell populations.Eight pts had high-risk and 3 intermediate-risk disease according to the modified Rai staging system; 8/11 had 17p deletion or TP53 mutation. Three pts had mutated IgHV genes, 3 unmutated, 2 had borderline mutational status (97-98% identity), and 3 were not assessed. Median number of previous treatments was 1 (range 1-4) and median lymphocyte count 45 x 109/L (range 3.1-109.5). At 12 mo, overall response rate was 100%. All pts achieved partial response, with 63% fulfilling all complete response criteria (Hallek M et al, Blood 2018), but for minimal residual disease negativity. Eight pts were still on treatment at 24 mo, 7 pts at 36 mo. Treatment discontinuation was due to: death (n=1), allogeneic transplantation (n=1), disease progression (n=1) and toxicity (n=1). In 3 pts, ibrutinib dose was reduced due to toxicity; the remaining pts stayed on full-dose (420 mg/day p.o.) during the whole treatment period. At study entry, eight pts had hypogammaglobulinemia but none had ongoing IgG replacement therapy. Of these, 5 started IgG replacement therapy during treatment. All 11 patients experienced G2 infections.After an initial increase in circulating CLL cells due to redistribution lymphocytosis, a gradual reduction occurred during treatment becoming significant at wk 16 (p<0.005) (Fig. 1A). As we previously reported (Palma M et al, ASH abs 4322, 2017), at study entry CD8+cells were significantly higher compared to controls (p=0.0005). CD8+cells started to decrease at wk 10 (p=0.02) and normalized (i.e. reached values not significantly different from controls) by 12 mo (Fig. 1B). On the other hand, CD4+T cells, at baseline not significantly different from controls, decreased also gradually from wk 16, reaching by 24 mo levels below normal (p=0.02) (Fig.1D).We show here that the reduction of CD8+ cells correlated with that of CLL cells (r=0.45, p< 0.0001) (Fig. 1C) and that the correlation between the number of CD4+ cells and CLL cells was even more significant (r=0.66, p< 0.0001) (Fig.1E). The expression of the immune checkpoints PD-1 and CTLA-4, at baseline aberrantly high on both CD4+ and CD8+ cells and mainly in the antigen-experienced subsets, decreased gradually during treatment.Among the helper subsets, Th1 (CCR6-/ CXCR3+) cells were at baseline higher compared to controls (p=0.0003), while Th2 (CCR6-/ CXCR3-) did not significantly differ (Fig.2A-B). As expected, this resulted in a Th1/Th2 ratio significantly higher than in controls (median 2.6 vs 0.26, p<0.0001) (Fig.2C). During treatment, both Th1 and Th2 cells decreased. Th1 normalized by 12 mo, while Th2 cells decreased below normal levels at 12 mo (p=0.006). However, Th1/Th2 ratio never normalized during 36 mo of treatment. The strongest direct correlation was between the decrease of Th1 cells and CLL cells (r=0.68, p< 0.0001). During treatment, NK cells decreased and also Tregs (CD4+/CD25+/ CCR4+/CD127low), in particular the Ag-experienced non-activated subset (CD45RO+HLA-DR-).In conclusion, the reduction of tumor burden directly correlated with the reduction of all T-cell subsets, but most significantly with those subsets involved in the antitumor immune response, i.e. CD8+cells and, among the CD4+subset, Th1 cells. Whether these changes also compromised the anti-infection immunity is difficult to assess, due to the fact that most of the pts had also underlying hypogammaglobinemia. [Display omitted] DisclosuresPalma:Takeda Pharma AB: Research Funding. Österborg:Gilead: Consultancy, Research Funding; Beigene: Research Funding; Pharmacyclics: Research Funding; Janssen: Research Funding; Abbvie: Research Funding. Mellstedt:Kancera AB: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sandoz: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria; Astra-Zeneca: Equity Ownership; Novo Nordisk: Equity Ownership; Global Health Sciences: Speakers Bureau.

Prognostic Significance of Serum BAFF, APRIL, TACI and BCMA Levels in Chronic Lymphocytic Leukemia.

As chronic lymphocytic leukemia (CLL) has a variable disease course, novel prognostic markers and risk assessment models are being developed in order to identify high-risk patients who may need early treatment. The two tumor necrosis factor family proteins BAFF and APRIL and their receptors BAFF-R, TACI and BCMA are considered to play a critical role in the survival of normal B cells. In order to highlight the pathophysiological role of this complicated biological network, we aimed to analyze the potential prognostic effects of BAFF, APRIL, TACI and BCMA in CLL patients. We investigated the prognostic impact of serum BCMA, TACI, BAFF and APRIL levels in 129 newly diagnosed CLL patients [median age: 64 (39-88) years; male/female: 85/44]. Serum BAFF, TACI and BCMA levels were significantly lower in the patient group compared to the control group (p < 0.001), while serum APRIL level did not differ significantly between two groups (p > 0.05). Serum BCMA [(p = 0.029; r = 0.208)] and TACI levels [(p = 0.011; r = 0.241)] were positively correlated with serum free light chain ratio. Serum BAFF [(p = 0.008; r = - 0.236)] and BCMA [(p = 0.042; r = - 0.183)] levels were negatively correlated with Rai stage. Overall survival (OS) was relatively better in patients with low serum BAFF levels [60 (1-187) months vs 39.5 (0-256) months; p = 0.063]. Probability of OS was higher in patients with low BAFF levels when compared to patients with normal levels, without statistical significance (53.6% vs 23.6%; p > 0.05). Large prospective studies are needed to validate the prognostic role of this essential biological pathway in CLL.

CD49 Expression in Chronic Lymphocytic Leukemia B-Cell and Non-Hodgkin Lymphoma

AbstractBackground: B-Chronic lymphocytic leukemia (B-CLL) is a monoclonal disorder which is characterized by the accu-mulation of small mature-appearing B lymphocytes in the blood, bone marrow, and lymphoid tissues. Several reliable prognostic markers were found to be capable of predicting the progression and outcome of the disease from its early stages. these markers including age, sex, lymphocyte mor-phology and number, lymphocyte doubling time (LDT), serum factors and recently immunophenotyping and cytogenetic findings.CD49d expression consistently identifies a subgroup of CLLwhich is characterized by poor outcome in many recent studies. These observations, recommended its use as a new marker that could be included in the initial prognostic assess-ment of all survival in patients with CLL .Aim of Study: The aim of this work is to assess the expression pattern of CD49 in patients with (B-CLL) and to evaluate their correlation with different clinical and laboratory data as well as the relation to disease staging and progression. We also aim to find if there is a difference in the expression between patients with B-CLL and B-cell non-Hodgkin lym-phoma (B-NHL).Material and Methods: This study was done at National Cancer Institute, during the period from January 2018 to March 2018, conducted on 6 B-CLL patients and 5 NHL patients who were evaluated for CD49 to detect their role and specificity in B-CLL. Patients were selected for the study on the basis of standard clinical, hematological and immu-nophenotypic criteria for diagnosis of B-CLL, Their ages ranged from 42–79 years, and 5 healthy, hematologically normal individuals, were chosen as control group. Measuring of CD49 by flow cytometry (Coulter EPICS-XL, USA)Results: In this study, there was a high significant increase in CD49d expression in CLL cases (p.004) when comparing with control group, A significant correlation was found between positive CD 49d pts. and Rai staging (p 0.015) in which there was an advanced Rai stage (III, IV) among positive CD49d patients in comparison to negative CD49d patients. While for LDH, its level was high significant (p.005) among positive CD49d patients compared to negative CD49d patients.High significant difference was found between the NHL group and control as regards expression of CD49d (p.0001).Most NHL (95%) expressed positive CD 49d while only 58% of CLL expressed positive CD 49d which is of statistically significant difference (p<0.05).Conclusion: The present study confirmed that CD49d expression is a powerful prognostic parameter in patients with CLL. when correlates with RAI stage and LDH level. So CD49d showed association with poor disease outcome in CLL patients. CD49d showed a significant high expression in NHL Thus CD49d is not specific for CLL. But it could be used as a prognostic marker.

Study of CD184 (CXCR4) Expression in Chronic Lymphocytic Leukemia Patients

Background: Chronic lymphocytic leukemia ,most common leukemia in western countries ,is characterized by accumulation of mature CD19,CD5,CD23 positive B cells. Indeed, microenvironments in the bone marrow, lymph node and other secondary lymphoid organs have been shown to inhibit spontaneous CLL cell apoptosis and enhance chemoresistance. Aim of the work: To study the clinical utility of CD184 in CLL patients,so that it can be used as routine investigation to predict the outcome of the disease and to help in choosing treatment regimen. Patients and methods: 33 newly diagnosed CLL patients at Nasser institute and Ainshams university hospitals, have undergone CBC , LFt, KFT; Virology, LDH,B2microglobulin, bone marrow aspiration and biobsy, immunophenotyping , cytogenetics, radiological investigation, And CD184 by flowcytometry which has been correlated with these diagnostic and prognostic parameters. Results: Positive correlation was found between CD184 and TLC, Lymphocytic count ,LDH,B2microglobulin,RAI stage and revised RAI score, while negative correlation was found between CD184 and both HB and platelet count. Conclusion: High CD184 expression associated with poor prognosis of CLL patients,so that it can be used as routine investigation to predict outcome of disease/

Chronic lymphocytic leukemia with proliferation centers in bone marrow is associated with younger age at initial presentation, complex karyotype, and TP53 disruption

The presence of expanded proliferation centers (PCs) in lymph nodes involved by chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma has been associated with adverse clinical outcomes, but the frequency and significance of PCs in bone marrow (BM) remain unclear. The study group included 36 patients with BM involvement by CLL in which PCs were present. We compared this group with 110 randomly selected BM samples involved by CLL without morphologically discernable PCs. Patients with PCs in BM were younger (median age, 53 years [range,18-71 years] versus 58 years [range, 31-82 years]; P = .007), more frequently experienced B symptoms (27.8% versus 8.2%, P = .0076), more often had Rai stage IV disease (30.6% versus 17.3%, P = .02) and higher serum lactate dehydrogenase (P = .0037) and β2-microglobulin (P = .0001) levels, and lower hemoglobin (P = .026) and platelet counts (P = .0422). TP53 alterations were more common in patients with PCs in BM (45.4% versus 18.7%; P = .0049), as was a complex karyotype (26.4% versus 9%; P = .019). There were no significant differences in the frequency of ZAP70 or CD38 positivity or IGHV mutation status. The median time to first treatment was shorter in patients with PCs in BM (7 months versus 19 months, P = .047), and the frequency of Richter syndrome was higher (14% versus 4%, P = .041). Patients with PCs in BM had significantly shorter overall survival compared with the control group (median, 249.3 months versus undefined; P = .0241). These data suggest that identification of PCs in BM samples involved by CLL is associated with adverse prognostic features.

Relationship between the expression of CD25 and CD69 on the surface of lymphocytes T and B from peripheral blood and bone marrow of patients with chronic lymphocytic leukemia and established prognostic factors of this disease.

Chronic lymphocytic leukemia (CLL) is a condition characterized by the accumulation of morphologically mature monoclonal lymphocytes B with the CD19+/CD5+/CD23+ phenotype in lymphoid tissue, peripheral blood and bone marrow. The clinical course of patients with CLL is heterogeneous, ranging from indolent to aggressive. The role of lymphocyte activation in the natural history of CLL is still a matter of discussion. The aim of this study was to determine the percentages and absolute numbers of lymphocytes B and T in peripheral blood and bone marrow of CLL patients. Moreover, we analyzed the relationship between the number of CD25-positive and CD69-positive lymphocytes and the established prognostic factors in CLL. The study included 80 untreated patients with CLL and 20 healthy subjects. The immunophenotype of peripheral blood mononuclear cells (in both groups) and bone marrow cells (solely in the CLL group) was determined by means of flow cytometry. Patients with CLL showed a higher absolute number of activated lymphocytes B with phenotypes CD19+CD25+ and CD19+CD69+, as well as a higher absolute number of CD3+CD25+ lymphocytes T than the controls. The enhanced activation of peripheral blood and bone marrow lymphocytes was associated with higher Rai stages, an increased concentration of lactate dehydrogenase and beta-2 microglobulin and the progression of the disease. The number of lymphocytes B CD19+ZAP-70+ correlated positively with the number of CD19+CD25+ B cells and CD3+CD69+ T cells. The study confirmed the association between an unfavorable prognosis and a high expression of activation markers in CLL patients. The determination of CD25+ and CD69+ lymphocytes T and B constitutes a valuable diagnostic tool, completing the cytometric evaluation of CLL.

Adherence to the Western, Prudent, and Mediterranean dietary patterns and chronic lymphocytic leukemia in the MCC-Spain study.

Diet is a modifiable risk factor for several neoplasms but evidence for chronic lymphocytic leukemia (CLL) is sparse. Previous studies examining the association between single-food items and CLL risk have yielded mixed results, while few studies have been conducted on overall diet, reporting inconclusive findings. This study aimed to evaluate the association between adherence to three dietary patterns and CLL in the multicase-control study (MCC-Spain) study. Anthropometric, sociodemographic, medical and dietary information was collected for 369 CLL cases and 1605 controls. Three validated dietary patterns, Western, Prudent and Mediterranean, were reconstructed in the MCC-Spain data. The association between adherence to each dietary pattern and CLL was assessed, overall and by Rai stage, using mixed logistic regression models adjusted for potential confounders. High adherence to a Western dietary pattern (i.e. high intake of high-fat dairy products, processed meat, refined grains, sweets, caloric drinks, and convenience food) was associated with CLL [ORQ4 vs. Q1=1.63 (95%CI 1.11; 2.39); P-trend=0.02; OR 1-SD increase=1.19 (95%CI: 1.03; 1.37)], independently of Rai stages. No differences in the association were observed according to sex, Body Mass Index, energy intake, tobacco, physical activity, working on a farm, or family history of hematologic malignancies. No associations were observed for Mediterranean and Prudent dietary patterns and CLL. This study provides the first evidence for an association between a Western dietary pattern and CLL, suggesting that a proportion of CLL cases could be prevented by modifying dietary habits. Further research, especially with a prospective design, is warranted to confirm these findings.

Genomic characterization of chronic lymphocytic leukemia (CLL) in radiation-exposed Chornobyl cleanup workers

BackgroundChronic lymphocytic leukemia (CLL) was the predominant leukemia in a recent study of Chornobyl cleanup workers from Ukraine exposed to radiation (UR-CLL). Radiation risks of CLL significantly increased with increasing bone marrow radiation doses. Current analysis aimed to clarify whether the increased risks were due to radiation or to genetic mutations in the Ukrainian population.MethodsA detailed characterization of the genomic landscape was performed in a unique sample of 16 UR-CLL patients and age- and sex-matched unexposed general population Ukrainian-CLL (UN-CLL) and Western-CLL (W-CLL) patients (n = 28 and 100, respectively).ResultsMutations in telomere-maintenance pathway genes POT1 and ATM were more frequent in UR-CLL compared to UN-CLL and W-CLL (both p < 0.05). No significant enrichment in copy-number abnormalities at del13q14, del11q, del17p or trisomy12 was identified in UR-CLL compared to other groups. Type of work performed in the Chornobyl zone, age at exposure and at diagnosis, calendar time, and Rai stage were significant predictors of total genetic lesions (all p < 0.05). Tumor telomere length was significantly longer in UR-CLL than in UN-CLL (p = 0.009) and was associated with the POT1 mutation and survival.ConclusionsNo significant enrichment in copy-number abnormalities at CLL-associated genes was identified in UR-CLL compared to other groups. The novel associations between radiation exposure, telomere maintenance and CLL prognosis identified in this unique case series provide suggestive, though limited data and merit further investigation.

Coexistence of chronic myeloid leukemia and diffuse large B-cell lymphoma with antecedent chronic lymphocytic leukemia: a case report and review of the literature

BackgroundChronic lymphocytic leukemia and chronic myeloid leukemia are the most common types of adult leukemia. However, it is rare for the same patient to suffer from both. Richter’s transformation to diffuse large B-cell lymphoma is frequently observed in chronic lymphocytic leukemia. Purine analog therapy and the presence of trisomy 12, and CCND1 gene rearrangement have been linked to increased risk of Richter’s transformation. The coexistence of chronic myeloid leukemia and diffuse large B-cell lymphoma in the same patient is extremely rare, with only nine reported cases. Here, we describe the first reported case of concurrent chronic myeloid leukemia and diffuse large B-cell lymphoma in a background of chronic lymphocytic leukemia.Case presentationA 60-year-old Saudi man known to have diabetes, hypertension, and chronic active hepatitis B was diagnosed as having Rai stage II chronic lymphocytic leukemia, with trisomy 12 and rearrangement of the CCND1 gene in December 2012. He required no therapy until January 2016 when he developed significant anemia, thrombocytopenia, and constitutional symptoms. He received six cycles of fludarabine, cyclophosphamide, and rituximab, after which he achieved complete remission.One month later, he presented with progressive leukocytosis (mostly neutrophilia) and splenomegaly. Fluorescence in situ hybridization from bone marrow aspirate was positive for translocation (9;22) and reverse transcription polymerase chain reaction detected BCR-ABL fusion gene consistent with chronic myeloid leukemia. He had no morphologic or immunophenotypic evidence of chronic lymphocytic leukemia at the time. Imatinib, a first-line tyrosine kinase inhibitor, was started. Eight months later, a screening imaging revealed new liver lesions, which were confirmed to be diffuse large B-cell lymphoma.ConclusionsIn chronic lymphocytic leukemia, progressive leukocytosis and splenomegaly caused by emerging chronic myeloid leukemia can be easily overlooked. It is unlikely that chronic myeloid leukemia arose as a result of clonal evolution secondary to fludarabine treatment given the very short interval after receiving fludarabine. It is also unlikely that imatinib contributed to the development of diffuse large B-cell lymphoma; rather, diffuse large B-cell lymphoma arose as a result of Richter’s transformation. Fludarabine, trisomy 12, and CCND1 gene rearrangement might have increased the risk of Richter’s transformation in this patient.

Serum Immunoglobulin Paraprotein in Patients with Chronic Lymphocytic Leukemia

AbstractBackground: Chronic Lymphocytic Leukemia (CLL) is the most common chronic lympho-proliferative disorder. This study was done to explore the frequency of serum Immu-noglobulin (Ig) paraprotein in patients with CLL and its correlation with other prognostic factors as expression of CD38 and zeta-chain-associated protein kinase 70 (ZAP70) on malignant lymphocytes.Aim of Study: To study the frequency of serum immu-noglobulin paraprotein in patients with CLL and its correlation with other prognostic factors as expression of CD38 and ZAP70 on malignant lymphocytes.Patients and Methods: Clinico-hematological profiles were done to 30 CLL patients. Serum Protein Electrophoresis (SPE) and Immunofixation Electrophoresis (IFE) were per-formed to measure serum Ig paraprotein. ZAP-70 and CD 38 were tested by flow cytometry.Results: In this study CLL patients were classified accord-ing to modified Rai staging system into: Low risk 10%, intermediate risk 40% and high risk 50%. Four CLL patients (13.4%) had monoclonal gammopathy (M-band), the type of M-band in the 4 patients was IgG-Kappa. One patient (3.3%) had diffuse band (polyclonal gammopathy). ZAP-70 and CD 38 positivity were detected in 7 patients (23.3%) and 15 patients (50%), respectively. There was significant relation between presence of Ig paraprotein in CLL patients and ZAP 70 expression (p=0.034). On the other hand there was no relation between presence of M-band in CLL patients and CD38 expression nor modified Rai staging system.Conclusion: Monoclonal gammopathy was found in 13.4% of CLL patients. The detection of Ig paraprotein might be applied for the assessment of prognosis in patients with CLL.

Sustained long-lasting responses after lenalidomide discontinuation in patients with chronic lymphocytic leukemia.

Sustained long-lasting responses after lenalidomide discontinuation in patients with chronic lymphocytic leukemia.

Pentoxifylline-Induced Apoptosis in Chronic Lymphocytic Leukemia: New Insights into Molecular Mechanism.

Chronic lymphocytic leukemia (CLL) is an indolent B-lineage neoplasm, characterized by clonal expansion of CD5 positive B cells with constitutive activation of survival pathways including NF-kB. Pentoxifylline, a xanthine-derivative compound indicated for the treatment of microvascular disturbancies, has been suggested to have anti-proliferative and anti-metastatic activities in various types of cancer. In the present study we extend these data showing one of the potential molecular mechanisms through which Pentoxifylline may promote apoptosis in CLL clonal lymphocytes. Peripheral blood mononuclear cells were isolated from 15 CLL patients 0 RAI stage and 15 healthy volunteers and treated for 24 and 48 hours with Pentoxifylline. Apoptosis induction was evaluated through Annexin V and TUNEL assays. Mitochondrial membrane potential depolarization analysis, active Caspase-3 assay, reactive oxygen species generation and Western Blot were assessed to further investigate the alterations induced by Pentoxifylline. We observed a statistically significant occurrence of apoptosis, DNA fragmentation and active Caspase-3 in lymphocytes from CLL patients compared to healthy volunteers after 48 hours of Pentoxifylline treatment. To clarify the molecular mechanism of the drug, we also evaluated the expression levels of NF-kB/p65 and its related proteins. In treated CLL cells, NF-kB/p65 was significantly decreased in comparison to normal cells, whereas we observed a less marked reduction of Bcl-2 expression. The treatment also induced a decrease of Mcl-1 in CLL cells with a greater down-regulation of the anti-apoptotic alternatively spliced isoform. These findings showed that Pentoxifylline induced apoptosis in leukemic cells through a molecular mechanism that involves the NF-kB signaling.

Disease characteristics and clinical outcomes in patients aged less than 40 with chronic lymphocytic leukemia

Outcomes in very young CLL patients (age ≤40) are not well characterized. We compared 71 consecutive patients aged ≤40 with 142 “older” matched patients >40 from our institution and used SEER database as an independent comparison group. Patients in the two age groups were diagnosed at similar Rai stage. At diagnosis, very young patients had a similar rate of adverse cytogenetics, IGHV mutation and ZAP70 expression and had lower beta-2-microglobulin and a lower incidence of second malignancies. There was no difference between the groups with respect to incidence of autoimmune manifestations, family history of lymphoma, time to initiation of CLL therapy, response to therapy, or Richter’s transformation. Variables including un-mutated IGHV and elevated LDH were associated with shorter times to treatment initiation in both groups. A trend to longer 5-year survival for very young patients in our institution (93% v 82%, p = 0.082) was validated by SEER data.

Smudge cell percentage as a surrogate marker for ZAP-70 expression in patients with chronic lymphocytic leukemia.

BackgroundThis study aimed to evaluate the prognostic value of smudge cell percentage as a surrogate marker for zeta-chain-associated protein kinase 70 (ZAP-70) expression in chronic lymphocytic leukemia (CLL) patients.MethodsSixty three newly diagnosed CLL patients were investigated at the Hematology Department of the Medical Research Institute of Alexandria University with complete blood count, lactate dehydrogenase, β2 microglobulin levels, ZAP-70 expression, and estimation of the percentage of smudge cells.ResultsThe percentage of smudge cells ranged from 2 to 58% with a mean of 24.03±13.98%. Higher percentages of smudge cells (>30%) were statistically significantly associated with markers of better prognosis (negative ZAP-70, early-stage disease according to the Binet and Rai staging systems, as well as low and intermediate risk CLL prognostic index). The percentage of smudge cells showed significantly negative correlation with the ZAP-70 expression and higher area under the curve for prediction of ZAP-70 positivity with better survival for 36 months in patients with >30% smudge cells.ConclusionThe percentage of smudge cells at presentation of newly diagnosed CLL patients could be used as a surrogate marker for ZAP-70 expression and an additional prognostic marker for disease progression.

Association of CD49d and CD44 in chronic lymphocytic leukemia patients and their effects on response to fludarabine, cyclophosphamide, and rituximab therapy

IntroductionChronic lymph ocytic leukemia (CLL) is a clonal expansion of small mature lymphocytes accumulating in the blood, bone marrow, and lymphoid organs. The interactions of the CLL microenvironment are known to promote CLL cell survival and proliferation. CD49d, an adhesion molecule belonging to the integrin family mediates cell-to-extracellular matrix and cell-to-cell interactions. CD44 is a glycoprotein and also a major hyaluronan receptor that mediates the response of cells to their cellular microenvironment. AimWe aimed to evaluate the value of CD49d and CD44 in CLL patients, and their effect on response to fludarabine, cyclophosphamide, and rituximab therapy, to study the relation of each to the other and its relation to the poor prognostic marker CD38. Patients and methodsThe study included 30 newly diagnosed CLL patients with measurement of the expression of CD49d and CD44 by flow cytometry before and after therapy. ResultsCD49d expression was increased in advanced risk stages according to modified Rai staging, but CD44 had no different expression in the different stages. There is a positive correlation between CD44 with CD49d, CD38 (documented poor prognostic marker) with CD49d, and CD38 with CD44. Lymphadenopathy showed a significant relation with CD49d only. Patients without complete response had a significant higher expression of CD49d, CD38, and CD44 before and after therapy. ConclusionThe study has shown that the expression of CD49d and CD44 in newly diagnosed CLL patients is related to resistance to the fludarabine, cyclophosphamide, and rituximab therapy and their expression is related to each other and to CD38.