Early Chronic Lymphocytic Leukemia Research Articles

Positron emission tomography detection of synchronous colon cancer in a patient with chronic lymphocytic leukemia

Positron emission tomography detection of synchronous colon cancer in a patient with chronic lymphocytic leukemia

Aerobic Glycolysis Predicts Outcome in Early Chronic Lymphocytic Leukemia.

AbstractAbstract 2482Despite the advent of prognostic factors such as cytogenetics and IGH mutational status, the natural history of chronic lymphocytic leukemia (CLL) remains heterogenous and difficult to predict in individual patients. We used the carbocyanine dye, JC-1, to investigate mitochondrial membrane potential in patients with untreated, predominantly early stage CLL. We found that JC-1 staining separated CD5+/CD19+ CLL cells into two discrete populations, Low Red (R)/Green (G) and High R/G, which mainly differed with regard to JC-1 green fluorescence (530 nm) in all patients (Fig. 1). In evaluating CLL patients, we found substantial variation in the proportion of cells in the two populations, with a range of 1.1% to 96.7% (median=79.9%) in the High R/G population. CD19+ B-cells from normal individuals typically had few cells with high JC-1 red fluorescence and had a JC-1 staining pattern that was distinct from CLL patients. Electron microscopy revealed disorganized mitochondrial christae, particularly in patients with elevated High R/G cell populations, compared with mononuclear cells from normal donors. Consistent with the morphologic disorganization, we found that the two populations of CLL cells had differential survival in culture with pyruvate and glucose as primary energy sources; cells in the High R/G population had inferior survival (p=0.016), while cells in the Low R/G population had equal survival in pyruvate compared with glucose (Fig. 2). This result indicates that the CLL clonal cell population can be subdivided into cells able to use oxidative phosphorylation and those dependent on aerobic glycolysis fro metabolic energy needs. Interestingly, patient samples with High R/G cell populations >90% had better cell survival in glucose-containing medium than samples derived from patients with < 75% High R/G cells (p=0.0085). We found that the mononuclear cells from patients with CLL expressed the M2 isoform of pyruvate kinase, which has been linked to the regulated utilization of aerobic glycolysis.We assessed the outcome of 100 patients with untreated CLL with more than 1 month of follow up (median 36 months) after measuring the proportion of cells in the mononuclear fraction of the peripheral blood with in the High R/G population. The clinical characteristics of the patients at the time of JC-1 staining are characteristic of patients with early stage, untreated CLL. The population was divided into approximate quartiles based on the percentage of mononuclear cells in the High R/G population, and progression-free survival (PFS) was estimated within each quartile. Patients with 50% or fewer cells in the High R/G gate appeared to have an indolent natural history; an estimated 88% (95% CI: 73%, 99%) of the patients had no evidence of significant clinical progression at two years. In contrast, patients with more than 90% of cells in the gate with High R/G cells had rapid progression with an estimated PFS of 50% (95% CI: 31%–82%) at two years (Fig. 3). Patients with 50–90% cells in the High R/G gate had an intermediate disease course. There was strong evidence of a difference in survival distributions across the four JC-1 categories (log-rank p-value < 0.001). The percentage of cells in the High R/G gate provided prognostic information that was independent of good risk or poor risk cytogenetic changes.In summary, we found that the clonal, neoplastic cells in patients with CLL comprise discrete populations separated on their metabolic dependence on aerobic glycolysis and aerobic glycolysis. We also show that the percentage of cells utilizing glycolysis has a significant adverse impact on the natural history of patients with CLL. Aerobic glycolysis, the so-called Warburg effect, is a common and incompletely understood aspect of cancer biology. These data tie the acquisition of aerobic glycolysis to more aggressive cancer biology, identify an important new prognostic measure for CLL and could lead to the development of therapies that target metabolic differences in cancer cells. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

BOTH MD Anderson Cancer Center MODEL and German Score Work in Predicting Time to First Treatment in EARLY Chronic Lymphocytic Leukemia: Results of an External Validation Analysis

AbstractAbstract 3934In chronic lymphocytic leukemia (CLL) there has been a limited published work on the development of models predicting time to first treatment that incorporate traditional and novel prognostic factors. We wondered whether the nomogram recently proposed by M D Anderson Cancer Center (MDACC) investigators that includes clinical and newer prognostic variables (i.e., three or more involved lymph node sites, increased size of cervical lymph nodes, presence of 17p deletion or 11q deletion by FISH, increased serum LDH, and unmutated IGHV mutation status) could work when applied to a prospective community-based CLL patient cohort.We considered suitable for the purpose of this study 328 previously untreated patients with newly diagnosed asymptomatic Binet stage A CLL from different primary hematology centers registered on a prospective basis during the period 2006–2010 on an observational database of Gruppo Italiano Studio Linfomi (GISL).Using the formula proposed by Wierda et a total point score was calculated for each patient. A value ranging between 0 and 69.5 (median, 19.9) - therefore similar to total point scores reported by the MDACC group (median, 21.0; range, 0 – 87.4) – was found. As a continuous variable (i.e., measuring the risk of each point increase), total point score was associated with time to first treatment (HR 1.04; 95% CI 1.02–1.05; P < 0.0001). Receiver Operating Characteristic analysis identified a point score of 25 [area under curve (AUC) 0.64, sensitivity 61.5 specificity 72.1; P<0.0001)] as the best threshold capable of separating patients who needed therapy from patients who did not (HR, 3.27; 95% CI, 2,07–5.18; P<0.0001). The prognostic index category remained a predictor of time to first treatment also when analysis was limited to patients in Rai stage 0 (HR,4.05; 95% CI: 2.25–7.52; P<0.0001). Finally, the goodness of fit showed that the nomogram model had significant good fit at 2 years (r2=0.966; P=0.002).We next wondered whether the German score based on 7 prognostic variables (i.e., age, gender, b2-microglobulin, sTK, IGVH mutational status, ECOG performance status and hierachic type of FISH abnormalities) and originally designed for predicting overall survival could forecast time to first treatment in patients with early disease. Since sTK was not available a slightly modified version of German model based on 6 prognostic variables was used. The risk score calculated in each patient by summing single ratings of adverse factors, allowed the identification of two different risk categories scoring respectively 0–2 and 3–5 whose clinical outcomes were different with respect to time to first treatment (HR 2.19, 95% CI: 1.15–2.20; P=0.005).In conclusion, this analysis shows that both models proposed by MDACC investigators and German group provide interchangeable tool for assessing time to first treatment. Interestingly, in this validation analysis the pattern of distribution of patients into a low and high-risk category revealed a high degree of concordance between models (P<0.0001; Mann-Whitney test). Disclosures:Morabito:Celgene: Honoraria.

The Pro-Inflammatory IL23/IL23R/IL17 Axis Is Active in IL23R-Expressing Circulating CLL Cells in Patients with Poor Prognosis

AbstractAbstract 3889Inflammatory cytokines play a biological role in the pathogenesis of Chronic lymphocytic leukemia (CLL). IL23 is a pro-inflammatory cytokine involved in T-cell responses and in tissue remodeling. It has been shown that the IL23 receptor (IL23R) is up-regulated in primary acute lymphoblastic leukemia (ALL) cells, and that IL23 inhibits ALL cell growth. Nevertheless, the anti-tumor function of IL23 still remains controversial. The role of the IL23R/IL23 axis in CLL has not been investigated so far. Herein we evaluated the expression pattern of IL23R/IL23 axis and its correlation with progression free survival (PFS) in CLL patients.A total of 233 newly diagnosed Binet stage A CLL cases from Italian institutions (clinicaltrials.gov NCT00917540) were studied for IL23R expression by flow-cytometry (FC) (median percentage IL23R expression=22.7, range 1.2–91.1). The median follow-up was 23 months (range 1–47). PFS information was obtained in 203 patients. Using the median value of 23% of IL23R as threshold, 8/102 IL23Rneg and 23/101 IL23Rpos CLL cases progressed with therapy requirement. The 2-year PFS probability of IL23Rneg patients was 89.7% as compared to 80.7% of IL23Rpos cases [χ2 7.7, P=.006; HR=3.0, 95%CI (1.3–6.6)]. Cases were then stratified according to IL23R positivity [IL23Rneg (102 cases) versus IL23Rpos (101 cases)]. No significant difference in terms of CD38 and ZAP-70 positive cases was observed, however, the IGVH mutational status could distinguish the two groups: IGHV-mutated in 92 (78.6%) of IL23Rneg vs 70 (61.9%) IL23Rpos and IGHV-unmutated in 25 (21.4%) vs 43 (38.1%), p=.006]. FISH analysis showed that IL23Rneg and IL23Rpos cases carrying 13q14.3 were respectively 53 (51.4%) and 44 (42.7%), while the number of patients with trisomy 12 were 8 and 10 respectively in cases with low and high IL23R expression. Deletion of 11q was detected in 3.9% (4/103) of IL23Rneg and in 8.7% (9/103) of IL23Rpos cases. Only 3 cases with 17p deletion were seen in this cohort of early CLL patients and all belonged to the IL23Rpos group. Overall, no significant differences in the incidence of the major genetic lesions were observed between the two groups. Il23R expression still remained independently associated with PFS also in multivariate analysis.In situ expression analysis of IL23R and of its ligand IL23 was then performed by immunohistochemistry (IHC) in 16 CLL samples [10 lymph node (LN) and 6 bone marrow (BM) biopsies] collected on diagnosis and in 8 control biopsies (4 lymph nodes with reactive follicular hyperplasia and 4 normal BM biopsies). IL23R was variably expressed in CLL and significantly expressed in the neoplastic clones of 9 (6 lymph nodes and 3 BM biopsies) of the 16 cases tested; IL23R was diffusely present along the membrane and cytoplasm of neoplastic cells effacing the lymph node or BM architecture (Fig. 1, upper-left). In CLL cases with low IL23R expression, IL23R was detected in few scattered lymphoid cells intermingling with neoplastic lymphocytes (Fig. 1, upper-right). IL23 was also detected, with a variable staining intensity (Fig. 1, middle-left), paralleling in part that of IL23R. Double-marker analysis confirmed the concomitant expression of IL23 and IL23R in CLL neoplastic infiltrates highlighting the co-localization of the two markers (Fig.1 middle-right) and suggesting the possibility of an autocrine IL23/IL23R loop in CLL clones.We speculated that the microenvironment of CLL cases rich in IL23R and IL23 could be enriched in IL17-producing cells. The IHC expression of IL17 in CLL cases with low or high IL23R and IL23 expression showed that CLL cases rich in IL23Rpos cells, also characterized by high IL23 expression, displayed significantly higher numbers of IL17pos infiltrating cells (Fig. 1 bottom-left), as compared with CLL cases with no or low expression of IL23R or IL23 (Fig. 1 bottom-right).In conclusion, our study shows that high IL23R expression predicts a worse PFS. Furthermore, we linked this picture with, the in situ engendering of a clone-related microenvironment characterized by the preponderancy of pro-inflammatory signals such as those of the IL23/IL23R/IL17 axis, and its correlates in the peripheral blood (i.e. IL23R expression on circulating CLL cells), may endorse its strong prognostic significance. This analysis prompts further investigation into the specific function of the IL23/IL23R/IL17 axis and its targets in the context of CLL. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

The Initial Approach to Patients with Chronic Lymphocytic Leukemia Diagnosed in Day-to-Day Practice

SUMMARY As chronic lymphocytic leukemia (CLL) research increases and new technologies and drugs become available, the landscape of this disease becomes more complex. It is this complexity that suggests the use of practical recommendations in day-to-day practice. The close relationship between early CLL and clinical monoclonal B lymphocytosis regarding clinically relevant parameters does not provide evidence to strictly separate these entities by a distinct threshold of clonal B cells. Prognostic testing and staging procedures based on costs and logistics should be favored at diagnosis, while assessment of more specific markers predicting treatment-related outcome (i.e., 17p del/P53 mutation) should be reserved to patients for whom treatment is required. A diagnosis of CLL, especially in younger patients (&lt;55 years), may cause a number of fears and worries with regard to the course–outcome of the disease. Therefore, communication between the patient and doctor should be informative with respect to the impact of disease on the patient’s affective, social and economic spheres. Finally, current available guidelines should drive the choices of physicians who currently treat CLL patients, with room allowed for consideration of emerging new evidence, physician practice style, patient preference, clinical judgment and patient quality of life. Doctors should also keep in mind that people with CLL want to live their lives as normally as possible; therefore, every effort should be made to ensure the achievement of this goal.

Do Regulatory T Cells Have a Role in Disease Progression and Autoimmune Cytopenias of Chronic Lymphocytic Leukemia?,

Abstract 3864 Introduction:Chronic lymphocytic leukemia (CLL) originates from mature B lymphocytes which avoid death through the intercession of external signals, including T cells. There is improved understanding of the relationship between different cell types in the normal immune response and the dynamic cross talk between tumors and immune system regulating tumor growth. Cancer and autoimmunity are considered as anti-thesis of each other. Regulatory T cells (Tregs) are shown to have a protective role in autoimmune diseases and conversely known to promote oncogenesis. The exact role of Tregs in disease biology of CLL is still unclear. Aims and Objectives : to define the role of Tregs in clinical presentation, disease progression and autoimmune cytopenias in CLL. Materials and Methods : 32 treatment naive CLL patients and 10 normal healthy volunteers were evaluated in this prospective study. Investigations included clinical examination, biochemical tests, complete blood counts, bone marrow examinaton and multicolor flowcytometry (FCM). More stringently defined specific markers (CD4+CD25highCD127low FoxP3+) were used to enumerate Tregs. Routine CLL panel for diagnosis and prognosis included 17 monoclonal antibodies (MoAbs) labeled with fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC) or peridinin chlorophyll protein (PerCP). FCM analysis was performed on FACS Calibur flowcytometer (BD Biosciences) using CellQuest® software. Diagnosis of autoimmune hemolytic anemia (AIHA), autoimmune thrombocytopenia (ITP) and pure red cell aplasia (PRCA) was made according to standard criteria. Results : Study group included 20 male and 12 female CLL patients. The mean age was 64.2 years; with 8/32 (25%) patients being <55 years. 17/32 (53.12%) patients were categorized to Binet stage A and 20/32 (62.5%) were asymptomatic at diagnosis. Mean total leukocyte counts (TLC) and absolute lymphocyte counts (ALC) were 69,731/μl and 62,697/μl respectively. Mean hemoglobin and platelet counts were 12.1 g/dl and 178,087/μl respectively. 25 patients completed follow-up investigations at 6 months. Disease progression occurred in 12/25 (48%) patients, of which 5/25 (20%) patients progressed to Binet stage C and had symptoms other than lymphadenopathy while the number of asymptomatic patients decreased to 10/25 (40%). Disease progression was assessed according to NCI-WG guidelines. Autoimmune cytopenias occurred in 8/32(25%) patients. 3 of them at disease presentation and remaining 5 were diagnosed on follow-up. AIHA was diagnosed in one, ITP in 4 and PRCA in 3 patients. Mean Tregs % was lower in CLL patients (0.98+0.94) as compared to the controls (2.89+1.46) (p=0.0026). However, CLL patients had significantly higher absolute Treg cell count than the controls (581.63+734.5 vs. 83.89+55.45) (p=0.0006). While the mean % Treg increased by 46% (not significant, p >0.05) from stages A and B (early CLL) to stage C (advanced CLL), increase in mean ALC was 172% (p<0.005). The increase in mean absolute Treg count was further significantly higher 384% (p<0.005). There was no significant difference between the mean absolute Tregs count at baseline of CLL patients with disease progression (progressors) and without disease progression (nonprogressors) at follow-up. Absolute Treg count was significantly higher in patients having autoimmune cytopenias at presentation as well as on follow-up. Mean absolute Treg counts were not significantly different between ZAP70 positive and negative CLL patients (p=0.1), as well as between CD38 positive and negative CLL (p=0.15). There was a strong inverse correlation between lymphocyte doubling time and absolute Treg count (p=0.03) as well as lymphocyte doubling time and % Treg cells (p=0.006). No correlation could be found between absolute Treg count and CD4: CD8 ratio, r= 0.29 (p=0.1). Conclusion : Absolute Treg cell counts (rather than % Treg) are increased in CLL patients compared to controls and also increased with disease severity. Absolute Treg counts may be more important than % Tregs in CLL pathogenesis. Tregs also have a strong inverse relation with lymphocyte doubling time, also indirectly confirming the role of Tregs in disease progression. We also found Tregs to be increased in patients with autoimmune cytopenias. Autoimmune cytopenias may be viewed as a continuum of CLL disease itself rather than a complication of advanced disease. Disclosures:No relevant conflicts of interest to declare.

Enhanced levels of both the membrane-bound and soluble forms of IgM Fc receptor (FcμR) in patients with chronic lymphocytic leukemia

AbstractThe association of an IgM-Fc receptor (FcμR) with chronic lymphocytic leukemia (CLL) was suggested more than 30 years ago, but its authenticity has never been formally addressed. We examined the expression of the recently identified FcμR by B and T cells in CLL patients using receptor-specific monoclonal antibodies. CLL B cells (CD5+/CD19+) expressed much higher levels of FcμR on their cell surface than B cells from healthy donors. Such enhanced expression was more evident in immunoglobulin heavy chain variable region (IGHV)–mutated, CD38− or early Rai-stage CLL than in IGHV-unmutated, CD38+, or advanced Rai-stage CLL. Intriguingly, surface FcμR levels also were significantly elevated in the non-CLL B cells (CD5−/CD19+) and T cells (CD5+/CD19−), especially in IGHV-mutated CLL. CLL patients also had high serum titers of FcμR compared with healthy donors, and serum FcμR levels correlated significantly with circulating lymphocyte numbers but not with the IGHV mutation status or Rai stage. The serum FcμR was resolved as an ∼ 40-kDa protein, distinct from the cell surface FcμR of ∼ 60 kDa, and it was produced by both CLL B and non-CLL B cells. Mass spectrometric analysis revealed that the serum FcμR is a soluble form of the receptor encoded by an alternatively spliced FcμR transcript. These findings indicate enhanced levels of both membrane-bound and soluble forms of FcμR in CLL patients.

Vitamin D insufficiency predicts time to first treatment (TFT) in early chronic lymphocytic leukemia (CLL)

Although vitamin D insufficiency is related to inferior prognosis in some cancers, limited data exist in hematologic malignancies. We evaluated the relationship between 25(OH)D serum levels and time to first treatment (TFT), a disease-specific end point, in 130 previously untreated Binet stage A chronic lymphocytic leukemia (CLL) patients. Measurement of 25(OH)D was performed by means of a direct, competitive chemiluminescence immunoassay using the DiaSorin LIAISON 25(OH)D TOTAL assay (DiaSorin, Inc., Stillwater, Minnesota). Overall, 41 patients (31.5%) had severe vitamin D insufficiency (<10ng/mL), 66 (50.7%) had mild to moderate insufficiency (10–24ng/mL), and 23 (17.6%) had 25(OH)D levels within the optimal range (25–80ng/mL), with no relationship with between the season of sample collection and 25(OH)D level (P=0.188). A patient stratification according to these 3 groups led to significant difference in terms of TFT, with vitamin D insufficient patients having the shortest TFT (P=0.02). With respect to continuous 25(OH)D levels and clinical outcome, TFT was shorter as 25(OH)D decreased until a value of 13.5ng/mL at which point the association of 25(OH)D and TFT remained constant. As a matter of fact, the 25(OH)D value of 13.5ng/mL identified two patients subsets with different TFT risk (HR=1.91; 95% CI=1.06–3.44; P=0.03). In multivariate analysis the variable entering the model at a significant level were mutational status of IgVH (P<0.0001), serum thymidine kinase (P=0.02) and absolute lymphocyte count (P=0.03). Thus confirming the Mayo clinic experience, our data provide further evidence that 25(OH)D levels may be an important host factor influencing TFT of Binet stage A patients. Whether normalizing vitamin D levels may delay disease-progression of patients with early disease will require testing in future trials.

Phase I clinical trial of CpG oligonucleotide 7909 (PF-03512676) in patients with previously treated chronic lymphocytic leukemia

CpG oligonucleotide 7909 (CpG 7909, PF-03512676), a synthetic 24mer single stranded agonist of TLR9 expressed by B cells and plasmacytoid dendritic cells, is immunomodulatory and can cause activation-induced death of chronic lymphocytic leukemia (CLL) cells. We report a phase I study of CpG 7909 in 41 patients with early relapsed CLL. A single intravenous dose of CpG 7909 was well tolerated with no clinical effects and no significant toxicity up to 1.05 mg/kg. Single dose subcutaneous CpG 7909 had a maximum tolerated dose (MTD) of 0.45 mg/kg with dose limiting toxicity of myalgia and constitutional effects. Multiple weekly subcutaneous doses at the MTD were well tolerated. CpG 7909 administration induced immunologic changes in CLL and non-malignant cells that were dose and route dependent. We conclude that multidose therapy with subcutaneous CpG 7909 (0.45 mg/kg) could be used in future phase II combination clinical trials for CLL.

Lymphocytes with crystalline inclusions in early chronic lymphocytic leukaemia

Lymphocytes with crystalline inclusions in early chronic lymphocytic leukaemia

Sequence analysis of 515 kinase genes in chronic lymphocytic leukemia

The pathogenesis of chronic lymphocytic leukemia (CLL) remains incompletely understood.1 Although acquired chromosomal aberrations have been demonstrated to influence CLL biology and clinical behavior, it remains unclear what single gene defects other than p53 or ATM mutations cause or contribute to the CLL phenotype.2 In particular, recurrent gene mutations that are increasingly found in other hematological malignancies have not yet been identified in CLL. One recent CLL gene re-sequencing study reported the analysis of selected exons of 70 tyrosine kinase genes in 95 CLL patients and reported no somatically acquired mutations.3 Given the frequent identification of stereotypical immunoglobulin receptor genes in CLL, it has been suggested that antigen engagement of the B-cell receptor on CLL cells serves a critical role in CLL cell survival and CLL disease etiology. Further, the reduced expression of del(13q)(14)-resident microRNAs has been implicated in early CLL pathogenesis in a subset of cases.4 It is unknown whether CLL is driven by high-frequency recurrent gene mutations in one or a few genes.

Definition and characterization of the systemic T-cell dysregulation in untreated indolent B-cell lymphoma and very early CLL

AbstractEpidemiologic data show that the immune system may control or promote the emergence and growth of neoplastic lymphomatous clones. Conversely, systemic lymphomas, especially myeloma and chronic lymphocytic leukemia (CLL), are associated with clinical immunodeficiency. This prospective controlled study demonstrates substantially reduced circulating T helper cells, predominantly naive CD4+ cells, in patients with nonleukemic follicular lymphoma and extranodal marginal zone lymphoma, but not in monoclonal gammopathy and early CLL. These changes were correlated with a preactivated phenotype, hyperreactivity in vitro, presenescence, and a T helper 2 shift of peripheral T helper cells. No prominent alterations existed in the regulatory T-cell compartment. Gene expression profiling of in vitro–stimulated CD4+ cells revealed an independent second alteration of T helper cell physiology, which was most pronounced in early CLL but also detectable in follicular lymphoma/extranodal marginal zone lymphoma. This pattern consisted of down-regulation of T-cell receptor signaling cascades and globally reduced cytokine secretion. Both types of T-cell dysfunction may contribute to significant immunodeficiency in nonleukemic indolent B-cell lymphomas as demonstrated by unresponsiveness to hepatitis B vaccination. The precise definition of systemic T-cell dysfunction serves as the basis to study its prognostic impact, its relationship to the established influence of the lymphoma microenvironment, and its therapeutic manipulation.

Chronic Lymphocytic Leukemia in Kidney Recipients

To the Editor: In France, a recent epidemiologic report (1) stated that among the 31 436 patients with end-stage renal disease (ESRD) 18 848 were ≥ 65 years old. At present the incidence after 65 years of age of chronic lymphocytic leukemia (CLL) is estimated to be around 20 cases/100 000 persons per year (2), implying that roughly four new cases of CLL should be diagnosed every year in the population of French ESRD patients. CLL is incurable, but also very heterogeneous: some patients live for decades with no therapeutic needs, whereas others have a rapidly aggressive clinical course (3). All CLL patients taken together, the 5-year survival rate exceeds 80% (2), which is probably why some transplantation centers have circumvented a theoretical contra-indication to solid organ transplantation and allowed some of these patients to become potential kidney recipients. In 2008 we had reported in this journal the evolution (up to four years post-transplantation) of four ESRD patients with CLL who had been engrafted with a deceased donor kidney, and treated with conventional immunosuppressive drugs (4). Our aim was to warn the scientific community of this high-risk situation: all four patients had developed severe infectious episodes, and two had developed tumoral complications (namely, the rapid progression of CLL and a Kaposi sarcoma). In addition, two patients had a prominent infiltration of the graft by monoclonal B cells, along with renal failure. The aim of the present letter is, first, to report the extended follow-up on these patients, and second, to discuss the measures that could improve the outcome of future patients, both prior and post-transplantation. At present, three patients out of four are dead. One (formerly patient [fp]#4) died 46 months post-tx from CLL progression and numerous septic complications, as previously reported (4). Another one (fp #1) died 47 months post-tx, with generalized lymph node enlargement and a metastatic cancer of a native kidney. A third one (fp #2) died 53 months post-tx from refractory CLL (of note, his graft had ceased to function 1 month earlier, due to a massive B-cell graft infiltration). Eventually, only one patient (fp #3) is still alive 75 months post-tx, but she has lost her graft 54 months post-tx. The last graft biopsy (indicated by chronic graft dysfunction) revealed a diffuse lymphocytic infiltration with severe interstitial fibrosis. All these patients experienced a high morbidity, and frequent hospital admissions. Although the long-term survival rate of ESRD patients with CLL is unknown, it is not immediately obvious that dialysis increases the morbidity or the mortality consecutive to CLL. To us, the contrasting observation that both the quality and the quantity of life of the four reported patients were dramatically reduced after a kidney transplantation should lead to consider the exclusion of these patients from the waiting list. At the very least, a discussion on a case-by-case basis should be undertaken between transplant physicians and hematologists/oncologists, taking into account the new molecular prognostic markers available for CLL such as the expression of CD38, cytogenetic abnormalities and ideally the mutational status of genes coding for immunoglobulin heavy chain variable region (5). In addition, since some of the drugs used to prevent rejection potentially increase the risk of an exacerbation of CLL by reducing the immunosurveillance against tumoral cells, it might be sound to favor drugs that target the cell cycle (such as mammalian target of rapamycin inhibitors). We urge physicians to report additional cases, both with good and poor outcome, to the transplant society. Of interest, a recent case of early CLL, diagnosed post-transplant, with good prognostic markers, was reported to be uncomplicated with a 3-year follow-up, after mycophenolate mofetil discontinuation (6). A year and a half later, under a ‘weak’ immunosuppression (low dose tacrolimus and steroids), this patient has a normal performance status, a preserved graft function, and no recent history of septic or tumoral complication. Although his graft is also infiltrated by specific B cells, this infiltration is not destructive and his CLL remains remarkably stable. Prognostic markers could therefore help to sort out some ESRD patients with early-stage CLL in whom a kidney transplant is eventually a reasonable option. Regardless, these patients should come under very close scrutiny. The authors of this manuscript have no conflicts to disclosed as described by the American Journal of Transplantation.

Differentiation on Biological Basis of Monoclonal B-Cell Lymphocytosis (MBL) From Chronic Lymphocytic Leukemia (CLL): Results of a Prospective GISL (Gruppo Italiano Studio Linfomi) Trial

AbstractAbstract 1360The arbitrary cut-off of 5000/μL chronic lymphocytic leukemia (CLL)-phenotype cells in peripheral blood is generally used to separate monoclonal B-cell lymphocytosis (MBL) from CLL. However, a major concern is the biological differentiation, if any, between MBL and CLL. We tried to address the issue therefore analyzing 261 Rai stage 0 patients enrolled in a Gruppo Italiano Studio Linfomi (GISL) prospective multicentre trial designed to validate biological parameters in early CLL as well as to assess the impact on clinical outcome of an early versus delayed policy of treatment with subcutaneous alemtuzumab in the high biological risk. In this cohort, biological characteristics of 105 (40.2%) patients who would be reclassified as MBL using the 2008 CLL diagnostic criteria were compared with those of the remaining 156 patients who had more than 5000/μL CLL-phenotype cells in peripheral blood and fulfilled diagnostic criteria of CLL.Male to female ratio was similar for MBL and CLL (54/53 vs. 92/66, P=0.21) as was median age (58.18 vs 58.18, P=0.98). Median absolute number of cells with CLL phenotype in peripheral blood was 3120/μL (range,400-4959) in MBL and 9925/μL (range, 5020–110000) in CLL (P<0.0001). No difference in the CD38 status (P=0.48),ZAP-70 expression (P=0.29) or cytogenetic abnormalities as detected by FISH [trisomy 12 (P=0.24); deletion 11q (P=0.68); del17p (P=0.09)] was found between patients with MBL and CLL. The only feature differentiating CLL from MBL was represented by an excess of patients with unmutated IgVH disease in the former group (CLL,69.2% vs. MBL, 30.8%: P=0.04). In addition, patients with CLL had an about 2-fold risk of having IgVH germline status in comparison to patients with MBL (OR,1.80; 95% CI, 1.02–3.13; P=0.04). Since the arbitrary cut-off of 5000/μL CLL-phenotype cells in peripheral blood failed to identify a peculiar biological profile for either MBL or CLL, we wondered whether a different B-cell threshold based on disease clinical outcome better stratified patients according to biological risk. In an independent cohort including 818 Rai stage 0 patients registered in a GIMEMA (Gruppo Italiano Malattie EMatologiche Maligne dell'Adulto) database, we demonstrated that a count of 10000/ μL B-cells is the best lymphocyte threshold to predict time to first therapy (TFT). When this cut-off was applied to the GISL series we found that the distribution of main high-risk features [CD38, P=0.83; trisomy 12,P=0.36; del11q,P=0.85; del17,P=0.37) was similar between patients with B-cell lymphocytes higher and lower than 10000/ μL. Only an excess of cases with unmutated IgVH (P=0.04) and slightly increase of ZAP-70 (P=0.06) characterized patients B-cell higher than 10000 μL. In conclusion, present data obtained from a prospective multicentre study indicate that biological characteristics of CLL are found also in MBL and there is no general predominance of good risk variables in MBL in comparison to CLL. This implies that MBL may not be considered a distinct disease but as an early stage of CLL. Disclosures:Musto:Celgene: Honoraria; Janssen Cilag: Honoraria.

Baff serum level predicts time to first treatment in early chronic lymphocytic leukemia

We analyzed the correlation between well-established biological parameters of prognostic relevance in B-cell chronic lymphocytic leukemia (CLL) [i.e. mutational status of the immunoglobulin heavy chain variable region (IgVH), ZAP-70 and CD38 expression] and serum levels of B cell-activating factor (BAFF of the TNF family) by evaluating the impact of these variables on the time to first treatment (TFT) in a series of 169 previously untreated CLL patients in Binet stage A. Higher levels of BAFF were more frequently associated with female gender (P=0.02), younger age (P=0.01), Rai stage 0 (P=0.002), higher platelet count (P=0.005), mutated IgVH disease (P=0.002), higher occurrence of normal cytogenetic profile or presence of 13q deletion (P=0.02), low ZAP-70- (P=0.003), and CD38-expression (P=0.02). Maximally selected log-rank statistic plot identified a serum BAFF concentration of 0.313 ng/mL as the best cut-off (P<0.0001). This threshold recognized two subsets of patients with different TFT (P<0.0001). Because in multivariate analysis soluble BAFF [Hazard ratio (HR), 8.23; confidence Interval (CI) 95%,3.0-22.6, P<0.0001] and mutational status of IgVH (HR=2.60; CI 95% 1.10-6.14, P=0.03) maintained the discriminating power their combined effect on clinical outcome was assessed. When three groups were considered: (1) low-risk (n=93), patients with concordant IgVH(mut) and higher soluble BAFF; (2) intermediate-risk (n=50), patients with IgVH(mut) and low BAFF levels or IgVH(unmut) and soluble higher BAFF;(3) high-risk (n=26), patients with concordant IgVH (unmut) and low soluble BAFF, the 2-yr TFTs were, respectively, 95%, 85%, and 41% (P<0.0001). In conclusion, our results indicate that in early B-cell CLL, the biological profile including among other parameters soluble BAFF may provide a useful insight into the complex interrelationship of prognostic variables.

Chronic Lymphocytic Leukemia Presenting With Localized Gynecologic Symptoms

Although no cure exists for chronic lymphocytic leukemia (CLL), treatment can reduce morbidity and/or improve survival in advanced disease. Identification of patients in an early disease state allows monitoring and treatment as soon as appropriate. We were unable to find previous reports of early CLL disease presenting with gynecologic symptoms. This report seeks to alert gynecologists of their potential role in identifying CLL patients. A 76-year-old woman presented with rectovaginal pressure. Cervical cone biopsy showed a lymphoproliferative process. Peripheral blood flow cytometry confirmed a diagnosis of early-stage CLL. The patient remains asymptomatic and is currently monitored for disease progression. Early CLL can present with localized gynecologic symptoms or cervical lesions. The recognition of early disease can assure optimal treatment.

Prognostic Value of Angiogenesis Assessment in Chronic Lymphocytic Leukemia: More Data Needed.

Abstract 4396 IntroductionThe association between angiogenesis and cancer progression in solid tumors has been documented, but its significance in chronic lymphocytic leukemia (CLL) has not been completely evaluated. Vascular endothelial growth factor /VEGF/ is the major pro-angiogenic factor in humans and his transduction pathway may be very active in CLL cells contributes to their enhanced survival. In this context VEGF can be additional tool for predicting the clinical course in early CLL. Patients and MethodsTo predict the risk of disease progression, we analyzed serum levels VEGF (sVEGF) using ELISA tehnique in 33 Binet stage A de novo CLL patients. In addition, We analyzed microvessel density (MVD) of the same patients using immunohistochemical staining with CD34 and vWf. Finally, we explored wheather changes of circulating VEGF concentrations and MVD reflected clinico-biological features of CLL (peripheral blood lymphocytosis (PBL), bone marrow (BM) histology, beta-2 microglobulin (β2m) level, lactat dehidrogenase (LDH) level) and kariotype abnormalities. ResultsThe VEGF serum levels (sVEGF) was not significantly elevated (p=0.31) in CLL patients (mean: 70.9 pg/mL; range: 16–483) compared with and age- and sex-matched healthy controls (mean: 44.7 pg/mL; range: 22–69.2). sVEGF level positively correlated with elevated LDH level (p=0.008), but no correlation with other clinico-biological features was found.Bone marrow MVD was significantly higher (p<0.0001) in CLL patients (mean: 35.91 vessels/field ± 15.71) compared to controls (mean:8.27 vessels/field ±6.19). Also, there was a significant difference between MVD counts according to the antibody used. MVD was higher using CD34 vs vWF (CD34, 35.91 vessels/field ±15.7, vs vWF, 8.15 vessels/field ±4.65, p<0.0001). Bone marrow MVD detected by CD34 was significantlyhigher in patinents with CD38 expression more than 30% (p=0.006). However, no significant MVD differences were detected between CLL subgroups with regard to clinical course, pattern of marrow infiltration, Rai stage and FISH abnormalities.In univariate analysis sVEGF (p=0.018), Rai substages (p=0.027), PBL (p=0,030), β2m level (p=0.008) and diffuse bone marrow infiltration (p=0.006) were significantly associated with increased risk of disease progression. But, in multivariate analysis only sVEGF (p=0.002) and β2m level (p=0.008) retained their prognostic significance. ConclusionsSerum VEGF level although not increased in comparasion with healthy controls, may improve the assessment of individual prognosis of patients with early CLL. Assessment of real clinical significance of bone marrow angiogenesis in CLL is required. Disclosures:No relevant conflicts of interest to declare.

External Validation On Biological Basis of New Prognostic Index in Early Asymptomatic Chronic Lymphocytic Leukemia (CLL) Patients: The Gruppo Italiano Studio Linfomi (GISL) Experience.

Abstract 2375Poster Board II-352A prognostic index based on widely available clinical and laboratory features was recently proposed to predict survival in patients with previously untreated patients with chronic lymphocytic leukemia (CLL) by MD Anderson investigators. However, whether proposed clinical risk categories may surrogate new biological variables of prognostic relevance (i.e., mutational status of the IgVH gene regions, ZAP-70 or CD-38 expression, cytogenetic abnormalities) is unclear thus far.In a series of 160 asymptomatic Binet stage A patients enrolled in a Gruppo Italiano Studio Linfomi (GISL) multicentre trial designed to validate prospectively biological parameters in early CLL as well as to assess the impact on clinical outcome of an early versus delayed policy of treatment with subcutaneous alemtuzumab in the high biological risk, we evaluated whether clinical categories derived from newly proposed prognostic index reflected biological risk.Since the original prognostic index was derived from a database including cases with more advanced disease we used an optimal cutoff search to determine how to best split Binet stage A patients in different prognostic groups. To this purpose an independent patient cohort consisting of 310 Binet stage A patients included in a GIMEMA (Gruppo Italiano Malattie EMatologiche Maligne dell'Adulto) database was used. According to recursive partitioning (RPART) model, a classification tree was built that identified two subsets of patients who scored respectively: 0-3 (low risk) and 4-7 (high risk). Therefore, by prognostic index, 48.7% and 51.2% of 160 asymptomatic stage A patients, respectively, met criteria of low risk and high risk disease.In our prospective series high- risk score was more frequently associated with both unmutated IgVH status (P=0.009) and higher CD38-expression (P=0.002); in contrast only a trend towards an increased ZAP-70 expression could be found (P=0.06). As far as cytogenetic abnormalities are concerned, we observed that 11q deletion occurred more frequently among patients belonging to high-risk score (P=0.005), while cases with 13q deletion or trisomy 12 were homogeneously distributed among low- and high-risk patient category(P=0.151 and P=0.452, respectively). We did not consider suitable for correlation analysis 17p deletion since observed only in 2 out of 160 Binet stage A patients.In conclusion, our results demonstrate in a prospective cohort of patients with early CLL that clinical categories of a revised score index may surrogate biological parameters of prognostic relevance. The observation reinforces the revised IWCLL guidelines recommendations to assess the risk of CLL patients on clinical basis and to deserve biological studies to patients eligible for clinical trials. Disclosures:No relevant conflicts of interest to declare.

The utility of a prognostic index for predicting time to first treatment in early chronic lymphocytic leukemia: the GIMEMA experience.

A prognostic index based on widely available clinical and laboratory features was recently proposed to predict survival in patients with previously untreated chronic lymphocytic leukemia. We assessed the utility of this index for predicting time to first treatment in early chronic lymphocytic leukemia. An observational database of the GIMEMA (Gruppo Italiano Malattie EMatologiche dell'Adulto), which included 310 patients with newly diagnosed Binet stage A chronic lymphocytic leukemia who were observed at different primary hematology centers during the period 1991 - 2000, was used for the purpose of this study. The new prognostic index enabled Binet stage A patients to be divided into two subgroups that differed with respect to time to first treatment (P=0.003). The original prognostic index was derived from a database that included cases observed at a reference academic center; these patients were younger (P<0.0001) and had more advanced disease (P<0.0001) than those in the current investigation, which studied community-based patients whose data were recorded at presentation. With this in mind, we used an optimal cut-off search to determine how best to split patients with Binet stage A disease into different prognostic groups. According to the recursive partitioning (RPART) model, a classification tree was built that identified three subsets of patients who scores were 0-2 (low risk), 3-4 (intermediate risk) and 5-7 (high risk). The probability of remaining free from therapy at 5 years was 100% in the low risk group, 81.2% in the intermediate risk group and 61.3% in the high risk group (P<0.0001). The results of this study confirm the utility of a new prognostic index for predicting time to first treatment in a large sample series of community-based patients with early stage chronic lymphocytic leukemia at presentation. Our effort to develop a revised scoring method meets the need to separate Binet stage A patients into different prognostic groups in order to devise individualized and tailored follow-up during the treatment-free period.

Serum level of CD26 predicts time to first treatment in early B‐chronic lymphocytic leukemia

We analyzed the correlation between well-established biological parameters of prognostic relevance in B-cell chronic lymphocytic leukemia (CLL) [i.e. mutational status of the immunoglobulin heavy chain variable region (IgV(H)), ZAP-70- and CD38-expression] and serum levels of CD26 (dipeptidyl peptidase IV, DPP IV) by evaluating the impact of these variables on the time to first treatment (TFT) in a series of 69 previously untreated Binet stage A B-cell CLL patients. By using a commercial ELISA we found that with exception of a borderline significance for ZAP-70 (P = 0.07) and CD38 (P = 0.08), circulating levels of CD26 did not correlate with either Rai substages (P = 0.520) or other biomarker [beta2-microglobulin (P = 0.933), LDH (P = 0.101), mutational status of IgV(H) (P = 0.320)]. Maximally selected log-rank statistic plots identified a CD26 serum concentration of 371 ng/mL as the best cut-off. This threshold allowed the identification of two subsets of patients with CD26 serum levels higher and lower that 371 ng/mL respectively, whose clinical outcome was different with respect to TFT (i.e. 46% and 71% at 5 yr respectively; P = 0.005). Along with higher serum levels of CD26, the univariate Cox proportional hazard model identified absence of mutation in IgV(H) (P < 0.0001) as predictor of shorter TFT. As in multivariate analysis all these parameters maintained their discriminating power (mutational status of IgV(H,)P < 0.0001; soluble CD26, P = 0.02) their combined effect on clinical outcome was assessed. When three groups were considered: (1) Low-risk group (n = 31), patients with concordant IgVH(mut) and low level of soluble CD26; (2) intermediate risk group (n = 26), patients with discordant pattern; (3) high-risk group (n = 12), patients with concordant IgVH(unmut) and high level of soluble CD26, differences in the TFT were statistically significant, with a TFT at 5 yr of respectively 88%, 51% and 43% (P < 0.0001). Our results indicate that in early B-cell CLL biological profile including among other parameters soluble CD26 may provide a useful insight into the complex interrelationship of prognostic variables. Furthermore, CD26 along with mutational status of IgV(H) can be adequately used to predict clinical behavior of patients with low risk disease.