Comparison of Clinical and Laboratory Features, Drug Availability, and Outcomes of CLL Patients Treated in Public or in Private Hospitals in Brazil: A Retrospective Analysis of the Brazilian Registry of CLL

Introduction: Chronic lymphocytic leukemia (CLL) typically occurs in elderly patients and has a highly variable clinical course. It is important to understand the aspects that affect the outcomes of CLL in a real-world setting. Besides biological factors, other socioeconomic and health system factors may influence the clinical course of CLL. Hence, data from the Brazilian Registry of CLL was analyzed to compare clinical and treatment-related characteristics in patients with CLL treated in public or in private institutions in Brazil. Objective: To describe the outcomes of a series of CLL patients followed in public or in private hospitals in Brazil. Methods: The Brazilian Registry of CLL was started in 2004 as a prospective non-interventional data collection tool. Inclusion criteria for enrollment followed the IWCLL guidelines. For this analysis, we included all patients with minimum available data for analysis of patient and disease characteristics and survival. Results/discussion: From January 2004 to July 2020, 2927 patients from 37 centers met eligibility criteria for this analysis: 2324 (79%) were followed at public hospitals and 603 (21%) at private hospitals. The majority were male (57%), with median age of 65 years (ranging from 23 to 106). Binet stage was A in 1618 (58%) patients, B in 628 (23%) and C in 525 (19%). FISH for del(17p) was performed in only 479 patients (16%), while FISH for the most common aberrations [del(13q), +12, del(11q), and del(17p)] was performed in only 445 patients (15%). IGVH mutational status was performed in only 211 patients (7%), and karyotype in only 140 patients (5%). Comparing public and private hospitals, we observed that patients in public hospital are slightly older (median age 66 years vs. 64 years for private hospitals, P=0.04), had more advanced diseases at diagnosis (frequency of Binet B or C was 44% in public vs. 32% in private hospital, P<0.0001), and there were more patients with elevated creatinine levels (14% vs. 10%, P=0.03). All prognostic markers were significantly more available in private than in public hospitals: FISH for del17p (42% of cases vs. 10%, respectively, P<0.0001), IGVH mutational status (13% vs. 6%, respectively, P<0.0001) and karyotype (16% vs. 2%, respectively, P<0.0001). The frequency of del(17p) was similar between public and private hospitals (10% vs. 11%, P=NS), while the frequency of unmutated IGHV status was more common in private hospitals, although not statistically different (60% vs. 48%, P=0.09). Analyzing 2102 diagnosed after 2010, we observed that 864 patients (41%) were treated after a median time of 7 months (range: 0-267) after diagnosis. First line treatment was predominantly based chlorambucil (45%) or fludarabine (40%). Anti-CD20 monoclonal antibody was used in only 39% of cases: (rituximab in 35% and obinutuzumab in 4%). Novel agents were used in first line in only 2% of patients, and in most cases in the context of a clinical trial. Of note, most patients (86%) with del(17p) detected by FISH were treated with chemoimmunotherapy. When comparing treatments between public or private hospitals we observed striking differences: in public hospitals there were significantly less patients receiving fludarabine-base regimens (36% vs. 54%, P<0.0001), and anti-CD20 monoclonal antibodies (28% vs. 78%, P<0.0001). Overall survival at 6 years was significantly worse in public than in private hospitals (72% vs. 93%, respectively, P<0.0001). After a multivariate analysis, survival in patients from public hospitals remained significantly worse than in private hospitals (hazard ratio 3.4, 95% confidence interval 2.4 - 4.8), after correcting for age, Binet staging and renal function. Conclusion: Our data indicate that are striking differences between patients treated in public or private hospitals in Brazil. The lack of accessibility to basic laboratory tests for prognostic factors and adequate therapies probably explains the worse outcome of patients treated in public institutions. In fact, prognostic testing rates were poor in both contexts and most high-risk patients received chemoimmunotherapy first-line. Urgent strategies are needed to increase accessibility to prognostic testing and to novel agents for quality improvement in health care in CLL patients in Brazil. Disclosures No relevant conflicts of interest to declare.

Clinical Prognostic Biomarkers in Chronic Lymphocytic Leukemia and Diffuse Large B-Cell Lymphoma

Context Diffuse large B-cell lymphoma and chronic lymphocytic leukemia are 2 of the most common B-cell lymphomas in adults. Both diffuse large B-cell lymphoma and chronic lymphocytic leukemia share heterogeneous outcomes, and the use of prognostic biomarkers to better stratify risk in these patients has now become commonplace. Objective To review chronic lymphocytic leukemia and diffuse large B-cell lymphoma biomarkers commonly used in the clinical laboratory, which can be divided into the following 3 main groups by testing methodology: chromosomal based (including fluorescence in situ hybridization and cytogenetics), expression based (including immunohistochemistry and flow cytometry), and DNA based (including gene sequencing for somatic mutations and IGVH mutational status). Data Sources Review of recent literature. Conclusions In chronic lymphocytic leukemia, important biomarkers include expression of CD38 and ZAP-70, IGVH mutational status, somatic mutations in TP53 and NOTCH1, and abnormalities in chromosomes 11, 12, 13q, and 17. In diffuse large B-cell lymphoma, important biomarkers include chromosomal rearrangement of BCL2, BCL6, and MYC and expression of CD5, BCL2, and CD43, as well as somatic mutations in TP53 and BCL6.

Cancer testicular antigens as a prognostic markers of chronic lymphocytic leukemia.

6575 Background: CLL is the most common leukemia in adults and has a variable course and prognosis. Clinical staging systems and the use of biomarkers such as cytogenetics and the IGVH mutational status remain the gold standard for stratification. Cancer testis antigens (CTAs) expression analyses have shown prognostic impact in other hematological malignancies such as acute leukemias and multiple myeloma. With the analysis of CTAs we aim to discover additional molecular markers that can help us predict the clinical course and outcome in CLL patients. Methods: We analyzed the expression by RT-PCR of 39 known CTAs including members of the MAGE, GAGE, MAD-CT and SSX CTAs families in a group of 59 CLL patients seen at our institution. The Moffitt Cancer Center Total Cancer Care (TCC) database was used to correlated these findings with relevant clinical and molecular markers such as Rai stage, demographic data, initial WBC count, IGVH mutational status, CD38 expression, cytogenetics by FISH analysis and treatment outcomes. Overall survival (OS) and progression free survival (PFS) were calculated using Kaplan Meier curves with a SPSS statistical software Results: Deletion 13q was the most common gene abnormality in 35/59 (59.3%). 29/59 (49.1%) required at least one line of treatment. MAGE family CTAs were present in 53/59 (83.9%), MAD-CT in 24/59 (40.7%), SSX in 14/59 (23.7%) and GAGE in 12/59(20.3%). 35/59 (59.3%) CLL patients expressed 2 or more MAGE family CTAs. No differences were noted in the Rai staging, initial white blood cell count, B symptoms, extranodal disease, presence of CD38, or unmutated IVGH. The OS was not significantly different among all CTAs families with a tendency towards a better OS those expressing the SSX family (p=0.63). Additionally, CLL patients with single expression of MAD-CT1 (p=0.05) and MAGE-B2 (p=0.038) antigens correlated with improved survival. Conclusions: Patients with SSX family have a trend towards improved survival. When analyzed by individual antigen, MAD-CT1 and MAGE-B2 expression was associated with improved survival. Further studies with a larger number of patients are needed to assess the real value of the expression of CTAs in CLL.

Heat Shock Protein 90 (Hsp90) Activity Status Is An Independent Prognostic Marker in Chronic Lymphocytic Leukemia (CLL) and Correlates with IgVH Mutational Status, ZAP-70 Expression and Time to First Treatment

Abstract 2544 The clinical presentation of chronic lymphocytic leukemia (CLL) is variable and includes at least two distinct clinical subtypes: indolent or progressive disease. Several prognostic markers such as the mutational status of the IgVH genes or ZAP-70 expression identify patients that will have more aggressive clinical courses. We have previously reported that ZAP-70 expression in CLL requires the support and stability provided by the Hsp90 activated complex (Hsp90-AC) and that Hsp90 inhibitors induce apoptosis in CLL cells preferentially in patients with high–risk/rapid progressive disease. Hsp90 requires the conformation of a multimeric complex with other co-chaperones (Hop, p23) to become active and function as a chaperone for mutated, over-expressed, misfolded or constitutively activated proteins. However, to measure Hsp90-AC activity is challenging and requires protein assays that are semi-quantitative and not always can provide a functional read out. To address this problem we designed a test to measure Hsp90-AC by taking advantage of the higher avidity of this complex to bind Hsp90 inhibitors such as 17-AAG. We hypothesize that Hsp90-AC activity is an independent prognostic marker of disease progression in CLL and to test this we measured the ability of 17-AAG to inhibit Hsp90-AC and promote apoptosis in vitro in a cohort of previously untreated CLL patients. This evaluation was performed in samples collected at the time of diagnosis and the results were correlated with the time from diagnosis to first treatment (t-Tx1) as well as the presence of other known prognostic markers. Ninety-five previously untreated CLL patients were included in this cohort. In vitro sensitivity/apoptosis to Hsp90-AC inhibition using 17-AAG (1μg/ml) was measured by flow cytometry using DiOC6 and PI after 48 hours of incubation. Only samples with more than 60% viability were included and we selected a cut-point of ≥ 53% induction of apoptosis to determine if a sample was sensitive or not to Hsp90-AC inhibition (This cut-point was selected using a ROC analysis for optimal diagnostic performance). We found that 37 patients (39%) were sensitive to 17-AAG. These patient had a median time from diagnosis to first treatment (t-Tx1) of 7 years compared to 4.2 years in patients that were resistant to 17-AAG (p=0.04). In addition, 37 patients (39%) had unmutated IgVH genes with a median t-Tx1 of 8.7 years compared to 4.2 years in patients with mutated IgVH genes (p=0.02) and 41 patients (43%) had high levels of ZAP-70 expression (>20% by flow cytometry) and this group of patients had a median t-Tx1 of 7 years vs. 4.2 years in ZAP-70 negative patients (p=0.03). We evaluated the association between the degree of Hsp90-AC inhibition (17-AGG sensitivity) and IgVH mutational status and found a strong correlation between these two variables with shorter t-Tx1 in patients that were IgVH unmutated and sensitive to Hsp90 inhibitor (p=0.0003). We found a similar strong correlation between ZAP-70 expression and sensitivity to Hsp90-AC inhibitor (p=0.003). In our patient cohort, we saw that IgVH, ZAP-70 and Hsp90 inhibitor sensitivity are independent prognostic markers in CLL (Cox regression, p=0.002). We found that the sensibility and specificity of ZAP-70 test for high-risk CLL (sensitivity 81.1% [95% IC 67–95%] and specificity 77.6%[95% IC 65–90%]) was increased when the Hsp90-AC inhibitor sensitivity test was added (sensitivity 87% [95% IC 71–100%] and specificity 91%[95% IC 79–100%]). In conclusion, in vitro inhibition of Hsp90-AC in CLL using 17-AAG is an independent prognostic maker and strong predictor of the need for treatment. Hsp90-AC inhibition showed a high correlation with IgVH mutational status and ZAP-70 expression. Additionally, patients sensitive to 17-AAG whose leukemic cells also highly express ZAP-70 appear to have the worst prognosis with the shortest t-Tx1. Our data suggest that in vitro sensitivity to 17-AAG at the time of diagnosis serves as a quantitative surrogate marker for Hsp90-AC activity and correlates with disease progression in CLL. This is the first time that such correlation has been established in a cohort of cancer patients and shows the relevance of the Hsp90 chaperone system in cancer biology and disease progression. Disclosures: No relevant conflicts of interest to declare.

Disruption of TP53 function by Point Mutations and Deletions Is Associated with An Increased Risk of Disease Progression within Previously Treated, Relapsed Chronic Lymphocytic Leukemia Patients

Abstract 2445 Chronic lymphocytic leukemia (CLL) patients with a deletion of the TP53 tumor supressor gene located at 17p13 have a poor prognosis in first line chemotherapy regimens. Recent studies indicated somatic TP53 mutations as a prognostic factor in CLL independent of 17p13 deletion status. We aimed to further characterize the prognostic value and the impact of TP53 mutations on progression-free survival (PFS) in the presence and absence of a 17p13 deletion in previously treated and relapsed CLL patients within an international phase III clinical study comparing Fludarabine and Cyclophosphamide with or without Rituximab (FC versus R-FC: REACH trial). We analyzed 457 patients at diagnosis for mutations in the TP53 gene using a combination of a microarray-based resequencing assay (AmpliChip p53 Test, Roche Molecular Systems, USA.) and Sanger sequencing of TP53 exons 2–10. The data were correlated with clinical and biologic markers as well as with interphase fluorescence in situ hybridization (FISH) and with PFS. Association of the clinical data with PFS was assessed by Cox proportional hazard models. To estimate the functional significance of the individual TP53 mutations we used the IARC TP53 database. TP53 mutations (n=60) were detected in 52 of 457 patients (11.4%) and included 42 missense, 4 nonsense, 8 frameshift mutations, 2 in-frame deletions and 4 mutations in splice sites. Among other clinical variables, only 17p13 deletion was associated with TP53 mutations: 27 of 52 TP53 mutated patients had a 17p13 deletion (concordance rate: 52%, Fisher's test p<0.001). Median PFS for patients with TP53 mutations (n=52, 13 months, HR=1.9 (1.4–2.7), p<0.001) was significantly shorter as compared to patients without TP53 mutations (n=480, 27 months). In a sub-group analysis, chemoimmunotherapy including Rituximab did not significantly improve the PFS of patients with TP53 mutations. Multivariate analysis including treatment arm, Binet stage, age, IGVH mutational status, 17p13 deletion and TP53 mutation status confirmed TP53 mutation status (HR-TP53=1.7 (1.1–2.6), p=0.009) as a prognostic factor for PFS independent of 17p13 deletion status (HR-17p=1.7 (1.1–2.7), p=0.024) and with a similar effect size. The other independent prognostic factors were treatment (HR=0.61 (0.48–0.76), p<0.001), Binet stage (HR=1.64 (1.3–2.1), p<0.001) and IGVH mutational status (HR=2.4 (1.85–3.1), p<0.001). To further dissect the contribution of TP53 mutation and 17p13 deletion on PFS, we considered a multivariate analysis comparing patients with both TP53 mutation and 17p13 deletion (n=28), with only 17p13 deletion (n=9), with a dominant negative TP53 mutation or multiple TP53 mutations (n=8) or with a single TP53 mutation (n=16) against patients without TP53 abnormalities (n=271), adjusted for treatment, Binet stage, age and IGVH mutational status. Patients with a predicted biallelic disruption of TP53 either by a TP53 mutation in combination with a 17p13 deletion (HR: 2.8 (1.8,4.2), p=<0.001) or patients with a dominant negative TP53 mutation as predicted by the IARC TP53 database or multiple TP53 mutations (HR=3.26 (1.5,7.1), p=0.003) had a risk similar in size and which was quite high for disease progression (the reference to calculate the risk, here and in the following, is always the group of patients without TP53 abnormalities). The risk slightly decreased for patients with only a deletion 17p13 (HR=2.2, (1.1–4.3), p=0.021). Very interestingly, single TP53 mutations showed a much lower risk for disease progression (in this case not even significant) (HR=1.61 (0.9–2.8), p=0.084) especially compared to the risk conferred by a biallelic disruption. In this large cohort of previously treated CLL patients, complete disruption of TP53 function (by a combination of a 17p13 deletion and a TP53 mutation, through dominant negative TP53 mutations or through multiple TP53 mutations) was associated with a higher risk for disease progression. Prognosis of patients with a single TP53 mutation was not significantly different from patients without TP53 aberrations. It remains to be shown whether CLL patients with a single TP53 mutation are at a higher risk of acquiring additional mutations of TP53 during disease progression. Prognostic stratification of previously treated CLL patients should include a routine molecular TP53 mutational analysis in addition to deletion analysis of the TP53 locus by FISH. Disclosures: Dufour: Roche: Research Funding. Bohlander:Roche: Research Funding. Spiekermann:Roche: Research Funding. Schneider:Roche: Research Funding. Hiddemann:Roche: Research Funding. Truong:Roche: Employment. Patten:Roche: Employment. Wu:Roche: Employment. Dmoszynska:Mundipharma:; Roche: Honoraria. Robak:Centocor Ortho Biotech Research & Development: Research Funding. Geisler:Roche: Speakers Bureau. Dornan:Genentech: Employment. Lin:Genentech: Employment. Yeh:Genentech: Employment. Weisser:Roche: Employment. Duchateau-Nguyen:Roche: Employment. Palermo:Roche: Employment.

A Phase II Study of Chlorambucil Plus Rituximab Followed by Maintenance Versus Observation In Elderly Patients with Previously Untreated Chronic Lymphocytic Leukemia: Results of the First Interim Analysis

Abstract 2462 Background: Two of the largest trials ever conducted in patients with chronic lymphocytic leukemia (CLL) have shown that the addition of rituximab to fludarabine plus cyclophosphamide (R-FC) significantly improves outcome. However, myelotoxicity and immunosuppression limit the use of this regimen in patients with impaired performance status and pre-existing co-morbidities, predominantly in the elderly. Chlorambucil (CLB) remains a first-line treatment option for such patients. The use of CLB in combination with R is thus an attractive therapeutic option in view of the potentially increased activity compared to CLB alone and the likely good tolerability. This study was designed to determine whether the R-CLB combination is feasible and beneficial as first-line treatment for elderly patients with CLL and to define the role of maintenance R. Patients and Methods: Between October 2008 and January 2010, 97 elderly patients with untreated CD20+ CLL requiring therapy according to the IWCLL criteria were enrolled into the protocol. CLB treatment was administered every 28 days for up to 8 courses at a dose of 8 mg/m2/day p.o. on days 1–7 combined with 375 mg/m2 R for cycle 3 and 500 mg/m2 for cycles 4–8. Responsive patients were randomized to R maintenance (375 mg/m2 every 2 months for 2 years) versus observation. At baseline, blood samples were taken for FISH analysis, IgVH mutational status and expression of Zap-70 and CD38. Minimal residual disease (MRD) was planned to be evaluated on peripheral blood (PB) and bone marrow (BM) cells by four-color flow cytometry and, when required, by PCR. The primary endpoint was the overall response rate at the end of the induction phase defined according to the IWCLL 2008 on the intention-to-treat (ITT) population (all enrolled patients who received at least 1 dose of R). Secondary endpoints included the adverse event (AE) profile, progression-free and overall survival. Results: These are the data of the planned interim analysis based on the first 54 evaluable patients from 19 Italian centers, including tumor response at the end of the induction phase and safety. The median age of patients was 70.5 years (range 61–84): 14.8% were between 61 and 64, 31.5% between 65 and 69, 31.5% between 70 and 74, 16.7% between 75 and 79, and 5.6% were ≥80 years; thus, 53.8% of patients were over the age of 70; 70.4% were males; 25.9% were Binet stage A, 57.4% stage B and 16.7% stage C. The overall incidences of trisomy 12 and abnormalities of 13q, 11q23 and 17p13 were 24.5%, 52.8%, 20.8% and 5.7%, respectively; 7.5% of patients had p53 mutations. Of the 51/54 patients analyzed for the IgVH mutational status, 64.7% were unmutated; of the 53/54 patients studied, 39.6% were CD38+ and 71.7% were Zap-70+. The overall response rate on an ITT analysis was 81.4% (44/54 patients); a CR assessed by CT scan and trephine immunohistochemistry was found in 16.7% of cases (9 patients: 4 in Binet stage A, 3 in stage B and 2 in stage C), a CRi in 3.7% (2 patients), a nPR in 1.9% (1 patient) and a PR in 59.3% (32 patients). Eight of the 9 CR cases were investigated for MRD by flow cytometry and all proved positive: 6/8 had MRD levels <10−3 in the PB and 2/8 in the BM. A progressive disease was recorded in 2 patients (4%) and a stable disease in 2 (4%). Six patients (11%) were not evaluable for response: 1 investigator's decision, 2 AEs (1 R infusion-related reaction and 1 unrelated episode of dyspnea) and 3 SAEs (1 CLB-related anemia, 1 endometrial in situ carcinoma and 1 anaplastic oligoastrocytoma). Seven SAEs occurred in 7 patients during courses 3–8. Only 1 SAE was related to treatment (1 CLB-related anemia). The most common toxicities were neutropenia (31.5% of patients, 8.9% of cycles) and thrombocytopenia (14.8% of patients, 5.7% of cycles). Grade III-IV neutropenia was present in 16.7% of patients and in 3.8% of cycles. No grade III-IV infections occurred. A median of 85.4 R-CLB courses was administered with 85.1% of patients completing the planned treatment; 15.3% of cycles needed a CLB dose reduction (12.9% due to toxicity, mainly neutropenia and thrombocytopenia). Conclusions: Overall, the results of the interim analysis indicate that R-CLB is active and well tolerated in elderly patients with previously untreated CLL. Disclosures: Foa: Roche: Consultancy, Speakers Bureau. Montillo: Roche: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Runggaldier: Roche S.p.A. Monza: Employment. Gamba: Roche S.p.A. Monza: Employment.