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Abstract Myeloid cell factor-1 (Mcl-1) is an anti-apoptotic member of the Bcl-2 protein family that associates with tumor cell survival and drug resistance in chronic lymphocytic leukemia (CLL). Increased Mcl-1 expression is associated with failure to achieve remission after chemotherapy with fludarabine and chlorambucil in CLL patients. However, the influence of Mcl-1 expression has not been examined in CLL trials using treatment regimens that contain rituximab, which has shown substantially improved outcomes for a large subset of patients and have become a mainstay in the therapy of newly diagnosed CLL. We previously reported that combination chemoimmunotherapy with pentostatin, cyclophosphamide, and rituximab (PCR) has significant clinical activity with low accompanying toxicity in previously untreated CLL patients, and is especially well tolerated in older patients in whom the use of fludarabine may be associated with prohibitive toxicities. As part of this study, we prospectively investigated association of Mcl-1 protein expression with response and progression-free survival. Eligible patients received a regimen consisting of pentostatin (2 mg/m2), cyclophosphamide (600 mg/ m2), and rituximab (375 mg/m2) given intravenously on day 1 of a 21-day cycle for a maximum of 6 cycles. Responses were assessed according to NCI 1996 criteria and included bone marrow evaluation and two-color flow cytometry two months after completion of therapy. Flow cytometry-negative status was defined as less than or equal to 1% CD5+/CD19+ cells. Of the 64 patients evaluated in this trial, clinical responses were seen in 58 (91%), with 26 (41%) complete responses (CR), 14 (22%) nodular partial responses (nPR), and 18 (28%) partial responses (PR). Lysates from peripheral blood cells obtained pre-treatment were analyzed for Mcl-1 expression by immunoblot. Mcl-1 as a continuous variable was not found to be associated with other commonly described risk factors. However, Recursive Partitioning Analysis (RPA) was devised employing Mcl-1 expression as a continuous variable, which revealed an optimal cut point of 0.85. At this cutoff, flow cytometry-negative status was significantly different between the two groups (p=0.01) and median PFS was significantly higher (p=0.02) in patients with Mcl-1 levels of <0.85 (50.8 vs. 18.7 months). No difference by Mcl-1 expression was noted in other pre-treatment or response parameters. Our data describes the first prospective validation of Mcl-1 over-expression adversely influencing both the likelihood of attaining a flow cytometry-negative complete remission and extended progression free survival. Mcl-1 expression was also independent of other risk factors, indicating additional benefit for this measure in assessing patients. Mcl-1 expression may therefore predict poor response to chemoimmunotherapy, and these findings advocate pursuing therapeutic agents targeting this important anti-apoptotic protein.

Abstract BACKGROUND: The diagnosis of monoclonal B-cell lymphocytosis (MBL) is used to characterize patients who have a circulating population of clonal B-cells, a total B-cell count of <5 x 109/L, and no other features of another B-cell lymphoproliferative disorder. In most patients the immunophenotype is similar to that of chronic lymphocytic leukemia with co-expression of CD19 and CD5. The natural history of CLL-phenotype MBL is unclear. We used the Mayo Clinic hematopathology database to identify patients with CLL-phenotype MBL and abstracted clinical information on natural history and prognostic parameters from clinical and research records. METHODS: Using hematopathology records, we identified a cohort of 1820 patients seen at Mayo Clinic between August 1998 and May 2006 who underwent investigation using peripheral blood flow cytometry and who were found to have a clonal B-cell population of CLL phenotype. After elimination of patients with advanced stage (Rai I-IV), previously treated CLL, and individuals with an established diagnosis of CLL/MBL >1 year prior to flow, 636 unique patients with isolated lymphocytosis were identified. Based on our prior analysis (Leukemia Research 32:1458) demonstrating that nearly all patients with an ALC >10x109/L have a B-cell count >5x109/L, we focused the remaining analysis on the 405/636 patients who had an ALC <10x109/L and the necessary raw data from flow cytometry analysis to determine the B-cell count. RESULTS: Among the 405 individuals with an ALC <10x109/L, 306 (76%) had a B-cell count <5x109/L at the time of flow and met the current criteria for the diagnosis of MBL (BJH 130:325). Median age at the time of MBL diagnosis was 70 years (range 34 – 93). A slight male preponderance was observed (59% male). B-cell counts ranged from 0.02 to 4.99x109/L and ALC ranged from 0.3–9.6 x109/L. CD38 status was available for 268 (88%) MBL patients of whom 20% were CD38 positive using the 30% threshold. Cytogenetic analysis by fluorescent in situ hybridization (FISH) was available in 130 (42%) MBL patients. Using the hierarchical classification system of Dohner and colleagues, 57 (44%) were 13q-, 23 (18%) were trisomy 12, 5 (4%) were 11q-, 4 (3%) were 17p-, and 39 (30%) had no defect detected. ZAP-70 status was available for 77 (25%) patients of whom 13% were ZAP-70+ positive. Patients underwent sequential monitoring with a median follow-up of 1.7 yrs (range 0–8.1 yrs). Time from diagnosis to treatment for patients meeting criteria for MBL (n=306) as compared to Rai stage 0 CLL patients with B-cell counts between 5.0 and 10.0 x109/L (n=99) was not significantly different and is shown in Figure 1 (p=0.87). For individuals with MBL, survival free of treatment was 98% (95%CI: 96–100%) at 1 year, 93% (95%CI: 89–98%) at 2 years, and 77% (95%CI: 67–89%) at 5 years. While gender (p=0.10), and B-cell count as a continuous variable (p=0.15) were not found to be associated with time to treatment (TTT) among MBL patients, age (p=0.02, HR=0.94, 95% CI: 0.89–0.99) and CD38 status (p<0.02, HR: 3.3, 95% CI: 1.2–9.2) did show a relationship with TTT. CONCLUSIONS: Although we observed a low rate of progression among individuals meeting criteria for MBL, TFS was similar to individuals meeting current criteria for Rai stage 0 CLL who had B-cell counts between 5.0 – 10.0 x109/L . Among individuals meeting criteria for MBL, younger patients and those who were CD38 positive had a shorter time to treatment. Additional studies are needed to determine what B-cell count or other characteristics should be used to distinguish between MBL and Rai stage 0 CLL. FIGURE: FIGURE:.

Abstract Figure Figure Background: An isolated 13q deletion in patients with chronic lymphocytic leukemia (CLL) is the single most common cytogenetic abnormality and is a favorable prognostic parameter (Dohner et al., NEJM3432:1910, 2000). Although 13q- can occur as either a heterozygous (13q-x1) or homozygous (13q-x2) deletion, it is unknown whether these two genetic types differ in their impact on clinical outcome. We previously postulated that 13q-x2 may be a more aggressive anomaly than 13q-x1 (Dewald et al., BJH121:287, 2003) and have undertaken a long-term clinical follow-up of patients with an isolated FISH detectable 13q- to test this hypothesis. Methods: We identified patients diagnosed with CLL between October 1992 to May 2008 and who had 13q deletion as their sole FISH abnormality. FISH was performed within 2 years of diagnosis and prior to treatment in all patients. Patients were sorted into three groups: 13q-x1 only, 13q-x2 only, or mosaic with both 13q-x1 and 13q-x2 cells. We assessed differences in percentage of abnormal cells, demographic characteristics, and clinical outcome among the three groups. Results: We identified 259 patients with isolated 13q deletion. Age ranged from 38 to 90 years (median=61) and included 99 women and 160 men. With respect to Rai stage, 142 patients (60%) had low risk [Rai 0], 81 (34%) intermediate risk [Rai I-II], and 13 (6%) high risk [Rai III-IV] disease. After a median follow-up of 1.9 years, 40 patients (15%) have been treated and 17 (7%) have died. Median TFS and OS for all patients were 6.9 and 9.3 years, respectively. The mean percentage of abnormal nuclei in the three groups was 44%, 53%, and 47% for 13q-x1 only, mosaic 13q-x1/13q-x2, or 13q-x2 only, respectively (p=0.22). No significant difference in age or gender was observed based on whether 13q- occurred as a heterozygous or homozygous deletion, or was mosaic. Likewise, no significant difference in survival (p=0.313, see Table) or time to treatment (p=0.53) was found among the three groups. Five year OS rates for the 13q- x1 only, 13q- x2 only, and mosaic 13q- x1/13q- x2 are 85%, 95%, and 83%, respectively Median time to treatment for 13q-x1 was not reached, and for the mosaic13q-x1/13q-x2 and 13q-x2 groups median time to treatment was 6.9 and 4.9 years, respectively. Conclusions: The presence of deletion in one versus two 13q- chromosomes in CLL patients appears to have no significant impact on treatment free or overall survival. This observation supports the grouping of CLL patients with isolated 13q- of one or both alleles into a single risk category as originally proposed in the risk categorization by Dohner et al. Taking the present findings together with those of Dohner et al and Dewald et al, we propose a hierarchical risk model of FISH abnormalities in CLL as 17p- à 11q- à 6q- à +12 à normal à 13q-, from most to least aggressive. The 13q- group would now include 13q-x1, 13q-x2, and the mosaic subgroups.

Abstract BACKGROUND: The diagnosis of CLL among asymptomatic patients without lymphadenopathy or cytopenias has historically been based on documenting a characteristic lymphocyte clone and the presence of lymphocytosis. Currently, there are no data regarding what lymphocyte type (absolute lymphocyte count [ALC] or B-cell count) or threshold should be used for this diagnosis. We analyzed the relationship of these lymphocyte counts to clinical outcome in a cohort of 459 patients to determine whether the CLL diagnosis: should be based on ALC or B-cell count what lymphocyte threshold should be used for diagnosis whether any lymphocyte counts has independent prognostic value after accounting for molecular prognostic markers. METHODS: We used the Mayo Clinic CLL database to identify all Rai stage 0 patients diagnosed with CLL between 1/1/00 and 12/31/07 who were evaluated and had flow cytometry at Mayo within 12 months of diagnosis. All patients had lymphocytosis (ALC ≥5 x 109/L) and fulfilled the 1996 criteria for CLL. Raw data from the peripheral blood flow analysis was used in conjunction with blood counts to determine ALC and absolute B-cell count at diagnosis. Estimates of survival were calculated using the Kaplan-Meier method. RESULTS: To assess whether B-cell count or ALC more strongly related to clinical outcome, we evaluated the relationship of these variables with treatment free survival (TFS) and overall survival (OS; n=459). When treated as continuous variables (i.e. measuring risk of each 1.0 x 109/L increase in cell count), both ALC and B-cell count were related to TFS (hazard ratio [HR] ALC=1.02; p<0.0001; HR B-cell count=1.02; p<0.0001) and OS (HR ALC=1.02; p=0.04; HR B-cell count=1.02; p=0.02). Because B-cell count and ALC related to TFS and OS as a continuous variables, we next evaluated what threshold (nearest 1.0 x109/L) at diagnosis best related to an individual’s risk of requiring chemotherapeutic treatment and/or dying of CLL using the HR (log rank statistic) and c-statistic [c=1 indicates perfect discrimination between poor survivors and good survivors; c=0.5 equivalent to chance]. The B-cell threshold that best predicted OS was 11 x 109/L (HR=2.36, p=0.01; c=0.60). This threshold also predicted TFS (HR=3.02; p<0.0001; c=0.64). With respect to ALC, a threshold of 12 x 109/L was able to predict TFS (HR=2.28, p=0.003; c=0.62) but not OS (HR=1.62, p=0.12; c=0.56). The B-cell threshold used in the current diagnostic criteria (5 x 109/L) was able to predict TFS (HR=3.24, p<0.0001; c=0.63), but not OS (HR=1.64, p=0.13; c=0.55) Finally, we evaluated the ability of B-cell count to predict TFS independent of IGHV mutation status, ZAP-70 status, CD38 status, and FISH. Since not all patients had all prognostic tests performed, the predictive value of B-cell count (< or ≥11 x 109) independent of the other prognostic variable was assessed for each variable independently. B-cell count retained prognostic value independent of IGHV mutation status, ZAP-70 status, CD38 status, and FISH (all p≤0.001) [Table]. CONCLUSIONS: Although B-cell count and ALC have similar ability to predict TFS and OS as continuous variables, B-cell count may be a better predictor of TFS and OS when a defined lymphocyte threshold is used. These findings provide evidence to justify the recent proposal by Hallek et al (Blood 111:5446) to base the diagnosis of CLL on B-cell count rather than ALC. The results also provide justification for retaining the size of the B-cell count as part of the diagnostic criteria even in the era of molecular/biologic prognostic markers, but imply a threshold of 11 x 109 is the B-cell count that best predicts patient’s TFS and risk of death. Differentiating between CLL and MBL based on a patient’s likelihood of developing clinical symptoms and dying of disease could minimize unnecessary psychologic distress caused by labeling asymptomatic individuals at low risk for adverse clinical consequences as having leukemia. TABLE: TREATMENT FREE SURVIVAL HR p-value CD38 + 3.20 0.0002 B-cell ≥11 3.10 0.0001 ZAP + 8.33 < 0.0001 B-cell ≥11 6.86 < 0.0001 Unmutated 5.26 < 0.0001 B-cell ≥11 3.06 0.001 FISH (17p- or 11q-) 3.97 0.01 B-cell ≥11 3.67 < 0.0001

Abstract Next‐generation sequencing identified about 60 genes recurrently mutated in chronic lymphocytic leukemia (CLL). We examined the additive prognostic value of the total number of recurrently mutated CLL genes (i.e., tumor mutational load [TML]) or the individually mutated genes beyond the CLL international prognostic index (CLL‐IPI) in newly diagnosed CLL and high‐count monoclonal B‐cell lymphocytosis (HC MBL). We sequenced 59 genes among 557 individuals (112 HC MBL/445 CLL) in a multi‐stage design, to estimate hazard ratios (HR) and 95% confidence intervals (CI) for time‐to‐first treatment (TTT), adjusted for CLL‐IPI and sex. TML was associated with shorter TTT in the discovery and validation cohorts, with a combined estimate of continuous HR = 1.27 (CI:1.17‐1.39, P = 2.6 × 10 −8 ; c ‐statistic = 0.76). When stratified by CLL‐IPI, the association of TML with TTT was stronger and validated within low/intermediate risk (combined HR = 1.54, CI:1.37‐1.72, P = 7.0 × 10 −14 ). Overall, 80% of low/intermediate CLL‐IPI cases with two or more mutated genes progressed to require therapy within 5 years, compared to 24% among those without mutations. TML was also associated with shorter TTT in the HC MBL cohort (HR = 1.53, CI:1.12‐2.07, P = .007; c ‐statistic = 0.71). TML is a strong prognostic factor for TTT independent of CLL‐IPI, especially among low/intermediate CLL‐IPI risk, and a better predictor than any single gene. Mutational screening at early stages may improve risk stratification and better predict TTT.

Abstract Aims Wound infections involving Candida albicans can be challenging to treat because of the fungus’ ability to penetrate wound tissue and form biofilms. The goal of this study was to assess the activity of a hypochlorous acid (HOCl)-generating electrochemical scaffold (e-scaffold) against C. albicans biofilms in vitro and on porcine dermal explants (ex vivo). Methods and Results C. albicans biofilms were grown either on acrylic-bottom six-well plates (in vitro) or on skin tissue excised from porcine ears (ex vivo), and the polarized e-scaffold was used to generate a continuous supply of low concentration HOCl near biofilm surfaces. C. albicans biofilms grown in vitro were reduced to undetectable amounts within 24 h of e-scaffold exposure, unlike control biofilms (5·28 ± 0·034 log10 (CFU cm-2); P < 0·0001). C. albicans biofilms grown on porcine dermal explants were also reduced to undetectable amounts in 24 h, unlike control explant biofilms (4·29 ± 0·057 log10 (CFU cm-2); P < 0·0001). There was a decrease in the number of viable mammalian cells (35·6 ± 6·4%) in uninfected porcine dermal explants exposed to continuous HOCl-generating e-scaffolds for 24 h compared to explants exposed to nonpolarized e-scaffolds (not generating HOCl) (P < 0·05). Conclusions Our HOCl-generating e-scaffold is a potential antifungal-free strategy to treat C. albicans biofilms in chronic wounds. Significance and Impact of the Study Wound infections caused by C. albicans are difficult to treat due to presence of biofilms in wound beds. Our HOCl producing e-scaffold provides a promising novel approach to treat wound infections caused by C. albicans.

The World Health Organization (WHO) has declared coronavirus disease 2019 (COVID-19), the disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a pandemic. Global health care now faces unprecedented challenges with widespread and rapid human-to-human transmission of SARS-CoV-2 and high morbidity and mortality with COVID-19 worldwide. Across the world, medical care is hampered by a critical shortage of not only hand sanitizers, personal protective equipment, ventilators, and hospital beds, but also impediments to the blood supply. Blood donation centers in many areas around the globe have mostly closed. Donors, practicing social distancing, some either with illness or undergoing self-quarantine, are quickly diminishing. Drastic public health initiatives have focused on containment and “flattening the curve” while invaluable resources are being depleted. In some countries, the point has been reached at which the demand for such resources, including donor blood, outstrips the supply. Questions as to the safety of blood persist. Although it does not appear very likely that the virus can be transmitted through allogeneic blood transfusion, this still remains to be fully determined. As options dwindle, we must enact regional and national shortage plans worldwide and more vitally disseminate the knowledge of and immediately implement patient blood management (PBM). PBM is an evidence-based bundle of care to optimize medical and surgical patient outcomes by clinically managing and preserving a patient’s own blood. This multinational and diverse group of authors issue this “Call to Action” underscoring “The Essential Role of Patient Blood Management in the Management of Pandemics” and urging all stakeholders and providers to implement the practical and commonsense principles of PBM and its multiprofessional and multimodality approaches.

Abstract To study the impact of dose modification and temporary interruption of ibrutinib in routine clinical practice, we conducted a retrospective study of consecutive CLL patients treated with ibrutinib outside the context of a clinical trial at Mayo Clinic, (Rochester, MN) from 11/2013 to 12/2017. Of 209 patients, 131 (74%) had unmutated IGHV , 38 (20%) had TP53 disruption, and 47 (22%) were previously untreated. A total of 87/209 (42%) patients started reduced dose ibrutinib (<420 mg daily; n = 43, physician preference; n = 33, concomitant medications; and n = 11, other). During 281 person‐years of treatment, 91/209 patients had temporary dose interruption (54%, nonhematologic toxicity; 29%, surgical procedures; 10%, hematologic toxicity; and 7%, other). After a median follow‐up of 24 months, the estimated median event‐free survival (EFS) was 36 months, and median overall survival (OS) was not reached. On multivariable analyses, temporary ibrutinib interruption (hazard ratio [HR]: 2.37, P = .006) and TP53 disruption at ibrutinib initiation (HR: 1.81, P = .048) were associated with shorter EFS, whereas only TP53 disruption (HR: 2.38, P = .015) was associated with shorter OS. Initial ibrutinib dose and dose modification during therapy did not appear to impact EFS or OS. These findings illustrate the challenges associated with continuous oral therapy with ibrutinib in patients with CLL.

7526 Background: Stem cell transplant (SCT) was considered to be beneficial in CLL patients with Richter transformation (RT). We report a single center experience of SCT for RT. Methods: CLL patients with biopsy-confirmed RT to DLBCL who underwent SCT were identified from Mayo Clinic CLL database, and clinical information was abstracted by chart review. Survival analysis was done with the Kaplan-Meier method. Results: Twenty-four of 204 RT patients underwent SCT, 20 autologous and 4 allogeneic. The median lines of prior CLL therapy was 1 (range 0-4). Only 4 patients were exposed to ibrutinib prior to RT. The median age at RT diagnosis was 62 (range 41-73). Five of 17 (29%) patients had bulky disease (≥ 5 cm), 12 of 20 (60%) had elevated LDH, and 6 of 15 (40%) had TP53 disruption (del(17p) or TP53 mutation). The median lines of RT therapy prior to SCT was 2 (range 1-4); treatments included R-CHOP-like alone (n = 7), platinum-based alone (n = 1), both R-CHOP-like and platinum-based (n = 11), novel agents (n = 2), and high dose MTX-based (n = 3). Response prior to SCT was CR in 12 (50%) and PR in 12 (50%) patients. The median time from RT diagnosis to SCT was 6.8 months (range 3.3-42.3). After a median follow-up of 52.7 months after SCT, there were 10 RT relapses, 4 CLL progression, and 11 deaths (7 RT, 1 CLL and 3 unrelated). The median progression-free survival (PFS) was 28.5 months (95% CI 8.3-NA; 1-year PFS 61.8%, 2-year PFS 50.5%). The median post-SCT survival was 56.3 months (95% CI 30.6-76.6; 2-year survival 82.0%, 4-year survival 65.6%). Elevated LDH was associated with worse PFS (median 8.9 months vs NA, P= 0.014) and a trend of worse post-SCT survival (53.7 vs 67.4 months, P= 0.153). Other factors including age, prior CLL treatment (untreated vs treated), bulky disease, response prior to SCT, and TP53 disruption were not associated with PFS or post-SCT survival. Among the 4 allogeneic SCT patients, 1 had RT relapse 10.5 months after SCT and was treated with venetoclax, ibrutinib and obinutuzumab and remained alive at last follow-up (12.9 months after SCT). All 3 other patients remained in remission and alive at last follow up (15.6, 16.1 and 108.5 months after SCT, respectively). Conclusions: SCT may benefit select RT patients. The role of SCT in the novel agent era needs further study.

7522 Background: Ibrutinib therapy is associated with an increased risk (~15%) of AF. An accurate risk assessment model for the development of AF in pts starting ibrutinib is not established, and outcomes after AF are not well described. Methods: An IRB approved retrospective review of CLL pts treated with ibrutinib at Mayo Clinic between Oct 2012 and Nov 2018 was conducted. Results: 299 pts were identified with a total of 565 years of ibrutinib exposure (Table). After a median follow-up of 24 months (range 0–70 months), 51 pts developed treatment-emergent AF (13 [25%] CTCAE 3 or higher). Our study assessed 3 clinical prediction models, the Framingham (Schanbel:Lancet 2009), Italian (Visentin:Blood 2018;132:3118), and Shanafelt (Shanafelt:Leuk Lymp; 2017) risk scores. Based on a lower Akaike information criteria, the Italian score was best able to predict risk of treatment emergent AF (2-year risk of AF with score 0 6%; 1-2 8%; 3-4 26%; 5+ 47%). Thirty (61%) pts were treated with medical therapy for AF (27 rate control; 2 rhythm control; 1 both). 16 (31%) pts underwent interventional therapy (3 AV node ablation; 11 cardioversion; and 2 pacemaker). Twelve (23%) pts temporarily held ibrutinib and resumed their original dose, 22 (43%) pts continued reduced dose ibrutinib and 11 (22%) continued their initial ibrutinib dose. Six (12%) pts permanently discontinued ibrutinib. Of 51 pts with treatment-emergent AF, 41 (80%) had a CHA2DS2-VASc score of ≥2 (41% received anticoagulation alone; 12% received antiplatelet therapy alone; 10% received both). No pt developed a thrombotic stroke after treatment-emergent AF. Two major bleeds, (1 GI and 1 intracranial) occurred, one in a pt on concomitant antiplatelet and anticoagulation therapy, and one in a pt receiving neither. The development of AF was associated with shorter event-free survival (HR 2.5, 95%CI 1.5-4.2, p<.001) and shorter overall survival (HR 3.5, 95%CI 2.0-6.3, p<.001). Conclusions: The Italian score was the best predictor of AF development in this cohort of ibrutinib treated pts. Although the vast majority of pts who develop AF after ibrutinib are able to continue therapy, the occurrence of AF appears to be associated with shorter EFS and OS. [Table: see text]