Grever, Michael R.

Abstract Hairy cell leukemia (HCL) and HCL variant (HCLv) are distinct, rare, and chronic splenic B-cell lymphomas/leukemias that partially overlap in clinicopathologic presentation but differ in genetic basis, prognosis, and management. HCL is caused by the BRAF-V600E kinase–activating mutation in >95% of the patients, usually has excellent responses to chemotherapy with purine analogues, and is also amenable to BRAF inhibitor–based targeted treatments. In contrast, HCLv lacks BRAFV600E mutation, requires combined therapy with purine analogues in addition to rituximab, and generally shows less durable responses. Here, an international team of hematologists, experts on these rare diseases, was convened by the Hairy Cell Leukemia Foundation to update the previous guidelines (published in 2017) by providing a summary of current methods to diagnose and manage patients with HCL and HCLv as well as a prospective on newer targeted therapies to further improve outcomes.

Abstract Hairy cell leukemia (HCL) is a rare B-cell malignancy, and there is a need for novel treatments for patients who do not benefit from purine analogs. Ibrutinib, an oral agent targeting Bruton tyrosine kinase in the B-cell receptor signaling pathway, is highly effective in several malignancies. Its activity in HCL was unknown, so we conducted a multisite phase 2 study of oral ibrutinib in patients with either relapsed classic or variant hairy cell leukemia. The primary outcome measure was the overall response rate (ORR) at 32 weeks, and we also assessed response at 48 weeks and best response during treatment. Key secondary objectives were characterization of toxicity and determination of progression-free survival (PFS) and overall survival (OS). Thirty-seven patients were enrolled at 2 different doses (24 at 420 mg, 13 at 840 mg). The median duration of follow-up was 3.5 years (range, 0-5.9 years). The ORR at 32 weeks was 24%, which increased to 36% at 48 weeks. The best ORR was 54%. The estimated 36-month PFS was 73% and OS was 85%. The most frequent adverse events were diarrhea (59%), fatigue (54%), myalgia (54%), and nausea (51%). Hematologic adverse events were common: anemia (43%), thrombocytopenia (41%), and neutropenia (35%). Ibrutinib can be safely administered to patients with HCL with objective responses and results in prolonged disease control. Although the initial primary outcome objective of the study was not met, the observation of objective responses in heavily pretreated patients coupled with a favorable PFS suggests that ibrutinib may be beneficial in these patients. This trial was registered at www.clinicaltrials.gov as #NCT01841723.

Abstract Approximately 10–15% of patients with CLL report a family history (FH) of CLL or a related lymphoproliferative disorder. Whether the biologic features of CLL that occurs within families differ from those that occur sporadically is unknown. A recent study addressing this question has reported that mutated IgVH is significantly more common among familial than sporadic CLL, 68% vs 47%, although the distribution of VH genes used was similar (Blood111:5691, 2008). Therefore we compared IgVH mutational status and gene usage, as well as other biologic features, between familial and sporadic CLL derived from patients enrolled on the CLL Research Consortium (CRC) Tissue Core. Of 4533 patients enrolled in the CRC database and tissue bank, 3328 had adequate family history information, and these were subdivided into 4 groups: FH of CLL (n=468), FH of other heme cancer but not CLL (n=421), FH of non-heme cancer (n=1444), and sporadic cases (no FH of any cancer) (n=995). Of those patients reporting a FH of CLL, 63% had at least one affected 1st degree relative, 20% at least one affected 2nd or greater degree relative, and 17% unknown. 68% of those reporting a FH of CLL also had other cancers in their family. Patients reporting a FH of CLL or non-CLL heme cancers were more frequently white (91%), compared to 87% of those with FH non-heme cancer and 84% of sporadic CLL patients (p=0.005). The proportion of CLL cases that were female was similar among groups, at 37.6% of patients with FH CLL, as compared to 32.5% of sporadic patients (p=0.12). The age at diagnosis of CLL also did not differ among the four groups, although a trend toward a difference was observed between the FH CLL patients, whose mean age at diagnosis was 54.9 (range 30, 93), and the sporadic patients, whose mean age at diagnosis was 56.1 (range 30, 84) (p=0.08). No significant difference was observed among groups in the frequency of IgVH mutation (defined as <98% homology to the most similar germline VH gene), which was present in 50.3% (95% CI 44.5–56.2) of patients with FH CLL, 56.3% (95% CI 49.7–62.7) of patients with FH other heme cancer, 51.9% (95% CI 48.5–55.3) of patients with FH non-heme cancer, and 51.3% (95% CI 47.0–55.5) of sporadic patients (p=0.53). The pattern of IgVH family gene usage also did not differ among the groups; the VH3 subgroup was most commonly used, at 45%, 52%, 46% and 47% of patients with FH CLL, FH other heme cancer, FH non-heme cancer and sporadic, respectively. The VH1 subgroup was next most common in all 4 groups, at 28%, 24%, 26% and 25%, respectively, followed by the VH4 subgroup, at 19%, 17%, 21% and 21%. In addition, no significant difference was observed in the distribution of ZAP-70 expression amongst the four family history subgroups (mean %ZAP-70 expression, 22.8% for FH CLL patients and 24.4% for sporadic patients, p=0.31), or in the fraction considered ZAP-70 positive (>20% expression), which was 40% for FH CLL patients and 45% for sporadic patients (p=0.16). The relative expression of CD23 and CD38 did not differ among groups, nor did levels of beta-2-microglobulin or IgG determined at time of CRC enrollment. Time from diagnosis to initial treatment for each of the subgroups is currently being analyzed. These data on patterns of biologic risk factors in this large group of familial CLLs compared to sporadic CLLs suggest that familial and sporadic CLL are biologically similar and therefore likely to have similar pathogenetic mechanisms, including perhaps an underlying genetic susceptibility.

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 We previously demonstrated that apoptosis-inhibiting proteins (e.g., Bcl-2, Mcl-1, and XIAP) are important in predicting response to chemotherapy and antibody therapy for CLL. Additionally, genetic abnormalities and epigenetic control of gene expression have become increasingly important in understanding the prognosis and potential therapeutic opportunities in this disease. Greater than 80% of patients with CLL have genetic abnormalities detectable by FISH. Important subsets include those with a less favorable prognosis (deletions of 17p, 11q), an intermediate prognosis (trisomy 12), or a favorable outcome (deletion of 13q). PTPRO, encoding a receptor-type tyrosine phosphatase, is a specific tumor suppressor gene located in chromosomal region 12p12.3. PTPRO has been highly characterized as inhibiting anchorage-independent tumor growth in human lung carcinomas. This gene has been found to be silenced by promoter methylation, and can be re-expressed by hypomethylating agents in human lung tumor models. Thus, we examined the methylation profile of the promoter region of PTPRO in primary leukemic cells from patients with CLL, and in B cells obtained from normal volunteers. Analysis of the PTPRO CpG island by methylation-specific PCR and combined bisulfite restriction analysis (COBRA) demonstrated that this region was methylated in 91% of samples from 46 patients, whereas it was unmethylated in control B cells from normal volunteers. Specific quantitative determinations of important proteins related to apoptosis in the leukemic cells from 20 of these patients showed that, in the group with methylation of the PTPRO promoter, there was a significant elevation of Bcl-2 (p=0.079), Mcl-1 (p=0.004), XIAP (p=0.048), Bcl-2/BAX ratio (p=0.0024), and Mcl-1/BAX ratio (p=0.0017). There was no apparent relationship with FISH category or immunoglobulin VH mutational status. Thus, PTPRO promoter methylation analysis may identify a subset of CLL patients less likely to respond to therapy, and provide a potential reversible target for new therapeutic approaches to this disease. Finally, analysis of methylation of CLL samples by restriction landmark genome scanning also revealed methylation of genes for three additional protein tyrosine phosphatases: PTPRN2, PTPRZ2, and PTPN11. Increases in methylation of two of these (PTPRN2 and PTPRZ2) in CLL samples relative to control B cells was confirmed by COBRA. These results provide additional potential molecular targets for CLL therapy.

Abstract Background : Effective therapy for classical hairy cell leukemia (c-HCL) refractory to purine nucleoside analog (PNA) treatment is limited, and there are no accepted treatment standards for variant hairy cell leukemia (v-HCL). Ibrutinib, an oral small molecule inhibitor of Bruton tyrosine kinase (BTK), has shown single-agent efficacy and acceptable tolerability in patients with various B-cell malignancies, including chronic lymphocytic leukemia, mantle cell lymphoma, and Waldenstrom's. While BTK is expressed in HCL cells, the clinical activity of ibrutinib in this disease has not been previously assessed. We conducted a single-arm, multi-center phase 2 study (NCI #9268) of single-agent ibrutinib to characterize the overall response rate (complete + partial response) and safety of single-agent ibrutinib treatment for HCL. Methods : Patients with c-HCL (unfit for or relapsed after PNA) and v-HCL (relapsed or treatment-naïve) were eligible. Enrolled patients required treatment, had ECOG ≤ 2, no active infection, and preserved end-organ function. Patients received continuous once daily ibrutinib in 28-day cycles. Using a Simon 2-stage design, the first 13 patients were treated at 420 mg daily, and a second cohort of 13 patients was accrued at the 840 mg dose-level. Ultimately, the pre-specified number of objective responses was reached at the 420 mg dose-level, and accrual to the second stage of the design continues at that dose. Response, including bone marrow biopsy with immunohistochemistry for minimal residual disease (MRD), was assessed after 8 and 12 cycles. Patients experiencing clinical benefit may continue ibrutinib until unacceptable toxicity or progressive disease. Results: As of 15 May 2016, 28 patients had been enrolled and were evaluable for response: 420 mg/day (n=15) and 840 mg/day (n=13). Enrolled patients (1 treatment-naïve v-HCL, 10 relapsed v-HCL, 17 relapsed c-HCL) included 22 male and 6 female patients with median age 65 years (range: 43-78). Relapsed patients received median 4 prior therapies (range: 1-11); all had prior PNA, 81% had prior rituximab, and 3 c-HCL patients had prior vemurafenib. Response data are summarized by dose and histology in the table. To date, the ORR is 46% [4 CR (all c-HCL) and 9 PR (6 c-HCL)], with objective responses more commonly observed in patients with c-HCL. Eight additional patients (29%) with stable disease have experienced clinical benefit (resolution of symptoms, improvement in peripheral blood cell counts) not meeting criteria for PR, most commonly attributable to persistent thrombocytopenia, and continue on treatment. At median follow-up of 22 months, 20 patients (71%) remain on treatment, 3 patients (1 v-HCL, 2 c-HCL) have progressed, 2 patients (c-HCL) with severe neutropenia at baseline (c-HCL) had early deaths from pneumonia, 1 (c-HCL) discontinued during cycle 8 for failure to resolve baseline neutropenia, and 2 other patients (v-HCL) have discontinued for adverse events. Estimated 24-month progression-free survival (PFS) was 79% (95% CI: 57-91%) and the median PFS has not been reached. Redistribution lymphocytosis was observed in patients with circulating disease at baseline. Soluble interleukin-2 receptor (sIL2R) level was increased at baseline in all c-HCL patients for whom data were available and correlated with response. In general, toxicity did not differ by dose level or disease histology. The most frequent (>10%) grade ≥3 adverse events (AEs) were lymphopenia (21%), neutropenia (18%), lung infection (18%), thrombocytopenia (14%), hypophosphatemia (11%), and hypertension (11%). Common grade 1/2 non-hematologic toxicities included myalgia (57%), diarrhea (54%), fatigue (50%), nausea/vomiting (46%), bruising (46%), and rash (46%); grade 1/2 atrial fibrillation (no grade ≥3) was observed in 5 patients. Grade ≥3 infection was observed in 25%, but no grade ≥3 bleeding AEs were reported. Conclusions: Ibrutinib can induce remission in both c-HCL and v-HCL patients, including heavily pre-treated patients, but complete remissions have only been observed in c-HCL to date. The drug is generally well-tolerated during long-term administration, and durable clinical benefit is observed in the majority of treated patients even when objective response criteria are not met. Table 1. Table 1. Figure 1. Figure 1. Disclosures Jones: Pharmacyclics, LLC, an AbbVie Company: Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding. Lozanski:Stemline Therapeutics Inc.: Research Funding; Beckman Coulter: Research Funding; Genentech: Research Funding; Boehringer Ingelheim: Research Funding.

Abstract The nucleoside analogue pentostatin has clinical activity in B-Cell Chronic Lymphocytic Leukemia (CLL) and has shown significant activity and minimal toxicity when combined with cyclophosphamide for previously treated CLL. Building on this, we initiated a trial in 2002 of combined pentostatin (2mg/m2), cyclophosphamide (600 mg/m2) and rituximab (375mg/m2). Of the 33 enrolled eligible patients included in these analyses, seventeen patients were in the high Rai risk group, 25 were male, and median age for this cohort was 62 yrs (range: 40–79); 17 were non-mutated for the immunoglobulin heavy chain variable region gene, and the majority (67%) were CD38 negative. Only 5 patients had no detectable chromosomal abnormalities by FISH at baseline, 20 had a single FISH anomaly, and 8 had 2 or more FISH anomalies. Of all 28 pts with any anomaly, the following specific abnormalities were detected: 13q- (n=17), +12 (n=8), 11q- (n=7), 17p- (n=2), t(14;18) (n=1), 6q- (n=1), and MDM2 (n=1). Of the 33 patients, 22 had grade 3+ toxicity; 16 patients had non-hematologic toxicity wtih the most common symptoms being nausea (6) and vomiting (4). One patient died on study of grade 5 hypoxia as well as hypotension and this was deemed possibly related to treatment. Almost all patients (32/33; 97%) had a best response of PR or better. Including review of bone marrows done 2 months post-treatment, there were 11 CRs (complete response), 7 nPRs (nodal partial response) and 13 PRs. No differences were observed between type of response and mutation status or CD38+ status. Post-treatment FISH analyses were available on 27 patients; results for 25 patients became normal after treatment. Of the remaining 2, one patient was 13q- x1 and went from 94% to 27.5% abnormal nuclei after treatment; the other is the patient who died on study. To establish minimal residual disease (MRD) post-response, we used three color flow cytometry to detect CD5+/CD19+/CD79b− B cells. This approach found all patients had a reduction in CLL B cells with a median reduction of 91% (range: 5 – 100%). Of interest, all 3 response groups (CR vs. nPR vs. PR) had patients with significant reductions in CLL B cells (i.e., >90%). The nPR group was most variable in terms of MRD (median 47%; range: 5–93%) compared to the CR (median: 91%; range: 42–99.9%) and PR (median: 97%; range: 46–100%) groups. In conclusion this novel regimen of pentostatin, cyclophosphamide and rituximab has demonstrated significant clinical activity irrespective of risk stratification parameters with rapid induction of responses, achievement of minimal residual disease in some, and modest toxicity. Patients continue to be accrued to further explore correlative measures of response to treatment.