A Phase I/II Study of Vorinostat (SAHA), Cladribine (2-CdA), and Rituximab Shows Significant Activity in Previously Untreated Mantle Cell Lymphoma

Abstract Abstract 441 Background: Despite significant progress in the treatment of mantle cell lymphoma (MCL), relapse remains the norm and additional therapies are needed especially for patients who are not candidates for aggressive treatment approaches. Increasingly, it has become evident that epigenetic modifications, including DNA hypomethylation and histone deacetylase inhibition, are critical to the pathogenesis and treatment of hematologic malignancies; important to cancer biology; and may be essential to the development of treatment resistance in B-cell malignancies. Further development and understanding of new and effective treatment regimens that target the epigenome are needed. 2-CdA has activity in a variety of B and T cell malignancies. In addition to its cytotoxic effects, our preliminary work shows that 2-CdA has hypomethylating properties in lymphoid malignancies. When primary MCL and CLL cells -before and 96 hours after cladribine treatment-were analyzed by HELP (HpaII tiny fragment Enrichment by Ligation mediated PCR), an array based genome-wide methylation assay, 2-CdA affected DNA hypomethylation. One of the genes hypomethylated was identified as DUSP2, a dual specificity phosphatase gene that is a p53 target gene. DUSP2 dephosphorylates phosphoserine/threonine and phosphotyrosine residues, negatively regulating mitogen-activated protein (MAP) kinases ERK1 and ERK2, which are associated with cellular proliferation and differentiation in B-NHL. Vorinostat (SAHA) is a histone deacetylase inhibitor (HDACi), which has shown modest single agent activity in lymphoma and is FDA approved for use in cutaneous T cell lymphoma (CTCL). MCL cell lines treated with cladribine activated DUSP2 mRNA and when treated with the HDAC inhibitor SAHA synergistically increased transcription of DUSP mRNA. Furthermore, MCL treated with cladribine in vitro showed inhibition of global histone methylation. Our hypothesis is that cladribine and vorinostat synergistically activate silenced genes such as but not limited to DUSP 1 and 2 that are important for tumor cell death. The mechanism of rapid tumor cell death is under investigation, and does not appear to involve the classical apoptosis pathway. Given the need for novel therapies and the potential synergy seen with 2-CdA and SAHA, we initiated a Phase I/II trial combining SAHA, 2-CdA, and rituximab (SCR) for the treatment of B-cell non-Hodgkin's Lymphoma (NHL). The Phase I portion has been completed while Phase II is actively enrolling patients including those with newly diagnosed MCL. Methods: Phase I enrolled 10 patients with relapsed/refractory NHL. The MTD of vorinostat for the Phase I was 400 mg (D 1–14) combined with 2-CdA 5mg/m2 IV (D 1–5), and R 375 mg/m2 IV (weekly × 4 for cycle 1 and 1x/month) every 28 days for up to 6 cycles. Phase II eligibility includes relapsed NHL as well as previously untreated mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL). Primary outcome is response rate (ORR). Scientific correlatives include analysis of CD20 expression, histone acetylation, gene microarray and HELP methylation analysis, ERK phosphorylation, and Q-PCR of potential target genes. Results: 52 patients (Phase I/II) have been enrolled and 45 patients have been treated. The ORR in evaluable relapsed patients (3 DLBCL, 10 MCL, 1 FL, 1 MZL, 7 CLL) is 32% (7/22). Among these relapsed patients, complete remissions (CR) have been observed in MCL as well as follicular and marginal zone lymphomas. Of the 20 previously untreated MCL patients, 19 have completed ≥ 2 cycles and are evaluable for response. ORR is 100% (19/19) with 79% (15/19) CR. Toxicities by CTCAE 3.0 criteria have primarily included reversible myelosuppression, fatigue, dehydration, 1 gr. 4 thrombo-embolic event (probably related), and 1 grade 5 pulmonary hemorrhage in a patient with relapsed pulmonary lymphoma. One previously untreated mantle cell lymphoma patient has ongoing Gr. 3 thrombocytopenia six weeks after completing therapy. Preliminary analysis of ongoing correlative studies is available in 1 MCL patient and shows DUSP2 upregulation. Conclusions: The SCR regimen shows activity across a number of B-cell malignancies and shows particular therapeutic promise in patients with previously untreated mantle cell lymphoma. Correlative studies are ongoing and will be presented. Future studies should continue to explore this regimen in previously untreated mantle cell lymphoma. Disclosures: Off Label Use: vorinostat (SAHA) is not FDA approved for the treatment of B cell lymphomas. Okada:Merck: Speakers Bureau. Epner:Merck: Speakers Bureau.

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Phase I and pharmacokinetic study of bendamustine hydrochloride in relapsed or refractory indolent B-cell non-Hodgkin lymphoma and mantle cell lymphoma

Cancer Science ◽

10.1111/j.1349-7006.2010.01633.x ◽

2010 ◽

Vol 101 (9) ◽

pp. 2054-2058 ◽

Cited By ~ 30

Author(s):

Michinori Ogura ◽

Toshiki Uchida ◽

Masafumi Taniwaki ◽

Kiyoshi Ando ◽

Takashi Watanabe ◽

...

Keyword(s):

Phase I ◽

B Cell ◽

Mantle Cell Lymphoma ◽

Hodgkin Lymphoma ◽

Pharmacokinetic Study ◽

Cell Lymphoma ◽

Non Hodgkin Lymphoma ◽

Mantle Cell

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Co-Expression of SYK and ZAP70 Subverts Negative B-Cell Selection and Enables Oncogenic Signaling in Multiple B-Cell Malignancies

Blood ◽

10.1182/blood-2019-128999 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 295-295

Author(s):

Teresa Sadras ◽

Mickaël Martin ◽

Lauren Kim-Sing ◽

Jevon Cutler ◽

Gal Lenz ◽

...

Keyword(s):

T Cells ◽

Cell Death ◽

B Cells ◽

B Cell ◽

Cell Lymphoma ◽

Cell Selection ◽

B Cell Malignancies ◽

Close Proximity ◽

Mantle Cell ◽

Bcr Signaling

B-cells are under intense selective pressure to eliminate autoreactive or premalignant clones. B-cell receptor (BCR) signals are required for survival, however, BCR-signaling exceeding maximum thresholds often reflects signaling from an autoreactive BCR or a transforming oncogene and triggers negative selection and cell death. The tyrosine kinase SYK initiates BCR-downstream signaling in B-cells while its close relative ZAP70 is almost exclusively expressed in T-cells. Interestingly, the segregation of SYK to B-cells and ZAP70 to T-cells is less confined in malignant lymphopoiesis suggesting that the balance of these related kinases may alter signaling output in disease and contribute to development of leukemia. As previously shown in B-cell chronic lymphocytic leukemia (B-CLL), we identified aberrant ZAP70 expression as a frequent feature in multiple other B-cell malignancies that depend on survival signals from a functional (pre-) BCR (E2A-PBX1+ pre-B ALL, and mantle cell lymphoma) or harbor oncogenic mimics of the BCR (BCR-ABL1+ B-ALL). Studying SYK and ZAP70 expression by single-cell Western blot, co-expression of the two tyrosine kinases was extremely rare in normal B- and T-cell populations. In contrast, >50% of tumor B-cells in mantle cell lymphoma, pre-B ALL and CLL co-expressed SYK and ZAP70. Despite their structural similarities, genetic deletion and engineered reconstitution of SYK and ZAP70 in human B-cell lymphoma cells revealed striking functional differences. Proximity-dependent biotin identification (BioID) analyses identified that SYK, but not ZAP70, engaged the PI3K pathway via interaction with CD19. Consistent with this, reconstitution with SYK and SYK-ZAP70 but not ZAP70 alone promoted survival and proliferation. Detailed analysis of BCR-mediated cascades in lymphoma cells expressing SYK, ZAP70 or SYK-ZAP70 established that ZAP70 is only weakly efficient at propagating BCR-mediated calcium and downstream pathway activation in B-cells. Strikingly, co-expression of ZAP70 with SYK resulted in re-wired BCR-signaling of intermediate strength: compared to cells expressing only SYK, SYK-ZAP70 co-expressing cells had markedly reduced activation of the BLNK-BTK-PLCγ pathway, further reflected in BCR-induced Ca2+ signaling with delayed onset, lower amplitude but longer duration. In this way, we speculated that SYK and ZAP70 may be present within close proximity at the apex of BCR-initiated interactions, and hence compete for downstream substrates resulting in a re-wiring of classic signaling programs propagated normally by SYK. To explore this, we utilized proximity ligation assays (PLA) to monitor the proximity of SYK and ZAP70 in resting or BCR-stimulated B-cells, and found that SYK and ZAP70 co-exist within close proximity consistent with the view that varying levels of these kinases may alter B-cell signaling output. Functional experiments further showed that phosphomimetic activation of SYK, but not ZAP70, induced hyperactivation of PI3K-signaling and acute BTK-mediated cell death in pre-B ALL cells. In line with altered BCR-signaling strength and quality in SYK and ZAP70 co-expressing cells, over-expression of Zap70 in pre-B ALL cells rescued auto-immune checkpoint activation induced by hyper-activation of BCR-associated signaling. To study functional consequences of SYK-ZAP70 co-expression during normal B-cell development, we generated a novel knock in Zap-70+/Mb1-Cre+mouse model, to induce conditional expression of Zap70 in the B cell compartment from the proB stage. Consistent with compromised central tolerance checkpoints, Syk-Zap70 co-expressing pro/pre-B and immature B-cells had reduced spontaneous apoptosis rates and gave rise to autoantibody production against multiple self-antigens. Importantly, our findings highlight a previously unrecognized role for ZAP70 in oncogenic BCR-signaling and we conclude that the co-expression of ZAP70 mitigates the ability of SYK, downstream of an autoreactive BCR or a transforming oncogene, to trigger negative B-cell selection and cell death (Figure 1). Disclosures Weinstock: Celgene: Research Funding. Meffre:AbbVie: Consultancy, Other: Grant.

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Forodesine (Immucillin H, BCX-1777) Shows Activity on Mantle Cell Lymphoma Primary Cells

Blood ◽

10.1182/blood.v112.11.4997.4997 ◽

2008 ◽

Vol 112 (11) ◽

pp. 4997-4997

Author(s):

Andrea Rinaldi ◽

Emilia Ceresa ◽

Davide Rossi ◽

Gianluca Gaidano ◽

Shanta Bantia ◽

...

Keyword(s):

B Cell ◽

Mantle Cell Lymphoma ◽

Cell Lines ◽

Cell Lymphoma ◽

Annexin V ◽

B Cell Lymphoma ◽

B Cell Malignancies ◽

New Therapeutic Approaches ◽

Mantle Cell

Abstract Mantle cell lymphoma (MCL) represents a subtype of B-cell lymphoma associated with a very unfavourable clinical outcome. Currently no therapy can be considered as standard, and new therapeutic approaches are needed. Forodesine is a potent inhibitor of purine nucleoside phosphorylase (PNP), whose major role is to catalyze the cleavage of inosine, deoxyinosine guanosine, and deoxyguanosine (dGuo) to their corresponding base and sugar 1-phosphate by phosphorolysis. In the presence of deoxycytidine kinase, PNP inhibition leads to an increase in the concentration of dGuo triphosphate (dGTP), followed by inhibition of DNA synthesis and cell death by apoptosis. When combined with dGuo, forodesine has been shown to have in vitro cytotoxic activity on T-cell (T-ALL, T-PLL) and on B-cell malignancies (CLL, B-ALL), and Phase I/II trials are on going in CLL and CTCL patients. Here, we report the first data on in vitro activity of forodesine in MCL. Primary MCL cells, derived from six patients, were exposed to forodesine (0, 2, 20 μM) in combination with dGuo (0, 10, 20 μM), for 48 hrs. Cells were cultured in X-VIVO 10 medium (Cambrex) with 10% FBS. Cell viability was assessed by flow cytometry with the Annexin V - propidium iodide assay. Four patient samples (67%) showed an increase in the number of Annexin V positive cells ranging from 1.9 to 5.3 times compared to untreated cells. The effect was larger for 20 μM forodesine compared with 2 μM. There was no effect of dGuo alone and only a minimal effect of increasing dGuo concentration from 10 μM to 20 μM. Cell lines did not appear to be ideal models to evaluate the efficacy of forodesine in vitro. Three established MCL cell lines (Granta-519, Rec, JeKo1) were treated with escalating doses of forodesine, but the results were not reproducible, while the same cells showed expected IC50 values between 25–30 μM when exposed to bendamustine for 72 hrs. In conclusion, the in vitro data reported here with 4/6 MCL patients primary samples sensitive to forodesine and the results from various groups on other T- and B-cell malignancies suggest that clinical trials of forodesine in MCL may be warranted.

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Genome-Wide Array-Based Methylation Profiling Reveals Preferential Methylation of Homeobox Transcription Factor Genes In Mantle Cell Lymphoma and Pro-Apoptotic Genes In Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v116.21.536.536 ◽

2010 ◽

Vol 116 (21) ◽

pp. 536-536

Author(s):

Anna M Halldorsdottir ◽

Meena Kanduri ◽

Millaray Marincevic ◽

Hanna Göransson ◽

Anders Isaksson ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Mantle Cell Lymphoma ◽

Cell Lymphoma ◽

Lymphocytic Leukemia ◽

Aberrant Methylation ◽

B Cell Malignancies ◽

Genome Wide ◽

Mantle Cell ◽

Apoptotic Genes

Abstract Abstract 536 Introduction: Mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL) are B-cell malignancies of different postulated origin, genetics, clinical presentation and prognosis. Several studies have reported that both MCL and CLL individually exhibit aberrant methylation in comparison to normal B-cells. However, a comprehensive comparison of the methylation profiles of these two B-cell disorders has not been performed yet. This strategy has the potential to identify cellular pathways and genes that are specifically targeted in each disease. Methods: We applied the genome-wide Illumina Infinium HumanMethylation27 BeadChip array (Illumina, San Diego, USA) which measures methylation levels at 27,578 CpG dinucleotides covering 14,495 genes, to compare the methylation profiles in: (i) 20 MCL cases; and, (ii) 30 CLL cases, 15 each with unmutated stereotyped subset #1 (IGHV1-5-7/IGKV1(D)-39) B cell receptors (BCRs) or mutated stereotyped subset #4 (IGHV4-34/IGKV2-30) BCRs, where these two subsets represent prototypes of unmutated and mutated CLL. The methylation status for each detected CpG site ranged between 0.1 (completely unmethylated) to 1 (completely methylated). Results: As expected, major differences in methylation patterns between MCL and CLL were observed. When the methylation profiles of the two entities were compared, 51 genes were identified as differentially methylated in all comparisons (MCL versus both CLL subsets combined and each subset separately). Among the 19 genes highly methylated in MCL were six (32%) homeobox or homeodomain-containing transcription factors (e.g. POU4F1, PITX3), whereas genes enhancing cell proliferation and tumor progression such as MERTK and CAMP were hypomethylated in MCL. Of the 32 genes hypermethylated in CLL were six pro-apoptotic genes, including DYRK2 and CYFIP2, the tumor suppressor PRDM2 and the cell cycle regulator CCND1. Conclusions: We report for the first time disease-biased methylation profiles for different functional classes of genes in MCL or CLL. Homeobox genes were highly methylated in MCL, whereas CLL was characterized by methylation of apoptosis-related genes. The identified differences in global methylation profiles between MCL and CLL may assist in unfolding distinct epigenetic silencing mechanisms involved in the pathogenesis of these B-cell malignancies. Disclosures: No relevant conflicts of interest to declare.

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Egress of CD19+CD5+ Cells Into Peripheral Blood Following Treatment with the Bruton Tyrosine Kinase Inhibitor, PCI-32765, in Mantle Cell Lymphoma Patients

Blood ◽

10.1182/blood.v118.21.954.954 ◽

2011 ◽

Vol 118 (21) ◽

pp. 954-954 ◽

Cited By ~ 1

Author(s):

Betty Y Chang ◽

Michelle Francesco ◽

Padmaja Magadala ◽

Min Mei Huang ◽

Marcel Spaargaren ◽

...

Keyword(s):

Phase I ◽

B Cell ◽

Mantle Cell Lymphoma ◽

Peripheral Blood ◽

Phase I Study ◽

Cell Lymphoma ◽

Malignant Cells ◽

Mantle Cell ◽

Bruton Tyrosine Kinase ◽

Secondary Lymphoid Organs

Abstract Abstract 954 PCI-32765 is an orally administered, highly potent and specific inhibitor of Bruton tyrosine kinase (BTK) in clinical development for the treatment of B cell lymphoproliferative diseases. Patients with chronic lymphocytic leukemia (CLL) often have marked but transient increases of circulating CLL lymphocytes following treatment with PCI-32765, as has been seen with other inhibitors of the B cell receptor (BCR) pathway. In the course of the Phase I study of PCI-32765, we have noted similar effects among treated patients with other types of non-Hodgkin lymphoma (NHL) including mantle cell lymphoma (MCL). We here characterize the patterns and phenotypes of cells mobilized among patients with MCL, and further investigate the mechanism of this effect. Nine patients with MCL treated in the previously reported Phase I study (Advani et al, ASCO, 2010) had baseline absolute lymphocyte counts (ALC) of 1.04 ± 0.42 (x 109/L, Mean ± SD) and had maximal increases during the first 28 day cycle of 12 to 794% (188% increase ± 250, Mean, SD). The ALCs of four patients who were treated on a dosing schedule that included a 1 week drug holiday within each cycle were noted to show intra-cyclic increases of ALC from day 1 to day 15 of each cycle, and decreases following each week off of treatment, for up to 9 cycles (Fig. 1). Patients receiving continuous dosing exhibited gradually decreasing ALCs following the first cycle. The cyclically increasing B lymphocytes were confirmed to be CD5+ (and often also CD45lo), and thus likely to represent circulating, mobilized lymphoma cells. Patient, D005, who attained a complete response, had an easily identifiable CD19+CD45lo subpopulation of 0.47 ×109 cells/L at baseline. This subpopulation increased to 15.2 × 109/L at day 8 of the first cycle, but then decreased markedly as the patient responded clinically. One patient who failed to respond had, by contrast, few if any detectable mobilized cells. Peripheral blood CD19+CD5+ cells from MCL patients treated with PCI-32765 after 8 days were found to have reduced levels of CXC chemokine receptor 4 (CXCR4) levels, whereas pretreatment malignant cells were CXCR4hi. This likely reflects the differences in MCL surface membrane phenotype in solid tissues compared to peripheral blood. Mechanistically, we found that PCI-32765 inhibited BCR- and CXCL12-mediated adhesion and chemotaxis of MCL cell lines in vitro (EC50 = 10–100 nM), and dose-dependently inhibited BCR, stromal cell and CXCL12 stimulations of pBtk, pPLCg and pErk in MCL cells. Importantly, PCI-32765 dose-dependently inhibited the pseudoemperipoleisis of MCL in the presence of stromal cells.Figure 1:Lymphocytic peripheral mobilization of Mantle Cell Lymphoma patients treated with PCI-32765Figure 1:. Lymphocytic peripheral mobilization of Mantle Cell Lymphoma patients treated with PCI-32765 Conclusion: Lymphocyte mobilization into the peripheral blood is notable from MCL in response to treatment with PCI-32765. The majority of these cells are marked with a phenotype (CD19+CD5+ CXCR4lo) which is consistent with malignant cells from secondary lymphoid organs. This effect is likely to be related to PCI-32765 inhibition of BTK activation which results in inhibition of MCL cell chemotaxis, adherence and pseudo-emperipoleisis. We propose that Btk is essential for the homing of MCL cells into secondary lymphoid organs, and that its inhibition results in peripheral blood compartment shift. Disclosures: Chang: Pharmacyclics Inc: Employment. Francesco:Pharmacyclics: Employment, Equity Ownership. Magadala:Pharmacyclics: Employment. Huang:Pharmacyclics: Employment. Spaargaren:Pharmacyclics: Research Funding. Buggy:Pharmacyclics, Inc.: Employment. Elias:Pharmacyclics Inc: Consultancy.

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Detection of Cyclin D1 (bcl-1, PRAD1) Overexpression by a Simple Competitive Reverse Transcription-Polymerase Chain Reaction Assay in t(11; 14)(q13; q32)-Bearing B-Cell Malignancies and/or Mantle Cell Lymphoma

Blood ◽

10.1182/blood.v89.3.965 ◽

1997 ◽

Vol 89 (3) ◽

pp. 965-974 ◽

Cited By ~ 73

Author(s):

Kaoru Uchimaru ◽

Toshiyasu Taniguchi ◽

Miwa Yoshikawa ◽

Shigetaka Asano ◽

Andrew Arnold ◽

...

Keyword(s):

B Cell ◽

Cyclin D1 ◽

Mantle Cell Lymphoma ◽

Cell Lines ◽

Cell Lymphoma ◽

Cyclin D3 ◽

Chain Reaction ◽

B Cell Malignancies ◽

Mantle Cell ◽

Polymerase Chain

Abstract In mantle cell lymphoma, the t(11; 14)(q13; q32) and its molecular counterpart, bcl-1 rearrangement, are consistent features and lead to cyclin D1 (bcl-1, PRAD1) proto-oncogene overexpression. In order to detect cyclin D1 overexpression, we developed a simple assay involving a reverse transcription followed by competitive polymerase chain reaction (PCR). A single upstream primer was derived from a homologous region between cyclin D1 and the other D-type cyclins, cyclins D2 and D3, while three downstream primers were specific to their respective D-type cyclins. Because the upstream primer was shared in PCR amplification of the three sequences, each PCR product served as a competitor and the quantification of the target was made by comparison of the intensity of the three products. With this assay we analyzed 45 hematopoietic cell lines and 40 clinical specimens. Cyclin D1 was rarely expressed in lymphoid cell lines except in t(11; 14)(q13; q32)-bearing B-cell malignancies and/or mantle cell lymphoma, which expressed cyclin D1 predominantly. In myeloid cell lines, the levels of cyclin D1 expression varied and never exceeded the sum of cyclin D2 and D3 levels. Cyclin D3 was ubiquitously expressed while cyclins D1 and D2 were differentially used. The observations suggest that human cyclin D3 may play a fundamental role in hematopoiesis and that cyclins D1 and D2 may have different lineage- or differentiation-dependent functions. With this assay, small aliquots of clinical specimens such as 100 μL peripheral blood were enough to detect cyclin D1 overexpression without a well-controlled standard. The technique was validated as highly comparable with Northern analysis. This rapid and reliable detection of cyclin D1 overexpression may have practical clinical utility in the analysis and management of B-cell malignancies.

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A Phase I/II Study of a Hybrid Schedule of Flavopiridol in Relapsed/Refractory Mantle Cell Lymphoma and Diffuse Large B-Cell Lymphoma

Clinical Lymphoma Myeloma & Leukemia ◽

10.3816/clml.2010.n.037 ◽

2010 ◽

Vol 10 (3) ◽

pp. E27 ◽

Cited By ~ 2

Author(s):

Kieron Dunleavy ◽

Geoff Shapiro ◽

Megan Disinski ◽

Lucian Chirieac ◽

Stefania Pittaluga ◽

...

Keyword(s):

Phase I ◽

B Cell ◽

Mantle Cell Lymphoma ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Large B Cell Lymphoma ◽

Mantle Cell ◽

Large B Cell

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Phase I Dose Escalation Study of Flavopiridol in Combination with Fludarabine and Rituximab: Activity in Indolent B-Cell Lymphoproliferative Disorders and Mantle Cell Lymphoma.

Blood ◽

10.1182/blood.v104.11.2492.2492 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2492-2492

Author(s):

Thomas S. Lin ◽

Beth Fischer ◽

Mollie E. Moran ◽

David M. Lucas ◽

Roshini S. Shank ◽

...

Keyword(s):

Phase I ◽

B Cell ◽

Mantle Cell Lymphoma ◽

Dose Escalation ◽

Cell Lymphoma ◽

Lymphoproliferative Disorders ◽

Clinical Activity ◽

Dose Escalation Study ◽

Mantle Cell ◽

Grade 3

Abstract The cyclin-dependent kinase inhibitor flavopiridol was inactive when administered as a 72-hour infusion, but a 1-hr IV bolus dosing schedule demonstrated clinical activity in mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL). Flavopiridol induces apoptosis via a p53-independent mechanism. Thus, we hypothesized that flavopiridol may eliminate tumor cells resistant to fludarabine and rituximab. We report preliminary results of an ongoing phase I dose escalation study of flavopiridol in combination with fludarabine and rituximab in patients (pts) with MCL, CLL and other indolent B-cell lymphoproliferative disorders. Pts had adequate marrow function (ANC ≥ 1500, hemoglobin ≥ 9.0, platelets ≥ 100,000), organ function, and performance status (ECOG 0–2) and provided informed consent. Pts in all cohorts received fludarabine 25 mg/m2 IV on days 1–5 and rituximab 375 mg/m2 on day 1 of each 28-day cycle. The planned dose escalation of flavopiridol was 50 mg/m2 by 1-hr IV bolus on day 1 (cohort 1), days 1–2 (cohort 2), or days 1–3 (cohort 3) of each cycle. Treatment was for up to 6 cycles, and pts were placed on prophylactic Bactrim and Valtrex. Fifteen pts have been enrolled to date, and 9 pts are evaluable for toxicity and response. Median age of these 9 pts was 67 years (range, 43–72), and 4 pts were male. Pts had the following diagnoses: CLL (5), MCL (2) and follicular lymphoma (FL; 2). Four pts had received 1–2 prior therapies; 5 pts were previously untreated. CLL pts had Rai stage III/IV disease (2) or required treatment for Rai stage I/II disease (3) by NCI 96 criteria. MCL/FL pts were stage III/IV (3) or had progressive stage II disease (1). Three pts were treated in cohort 1; 2 pts completed 6 cycles, but 1 pt was removed from study after cycle 3 due to prolonged cytopenias. Six pts were treated in cohort 2. Two pts developed dose-limiting toxicity; 1 pt developed grade 3 confusion and grade 3 generalized seizures during cycle 2, and 1 pt developed nausea and diarrhea, which resulted in grade 3 acute renal failure. Infectious toxicity was limited to 1 pt who was hospitalized for 48 hrs with a grade 3 upper respiratory infection and febrile neutropenia. Three pts in cohort 2 were removed from study for prolonged cytopenias after 3, 3 and 4 cycles; only 1 pt in cohort 2 completed 6 cycles. Two of the 6 pts in cohort 2 did not receive flavopiridol after cycles 2 and 3, due to life threatening tumor lysis in our single agent flavopiridol study. Response was graded by NCI 96 criteria (CLL) or IWG criteria (MCL/FL). Overall response rate (ORR) was 100%; 7 pts (78%) achieved CR, and 2 pts achieved PR (22%). Two pts relapsed after 7 and 8 months; 7 pts remain in remission a median of 9 (range,7–12) months after therapy. Of note, all 4 MCL/FL pts remain in CR. An ongoing expansion of 12 pts at the cohort 1 dose level is being conducted, to better define toxicity and efficacy; 6 pts have been enrolled to date. In conclusion, flavopiridol, fludarabine and rituximab exhibited significant clinical activity in a small group of pts, with a 78% CR rate. This combination warrants further study, particularly with consideration to an altered flavopiridol schedule using our highly active 30-minute bolus followed by 4-hour infusion regimen.

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