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Inflammation is involved in the initiation and progression of several chronic lymphoid malignancies of B-cell type. Toll-like receptors (TLR) are transmembrane inflammatory receptors that on recognition of pathogen-associated molecular patterns trigger an innate immune response and bridge the innate and adaptive immune response by acting as costimulatory signals for B cells. Fine tuning of TLR and IL-1R–like (ILR) activity is regulated by TIR8 (SIGIRR), a transmembrane receptor of the TLR/ILR family which inhibits other family members. To test the hypothesis that TLR and/or ILR may play a role in the natural history of chronic B-cell tumors, we crossed Eμ-TCL1 transgenic mice, a well established model of chronic lymphocytic leukemia (CLL), with mice lacking the inhibitory receptor TIR8 that allow an unabated TLR-mediated stimulation. We here report that in the absence of TIR8 the appearance of monoclonal B-cell expansions is accelerated and mouse life span is shortened. The morphology and phenotype of the mouse leukemic expansions reproduce the progression of human CLL into an aggressive and frequently terminal phase characterized by the appearance of prolymphocytes. This study reveals an important pathogenetic implication of TLR in CLL development and progression.

Inflammation is involved in the initiation and progression of several chronic lymphoid malignancies of B-cell type. Toll-like receptors (TLR) are transmembrane inflammatory receptors that on recognition of pathogen-associated molecular patterns trigger an innate immune response and bridge the innate and adaptive immune response by acting as costimulatory signals for B cells. Fine tuning of TLR and IL-1R–like (ILR) activity is regulated by TIR8 (SIGIRR), a transmembrane receptor of the TLR/ILR family which inhibits other family members. To test the hypothesis that TLR and/or ILR may play a role in the natural history of chronic B-cell tumors, we crossed Eμ-TCL1 transgenic mice, a well established model of chronic lymphocytic leukemia (CLL), with mice lacking the inhibitory receptor TIR8 that allow an unabated TLR-mediated stimulation. We here report that in the absence of TIR8 the appearance of monoclonal B-cell expansions is accelerated and mouse life span is shortened. The morphology and phenotype of the mouse leukemic expansions reproduce the progression of human CLL into an aggressive and frequently terminal phase characterized by the appearance of prolymphocytes. This study reveals an important pathogenetic implication of TLR in CLL development and progression.

The pathophysiology of the occurrence and resolution of sickle cell crisis is unknown. The molecular abnormality is constant, while crisis is episodic. In the present study, red cell filterability and sickling with deoxygenation have been measured during sickle cell crises. Recovery from sickle crisis is associated with an increased filterability of the circulating red cell and a decreased susceptibility of the red cell to sickle with deoxygenation (p less than 0.05). The possibility that these changes are responsible for the resolution of crisis is suggested.

The pathophysiology of the occurrence and resolution of sickle cell crisis is unknown. The molecular abnormality is constant, while crisis is episodic. In the present study, red cell filterability and sickling with deoxygenation have been measured during sickle cell crises. Recovery from sickle crisis is associated with an increased filterability of the circulating red cell and a decreased susceptibility of the red cell to sickle with deoxygenation (p< 0.05). The possibility that these changes are responsible for the resolution of crisis is suggested.

No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

B-Cell Receptor (BCR) triggering and responsiveness play a crucial role in the survival and expansion of Chronic Lymphocytic Leukemia (CLL) clones. In the recent past, several groups including ours have investigated the activation status of the signaling pathways originating from the leukemic BCR. Specifically we found that around 50% of CLL patients display a biochemical signature characterized by constitutive phosphorylation of ERK1/2 (pERK(+)) and constitutive nuclear translocation of NF-ATc1. These cases are unable to respond in vitroto BcR stimulation and are resistant to spontaneous apoptosis, thus resembling B lymphocytes previously anergized in vivo. Similar biochemical and functional features have been recently demonstrated in B leukemic cells persisting in the blood in patients treated with the BTK inhibitor, Ibrutinib, thereby making anergy an attractive target on the way to obtain eradication of the disease.

No relevant conflicts of interest to declare.

Chronic Lymphocytic Leukemia (CLL) is a chronic lymphoid malignancy characterized by immune suppression that is responsible for an increase in infection susceptibility but also concurs to a reduced ability of the immune system to promote an effective response against the leukemic cells. Tumor-immunosuppressive mechanisms are essentially due to the capacity of CLL cells of modifying the surrounding microenvironment including immune effectors likely contributing to disease progression but also to limited effectiveness of current immunotherapy approaches.

The prognosis of patient with relapsed or refractory CLL/SLL is dismal with an overall response rate (ORR) to salvage therapy for refractory patients of 10–30%, and limited survival benefit with current treatment approaches. Phase II studies of single agent lenalidomide in patients with relapsed or refractory CLL revealed an ORR of 32–58% (7-17% CR). Recent in vitro studies have shown that lenalidomide enhances the rituximab-induced killing of NHL cell lines and B-CLL cells by enhancing ADCC activity and restoring the defective T-cell and NK-cell mediated tumor cell cytotoxicity.Patients with relapsed or refractory CLL/SLL received oral lenalidomide via dose escalation as follows: 2.5 mg on days 1–7, 5 mg on days 8–14 and 10 mg on days 15–21 followed by 7 days of rest in 28-day cycle; for cycle 2 and beyond 20 mg was given on days 1–21 on a 28-day cycle. Rituximab was dosed at 375 mg/m2 IV weekly for 4 weeks starting on day 15 of cycle 1. Treatment was continued until disease progression or toxicity. Primary objectives were ORR (CR+PR) and safety and tolerability of the combination regimen. CT scans, and bone marrow biopsies were done every 2 months to assess for response (NCI-WG 2008). Peripheral blood and bone marrow aspirates were collected for correlative studies before lenalidomide was initiated, before rituximab was initiated (between days 13–15), after finishing treatment with rituximab and then every two months until disease progression. Flow cytometry was performed using the following antibodies CD3, CD4, CD5, CD8, CD19, CD20, CD23, CD40, CD45RA, CD62L, CD80, CD86, CD95, IL-17A and FoxP3. Panels were created for the analysis of T-cell memory/naïve populations, B-cell populations, regulatory T-cells and Th17 cells. Data was collected to a limit of 10,000 events of the population of interest. Data is presented as total number of cells/ul instead as percentage to avoid misinterpretation due to the dramatic reduction in the number of B cell lymphocytes after initiation of therapy. Subpopulation of T cells memory/naïve were compared with an age matched population of normal controls.18 patients with CLL/SLL were enrolled on study. Median number of prior chemotherapies was 3 (range 1–5). Median age was 63 years (range 42–80). High risk cytogenetic abnormalities (del11q (11%), del 17p/p53 (11%), complex (22%)) were observed in 44% of the patients. 95% of the patients had received prior fludarabine therapy and 50% were fludarabine refractory. Overall clinical benefit was seen in 92% of patients (42% PR, 50% SD) with a median duration of response of 18 months for patients who achieved a PR and 12 months for patients with SD. Although all responses were PR, the PR rate improved with continued therapy suggesting increased responses with a longer duration of treatment with lenalidomide.Most common adverse effects were neutropenia (50% grade 3–4), tumor flare (28% grade 1–2, 11% grade 3–4), fatigue (11% grade 1–2, 6% grade 3–4), venous thromboembolic disease (11% grade 3–4), acute renal insufficiency (11%), rituximab related infusion reactions (11%), flu-like symptoms (11%), infections (11%), and hypercalcemia (11%).Correlative studies showed that peripheral blood CD4 and CD8 effector memory subpopulations decreased after initiation of lenalidomide therapy with subsequent elevation after rituximab treatment on the CD4 effector memory compartment. The Th17 compartment was minimally decreased after initiation of lenalidomide while the levels of regulatory T cells (Tregs) appeared to decrease with lenalidomide therapy and increase slightly after rituximab. The expression of CD20 from bone marrow samples decreased as expected with rituximab therapy; however shortly after the discontinuation of rituximab CD20 expression was regained by the B cells compartment. Later time points will be presented at the meeting.The combination of lenalidomide with rituximab is a promising with clinical activity in heavily pretreated patients with relapsed or refractory CLL. The combination appears tolerable with observed events consistent with the use of these two agents in other studies. The impact of lenalidomide on the T cell subpopulations in patients treated with rituximab remains unclear. A detailed analysis of the BM compartment at latter time points will be investigated.Lancet: Eisai: Consultancy; Celgene: Honoraria. Komrokji:Genentech: Research Funding.

Abstract 4630

In concert with emerging effectiveness data, we are learning that the side effect profile of lenalidomide (L) in CLL may be different from that observed with other malignancies, such as multiple myeloma (MM) or myelodysplastic syndrome (MDS). In particular, at lower doses than typically used for MM or MDS, a unique tumor flare response (TFR) has been observed for which the mechanism is still unclear, but may be related to a “cytokine release” phenomenon.Here we would like to describe 3 cases of hypercalcemia that occurred in 3 of 17 patients with fludarabine refractory CLL treated at the Moffitt Cancer Center: 2 patients who were on a clinical trial of lenalidomide plus rituximab (LR), and 1 patient who was treated with L outside of a clinical trial setting.Pt #1 was initiated on (LR) on 12/2007 for CLL with bulky LAN, elevated wbc (110K), and 90% bone marrow (BM) involvement. By day 6, his calcium peaked at 11.78 and was accompanied by a drop in wbc to 15K. He had no other evidence of TFR, and was specifically without complaints of back pain or tender/progressive LAN. He was treated with IV fluids and steroids which promptly corrected his calcium, after which L was resumed. His course has been complicated by neutropenia requiring G-CSF. Although LAN remains stable, he has had an excellent BM response, with only 40% CLL seen on reevaluation in 06/2009.Pt #2 was initiated on LR on 8/2008 for CLL with bulky LAN, mildly elevated wbc (20.5K) and 60% BM involvement. By day 4 his calcium peaked at 16.94, at which time he presented to our urgent care center with weakness, sweats, back pain, nausea and vomiting. No tender or progressive LAN was appreciated. He was treated with IV fluids, pamidronate, calcitonin and steroids which normalized his calcium. An attempt was made to restart L, which again produced hypercalcemia after 4 days. No further attempts were made to resume this medication. A workup performed during the first episode of hypercalcemia was notable for a slight elevation in the PTHrp (8.5).Pt #3 was initiated on L alone on 6/2008 for CLL with bulky LAN, elevated wbc (283.6K), and 95% BM involvement. By day 4, his calcium peaked to 14.64, and was associated with a TFR, including a rise in wbc (306K), LDH (1285), and enlarging/painful LAN. The hypercalcemia was treated with IV fluids, zoledronate, and steroids. Bendamustine was immediately initiated for the treatment of CLL with dramatic improvement on lymph nodes and WBC counts. Following 1 cycle of bendamustine, L was resumed, with stabilization of his disease.Workup in these patients was unremarkable, with normal VitD/PTH levels, and only a mild elevation of PTHrp in Pt#2 (see table), in whom hypercalcemia had reoccurred upon rechallenge with L. Pt#1 and #3 were successfully retreated without recurrent hypercalcemia, though, Pt#3 received prior cytoreductive therapy with bendamustine.Hypercalcemia is a rare complication of advanced B-cell malignancies seen with lymphokine/cytokine mediated bone disruption. Increased production of l,25-(OH) 2D by lymphocytes may also lead to hypercalcemia, and in CLL it is mainly associated with Ritcher's transformation or presence of osteolytic lesions. Here we report on 3 cases of hypercalcemia, which were identified during L treatment in CLL, and which had not been observed in other studies of L. We hypothesize that the hypercalcemia could be a direct reflection of increased cytokine levels, including IL-6, TNFα, and, MIP-1α, which are known to influence osteoclastogenesis. Interestingly, IL-6 is known to act synergistically with PTHrp in experimental models to drive hypercalcemia, which may be relevant for the patient who developed recurrent hypercalcemia on rechallenge with L. Levels of cytokines at different time points are now under investigation and are anticipated for presentation at the meeting. These events indicate that serum chemistries of patients initiating treatment with lenalidomide should be closely monitored during the first weeks of treatment.Off Label Use: Lenalidomide is an immune modulator drug currently being studied for efficacy in conjunction with Rituxan in chronic lymphocytic leukemia. This abstract summarizes a unique side effect, hypercalcemia, seen among a small cohort of patients on this study.. Lancet:Celgene: Research Funding. Pinilla:Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Research Funding; exelixis: Research Funding.

Due to significant activity of lenalidomide in chronic lymphocytic leukemia (CLL), research is ongoing to better characterize its mechanism of action. Recent laboratory studies of cells from CLL patients have shown that lenalidomide could exert its antiproliferative activity by decreasing the presence of survival-promoting cytokines. It has also been shown to facilitate immune-mediated ADCC by restoring the defective ability of T- and NK-cells to form immune synapses with tumor B-cells (Ramsay et al JCI 2008). However, no direct cytotoxic effect of lenalidomide has yet been demonstrated in CLL cells. Here, we suggest a new potential mechanism of action based on morphologic evaluation of bone marrow samples from patients with CLL receiving treatment with lenalidomide.Bone marrow biopsy and aspirations from 13 patients with relapsed or refractory CLL, who are participating in a phase II clinical trial of lenalidomide and rituximab, and 1 patient who received lenalidomide outside of study, were reviewed before initiation of therapy and every 2 months thereafter.Of 14 CLL samples evaluated, 3 samples contained cells with lymphoplasmacytoid features in both bone marrow (figure 1) and peripheral blood, which had not been present prior to initiation of treatment. Of the 3 samples containing lymphocytes with plasmacytoid features, 2 were obtained from patients who received more than 6 months of therapy with the lenalidomide/rituximab combination, and 1 was taken from the patient receiving lenalidomide, after one month of therapy. Long term morphologic effects of lenalidomide therapy on lymphocytes from patients with CLL remain unknown. However, we hypothesize that the observed changes in morphology after lenalidomide treatment could represent either a differentiation phenomenon, as a potential antitumor mechanism in CLL cells, or perhaps represent just a selection effect.An exact direct antitumor mechanism of lenalidomide on CLL cells remains unknown. Our observations suggest that lenalidomide may promote terminal differentiation of lymphocytes thus proposing an additional mechanism by which lenalidomide exerts its anti-tumor activity in CLL.No relevant conflicts of interest to declare.

Patients with relapsed or refractory CLL/SLL and patients with mantle cell lymphoma (McL) have a poor prognosis. Overall response rate (ORR) to salvage therapy for refractory patients is approximately 10-30%, and survival benefit with current treatment approaches is limited. Phase II studies of single agent lenalidomide in patients with relapsed or refractory CLL revealed an ORR of 32-58% (7-17% CR), depending on treatment dose, scheduled used and duration of treatment with lenalidomide. In patients with refractory or relapsed McL, lenalidomide treatment resulted in an ORR of 53% (CR 20%, PR 33%), and a 14-month median duration of response (Habermann et al 2009). Recent in vitro studies have shown that lenalidomide enhances the rituximab-induced killing of NHL cell lines and B-CLL cells via ADCC by restoring the defective T-cell and NK-cell mediated ability to form immune synapses to exert tumor cell cytotoxicity.Patients with relapsed or refractory CLL/SLL or McL received oral lenalidomide via dose escalation as follows, 2.5 mg on days 1-7, 5mg on day 8-14 and 10mg on day 15-21 followed by 7 days of rest in 28-day cycle; for cycle 2 and beyond 20mg was given on day 1-21 on a 28 day cycle. Rituximab was dosed at 375mg/m2 IV weekly for 4 weeks starting on day 15 of cycle 1. Treatment was continued until disease progression or toxicity. All patients were given allopurinol 300mg orally twice per day starting 3 days prior to first dose of lenalidomide. CT scans, and bone marrow biopsies were done every 2 months to assess for response. Primary objectives were overall response rate (CR+PR) and safety and tolerability of the combination regimen.17 patients were enrolled on study (13 patients with CLL/SLL and 4 patients with McL). Median number of prior chemotherapies was 3 (range 1-5). Median age was 64 years (range 42-80). Among patients with CLL, the most common cytogenetic abnormalities were trisomy 12 (isolated n=3, associated with other abnormalities n=4), del11q (isolated n=1, with others n=3), isolated del13q (n=1), complex cytogenetics with 3 or more abnormalities (n=4 including 1 patient with del 17p). Responses were assessed every 2 months after initiation of therapy. Response rate for 13 evaluable patients (10 with CLL and 3 with McL) relative to months on treatment with lenalidomide are summarized in the table. Although all responses were PR, the rate of PR improved with continued therapy suggesting increased responses with a longer duration of treatment with lenalidomide. Currently, 7 patients are still receiving active treatment on study, all with CLL (3 achieved a PR and 4 have SD). Of the 4 patients with McL enrolled on study, 1 achieved a PR after 2 months of therapy; 1 achieved SD after 2 months of therapy with a sustained SD after 6 months; 1 patient achieved SD after 2 months, but progressed after 6 months on treatment.The regimen was well tolerated. Most common (>5%) toxicities include neutropenia (35% grade 3, 6% grade 4), fatigue (17% grade 1-2, 6% grade 3), tumor flare (12% grade 2, 12% grade 3), acute renal insufficiency (6% grade 1, 12% grade 3), rituximab related infusion reactions (6% grade 2, 6% grade 3), flu-like symptoms (6% grade 2, 6% grade 3), venous thromboembolic disease (6% grade 2, 6% grade 3), infections (11% including 1 patient with fatal endocarditis), and hypercalcemia (11% grade 4). Correlative studies are ongoing.The combination of lenalidomide with Rituximab is a promising combination regimen in CLL patients with very poor prognosis who have undergone multiple lines of therapy. This treatment combination appears tolerable with observed events consistent with the use of these two agents in other studies. Further investigation is warranted, possibly in the front line setting and in combination with other agents.Lancet: Celgene: Research Funding.

Abstract 4413

Abstract 4412

Abstract 2376