ALBERT

Description: Key Points Anticoagulants inhibit release of angiogenic proteins from platelets.

Description: Inherited thrombocytopenias (ITs) are a heterogeneous group of disorders characterized by low platelet count that may result in bleeding tendency. Despite progress being made in defining the genetic causes of ITs, nearly 50% of patients with familial thrombocytopenia are affected with forms of unknown origin. Here, through exome sequencing of 2 siblings with autosomal-recessive thrombocytopenia, we identified biallelic loss-of-function variants in PTPRJ. This gene encodes for a receptor-like PTP, PTPRJ (or CD148), which is expressed abundantly in platelets and megakaryocytes. Consistent with the predicted effects of the variants, both probands have an almost complete loss of PTPRJ at the messenger RNA and protein levels. To investigate the pathogenic role of PTPRJ deficiency in hematopoiesis in vivo, we carried out CRISPR/Cas9-mediated ablation of ptprja (the ortholog of human PTPRJ) in zebrafish, which induced a significantly decreased number of CD41+ thrombocytes in vivo. Moreover, megakaryocytes of our patients showed impaired maturation and profound defects in SDF1-driven migration and formation of proplatelets in vitro. Silencing of PTPRJ in a human megakaryocytic cell line reproduced the functional defects observed in patients’ megakaryocytes. The disorder caused by PTPRJ mutations presented as a nonsyndromic thrombocytopenia characterized by spontaneous bleeding, small-sized platelets, and impaired platelet responses to the GPVI agonists collagen and convulxin. These platelet functional defects could be attributed to reduced activation of Src family kinases. Taken together, our data identify a new form of IT and highlight a hitherto unknown fundamental role for PTPRJ in platelet biogenesis.

Description: Platelets are specialized anucleate cells that circulate in the blood and serve to prevent bleeding and minimize blood vessel injury. In addition to their hemostatic functions, platelets participate in wound healing, angiogenesis, inflammation, and immunity, and are therefore central players in both maintaining normal physiology and in disease pathogenesis. Platelets are derived from their precursor cells, megakaryocytes (MKs), that reside principally in the bone marrow. During maturation, MKs undergo an altered cell cycle called endomitosis in which they replicate their DNA but avoid cell division, resulting in polyploid MKs with amplified microtubule (MT)-organizing centers called centrosomes. Subsequently, MT-dependent forces are responsible for extending long cytoplasmic protrusions called proplatelets into sinusoidal blood vessels, eventually giving rise to circulating platelets. Despite progress in elucidating key steps of platelet production, there is a conspicuous lack of understanding of what triggers mature, polyploid MKs to undergo the MT rearrangements required for proplatelet production. Using live cell imaging of mouse fetal liver-derived MKs expressing fluorescent b1-tubulin, we have identified a novel MT-based structure in MKs termed a monospindle. Our data suggest that monospindles result from polyploid MKs clustering multiple centrosomes into a centralized MT-organizing center during mitosis, leading to an enlarged array of MTs oriented towards the cell cortex. These structures were also apparent in mouse bone marrow- and human cord blood-derived MKs, suggesting that monospindle formation is a general phenomenon in MKs. Interestingly, a higher percentage of MKs contained monospindles at a timepoint directly prior to proplatelet production (50%) compared to when proplatelets were actively being produced (22%), indicating a possible role in initiating proplatelet formation. Centrosome clustering in cancer cells is mediated by the MT-based mitotic motor protein, KIFC1. Consistently, we found that small molecule inhibition of KIFC1 decreased the percentage of MKs containing monospindles (55% ctrl vs. 6% KIFC1 inhibitor). Strikingly, KIFC1 inhibitor treatment also drastically reduced the percentage of MKs producing proplatelets (peak proplatelet formation: 40% ctrl vs. 5% KIFC1 inhibitor), suggesting that KIFC1-mediated centrosome clustering into monospindles is important for proplatelet production. To test how KIFC1 contributes to these phenotypes, we assessed its expression at different timepoints by Western blot and detected increased KIFC1 levels in more mature MKs preceding proplatelet formation. Cell sorting of MKs into distinct ploidy populations followed by Western blot showed that KIFC1 expression increased with higher ploidy. Thus, our results lead us to suggest a working model in which elevated KIFC1 levels in mature MKs drive monospindle formation to trigger proplatelet formation. Investigating the role of KIFC1-mediated monospindle formation for initiating proplatelet formation could yield a coherent, molecular understanding of how mature, polyploid MKs reorganize MTs for proplatelet production. In addition, these data will help inform basic cell biology, as there are important parallels between centrosome clustering in MKs and cancer cells. Finally, our findings could yield novel therapeutic strategies for treating patients with thrombocytopenia (low platelet counts) by directly stimulating platelet production from mature MKs residing in the bone marrow. Disclosures Italiano: Platelet Biogenesis: Employment, Equity Ownership; Ionis Research Funding: Research Funding.

Description: Background We hypothesized lenalidomide (len) related diarrhea (LRD) may correlate with activation of an immune response against multiple myeloma (MM) based on colon biopsies that showed crypt apoptosis reminiscent of GVHD in 3 MM patients with severe LRD but without prior allogeneic stem cell transplantation or gastrointestinal disorder. To investigate whether survival may be influenced by the presence of LRD we performed a retrospective chart review. Methods Patients who developed symptomatic MM on or after 1/1/2005 as defined by the start of anti-MM therapy and had been on len for at least 6 consecutive months by 12/31/2010 were included in the analysis. Significant LRD was considered present if the treating physician attributed the diarrhea to len and recommended supportive therapy in at least two clinic notes.  The first time treatment for LRD was recommended was used as the date of onset of LRD. As possible confounding factors age at the start of len therapy, number of prior regimens, prior high dose chemotherapy with autologous stem cell transplant (ASCT), and use of antineoplastic agents other than corticosteroids at any time during the continuous len therapy were collected. The primary outcome was overall survival (OS), which was calculated from the start of len. Fisher’s exact test, Mann-Whitney test, and the logrank test were used to compare characteristics and duration of len between patients with and without LRD. Proportional hazards models were used to assess the impact of LRD on OS. The lag between the start of len and development of LRD was accounted for by treating LRD as a time-varying covariate in these models. Results 161 patients were identified, 47 (29%) had LRD, and 59 (37%) died during follow up. LRD and no LRD groups were balanced for gender, age, number of prior regimens, prior transplant, and use of antineoplastic agents other than corticosteroids with len, but len treatment duration differed; LRD patients had received a median of  43.4 consecutive months of len (range 5.8-82.9) compared to 14.6 (range 5.9-89) for patients without LRD (p=0.001). Onset of LRD occurred after a median of 17.7 months (range 0.3-75.4) of len therapy. In multivariable analyses, development of LRD (HR 0.46, 95% C.I. 0.21-1.00, p=0.05) was associated with improved OS as were the number of prior therapies (0-2, vs 〉2, HR 0.16, 95% C.I. 0.08-0.32, p

Description: With the advent of reduced intensity conditioning regimens, new treatment options are available to patients who suffer relapse of Hodgkin’s lymphoma (HL) following autoSCT, but the efficacy of this procedure is not proven. We retrospectively compared the cohort of adult patients who received alloSCT for relapse of HL following autoSCT to those who did not at a single institution. 64 patients underwent autoSCT for HL between 7/87 and 5/02: 41 for relapsed disease, 16 for primary refractory disease and 7 unclassified. Thirty-five patients (55%) relapsed at a median 12.5 months (range 0.9 to 140) after autoSCT. Eleven patients underwent alloSCT a median of 182 (35 – 645) days after relapse; 7/11 received reduced intensity conditioning, 5/11 received SCT from an unrelated donor. Of 24 patients who had relapsed after autoSCT (and did not receive an alloSCT), 16 survived at least 6 months and were considered in this analysis. Acute graft-versus-host disease (GVHD) 〉 grade 2 occurred in only one patient, 5 /9 evaluable patients developed chronic GVHD (2 extensive). Five recipients of alloSCT have died: 2 with relapsed disease, 2 with disseminated fungal infections and 1 with interstitial pneumonitis. 6 patients remain alive, 5 remain free of disease. Median event free survival is12.5 months for recipients of alloSCT with median follow up of 3.1 years. Median overall survival is 4.1 years (64 days – 12.5+ years) and estimated overall survival is 71% at 3 years. This compares favorably with a median overall survival of 1.4 years in patients who survived at least 6 months, but did not receive alloSCT after relapse. Improved survival in the alloSCT cohort demonstrates that potent graft-versus-lymphoma activity can be achieved against HL without significant GVHD.

Description: DVd in combination with Thalidomide (T) and the appropriate supportive care measures resulted in a high response rate (88%) as well as an improved quality response (50% CR & NCR) similar to what is achieved with high dose therapy. Immunomodulatory drugs (IMiDs) are potent T derivatives. R is 50 to 2000 times more potent than T in stimulating T-cell proliferation triggered via the T-cell receptor, and 50 to 100 times more potent than T in augmenting IL-2 and IFN-a production. A recent phase I trial showed responses of at least 25% reduction in paraprotein in 17 (71%) of 24. We therefore initiated a phase I/II trial to define MTD of R in combination with DVd, then we proceeded to expand the MTD dose level to evaluate the efficacy and safety of the combination in patients with Rmm. SWOG criteria were used to assess response, and NCR was defined as a decrease of the M-Protein by 〉90%. Refractory patients were defined as those patients progressing on active therapy. R was started a week prior to DVd in cycle 1 to evaluate different coagulation parameters, from there on R was started on day 1 of therapy. The regimen was given as follows: on day 1 D was given at 40 mg/m2 IVPB; V at 2 mg IVP; d at 40 mg PO daily X 4 days; R was started at 5 mg a day for 21 days with one week off. A standard phase 1 dose escalation of R was performed to identify the MTD. 3 pts were enrolled at each dose level, with up to 6 pts assigned to each dose level, depending on DLT. DVd was repeated q 4W, for a minimum of 4 cycles & 2 cycles after best response. Pts were maintained on R +/− prednisone 50 mg QOD. All patients received amoxicillin, acyclovir and aspirin 81mg prophylactically. 25 pts Rmm pts are enrolled with 21 evaluable for toxicity and mature data available for response on 21 pts (refractory: 15 (71%); relapsed: 6 (29%). 17/21 patients were stage 3, median age of 62 ± 9 years, baseline b2 microglobulin level (mean 5.04 ± 2) and serum albumin (mean 3.4 ± 0.7). 14/21 patients failed T containing regimens. The DLT was sepsis/septic shock that occurred at dose level 3 (R 15mg) with two of the patients developing non neutropenic sepsis. The MTD for R was defined at 10mg. Three patients started therapy with a neutrophil count 〈 500/mL and or platelet counts 〈 50k/mL; all 3 patients were responders. There was one grade 4 hyper-coagulation event in the form of a PE that has recovered. This event occurred in a refractory patient with renal failure performance status of 3 who achieved CR after 2 cycles. In the expanded cohort there was 2 grade 3 neutropenia & one grade 3 neuropathy requiring dose reductions of R and D in each pt with resolution of the neutropenia and neuropathy. 3/21 (14%) patients achieved CR, 4/21 (19%) NCR. All CR+NCR patients (33%) are refractory patients. An additional 7 pts achieved PR, and 6 SD 5 of whom showing greater than 25% decrease in the m-protein. All patients except for 4 achieved 〉 25% reduction of the m-protein after one cycle of therapy and 3/4 after 2 cycles. R at 10mg is the MTD in combination with the DVd in RMM. DVd-R is an extremely effective regimen with a SWOG response rate 〉66%, CR+NCR of 33% in refractory stage 3 patients with minimal toxicity.

Description: Background Information is limited on the efficacy and long-term tolerability of weekly ubcutaneous (SC) bortezomib (BTZ), especially when given alone or combined only with glucocorticoids. We implemented use of SC BTZ in 12/2010 and based on equal AUC and efficacy with twice a week SC as IV BTZ (Moreau et al. Lancet Oncology 2011) at reduced but still significant neurotoxicity allowed weekly SC, maintaining the BTZ starting dose at 1.3mg/m2. Methods Multiple myeloma (MM) and AL amyloidosis (ALA) patients (pts) who had received SC BTZ by February 2013 were identified from our plasma cell disorder registry. After IRB approval, their electronic medical records were reviewed for occurrence, severity, and evolution of PNP with each BTZ containing regimen, administration schedule of BTZ, presence of underlying PNP and neuropathy risk factors (diabetes mellitus, ESRD, spinal cord compression/disease, vitamin B12 deficiency, alcoholism, chronic liver disease, hyperlipidemia, hypothyroidism), concurrently used antineoplastic agents, physician assigned responses, and reasons for BTZ dose reductions or discontinuation. To compare first BTZ regimen administration schedules Fisher’s exact test and chi-square tests were used for categorical data, Kruskal-Wallis and Wilcoxon rank sum test for age and interval from diagnosis to treatment, and logrank test for treatment duration. Proportional hazards models were used to assess the impact of BTZ administration schedule on neuropathy and response. The impact of prior regimens before first BTZ administration on response was estimated by logistic regression models. Results 136 patients were identified, 12 were excluded due to insufficient data (not followed at our Center). The remaining 124 pts began their first BTZ regimen between 02/2005 and 02/2013. 81% had MM, 12 % ALA, and 7% both MM and amyloidosis. Patients received a median of 2 BTZ containing regimens (range 1-9); overall 312 BTZ regimens were analyzed. In 114 SC weekly, 32 SC twice a week, 59 IV weekly, 62 IV twice a week, and 11 twice a week SC/IV followed by weekly BTZ regimens, neuropathy led to BTZ discontinuation in 7.9% (n=9), 9.4% (n=3), 13.6% (n=8), 22.6% (n=14), 9.1% (n=1), respectively, and to dose reduction in 5.3% (n=6), 3.2% (n=1), 6.8% (n=4), 6.5% (n=4), 9.1% (n=1), respectively. Patients who received weekly SC BTZ as their first BTZ containing regimen (n=37) had received a median of 0 prior regimens (range 0-10), 27% (n=10) had mild (n=8) or severe (n=2) underlying neuropathy, and most (68%) received BTZ with only glucocorticoids (n=23) or alone (n=2), while lenalidomide (n=8) or other agents (n=4) were added to 32%. After a median treatment duration of 4.3 months (0.2-23.3+), 26 of these 37 pts (70%) developed no neuropathy (n=20) or no worsening of pre-existing neuropathy (n=6), but 7 (19%) required BTZ dose reduction (n=2) or supportive medications (n=5) for neuropathy and in 4 (11%) BTZ was discontinued because of neuropathy. In multivariable analyses for neurotoxicity and lack of response, use of schedules other than weekly SC as the first BTZ administration schedule caused more neuropathy (HR 2.3, 95% C.I. 1.0-5.3, p=0.05), while age and underlying disease associated with neuropathy had no impact (p=0.57 and 0.61, respectively); lack of response tended to be more common with schedules other than weekly SC (HR 2.0, 95% C.I. 0.9-4.5, p=0.09) but age and disease (MM vs. AL amyloid) did not affect response (p=0.33 and 0.32, respectively). A response rate of 71% (n=22) to the first SC weekly bortezomib containing regimen in 37 pts who had received a median of 0 (range 0-10) previous regimens was within the expected range for standard administration schedules; of 8 pts who received weekly SC BTZ with not more than a total of 40mg dexamethasone per week as upfront therapy for myeloma, 5 achieved VGPR, 1 PR, and one MR; in 6 evaluable AL amyloid patients this upfront treatment led to VGPR in 3 and PR in 1 patient. Conclusions Weekly SC BTZ, even if administered only with glucocorticoids, is effective and better tolerated than other BTZ administration schedules. However, neuropathy continues to impact therapy, affecting about a third of patients in our series who received BTZ for the first time. Disclosures: Off Label Use: Upfront weekly SC bortezomib. Faiman:Onyx: Consultancy, Speakers Bureau; Millennium: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau. Valent:Millennium: Speakers Bureau; Clegene: Speakers Bureau. Duong:Celgene: Honoraria, Research Funding. Reu:Onyx: Speakers Bureau; Celgene: Research Funding.