ALBERT

Description: Postmenopausal women have an increased risk of cardiovascular disease, and heart disease is the leading cause of death in postmenopausal American women. Conventional hormone replacement therapy has been shown to result in an increase in thrombotic events in large prospective clinical trials including HERS I, and the recently halted Women’s Health Initiative. One possible mechanism for this observed increase is the unfavorable net effects of conjugated equine estrogens and medroxyprogesterone acetate on the hemostatic balance and inflammatory factors. An estimated 50 million American women are peri or postmenopausal and clinical therapies for menopausal symptoms remain a significant challenge in light of the known thrombotic risks. In this prospective blinded study, we examined the short-term effect of topical progesterone cream on menopausal symptom relief in 30 healthy postmenopausal women. Potential adverse effects of topical progesterone on hemostatic and inflammatory factors and cortisol levels were also examined. Subjects were randomized to first receive either 20 mg of topical progesterone cream or placebo cream for 4 weeks. Following a subsequent 4-week washout period, subjects were crossed over to either placebo cream or active drug for an additional 4-week period. In each case, progesterone and cortisol levels were monitored by salivary sampling. Baseline values, 4-week follow-up values and end-of-study values were also obtained for the Greene Climacteric Scale, total factor VII:C, factor VIIa, factor V, fibrinogen, antithrombin, PAI-1, CRP, TNFα, and IL-6. For subjects receiving 20 mg of topical progesterone cream for 4 weeks, Greene Climacteric Scale scores were consistently and significantly improved (decreased) over baseline, demonstrating significant relief from menopausal symptoms. In addition, in a subpopulation of hypercortisolemic women, topical progesterone was associated with a favorable decrease in nocturnal cortisol. Surprisingly, and in sharp contrast to earlier studies with conventional hormone replacement therapy, topical progesterone had no effect on any of the hemostatic components examined: total factor VII:C, factor VIIa, factor V, fibrinogen, antithrombin, and PAI-1 levels were all unchanged. Levels of CRP, TNFα and IL-6 also remained unchanged. From this study we conclude that administration of topical progesterone cream at a daily dose of 20 mg significantly relieves menopausal symptoms in postmenopausal women without adversely altering prothrombotic potential. Since the thrombotic complications that are typically observed with conventional hormone replacement therapy do not seem to occur with topical progesterone, this treatment should be seriously considered as an effective and safe alternative clinical therapy for women suffering from menopausal symptoms.

Description: Introduction: Interphase FISH on CD138-selected bone marrow cells enables genetic risk stratification in newly diagnosed multiple myeloma (MM), however as MM remains incurable, most centres still treat newly diagnosed MM uniformly, utilising the most active regimens available. At relapse an increasing choice of regimens, coupled with co-morbidities and treatment-emergent toxicities, means no uniform approach is possible. Instead, therapy is tailored to disease and patient related risk factors. In this setting, FISH testing may be particularly useful if not done at diagnosis and to identify progression events that may alter prognosis. Aim: To evaluate the outcome of FISH analysis in consecutive patients with relapsed MM undertaken at our centre: success rate, frequency of abnormalities, incidence of progression events and correlation of FISH abnormalities with treatment outcomes. Methods: FISH analysis was performed on 192 samples from 154 relapsed patients (2012-13). Plasma cells were selected using magnetic CD138 MicroBeads and interphase FISH carried out using probes as recommended by the EMN (Ross et al, 2012). If patients had no prior results, a full FISH MM panel was performed, using probes for t(4;14), t(14;16), t(11;14), deletion 17p (17p-), Chr 1 abnormalities (1p-/1q+) and deletion 13q (13q-). If patients had been previously tested for an IgH translocation (Tx), a progression event panel was used: 1p-/1q+, 17p- and 13q-. Patients underwent FISH testing prior to starting the next line of therapy. Results: 79% of samples were successfully analysed, with analysis limited in 16% and failed in 5%. Common reasons for failure were poor quality/aged slides, insufficient material and poor hybridisation. 17% of patients had no cytogenetic abnormality. The most common abnormality was 13q- (43.1%), followed by 1q+ (41.4%), t(11;14) (18.3%), t(4;14) (12.4%), 17p- (12.0%) 1p- (8.9%), and t(14;16) (5.6%) Progression events were more common in t(14;16) and t(4;14) groups. All patients with t(14;16) and 82% with t(4;14) had an additional genetic lesion. Only 21% of patients with t(11;14) and 54% with no IgH Tx had an additional event. 80 patients (51.3%) had prior FISH results and 13 (16.3%) had developed a new abnormality on the later test. In 9 cases the progression event was 17p-, in 2 it was 1q+ and 2 cases developed 17p- and 1q+. The patients developing 1q+ were previously standard risk, so repeat testing altered risk group. Acquisition of 17p- indicates especially poor outcome, thus in all 13 cases repeat FISH analysis altered risk. Among patients with progression events none harboured t(11;14), 8 (64%) had no IgH Tx, 3 had t(14;16) and 2 had t(4;14). FISH results were correlated with clinical outcome. Patients were stratified as having high risk genetics [t(4;14), t(14,16), 17p- in ≥50% cells, 1p-/1q+] or standard risk [t(11;14), normal cytogenetics]. 63 (41%) patients were high risk, 83 (54%) standard risk, with no information available for 8 (5%). Both groups had received a median of 2 prior lines of therapy. Response rates (≥PR) to the next line of therapy were similar (60.4% standard risk vs 56.0% high risk). PFS from time of FISH was significantly longer in the standard risk group (9.8 months vs 5.9, p

Description: Abstract 2187 Plasma cells, the terminal effectors of the B-cell lineage include both short- and long-lived cells. The latter persist for extended periods in the absence of cell division, supported in niche environments. No model system has successfully recapitulated the function of the niche to allow the in vitro generation of long-lived plasma cells. This limits investigation into the factors controlling and targeting plasma cell populations. Here we describe the generation of mature human plasma cells with extended lifespan from peripheral blood B-cells. Cell division accompanies phenotypic maturation between plasmablasts and plasma cells. These cells then persist in the absence of cell division, remaining functional and viable in extended culture, currently limited solely by elective termination. Extended survival is accompanied by maturation to a phenotype consistent with human bone marrow plasma cells. By establishing a set of conditions sufficient to allow the development and persistence of mature human plasma cells in vitro, we recapitulate the essential function of the plasma cell niche. We definitively link phenotypic maturation to lifespan and provide the first platform with which to explore and manipulate the full trajectory of human plasma cell differentiation. Disclosures: No relevant conflicts of interest to declare.

Description: Multiple myeloma (MM) is the second most common hematologic malignancy. Presently, the majority of suitable MM patients who undergo high-dose melphalan therapy followed by autologous peripheral blood stem cell transplantation (PBSCT) fail to achieve a complete response (CR). This suggests that treatment options following autologous transplantation are needed. Moreover, there is a need to determine the optimal role of maintenance therapy following PBSCT in MM. Over time, Bortezomib (B) has been shown to be an active agent in the treatment of newly diagnosed, and relapsed or refractory MM. Therefore, the primary objective of this study was to determine the efficacy of B treatment after high-dose melphalan therapy followed by PBSCT in MM. Fifty patients (pts) were enrolled between March 2004 and November 2007, and 47 were evaluable (2 pts ineligible and 1 pt data pending). Pts received B 1.3 mg/m2 IV on Days 1, 4, 8, and 11 of each 21-day cycle. Pts were treated for 4 cycles or until evidence of disease progression or intolerable toxicity. If an improvement in response was noted after Cycle 4, pts could receive up to 4 additional cycles. To reduce the incidence of varicella zoster infection, antiviral prophylaxis (acyclovir 400 mg PO BID) was taken for the duration of the study. The median patient age was 56 years (range, 39–74), 82% were white, and 68% were male. The majority of pts (64%) had ECOG PS 0, 44% were Durie-Salmon Stage IIIA prior to induction therapy. Forty percent had symptomatic IgG-kappa multiple myeloma. Of all pts, 74% had a single transplant, while 24% had tandem transplants (2% [n=1 pt] data pending). Sixty-eight percent of pts had a PR and 18% had a MR following their transplant(s). While on study, pts received a median of 4 cycles (range, 2–8) of therapy with B. Efficacy results for the evaluable population are: CR 4%, unconfirmed (u) CR 4%, PR 21%, uPR 17%, MR 11%, and No Change 36%. Median time-to-treatment failure was 5.8 months (mos) (range, 0.2–19.4). There were 2 on-study deaths (sepsis and PD). Grade 3–4 treatment-related toxicities reported in 〉1 pt were thrombocytopenia (15%), asthenia (10%), neutropenia or neuropathy (8% each), peripheral neuritis (6%), and nausea (4%). Twenty patients discontinued study treatment due to toxicity (22%), pt request (6%), disease progression, ineligibility, and intercurrent illness/protocol deviation (4% each). 26 pts (52%) completed the study; 4 pts are still on study (8%). Sixteen pts started new treatment; median time from start of study treatment to the start of new treatment was 5.2 mos (range, 1.5–17.6 mos). The study was closed earlier than the planned due to the widespread availability of B, and the inability to find B-naïve patients. Bortezomib given after high-dose melphalan therapy and autologous PBSCT was well-tolerated with manageable adverse events. Updated cytogenetic analysis will be available for presentation.

Description: Background: Talabostat (PT-100), an orally available inhibitor of dipeptidyl peptidases, is in Phase 2 studies for B-cell malignancies and solid tumors. Talabostat increases production of cytokines and chemokines in lymph nodes and spleen, stimulating both adaptive and innate immune responses (Adams S, Cancer Research, 2004;64:5471). Talabostat may thus enhance the antibody-dependent cytotoxicity of MAbs such as rituximab. Methods: This is a Phase 1 study to evaluate the safety and activity of talabostat and rituximab in patients with indolent NHL who did not respond or progressed following rituximab. Rituximab 375mg/m2 was administered weekly x 4. Total daily doses of talabostat 400μg (n=6), 600μg (n=3), or 800μg (n=6) were administered BID for 6 days following each dose of rituximab. Cytokines and chemokines were assessed pre-, 2, and 6 hours post-talabostat on Days 1, 6, 13, 20, and 27. Flow cytometry was performed at baseline and Day 28. Clinical and laboratory evaluations were performed at specified times. Adverse events (AEs) were graded per NCI-CTC and recorded throughout the study. Disease assessments were performed on Days 28 and 84. Results: 11 men and 4 women aged 48–82 with NHL (n=10) or SLL/CLL (n=5) have been treated. 9 patients completed the 28-day study: 4 at 400μg, 1 at 600μg, and 4 at 800μg. Enrollment continues at 400μg/day. The most frequent AEs have been edema (67%), nausea (47%), dizziness (40%), hypotension (33%), fatigue (33%), vomiting (33%), constipation (33%), thrombocytopenia (27%), and weight gain (27%). Grade 3 toxicities include: dizziness, myopathy (400μg/day), and 2 events of thrombocytopenia (600μg/day). Grade 3 peripheral edema, myalgia, dehydration, electrolyte imbalance, hypereosinophilia, elevated CPK (primarily CK-MM), and rhabdomyolysis were seen in 2/6 patients at 800μg/day; these events were DLTs. One partial response (PR) lasting 7 months was seen in one patient (800μg/day). A PR was seen in a second patient at 800μg/day but did not meet the strict NCI-WG criteria for response. Elevations in cytokines 〉ULN were reported across all doses following talabostat: G-CSF (13/15), IL-1β (10/15), IL-2 (7/15), IL-6 (8/15), IL-8 (8/15), IL-10 (11/15), TNF-α (11/15), and IFN-γ (3/15). At Day 28 or early termination, CD20 was decreased in most (12/15) patients. Increases were seen in the percentage of CD3 (12/15), CD3/4 (11/15) and CD3/8 (9/15). In all 5 patients with SLL/CLL, CD5+/CD20+ was

Description: Background: T cells can be activated and expanded ex vivo using the Xcellerate™ Process, in which peripheral blood mononuclear cells (PBMC) are incubated with anti-CD3 and anti-CD28 antibody-coated magnetic beads (Xcyte™-Dynabeads®). In an ongoing trial of Xcellerated T Cells in subjects with chronic lymphocytic leukemia, marked and sustained reductions in lymphadenopathy and splenomegaly were observed (Wierda et al., ASCO 2004). Increases in neutrophil, platelet and NK cell counts were also documented. This study is designed to determine if similar effects can be observed in subjects with indolent non-Hodgkin’s lymphoma (NHL). Methods: Subjects must have indolent NHL (follicular, small lymphocytic, marginal zone, or mantle cell lymphoma), have relapsed or refractory disease, and have received at least 1 but not more than 4 prior treatment regimens. PBMC are collected by leukapheresis for the Xcellerate Process, and subjects subsequently receive two infusions of 20–60 x 109 Xcellerated T Cells separated by 6–8 weeks. Approximately 40 subjects will be treated. Results: Seven subjects have been enrolled and Xcellerated T Cells have been manufactured in 5 subjects to date. T cells expanded 181.8 ± 88.5 fold and the final product was 〉99.0 ± 0.0% T cells (mean + SD). One subject with small lymphocytic lymphoma has been treated with two infusions of 38.6 x 109 Xcellerated T Cells. There have been no serious adverse events to date. Following the first treatment, the lymphocyte count increased from 1.8 x 109/L to 2.9 x 109/L on Day 28. The neutrophil count also increased from 2.9 x 109/L to 5.5 x 109/L six weeks following infusion. The subject had a significant reduction in cervical lymphadenopathy and a slight decrease in bulky mesenteric lymphadenopathy six weeks following the first infusion. Conclusions: Xcellerated T Cells can be manufactured in subjects with indolent NHL. Treatment leads to significant increases in T cell and neutrophil counts. Preliminary data suggest a reduction in peripheral lymphadenopathy. Data on additional subjects will be presented.

Description: Background:Flow cytometric studies are useful in the diagnostic workup of patients with unexplained cytopenias and it has been demonstrated that bone marrow aspirates with immunophenotypic abnormalities by flow cytometry but not diagnostic morphologic or cytogenetic findings frequently evolve into myelodysplastic syndromes (MDS) (Kern 2013). Two flow cytometric scoring systems (FCSSs), the Wells FCSS and the Ogata FCSS, have diagnostic and prognostic utility. The Wells FCSS utilizes a difference from normal algorithm incorporating more than ten phenotypic parameters. The accumulation of these abnormalities is not only useful in diagnosis but is predictive of patient outcome (Wells 2003, Scott 2008, Alhan 2014). The recommended Ogata FCSS has evolved to include four cardinal parameters: (1) CD45 intensity on the myeloid progenitors, (2) frequency of lymphoblasts, (3) frequency of myeloid progenitors, and (4) granularity of the maturing myeloid cells. The Wells FCSS is more comprehensive as it uses more phenotypic characteristics, while the Ogata score is considered straightforward to implement in a routine setting (Della Porta 2012, Ogata 2009). This study compares the Wells FCSS and Ogata FCSS for sensitivity and specificity to detect clonal abnormalities documented by SNP/CGH microarray and conventional cytogenetics. Patients and Methods: The cohort included 99 patients with unexplained cytopenias whose bone marrow aspirates were submitted for SNP/CGH microarray and flow cytometry (HematoLogics). The immunophenotypic data were independently assigned a Wells FCSS (Cutler 2012) and an Ogata FCSS (Della Porta 2012). SNP/CGH microarray was assessed for MDS-associated genetic abnormalities. The findings were further correlated with conventional cytogenetic findings. Results: Of the 99 bone marrow aspirates, 20 exhibited clonal abnormalities associated with MDS. The Wells FCSS identified immunophenotypic abnormalities suggestive of MDS for 18 of 20 CGH positive specimens (sensitivity of 90%) and did not detect phenotypic abnormalities suggestive of MDS in 68 of 79 CGH negative specimens (specificity of 86%). In contrast the Ogata FCSS identified immunophenotypic abnormalities suggestive of MDS for 13 of 20 CGH positive specimens (sensitivity of 65%) and did not detect phenotypic abnormalities suggestive of MDS in 64 of 79 the CGH negative specimens (specificity of 81%). In an attempt to improve the sensitivity and specificity of the Ogata score, the granularity parameter was modified from side scatter channel mode of the granulocytes (compared to the side scatter mode of the lymphocytes) to the side scatter channel at the 15thpercentile of granulocytes (compared to the mean of lymphocytes). This modified parameter detected all specimens defined as hypogranular by the side scatter mode, and detected an additional 11 specimens as hypogranular. All of these specimens were detected as hypogranular by the Wells definition. This modified granularity method was then used along with the other three cardinal parameters to create a modified Ogata FCSS. The granularity modification resulted in improved sensitivity (70% versus 65%); specificity was unchanged. While the modified method outperformed the original, it did not match the performance of the Wells FCSS. Conclusions: In patients with unexplained cytopenias, the Wells FCSS demonstrates superior specificity and sensitivity than the Ogata FCSS for detecting myeloid immunophenotypic clones associated with SNP/CGH array and cytogenetic abnormalities. Modifying the Ogata granularity parameter marginally improves the sensitivity but does not improve the specificity. Implementation of the Wells FCSS requires a comprehensive understanding of phenotypic intensities and relationships in non-clonal hematopoiesis for patients with cytopenias. While the relative ease of implementing the Ogata FCSS is attractive, improvements are essential for diagnostic accuracy; improving the granularity parameter alone is not sufficient. Adding measurements for the maturing myeloid and erythroid compartments may increase the diagnostic utility of the Ogata FCSS but requires further study. Disclosures Brodersen: Hematologics Inc.: Employment. Menssen:Hematologics Inc.: Employment. Zehentner:HematoLogics Inc.: Employment, Equity Ownership. Stephenson:Hematologics Inc.: Employment. de Baca:Hematologics Inc.: Employment. Johnson:Hematologics Inc.: Employment. Singleton:Hematologics Inc.: Employment. Hartmann:Hematologics Inc.: Employment. Loken:Hematologics: Employment, Equity Ownership. Wells:HematoLogics Inc.: Employment, Equity Ownership.

Description: The concept of clonal diversity is becoming well accepted as a hallmark of cancer. As a tumor grows and progresses, the genetic landscape of the cell population can change. These changes are largely due to random errors occurring during each cell division or through mutational events stimulated by various exposures. When one of these random events occurs in the right locus it will result in a survival advantage for all of the subsequent offspring of that initial cell. Understanding the underpinnings of clonal diversity may prove to be an essential part of the treatment plan for patients, helping to guide drug selection and to determine the percentage of clones that may be responsive/resistant to specific treatments. Moreover, many of the therapies used to treat myeloma will likely induce mutations through their mechanisms of action or through unexpected secondary effects. Understanding the effects of individual therapies and specific combinations on the underlying mutation rates that drive the diversity of a tumor population will help to identify regimens that increase the underlying mutation rate and put the patient at an increased risk of developing an aggressive clone. These changes can be identified by next generation sequencing of the bulk tumor population compared to single cell clones that have been selected from that population. In order to identify the diversity of mutations found in the bulk tumor population, we propose that single cell cloning the parent population, and then sequencing and comparing across several individual clones will give a better idea of the random variety of mutations present in individual cells that originate from the same parent population. To identify the diversity present in a random population of myeloma cells we selected the human myeloma cell line KMS-18 as a model system. We sorted single cells from the KMS-18 parent population by FACS with the selection criteria based solely on the viable, single cells. These individually sorted cells expanded over a period of weeks until the population was large enough to be collected for analysis (target approximately 5E6 cells). Four of these single cell clones were selected (SCC_04, SCC_10, SCC_16, SCC_18) for analysis. We prepared whole genome libraries and captured a 3.2Mb region using the Agilent SureSelect Kinome capture kit. The final capture libraries were sequenced on the Illumina MiSeq platform to an average target region depth of 200X. Results were filtered to identify the number of mutations present exclusively in one subclone compared to another. Such events either existed in the original single cell or occurred early in the expansion of the single cell clone. To limit the analysis to events present in the original single cell or very early in the doubling process we identified the variants that were found at a frequency of 〉20%. Many of these events were present in multiple single cell clones that could define the clonal relationship of each original cell, however, 10% of these variants were unique to a single subclone. On average we observed 1.6 mutations per Mb of the target region. If this same mutation rate holds true across the entire genome, we would expect to see over 5000 unique mutations between any two random cells taken from a bulk tumor sample. Studies are currently ongoing to examine clonal diversity between generations of subclones. Further studies are also underway to look at changes in clonal diversity between different myeloma subtypes, with the hypothesis that more aggressive subtypes like t(4;14) and MAF may lead to a more diverse clonal population. If a more diverse clonal population correlates with more aggressive tumor subtype, then this returns full circle to the question of appropriate therapies, and if certain therapies may indeed increase diversity in the tumor population and result in a more aggressive relapse of the disease. Disclosures No relevant conflicts of interest to declare.

Description: INTRODUCTION: Perifosine (peri) is an oral, novel synthetic alkylphospholipid, with multiple effects on signal transduction pathways, including inhibition of Akt and activation of JNK. In vitro studies showed that peri induces cytotoxicity in both MM cell lines and patient (pt) MM cells resistant to conventional therapies, and augments dexamethasone (dex) and bortezomib-induced MM cell cytotoxicity (Hideshima T. et. al. BLOOD 2006). In vivo studies showed antitumor activity in a human plasmacytoma mouse model. Here we report the results of a phase II trial of peri, alone and + dex, in pts with relapsed or relapsed / refractory MM. METHODS: Pts received 150 mg of peri daily for a 21 day (d) cycle, and were assessed every cycle by serum- and/or urine-electrophoresis. Eligible pts had symptomatic relapsed or relapsed / refractory MM. Pts were permitted to receive bisphosphonate treatment. Concomitant steroids (prednisone 〉 10 mg/d), creatinine of 〉 3.0 mg/dL, and hemoglobin 〈 8.0 g/dL within 14 d of enrollment were exclusion criteria. Progressing pts, documented on 2 occasions at least one week apart, had dex 20 mg twice per week added to peri. Toxicities were assessed by NCI-CTCAE, v3.0. RESULTS: 64 pts (35 M/ 29 F, median age 62, range 38–79) have been treated to date. Median lines of prior treatment was 4 (range 1–11); 32 (50%) pts had relapsed and refractory MM. Prior therapy included dex (95%), thalidomide (89%), bortezomib (73%), lenalidomide (30%) and ASCT (61%). Among 48 pts currently evaluable for response, best response (EBMT criteria) to single agent peri after ≥ 2 cycles was MR in 1 pt, stable disease (〈 25% reduction in M-protein) in 22 pts (46%). Dex was added in 37 pts with PD, with 31 pts evaluable for response on the combination as follows: Peri + Dex N (%) Duration (wks) PR 4 (13%) 17, 24, 44+, 46+ MR 8 (25%) 3+, 12+, 19, 21, 25, 30, 32, 55+ Stable Disease 15 (47%) 6+ − 46 (median 12)* *4 pts ongoing at 6, 9, 11 and 24 wks Most common adverse events included nausea (74%, 8% G3); vomiting (61%, 5% G3); diarrhea (65%, 2% G3); fatigue (31%, 2% G3), increased creatinine (51%, 7% G3/4 in the context of PD and light chain nephropathy but reversible) and anemia (63%, 5% G3). 10 pts had G3/4 neutropenia which resolved. Dose reduction was required to 100 mg/d (n=16) or to 50 mg/d (n=4). 9 pts discontinued treatment due to side effects. Attributable toxicities otherwise proved manageable with supportive care and no peripheral neuropathy or DVT seen. CONCLUSION: Perifosine as monotherapy has modest activity, but in combination with dex showed significant activity in pts with relapsed/refractory MM, achieving PR + MR in 38%, and/or stabilization of disease in 47% of evaluable pts to date. It was generally well tolerated, although caution in pts with renal dysfunction is warranted. PK, IHC and gene array studies are ongoing. Other novel studies with peri in combination with bortezomib and with lenalidomide +/−dex are underway.

Description: Hemochromatosis is a common disorder characterized by excess iron absorption and accumulation of iron in tissues. Usually hemochromatosis is inherited in an autosomal recessive pattern and is caused by mutations in the HFE gene. Less common non-HFE–related forms of hemochromatosis have been reported and are caused by mutations in the transferrin receptor 2 gene and in a gene localized to chromosome 1q. Autosomal dominant forms of hemochromatosis have also been described. Recently, 2 mutations in theferroportin1 gene, which encodes the iron transport protein ferroportin1, have been implicated in families with autosomal dominant hemochromatosis from the Netherlands and Italy. We report the finding of a novel mutation (V162del) in ferroportin1 in an Australian family with autosomal dominant hemochromatosis. We propose that this mutation disrupts the function of the ferroportin1 protein, leading to impaired iron homeostasis and iron overload.