Citations

Abstract Background: Smoldering multiple myeloma (SMM) is an asymptomatic plasma cell proliferative disorder with a high risk of progression to symptomatic multiple myeloma. Identification of risk factors that predict progression of SMM to symptomatic MM could identify higher risk patients who might benefit from chemoprevention or more intensive surveillance. We hypothesized that increased monoclonal free kappa or lambda immunoglobulin light chains in smoldering myeloma (SMM), as detected by the serum free light chain (FLC) assay, indicates an increased the risk of progression to active myeloma. Methods: Of 276 pathologically confirmed SMM patients seen at the Mayo Clinic from 1970 to 1995, baseline serum samples obtained within 30 days of diagnosis were available in 273. Results: At a median follow-up of surviving patients of 12.4 years, transformation to active disease has occurred in 161 (59%) patients. An abnormal FLC ratio was present at baseline in 90% of patients. The best break-point for predicting risk of progression was a FLC ratio less than or equal to 0.125 or greater than or equal to 8 (hazard ratio, 2.3; 95% CI, 1.6–3.2) [Figure 1]. The extent of abnormality of FLC ratio was independent of SMM risk categories defined by number of plasma cells in the bone marrow and size of serum M-proteins (bone marrow plasma cells ≥ 10% and serum M protein ≥ 3 g/dL; bone marrow plasma cells ≥ 10% but serum M protein < 3 g/dL; and serum M protein ≥ 3 g/dL but bone marrow plasma cells < 10%). Incorporating the FLC ratio into the risk model, the division of patients into high-, intermediate-, and low-risk groups is 28, 42, and 30% with 5 year progression rates of 76, 51, and 25%, respectively [Figure 2]. Conclusions: The serum immunoglobulin FLC ratio is an important additional determinant of clinical outcome in patients with SMM. Figure Figure Figure Figure

Abstract The importance of bone marrow angiogenesis in multiple myeloma (MM) provided the initial rationale for using thalidomide in this disease, given its anti-angiogenic properties. Thalidomide (α-N-[phthalimido] glutarimide, C 13 H 10 N 2 O 4), a glutamic acid derivative, has proved useful in the treatment of both newly diagnosed and relapsed/refractory MM, however, its potential for teratogenicity lays a huge burden on its use as a therapeutic agent. Ongoing efforts have been directed towards development of analogues, which retain anti-MM activity without teratogenic effects. A product of cytochrome P450 2C19 isozyme biotransformation of thalidomide, 5′-OH-thalidomide, has been shown to be partly responsible for the anti-angiogenic effect of thalidomide. Based on the structure of this metabolite several N-substituted and tetraflourinated thalidomide analogues have been synthesized as potential anti-angiogenic agents and have shown pre-clinical activity in prostate cancer. In this study, we demonstrate potent anti-MM activity and delineate potential mechanisms of two such compounds, CPS11 (an N-substituted thalidomide) and CPS49 (a tetraflourinated thalidomide). Both CPS11 and CPS49 showed inhibition of proliferation of several MM cell lines as well as primary tumor cells from relapsed/refractory MM patients. These drugs had significant activity in myeloma cell lines resistant to conventional chemotherapy agents (dexamethasone, adriamycin, mitoxantrone and melphalan). Both the drugs were able to overcome the protective effects of growth factors like IL-6, IGF-1, and VEGF, and co-culture with bone marrow stromal cells (BMSCs), demonstrating their activity against myeloma cells in conditions simulating the natural microenvironment. In most of the cytotoxicity experiments, CPS49 was more potent compared to CPS11. An additive cytotoxic effect was noted when these agents were combined with dexamethasone and melphalan. Apoptosis was noted in MM1.S cells as evidenced by annexin and PI staining and a time- and dose-dependent increase in cleavage of poly ADP-ribosepolymerase (PARP) and caspase-8. Additionally, Z-VAD-FMK partially blocked these effects. Importantly, apoptosis triggered by these drugs was associated with down-regulation of anti-apoptotic proteins like Mcl1 and XIAP. In matrigel based angiogenesis assays both drugs demonstrated anti-angiogenic activity, CPS49 being more potent than CPS11. To further delineate their molecular mechanism of actions, we evaluated the effect of these drugs on various intracellular signaling pathways known to be important in myeloma. Both the drugs were able inhibit the PI3K/Akt and JAK/STAT pathways in MM1.S myeloma cells line. These encouraging in vitro data will provide the platform for testing these analogues initially in animal models and subsequently in the clinic.

Abstract Background: Cytotoxic T-cell infiltrates are a nearly universal finding in the bone marrow of patients with multiple myeloma. It has been postulated that presence of T-cells in the bone marrow of multiple myeloma (MM) patients represents an immune response against the tumor and therefore, might be associated with an improved prognosis. However, the impact of bone marrow T-cells on the prognosis of multiple myeloma patients has not been studied systematically. Methods: Bone marrow biopsies of patients with newly diagnosed multiple myeloma were stained by immnohistochemistry for the CD8 antigen and reviewed by a blinded hematopathologist. Three high power fields are reviewed for each biopsy and the total number of CD8 positive cells counted and reported. For patients with more than 300 cells per 3 fields, results were reported as >300. The number of bone marrow CD8 positive cells was then correlated with patients’ clinical data, including other prognostic factors and overall survival. Results: Bone marrow biopsy specimens from 100 patients, performed within the week of a diagnosis of multiple myeloma and collected between May 1998 and January 2001 were evaluated. The median number of CD8 positive cells was 270 (33 – >300). Patients’ characteristics are shown in Table 1. Median follow up was 30 months (0–80). The number of cytotoxic T-cells as a continuous variable was a risk factor for shortened overall survival, HR 1.86 (95% CI 1.11–3.35). Using minimal p value approach, the cutoff of 270 cells (the median) risk stratified patients into two groups: the median survival of patients with > 270 CD8 positive cells was 16 months vs. 48 months in patients with ≤270 cells, p=0.005 (Figure). In multivariate analysis including age, B2M, albumin, CRP, bone marrow plasma cell percentage and plasma cell labeling index, the number of cytotoxic T-cells was an independent predictor of overall survival was HR 3.1, p=0.0017. Conclusion: We show that the number of cytotoxic T-cells in the bone marrow is a strong and independent prognostic factor in patients with newly diagnosed multiple myeloma. Our observation does not contradict the hypothesis that cytotoxic T-cells participate in an immune response against the tumor since our findings may represent a higher level of immune response associated with baseline aggressive disease biology. However, our study suggests for the first time that increased marrow cytotoxic T-cells have an adverse effect on outcome in myeloma, and suggest that these cells may have a direct facilitating effect on tumor growth and on the marrow microenvironment. Further studies of the biology of behind this observation are warranted. Characteristic N Median (range) Gender male 61 CRP 81 0.4mg/L (0.01–11.2) Albumin 99 3.6 g/dL (2.6–5.4) B2microglobulin 94 4.0 (0.9–28) μg/mL Marrow PC% 90 45% (11–99) PC labeling index 90 high (>1%) 36 BM CD8 cells 100 270 (33 – >300) ISS 94 1 19 2 41 3 34 Figure Figure

Abstract Background: Syndecan-1 (CD138) is a heparan sulfate bearing proteoglycan found on various epithelial cells as well as on B lineage cells depending on its stage of development. Syndecan-1 (CD138) is abundantly expressed by plasma cells, especially myeloma cells. The extra cellular domain along with the heparan sulfate side chains can be cleaved off the cell surface and can be detected in the serum as soluble syndecan. Syndecan possibly plays a multifunctional role in the biology of myeloma. It has been shown to be an independent prognostic factor in patients with multiple myeloma. It has also been shown to promote myeloma cell growth through different mechanisms. Its expression has also been suggested to correlate with bone disease in MM. Methods: In this study we studied serum levels of soluble syndecan in newly diagnosed MM patients enrolled in the Eastern Cooperative Oncology Group (ECOG) E9486 and its associated correlative laboratory clinical trial E9487. We evaluated the prognostic value of syndecan in MM and its relationship to other known prognostic factors for this disease. In addition, syndecan levels were correlated with clinical and laboratory markers of bone disease. Results: A total of 501 patients were studied and the median serum syndecan-1 was 158 ng/mL. Syndecan levels correlated positively with other prognostic factors and markers of tumor burden such as β2-microglobulin (correlation coefficient 0.3; P <0.00001), labeling index (0.25; <0.0001), creatinine (0.23; <0.0001), soluble IL6 receptor (0.3; <0.0001), BM plasma cell percentage (0.16; <0.0006), and disease stage (P=0.0007). Significant differences in the overall and progression free survival was found between two groups of patient separated using the median value as cut-off. The High syndecan group had a median overall survival of 36.3 months compared to 49.3 months for the low syndecan group (P < 0.0001). Similarly, the high syndecan group had progression free survival of 25.4 months compared to 33.5 months for the low syndecan group (P < 0.0001). In a proportional hazards model including syndecan-1 as well as labeling index, β2M, Platelet count, IL-6R, syndecan-1 retained its prognostic value for overall survival (HR 1.3, P = 0.021). Syndecan levels were correlated with various bone markers including C-terminal telopeptide of type I collagen (ICTP), osteocalcin (OC), C-terminal type I procollagen (PICP), bone-specific alkaline phosphatase (BAP), and tartrate resistant alkaline phosphatase (TRAP) and were found to correlate only with ICTP (0.25, P < 0.0001). No correlation was found between clinical markers of bone disease including presence of lytic lesions, osteoporosis and pathologic fractures on X-rays or bone pain. Conclusion: In this large study, we once again confirm the prognostic value of serum syndecan-1 levels in large group of patients with newly diagnosed myeloma. Syndecan-1 level correlates with other disease markers. Syndecan levels also correlated with ICTP, a marker of bone turnover, though no strong correlation was found between syndecan levels and clinical markers of myeloma bone disease. The biological basis of these finding needs further evaluation.

Abstract PS-341 (bortezomib, Velcade™) is a promising novel agent for treatment of advanced multiple myeloma (MM); however, 65% of patients with relapsed refractory disease in a phase II study did not respond. Lysophosphatidic acid (LPA) is a phospholipid which mediates tumor cell migration and invasion. Recent studies have shown that inhibition of LPAAT-β inhibits both Ras/Raf/Erk and PI3K/Akt signaling cascades. We have previously shown that lysophosphatidic acid acyltransferase (LPAAT)-β inhibitor CT-32615 triggers caspase-dependent apoptosis, and can overcome resistance to conventional therapeutics (ie, dexamethasone, doxorubicin, melphalan) in MM cells. In this study, we determined whether CT-32615 could also overcome resistance to PS-341. We first characterized molecular mechanisms of resistance to PS-341 in DHL-4 lymphoma cells. DHL-4 cells express low levels of caspase-3 and caspase-8; furthermore, no cleavage in caspase-8, caspase-9, caspase-3, poly ADP-ribose polymerase (PARP), or DNA fragmentation factor (DFF) 45 is triggered by PS-341 treatment. We have previously shown that PS-341 treatment triggers phosphorylation of c-Jun NH2-terminal kinase (JNK), which subsequently induces caspase-dependent apoptosis; conversely, JNK inhibition blocks PS-341-induced apoptosis. Here we show that PS-341 does not induce phosphorylation of SEK-1, JNK, and c-Jun in DHL-4 cells, suggesting that it does not trigger a stress response. Importantly, CT-32615 inhibits growth of DHL-4 cells in a time- and dose-dependent fashion: a transient G2/M cell cycle arrest induced by CT-32615 is mediated via downregulation of cdc25c and cdc2. CT-32615 triggers swelling and cell membrane destruction in DHL-4 cells, without caspase/PARP cleavage or TUNEL-positivity, suggesting a necrotic response. Our studies therefore demonstrate that LPAAT-β inhibitor CT-32615 triggers necrosis even in PS-341-resistant DHL-4 cells, providing the framework for its evaluation to overcome clinical PS-341 resistance and improve patient outcome.

TPS8057 Background: Multiple myeloma (MM) is the second most prevalent blood cancer, representing approximately 1% of all cancers. Although overall survival has improved in recent years due to new approved agents, the vast majority of MM patients (pts) ultimately stop responding to treatment. Moreover, about a quarter of MM pts characterized as “high risk” experience limited benefit from existing treatments. Seminal genomic sequencing research efforts, such as the MMRF CoMMpass study, have highlighted that a large number of MM cases harbor potentially actionable oncogenic molecular alterations and published reports on small numbers of cases suggest that Precision Medicine (PM) interventions clinically targeting such actionable drivers may benefit MM pts. These results suggest that PM approaches in MM are possible and should be further studied clinically. To that end, we have launched MyDRUG, a master protocol aimed at developing new myeloma regimens based on individual pt’s genomics. Methods: This study is a Phase I-II, multicenter master protocol in functional high risk MM patients (i.e patients having progressed from baseline treatment within 18 months without maintenance or 3 years on maintenance) with 1-3 prior lines of therapy. Patients are screened for actionable genomic alterations on the CLIA-grade MI-ONCOSEQ platform. Patients with actionable alterations are assigned to the appropriate targeted agent used in combination with a backbone regimen ixazomib, pomalidomide and dexamethasone (IPd), whereas patients without such alterations go on an immune arm (see Table). Inclusion criteria include measurable disease (as measured by M-protein and FLC) and acceptable hematologic and metabolic functions. A maximum of 12 evaluable pts will be accrued onto the phase I portion of each arm and an additional 21 evaluable phase II pts will be accrued onto each arm for a total of 27 evaluable pts at the MTD combination dose. The primary objective of the study is to evaluate ORR per IMWG consensus criteria. Secondary objectives are to assess adverse events (AEs), progression-free survival, and overall survival (OS). Experimental correlative aims include assessing molecular or clonal response, molecular and immune signatures of resistance/response, MRD. Clinical trial information: NCT03732703. [Table: see text]

TPS8052 Background: The most common translocation in multiple myeloma (MM) is t(11;14)(q13;q32), present in approximately 20% of cases. MM cells with t(11;14) usually have a favorable high BCL-2 level and inferior outcomes compared to standard risk MM. Venetoclax (VEN) is a potent, selective, orally available small-molecule BCL-2 inhibitor that induces cell death in MM cell lines and primary samples. VEN has single agent activity in relapsed/refractory MM (RRMM) with an acceptable safety profile, especially in t(11;14) MM; however, non- t(11;14) MM patients may benefit from single agent VEN and VEN incorporated in multi-agent RRMM regimens. Dexamethasone (d) promotes Bcl-2 dependence in MM resulting in sensitivity to VEN and this combination with bortezomib (Vd-VEN) has an acceptable safety profile with high response rates in heavily pre-treated MM. Combination therapy with daratumumab and bortezomib (DVd) has become a standard of care in RRMM. Our hypotheses are that the addition of VEN will improve upon this standard and be most effective in the t(11;14) positive subset. Methods: Eligibility criteria include RRMM with measurable disease, not bortezomib refractory, platelet count > 100K. t(11;14) is an integral biomarker with status (positive or negative) established at registration. After a Ph1 study to determine the recommended phase 2 VEN dose, patients are randomized to DVd +/- VEN [stratified by prior lines of therapy and R-ISS]. The primary Ph2 objectives are to compare 8-cycle minimal residual disease (MRD) negative rate and to inform the role of t(11;14) as a biomarker. The Ph2 design proposed by Freidlin et al. follows a decision algorithm as outlined in the table below. Simulations were run to establish an optimal sample size given various parameters including biomarker prevalence and power to make appropriate decisions for a Ph3 design. Target Ph2 accrual is 240 patients with a 1/3 positive:2/3 negative t(11;14) split. Clinical trial information: NCT03701321. [Table: see text]

e19519 Background: NSMM is a rare subtype of myeloma that has not been well characterized in the era of novel agent induction therapy. Thus, we evaluated the clinical and cytogenetic features of patients with newly diagnosed NSMM evaluated at our institution. Methods: We evaluated all NSMM patients seen at the Mayo Clinic, Rochester from 2008-2017 based on the absence of a detectable monoclonal protein in their serum and urine electrophoresis and having a concurrent involved serum immunoglobulin light chain of less than 5 mg/dL. Survival analysis was performed by the Kaplan-Meier method and clinical and cytogenetic features were reported descriptively. Results: This cohort consisted of 30 consecutive patients with NSMM with a median age of 62 years (40-79) of which 16 (53%) were male. The median bone marrow plasma cell involvement was 70% (Range: 0 – 100). There were 3/25 (12%) patients who presented with a creatinine of 2 mg/dL or greater and 6/23 (26%) patients who presented with hypercalcemia (11 mg/dL or greater). The ISS classification of this cohort is as follows: Stage 1- 8 (36%) patients, Stage 2- 3 (14%) patients and Stage 3- 11 (50%) patients. There were 6 (21%) out of 28 patients with high risk cytogenetics (either del 17p, t(4;14), t(14;16) or t(14;20). The distribution of primary cytogenetic abnormalities among this cohort was as follows: t(11;14) – 16 (57%), t(4;14) – 1 (4%), trisomies – 6 (21%) and other - 5 (13%). The median overall survival (OS) for patients in this cohort was 59 months (95% CI: 34 – 64). In comparison to a control cohort of newly diagnosed secretory myeloma patients matched for age, gender and year of diagnosis in a 1:2 ratio, the median OS was worse for the NSMM cohort but this was not statistically significant (59 vs. 92 months, p = 0.257). Conclusions: Patients diagnosed with NSMM had a predisposition towards having a t(11;14) primary cytogenetic abnormality. In the era of novel agent induction therapy, they may have a survival outcome that may be worse than newly diagnosed secretory myeloma patients.

e19503 Background: The risk of venous thromboembolic events (VTE) despite mandatory thromboprophylaxis (TPx) in patients with multiple myeloma (MM) receiving contemporary lenalidomide (Len)-based regimens is not well-defined. Methods: Medline, Embase, and CENTRAL databases were queried to identify phase 1-3 clinical trials of Len-based regimens with mandatory TPx published until April 2018. Random effects meta-analysis was performed using CMAv3 software to obtain crude, and per 100 patient-cycle incidence rate (IR). Prespecified subgroup analyses were performed to obtain IR for VTE by (1) line of treatment (newly diagnosed [ND], relapsed/refractory [RR] and maintenance [Mx] phase) and (2) different Len-based regimens. Results: We screened 1069 citations and analyzed data from 43 trials with 8696 patients, including 17 in ND, 23 in RR and 3 in Mx phase. 12 out of 43 were Phase 3 randomized controlled trials. The overall incidence of VTE in 8696 patients was 4.6% (95% CI, 3.7-5.8%), with 1.0 event (95% CI, 0.7-1.4%) per 100 patient cycles. In patients with NDMM (n = 3295), 193 (5.9%) experienced VTE with a pooled IR of 0.9 (95% CI, 0.3-2.3) per 100 patient-cycle. Among RRMM patients (n = 3981), 251 (6.3%) experienced VTE with a pooled IR of 1.0 (95% CI, 0.4-2.3) per 100 patient-cycle. The incidence of VTE was highest with the following triplet regimens: Rd + Proteasome inhibitor (PIs) and Rd + Anthracycline (Ac) [+/- Vincristine], with the latter having a high risk of VTE regardless of the treatment phase (table). With Len Mx (n = 1420), 38 (2.7%) patients had VTE, with a pooled IR of 0.2 (95% CI, 0-2.1) per 100 patient-cycle. Conclusions: Patients with MM undergoing treatment with Len-based regimens remain at risk of VTE despite current TPx strategies. The estimates of VTE IR provided by this analysis can guide clinicians in assessing treatment-related risk of VTE in this setting. [Table: see text]