Lacy, Martha Q.

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 INTRODUCTION The bone marrow plasma cell labeling index (PCLI) and the beta 2 microglobulin (β2M) when combined provide a powerful staging system highly predictive of outcomes in myeloma patients (Greipp et. al., Blood, 81:3382–87, 1993). This staging system lacks information on cytogenetics and it has been demonstrated that deletion 13 (Δ13) by conventional cytogenetics is associated with poor outcomes. In conventionally treated patients Δ13 by FISH is an independent prognostic variable. We analyzed the importance of Δ13 by FISH in a cohort of patients receiving high dose therapy and assessed its value relative to known prognostic parameters, PCLI and β2M. PATIENTS AND METHODS 238 patients undergoing high dose therapy between January 1990 and September 2001 were studied. The minimum follow-up of patients was 34 months. The median age of the cohort 56 years. Interphase fluorescence in situ hybridization with Probes LSI13/D13S319 was performed with simultaneous immunofluorescent detection of bone marrow plasma cells by the presence of cytoplasmic immunoglobulin. PCLI was done by a slide-based immunofluorescence method with antibodies to bromodeoxyuridine. RESULTS Of the 238 patients studied 215 had a successful FISH analysis and 212 of these had a bone marrow labeling index. Deletion 13 was absent in 104 patients (48%) and present in 111 patients. The overall survival and time to progression for patients lacking and having deletion 13 was 47.8 vs 26.4 (OS) and 19.0 vs 11.7 (TTP)months respectively. A Cox model was constructed with the PCLI, the presence or absence of deletion 13 and serum β2M. Although all three variables were independently predictive of survival and time to progression, the β2M was the weakest of these three variables. The hazard function for survival for PCLI, deletion 13 and β2M was 1.4, 1.6 and 1.1 respectively. A staging system was constructed assigning 1 point for the presence of deletion 13 or a PCLI ≥ 1%. Patients fell into 3 categories of 0 points (N=69), 1 point (N=104) and 2 points (N=39). The overall survival for the three groups respectively were 56.5, 36.6 and 13.7 months (figure). The time to progression for the three groups were 22.3, 15.4 and 8 months (P<0.001). When analysis was limited to those patients transplanted within 12 months of diagnosis the results remained significant (P<0.003) with median survivals of stage 0, 1 and 2 of 72.5, 59.1 and 15.4 months respectively. TTP for stage 0, 1 & 2 in this same cohort of patients transplanted < 12 mos after diagnosis is 30.6, 21.5 & 8.2 months respectively. CONCLUSION Deletion 13 by FISH is an independent predictor of survival and time to progression providing information independent of the β2M and the PCLI. Combining the PCLI with FISH del 13 identifies patients who following transplant have a median time to progression of 8 months and a median OS of 13.7 months. This group may not benefit from autologous transplant and are candidates for novel therapeutic approaches. Figure Figure

Abstract Background: Primary systemic amyloidosis (AL) is an incurable plasma cell dyscrasia with limited therapeutic options. Based on the observed 25 to 50% partial response rates in myeloma patients, thalidomide has been tried in patients with AL. Two reports (Dispenzieri et al, Amyloid10:247; 2003; Seldin et al, Clinical Lymphoma3:241; 2003) have demonstrated that standard dose, single agent thalidomide (≥ 200 mg per day) has a higher than expected rate of adverse events in AL patients without hematologic or organ response, potentially due to rapid patient attrition. We therefore performed a prospective clinical trial using low dose, single agent thalidomide in patients with AL. Methods: In August 2001, we began accrual to a phase II trial of low dose thalidomide for patients with AL. Eligibility included: age ≥ 18 years, creatinine <3mg/dL, total bilirubin < 3 mg/dL, ANC ≥ 1 x 10(9)/L, left ventricular ejection fraction of >45%, histochemical proof of amyloid and a monoclonal plasma cell dyscrasia, symptomatic organ involvement, and ability to supply informed consent. Patients were started with thalidomide 50 mg per day, with a dose escalation to a maximum dose of 400 mg per day over a 19 week period as tolerated. Patients were evaluated monthly by their physician. These office visits were staggered with telephone toxicity checks monthly by the primary investigator. The endpoint of the study was hematologic and organ response by 12 months. Results: Eighteen patients were treated. The median age was 66 years (range 43–83). Eight were male. Twenty-eight percent had an ECOG performance status of 2 or 3. Five patients (28%) had received no prior therapy. Five had received one prior regimen; 6, two prior; and 2, ≥ three. Three had received prior PBSCT. Sixteen patients had a monoclonal lambda plasma cell disorder; two, monoclonal kappa. Immunoglobulin heavy chains were IgG (n=9), IgM (n=5), IgA (n=1), and none (n=3). Twelve patients (67%) had cardiac involvement; twelve (67%) renal; two liver; two symptomatic gastrointestinal; one pulmonary; and two nerve involvement. Study participants had significant symptomatology at baseline with dyspnea (n=12), light-headedness (n=6), and edema (n=13). Median number of organs involved was 1, range (1 to 4). Median time on study drug was 5.6 months, range 0.7–24 months. Reasons for discontinuation included: progression, 7 (39%); 3 each (17%) for adverse events, death, and patient refusal; and other causes in 2. The median tolerated dose was 100 mg. There were no hematologic responses observed. The overall organ response rate was 11%. There was one renal response at 1 year and a gastrointestinal response at 3 months with 24 hour fecal fat changing from 21 to 8.7 grams. The most common adverse effects attributable to thalidomide were: constipation (78%), edema (44%), sinus bradycardia (17%), dyspnea (11%), and paresthesias (11%). Light-headedness, rising creatinine, thrombosis, infection, syncope, and rash was attributed to the thalidomide in 5.6% of patients. Conclusions: Low dose thalidomide is tolerable in this fragile group of patients, but as a single agent efficacy is limited. Further study of combination therapy of thalidomide and low dose dexamethasone may be warranted. A study using the next generation immune modulatory drug Revlimid is scheduled to open to accrual. Funded by NIH grant CA 91561 and Celgene.

Abstract The infused autograft absolute lymphocyte count (A-ALC) is an independent prognostic factor for survival after autologous hematopoietic stem cell transplant (AHSCT) in non-Hodgkin’s lymphoma (NHL). Previous studies have shown that A-ALC directly correlates with the peripheral blood absolute lymphocyte count (PC-ALC) at the time of apheresis collection. However, factors affecting the PC-ALC at apheresis remain undefined. We hypothesized that one possible factor impacting PC-ALC may be the time interval from last chemotherapy to stem cell collection (TILC). Data from 160 patients who underwent AHSCT for treatment of relapsed NHL at Mayo Clinic between 1993 and 2001 were collected and analyzed. The primary end point of this study was correlation between TILC and PC-ALC, and our analysis revealed a strong correlation (r = 0.67, p < 0.0001). Further analysis revealed higher PC-ALC numbers in patients with TILC ≥ 60 days versus TILC < 60 days (median of 7.44 x 109/L in the ≥ 60 day group versus 3.87 x 109/L in the < 60 day group, p < 0.0001). Both the median overall survival (figure below) and the progression-free survival were longer in the TILC ≥ 60 days group versus the TILC < 60 days group (76 versus 21 months, p < 0.0037; 76 versus 11 months, p < 0.018, respectively). These findings were independent of other prognostic indicators for relapsed NHL patients undergoing APHSCT. The data supports our hypothesis that TILC affects PC-ALC and survival post-AHSCT in NHL. Figure Figure

Abstract Background: It has been hypothesized that multiple myeloma (MM) remains incurable due to a stemcell/proliferative component that responds only partially to standard treatment regimens. We have shown that abnormal production of IL-1beta in the myeloma microenvironment stimulates the generation of paracrine IL-6, the central myeloma growth factor, and that IL-1Ra will inhibit paracrine IL-6 production in vitro. Based on these preclinical studies, we have developed a Phase II trial using IL-1Ra. Purpose: To determine the biologic and clinical activity of IL-1Ra (Anakinra) in a subgroup of smoldering MM (SMM) patients that present with an elevated plasma cell labeling index (PCLI; a measure of myeloma cell proliferation). Methods: Patients that had ≥ 10% bone marrow plasma cells and/or an IgG or IgA M-spike ≥ 3 g/dL and did not require immediate chemotherapy were eligible. Patients received 100 mg of Anakinra (IL-1Ra) SQ qd for a total duration of 6 months. Results: Eleven of 29 patients enrolled on the protocol had an on-study PCLI > 0 and have completed six months of therapy with IL-1Ra. Seven of the 11 patients had a decrease in the PCLI of 75–100% (see Table), three had only modest changes (≤ 50%), and one had an increase. The decrease in the PCLI paralleled a decrease in the high sensitivity C-reactive protein (CRP) in all cases (43–90% decrease). The above results suggested that IL-1Ra inhibited IL-6 production in the myeloma microenvironment, as evidenced by a reduction in the CRP, resulting in suppression of myeloma cell growth. However, there was little effect on the M-protein. To investigate these clinical observations, we co-cultured IL-1beta transduced +/− myeloma cells with stromal cells +/− dexamethasone (DEX), IL-1Ra, or both for 48 hours and quantitated the percent apoptotic cells by flow cytometry and IL-6 production by ELISA. The results showed: 1) IL-1Ra was superior at inhibition of IL-6 but caused no increase in apoptosis; 2) the DEX apoptotic effect was eliminated by IL-6 3) DEX and IL-1Ra combined induced maximal IL-6 inhibition and apoptosis of myeloma cells. Based on these in vitro results, 5 of the 11 patients have been advanced to IL-1Ra + low dose DEX (20 mg/week) resulting in 4/5 minor responses (25–50% decrease M-spike) and 1/5 with stable disease. Conclusion: The use of IL-1Ra is a novel targeted therapeutic strategy that interferes with myeloma cell growth. By inhibiting IL-1beta induced IL-6 production, IL-1Ra specifically targets the proliferative myeloma fraction and also complements DEX induced apoptosis. Preliminary studies on the use of IL-1 inhibitors in SMM patients to delay/prevent progression to active MM and to minimize toxicity appear encouraging, however, more patients need to be studied. Patient # On Study 6 months % Change 1 PCLI 0.8% 0% −100% CRP (mg/L) 2.01 0.58 − 71% M-spike (g/dL) 2.5 2.4 − 4% 2 PCLI 0.2% 0% − 100% CRP 0.89 0.51 − 43% M-spike 2.8 2.7 − 3% 3 PCLI 0.2% 0% − 100% CRP 5.7 1.02 − 82% M-spike 2.9 2.9 0% 4 PCLI 4.1% 1% − 75% CRP 3.39 1.51 − 55% M-spike 3.8 3.6 − 5% 5 PCLI 0.4% 0% − 100% CRP 3.46 0.34 − 90% M-spike 2.7 3.1 15% 6 PCLI 0.3% 0% − 100% CRP 17 5.35 − 68% M-spike 4.4 3.9 − 11% 7 PCLI 1.2% 0% − 100% CRP 1.06 0.32 − 70% M-spike 3.1 2.9 − 6%

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.

8039 Background: Treatment of MM has evolved significantly over the past decade, with increasing use of multi-drug combinations for initial therapy. In addition, supportive care approaches have also improved. We examined how these improvements have translated to survival outcomes in patients with newly diagnosed MM. Methods: Patients (n=3449) with a diagnosis of MM made between 2004 and 2017 and seen at Mayo Clinic within six months of the diagnosis, were included in this analysis. Patients were divided into three groups based on the year of diagnosis; group 1- 2004-07 (n=831), group 2-2008-12 (n=1161), and group 3-2013-17 (n=1457). Survival of the groups were estimated using Kaplan-Meier method, and compared using log rank test. Results: The median age was 64 years (22 to 96); 60% were male and 40% were female. 14% were >75 years, 33% were aged 65-75 and 53% were <65 years. The median overall survival for the whole cohort was 5.7 years (95%CI; 5.4, 6.3). The median OS for the groups 1, 2 and 3 were 3.9, 6.3 and NR, respectively; p<0.001. The 4-year survival estimates were 50%, 62%, and 75%, respectively. We then explored the improvements in patient subgroups. While all patients experienced improvements in OS over time, improvement in group 3 was most prominent for those >75 years. In patients <65 years, the 4-yr OS for groups 1, 2 and 3 were 57, 71, and 79% respectively. In patients 65-75 years of age, the 4-yr OS for groups 1, 2 and 3 were 48, 60, and 75% respectively. In patients >75 years, the 4-yr OS for groups 1, 2 and 3 were 24, 35, and 56% respectively. While patients with high-risk disease did not see as much benefit in the earlier period, substantial progress was seen in the last group. The 3-yr OS for patients with high-risk cytogenetics were 52, 55, and 73% for groups 1, 2 and 3 compared to 67, 75, and 85% for standard-risk cytogenetics respectively. 2067 patients were staged according to the International Staging System and the median OS for stages 1, 2 and 3 were 6.5, 4.6 and 2.4 in group 1; 9.2, 6.6 and 3.5 in group 2 and NR for any of the stages in group 3. Conclusions: The results confirm continued improvement in survival of newly diagnosed multiple myeloma patients, including elderly and high-risk MM.

8034 Background: Minimal residual disease (MRD) assessment in multiple myeloma (MM) has improved our ability to assess disease activity, resulting in more advanced prognostication. While MRD assessment remains confined to the bone marrow (BM) plasma cell population, serum studies including immunofixation (IFE) are required to complete response evaluation. The significance of those who are MRDneg yet have detectable monoclonal protein through IFE remains unclear. Methods: We retrospectively studied 256 MM patients who had MRD assessment via the Euroflow multiparametric flow cytometry on the BM with concomitant serum IFE testing. Patients who were MRDneg were included in the study. Outcomes included probability of disease progression (PD) at 1 year. The Cox-proportional hazards model was used to compare probability of PD among different groups. Time to progression (TTP) was calculated as the difference from date of MFC analysis to PD in months. Results: Among the entire cohort, 178 (70%) patients were MRDneg and median follow-up from MRD assessment was 6.3 months. Among these patients, 74 (42%) had a positive IFE at the time of MRD analysis. Within the MRDneg/IFEpos group, 31 (42%) patients remained IFEpos after a median follow up of 5.5 mo from initial MRD/IFE testing while 34 patients eventually became IFEneg after a median of 2.8 mo with no subsequent IFE available in 9 patients. The 1 year probability of PD in the MRDneg/IFEneg group was 20% compared to 41% in the MRDneg/IFEpos group (P < 0.01, Wilcoxon test). When comparing subsequent IFE status in those who were MRDneg/IFEpos, those who remained IFEpos had a trend towards shorter TTP compared to patients who later became IFEneg. Conclusions: Persistent monoclonal protein in the face of MRD negativity predicts for a shorter TTP. This likely reflects persistent disease that was not sampled on the BM aspirate in many of these patients compared to those who eventually become IFEneg with a trend towards longer TTP owing to the prolonged half-life and therefore clearance of M protein. This supports the current strategy of assessing for MRD at the time of suspected complete response to reduce the chance of positive MRD tests and thus avoidance of multiple BM exams.

8031 Background: Our prior studies identified the prognostic significance of ≥400 cPCs/150,000 analyzed events quantified by MFC in NDMM. We evaluated if a similar quantification of cPCs using MFC can add prognostic value to the current R-ISS classification of NDMM pts. Methods: We evaluated all NDMM pts seen at the Mayo Clinic, Rochester from 2009-2017 who had their peripheral blood samples evaluated by 6-color MFC prior to therapy. The cPCs detected were reported as the number of clonal events/150,000 collected total events. Survival analysis was performed by the Kaplan-Meier method and differences assessed using the log rank test. Results: This cohort consisted of 566 consecutive pts with NDMM with a median age of 66 years (27-95). The distribution of the R-ISS classification of this cohort is as follows: Stage 1- 128 (23%) pts, Stage 2- 369 (65%) pts and Stage 3- 69 (12%) pts. The median number of cPCs was 59 (0-46,412) / 150,000 events. The median time-to-next-treatment (TTNT) and overall survival (OS) for pts with ≥400 cPCs (n = 140, 25%) was 19 months and 46 months compared with 34 months and 77 months for those with < 400 cPCs respectively (n = 426, 75%) (p < 0.001 for both). The median TTNT and OS for pts based on their R-ISS classification as well as with and without the presence of ≥400 cPCs by MFC was as follows in the following Table. Conclusions: Quantifying ≥400 cPCs/150,000 analyzed events by MFC can potentially upstage the R-ISS classification of a subset of NDMM pts with stage I and II disease and identify those pts with a worse than expected survival outcome.[Table: see text]

8019 Background: POEMS syndrome is a rare paraneoplastic syndrome caused by an underlying plasma cell disorder. Most of the information regarding treatment has been gleaned from retrospective data. The combination of a proteasome inhibitor, an IMiD and corticosteroid is known to be highly effective among patients with myeloma. Methods: We designed a pilot using a 28-day oral regimen of ixazomib (4 mg days 1, 8, 15), lenalidomide (25 mg days 1-21), and dexamethasone (20 mg days 1, 8, 15, 22). Aspirin and acyclovir were used for prophylaxis. Eligibility included a diagnosis of POEMS syndrome, a plasma VEGF > 200 pg/ml, a PS < 3. There were two groups [gp] (intended enrollment 15 per gp): Gp A, 13 cycles for patients (pts) who had relapsed or refractory disease; Gp B, 3 cycles for pts destined for high-dose chemotherapy with stem cell transplant. Primary endpoint was VEGF complete response (CR = normalization) after 3 cycles. Secondary endpoints included safety, hematologic response, and overall survival at 3 and 12 months. Other domains including PET response, clinical responses including neurologic response were also studied. To date, 13 pts enrolled since 10/31/2016—4 to Gp A and 9 to Gp B. 11 pts were analyzed (2 dropped out before receiving any therapy). Data were frozen as of 1/21/2019. Results: Median age was 55; 73% were male. So far, overall 64% met primary endpoint of VEGF CR (Table). The median follow-up of survivors is 12.4 mo (6, 24). 1 pt came off study for non-responsive disease and died thereafter. 38% of patients had grade 3+hematologic AE; 72% had grade 3-4 non-hematologic AE. These included: rash, respiratory infection and hypotension in 2 each; atrial fibrillation, diarrhea, edema, dyspnea, and thromboembolism in 1 each. 4 pts had non-objective worsening of their neuropathy. Conclusions: These preliminary results suggest that Ixa-Len-Dex is an effective and tolerable regimen for patients with POEMS syndrome. Clinical trial information: NCT02921893. [Table: see text]