Markovic, Svetomir N.

7605 Background: In the setting of autologous stem cell transplantation (ASCT) in multiple myeloma (MM), it has been shown that peripheral blood absolute lymphocyte count (ALC) on day 15 is an independent prognostic factor for clinical outcomes. Recently the International Staging System (ISS) for MM has been developed as a simple staging system to assess survival in newly diagnosed MM patients. The role of ALC on survival in newly diagnosed MM patients is unknown. Methods: Between 1994 and 2002, 1,835 consecutive MM patients were evaluated at the Mayo Clinic, Rochester. Of these patients, we retrospectively analyzed 584 MM patients that were originally diagnosed and followed at the Mayo Clinic. The primary end point was to assess the role of ALC at the time of MM diagnosis on overall survival (OS). OS was measured from the date of diagnosis to time of death or last follow-up. ALC was analyzed as a continuous variable and dichotomized based on finding the optimal cut point based on the log-rank statistic. ALC was then compared to the ISS. Results: The median age of the cohort was 67 years (range: 29–94 years), including 234 females and 350 males. The median follow-up was 32 months (range: 1–136 months). The median ALC at diagnosis was 1.2 x 109/L (range: 0.12–5.44 x 109/L). ALC, as a continuos variables was identified as a prognostic factor for OS (HR= 0.466, 95%CI= 0.396–0.547, p < 0.0001). MM patients with an ALC ≥ 1.3 x 109/L experienced a superior OS compared with MM patients with an ALC < 1.3 x 109/L (55.5 months versus 22.6 months, p< 0.0001). In the multivariate analysis, ALC was independent prognostic factor when compared to the ISS (HR = 0.580, 95%CI=0.518–0.647, p< 0.0001). Conclusions: Our study shows that ALC at diagnosis for MM is an independent prognostic factor for OS, suggesting how the host immune status plays a critical role in the survival of patients with MM. No significant financial relationships to disclose.

7586 Background: The immunologic mechanisms of action of rituximab have been described as complement mediated lysis, vaccine like effect, antibody-dependent cellular cytotoxicity (ADCC) and the cellular microenvironment. We hypothesized that in the treatment of follicular grade 1 lymphoma (FL), the presence of a stronger host immune status prior to rituximab therapy would result in a prolonged time to progression (TTP). As a surrogate marker for immune status, we evaluated the absolute lymphocyte count (ALC) prior to rituximab treatment. Methods: Between 1996 and 2002, 1,104 consecutive FL patients were evaluated at Mayo Clinic Rochester. Of these patients, we retrospectively analyzed a group of all FL patients who received rituximab (375 mg/m2 once a week for four weeks) alone at any time during their lymphoma treatment at the Mayo Clinic (n=79). The primary end-point was to assess the impact of ALC just prior to rituximab therapy on TTP for FL. Results: The median age of the cohort was 56.6 years (range: 25–98 years). The median follow-up was 12.5 months (range: 1–76 months). An ALC count of ≥ 890 cells/μL prior to rituximab therapy predicted a longer TTP compared with an ALC < 890 cells/μl (25 months versus 8 months, respectively, p < 0.0124). A higher complete response rate was observed in the ALC ≥ 890 cells/μL group compared with the ALC < 890 cells/μL group [15/40 (38%) vs 5/39 (13%), p < 0.035]. The groups were balanced regarding the Follicular Lymphoma International Prognostic Index (FLIPI) (p = 0.794). Multivariate analysis demonstrated ALC ≥ 890 cells/μL prior to rituximab therapy as an independent prognostic factor for TTP when compared to hemoglobin, LDH, and Ann Arbor stage. The ALC was independent of the FLIPI in multivariate analysis. Conclusions: This data supports the hypothesis that a higher lymphocyte count, as a marker of the immune status of the patient, predicts for a longer TTP following rituximab therapy. No significant financial relationships to disclose.

19002 Background: Preliminary data suggesting positive clinical outcome in patients treated with adjuvant granulocyte macrophage colony stimulating factor (GM-CSF) following surgical resection of stage III/IV melanoma already exist (Spitler et al). A compassionate use protocol for the treatment of such patients with the “Spitler” regimen (GM-CSF administered at 250 mcg/day s.c. for 14 days of a 28 day cycle) has been ongoing at Mayo Clinic. Herein we present a retrospective review. Methods: Between 1998 and 2006, data was collected from 30 patients rendered disease free by surgery who received adjuvant GM-CSF. GM-CSF was administered per the “Spitler” regimen, although one patient received a reduced dose of 125mcg/day. The Kaplan-Meier approach was used to estimate relapse free survival. Results: Thirty patients (14 Female) with a median age of 53 and excellent performance status were evaluated. Most patients had resected stage III melanoma (Stage II/III/IV: 1/20/9). Median followup was 33 months (3–96). Twenty one patients were treated with GM-CSF for at least 12 months. Overall, relapse-free survival at 1 year was estimated to be 69% (95% confidence interval (CI) of 54% to 88%), reducing to 44% (95% CI of 29% to 67%) at 3 years. According to stage, relapse free survival at 1 year was 70% (III) and 63% (IV), and at 3 years was 54% (III), and 13% (IV). Among the 21 patients completing at least 1 year of therapy, relapse free survival at 3 years was 52%. Seventeen patients relapsed (III/IV: 10/7). Six of these patients were retreated and only two relapsed. Toxicities were grade 1 with most common being injection site rash (23%), asthenia (17%), myalgia (17%), and fever (7%). One patient required dose modification for elevated liver function tests. Conclusions: Although limited in scope, our data further support a potential beneficial effect of adjuvant GM-CSF in resected stage III/IV melanoma. Recurrences in the treated population were often localized and amenable to further surgical treatment. The clinical relevance of this strategy is currently being prospectively tested (E4697, now closed to accrual). No significant financial relationships to disclose.

18505 Background: Besides radiation, the immunologic mechanisms of action of 90Y ibritumomab tiuxetan (Zevalin) radioimmunotherapy (RIT) have been attributed to complement mediated lysis and antibody-dependent cellular cytotoxicity (ADCC). We hypothesized that a stronger host immune system prior to Zevalin therapy for relapsed follicular (grade 1&2) lymphomas (FL) would result in an improved time to progression (TTP). As a surrogate marker of host immune status, we studied absolute lymphocyte count (ALC) prior to Zevalin therapy and its impact on TTP in relapsed FL patients. Methods: Between 1996 and 2006, 75 patients with relapsed FL were treated with single agent Zevalin (0.4 mCi/kg, maximum of 32 mCi) at the Mayo Clinic. ALC was obtained from the complete blood cell count prior to RIT. Results: The median age of the cohort was 60 years (range, 29–82 years). 36% (27/75) patients were rituximab refractory. The median TTP in all patients was 6.4 months (range, 1–99+ months). Univariately, ALC as a continuous (HR = 0.448, p < 0.006) or dichotomized [ALC = 1.0 x 109/l (HR = 0.684, p < 0.007)] variable was identified as a prognostic factor for TTP. Superior TTP was observed with an ALC = 1.0 x 109/l (N = 32) compared with an ALC < 1.0 x 109/l (N=43) (median: 12.4 months vs 6.5 months, respectively, p < 0.007). Both groups were balanced in regard to the Follicular Lymphoma International Prognostic Index (FLIPI). ALC, as a continuous (HR = 0.507, p < 0.02) or dichotomized [ALC = 1.0 x 109/l (HR = 0.697, p < 0.01)] variable was identified as an independent prognostic factor for TTP in the multivariate analysis when compared with FLIPI. In the ALC = 1.0 x 109/l group, 14/32 (44%) of FL patients achieved a TTP = 12 months compared to only 7/43 (16%) in the ALC < 1.0 x 109/l group, (p < 0.02). Conclusions: This study supports our hypothesis that a higher ALC, as a marker of immune status of the patient, predicts longer TTP following Zevalin therapy in relapsed FL. No significant financial relationships to disclose.

8565 Background: Previous studies using low-dose (250 ug/dose) aerosolized sargramostim (granulocyte macrophage colony stimulating factor: aero-GM-CSF) for the treatment of metastatic melanoma, revealed anecdotal clinical responses that appeared to correlate with an increase in the number of tumor specific cytotoxic T lymphocytes (CTL) in blood. Herein, we address whether or not further dose escalation of aero-GM-CSF is safe, results in greater increase of melanoma specific CTLs, and affects clinical outcomes. Methods: A 5+5 dose escalation clinical trial was conducted to determine the dose of aero-GM-CSF that would increase in the frequency of peripheral blood melanoma specific CTLs in HLA-A2+ patients with metastatic melanoma to the lungs. Aero-GM-CSF was administered twice daily on days 1–7 and 15–21 of a 28 day cycle at doses of: 500, 750, 1,000, 1,250, 1,500, 1,750, and 2,000 ug per cohort. If at most 2 of 5 patients at a dose level reported a 5-fold increase in CTLs, dose escalation occurred. If not, 5 additional patients were treated at that dose. Dose escalation continued until =2 of 5 patients at a dose level developed severe toxicity, at least 7 of the 10 patients at a dose level reported a 5-fold increase in CTLs, or all dose levels were exhausted. Results: The study accrued 40 patients from August 2002 to July 2006. Four patients cancelled participation prior to treatment. One patient was replaced (500 ug) as he died within 13 days of registration. Among the 35 remaining patients, median age was 58 (23–84), 72% were male, and 72% had a PS=0. Dose escalation was not terminated due to toxicity. Toxicities = grade 3 (at least probably treatment related) included: grade 3 fatigue (1) at 1,000 ug; and grade 3 cough (1), and grade 4 dyspnea (1) at 1,750 ug. At the 2,000 ug dose, most patients (4 of 5) exhibited a greater than 2-fold increase in melanoma specific CTLs. Two of these patients remain on treatment: one with partial response at 14.0 months and the other with stable disease at 6.5 months. A 5-fold increase in tumor specific CTLs was not achieved in any patient. Conclusions: Aero-GM-CSF therapy administered at up to 2,000 ug/dose appears safe and seems to correlate with increased frequencies of tumor specific CTLs in a dose-dependent fashion. Further dose escalation followed by clinical efficacy testing is warranted. No significant financial relationships to disclose.

8530 Background: RAD-001 (Everolimus) is an oral inhibitor of mammalian target of rapamycin (mTOR). Interim analysis results from a phase II study of RAD-001 in patients with metastatic melanoma (MM) were presented at ASCO 2005. This study was re-opened using a higher dose based on an improvement in the 16 week progression free survival (PFS) rate and good tolerability. Methods: A two- stage, phase II multi-institutional trial was conducted in patients with MM to assess that 16 weeks PFS rate was at least 50%. Inclusion criteria: measurable disease, ECOG performance score of 0–2. Exclusion criteria: presence of intracranial metastases, concurrent use of inducers of cytochrome 3A4 and abnormal organ function. The dose of RAD-001 in the second cohort was increased to 10 mg daily (increased from 30 mg weekly) based on evidence of safety of the higher dose. Results: Twenty-nine patients were enrolled; baseline information is available on 27. Median age was 63 yrs; 15 (56%) had >2 sites of metastatic disease. Most (48%) had stage M1c disease. PS was 0, 1 and 2 in 58%, 38% and 4%. All but 4 (15%) had received prior therapy. Grade 3 adverse events included stomatitis and fatigue (2 each), leukopenia, neutropenia, diarrhea, anorexia, dehydration, dyspnea, hyperglycemia, and hypersensitivity (1 each). Planned interim analysis was done after 20 patients were enrolled. 14 (70%) had progressed 16 weeks, failing to meet the decision rule needed (PFS >30%) to restart accrual. The median PFS for all 29 patients was 56 days. The median overall survival (OS) has not been reached. For the entire cohort of 53 pts treated on this study (at both dose levels), the median PFS, median OS were 59 and 286 days respectively. Conclusion: Interim analysis after enrollment of 20 patients at a higher dose of RAD-001 demonstrated significantly more toxicity and no added clinical efficacy. The 16 week PFS rate target was not reached, and accrual was suspended. No significant financial relationships to disclose.

8055 Background: mTOR inhibition with intravenous temsirolimus (Wyeth Pharmaceuticals) has been associated with responses in mantle cell lymphoma (J Clin Oncol 23;5347, 2005) as well as other lymphomas (Blood 108 (11) 2483; 2006). This phase II study tested the oral mTOR inhibitor everolimus (RAD001, Novartis Pharmaceuticals) in three simultaneous two-stage phase II lymphoma studies - aggressive (group 1), indolent (group 2), or uncommon (group 3). The goals were to learn the toxicity profile and to assess the anti-tumor response. Planned interim analysis for groups 1 and 3 have been completed and are the subject of this report. Methods: Patients (pts) received 10 mg PO daily for each 28 day cycle (up to 12) and restaged after 2, 6, and 12 cycles. The primary endpoint is the confirmed response rate, including CR, CRu or PR. 12 pts were enrolled in stage 1 of each study. At least 1 success in 12 is required to proceed to stage 2, to a total of 37 pts. Overall, the treatment will be considered promising if 4 or more successes are observed in all 37 pts in each group. Results: The median age of the 12 pts in group 1 was 68.5 yrs (range: 53–80), with a median of 3 (range, 1–15) prior therapies. Four pts had a prior stem cell transplant (SCT). Pts completed a median of 7 (range, 1–12) cycles of therapy. 6 confirmed responses have been achieved (1 CR, 5 PR), meeting the overall criteria for promising results in this study. Common grade 3 adverse events (AEs) include thrombocytopenia (3 pts) and anemia (2 pts). For group 3, the median age was 49 yrs (range, 27–78), with a median of 7 (range, 1–13) prior therapies and 6 pts had a prior SCT. Pts have completed a median of 6.5 cycles (range, 1–11). 5 confirmed responses have been achieved (5 PR), meeting the criteria for this regimen to be considered promising. Of these 5 patients, 3 had HD, 1 T-cell NHL, and 1 had macroglobulinemia. Common grade 3 AEs include anemia (3 pts) and thrombocytopenia (2 pts). No grade 4 AEs were reported. Conclusions: Oral everolimus has activity in a spectrum of lymphomas with acceptable toxicity. The responses observed in both group 1 and group 3 met the criteria to continue accrual. These results provide the rationale for additional studies with this novel class of agents and to integrate mTOR inhibitors into salvage treatment regimens. No significant financial relationships to disclose.

Background: A female survival advantage in cutaneous melanoma has been long recognized. However, whether this extends across all age groups, with risk stratification using the latest prognostic staging system or in the current era of efficacious systemic therapies is unknown. Therefore, we evaluated whether sex-based differences in melanoma survival persisted within a recent population-based patient cohort with consideration of these factors. Materials and Methods: We identified stage II−IV cutaneous melanoma patients from 2010 to 2014 Surveillance, Epidemiology, and End Results cancer registries data. We recalculated stage per American Joint Committee on Cancer 8th edition guidelines. Cancer-specific survival (CSS) was estimated by using the Kaplan–Meier method and multivariable Cox proportional hazards regression. Results: Of 16,807 patients (39.8% female), 8,990 were stage II, 4,826 stage III, and 2,991 stage IV at diagnosis. Unadjusted 3-/5-year CSS estimates for females versus males were 64.2% versus 59.7%, and 53.5% versus 49.9%, respectively, p ≤ 0.0001. Five-year CSS varied within each stage and across age strata of <45, 45 − 59, and ≥60 years. Within each stage, females <45 had better CSS than all other sex/age groups ( p < 0.0001). In multivariable analysis of stage II/III patients, female sex, younger age, and lower mitotic index retained favorable CSS prognostic significance ( p < 0.001). Conclusions: Sex-based differences in melanoma survival persist in a contemporary patient cohort staged with the latest prognostic system. These data may guide decision marking regarding adjuvant therapy, highlight the importance of including sex as a pre-specified clinical trial variable, and suggest that investigation of underlying biologic mechanisms may drive discovery of biomarkers and therapeutic targets to improve patient care.

Abstract Background Immunosuppression in glioblastoma (GBM) is an obstacle to effective immunotherapy. GBM-derived immunosuppressive monocytes are central to this. Programmed cell death ligand 1 (PD-L1) is an immune checkpoint molecule, expressed by GBM cells and GBM extracellular vesicles (EVs). We sought to determine the role of EV-associated PD-L1 in the formation of immunosuppressive monocytes. Methods Monocytes collected from healthy donors were conditioned with GBM-derived EVs to induce the formation of immunosuppressive monocytes, which were quantified via flow cytometry. Donor-matched T cells were subsequently co-cultured with EV-conditioned monocytes in order to assess effects on T-cell proliferation. PD-L1 constitutive overexpression or short hairpin RNA–mediated knockdown was used to determined the role of altered PD-L1 expression. Results GBM EVs interact with both T cells and monocytes but do not directly inhibit T-cell activation. However, GBM EVs induce immunosuppressive monocytes, including myeloid-derived suppressor cells (MDSCs) and nonclassical monocytes (NCMs). MDSCs and NCMs inhibit T-cell proliferation in vitro and are found within GBM in situ. EV PD-L1 expression induces NCMs but not MDSCs, and does not affect EV-conditioned monocytes T-cell inhibition. Conclusion These findings indicate that GBM EV-mediated immunosuppression occurs through induction of immunosuppressive monocytes rather than direct T-cell inhibition and that, while PD-L1 expression is important for the induction of specific immunosuppressive monocyte populations, immunosuppressive signaling mechanisms through EVs are complex and not limited to PD-L1.

362 Background: Most pts with mGEA do not respond to ICI or ramucirumab/paclitaxel (RAM/TAX). Emerging data suggest that ICI may potentiate subsequent therapy (Tx). In KN059 we unexpectedly observed durable responses in 2 pts on RAM/TAX after ICI (PMID 29674442). We examined if ICI impacts efficacy of subsequent RAM/TAX in a larger cohort and explored alterations in the tumor microenvironment. Methods: All pts with mGEA at Mayo Clinic (MN, AZ, FL) who received RAM/TAX (2014-19) were included. We compared best objective response rate (ORR: complete [CR] or partial response [PR]) per RECIST1.1 and overall survival (OS) in pts who received RAM/TAX with (Group A) vs without (Group B) immediately preceding PD-1 blockade. Chi square and multivariate logistic and Cox regression were used. We adjusted for total lines of Tx received and the line of Tx in which RAM/TAX was given, to control for potential differences in the biology of heavily treated pts or those receiving RAM/TAX as 2nd vs 3rd line Tx. We also adjusted for age and ECOG PS. Results: In total 87 pts met inclusion criteria. In the 19 pts (Group A) who received RAM/TAX (usually as 3rd line Tx) after progression on ICI, there was a 77% relative risk reduction of death after initiation of RAM/TAX compared to the 68 pts (Group B) who received RAM/TAX (usually as 2nd line) without preceding ICI (median OS 15.0 vs 7.6 mo; HR 0.23; P < .001). The ORR was also significantly higher (58% vs 18%; P < .001) including the CR rate (16% vs 1%; P = .006). Two CRs in Group A are ongoing (10 - 34 mo). Of note, 95% of Group A pts did not respond to ICI, and all had irRECIST progression on ICI. Immunofluorescent analysis from a responder showed 65-fold reduction (post vs pre-Tx) in intratumoral density of immunosuppressive FOXP3+ Tregs, with preserved density of antitumor CD8+ T cells. Conclusions: Higher than expected efficacy was observed on RAM/TAX when immediately preceded by ICI, suggesting ICI may enhance efficacy of subsequent anti-VEGFR/taxane therapy. This serial immunotherapy combination may be a novel option for pts with primary resistance to ICI and will be tested prospectively in a new randomized phase 2 trial (NCT04069273).