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Purpose:Ruxolitinib with lenalidomide and dexamethasone shows antimyeloma effects in vitro and in vivo. MUC1 leads to lenalidomide resistance in multiple myeloma cells, and ruxolitinib blocks its expression. Thus, ruxolitinib may restore sensitivity to lenalidomide. Therefore, a phase I trial was conducted to determine the safety and efficacy of ruxolitinib with lenalidomide and methylprednisolone for patients with relapsed/refractory multiple myeloma (RRMM) who had been treated with lenalidomide/steroids and a proteasome inhibitor and showed progressive disease at study entry.Patients and Methods:A traditional 3+3 dose escalation design was used to enroll subjects in four cohorts with planned total enrollment of 28 patients. Subjects received ruxolitinib twice daily, lenalidomide daily on days 1–21 of a 28-day cycle, and methylprednisolone orally every other day. Primary endpoints were safety, clinical benefit rate (CBR), and overall response rate (ORR).Results:Twenty-eight patients were enrolled. The median age was 67 years and received a median of six prior treatments including lenalidomide and steroids to which 93% were refractory. No dose-limiting toxicities occurred. The CBR and ORR were 46% and 38%, respectively. All 12 responding patients were refractory to lenalidomide. Grade 3 or grade 4 adverse events (AE) included anemia (18%), thrombocytopenia (14%), and lymphopenia (14%). Most common serious AEs included sepsis (11%) and pneumonia (11%).Conclusions:This phase I trial demonstrates that a JAK inhibitor, ruxolitinib, can overcome refractoriness to lenalidomide and steroids for patients with RRMM. These results represent a promising novel therapeutic approach for treating multiple myeloma (ClinicalTrials.gov number, NCT03110822).

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High-risk multiple myeloma (MM) continues to have a poor prognosis and remains a therapeutic challenge. This phase 2 study evaluated the efficacy and safety of elotuzumab in combination with pomalidomide, carfilzomib, and low-dose dexamethasone for patients with high-risk relapsed/refractory (RR)MM (NCT03104270). Of 13 enrolled patients, 11 were evaluable for efficacy. Overall response rate and clinical benefit rate were 45.4% and 54.5%, respectively. Deep responses were observed including two complete responses. The novel quadruplet combination was overall well-tolerated, with clinically manageable adverse events. Common adverse events of ≥ grade 3 included lymphopenia (15%), anemia (15%), sepsis (15%), pneumonia (15%), and hypophosphatemia (15%). The novel combination showed promising efficacy and was well tolerated in this heavily pretreated MM population. Even though the study was terminated early prior to completion of enrollment, the results indicate that this may be a promising therapeutic approach for high-risk RRMM patients, which warrants further study.

Background Multiple myeloma (MM) patients have variable responses to mRNA vaccination to COVID-19. Little is known regarding their vaccine-induced antibody levels over time. Methods We monitored spike IgG antibody levels over 24 weeks among a subset of 18 MM patients who showed a full response after two mRNA vaccinations. MM patients had a more rapid decline in antibody levels as compared to 8 healthy controls, with power law half-lives of 72 days (versus 107 days) and exponential half-lives of 37 days (versus 51 days). Results The patients with longer SARS-CoV-2 antibody half-lives were more likely to have undetectable monoclonal protein than those with shorter half-lives, suggesting better disease control may correlate with longer duration of vaccine-induced antibodies. Regardless, by 16 weeks post-second dose of mRNA vaccination, the majority of patients had antibody levels below 250 binding arbitrary units per milliliter, which would be unlikely to contribute significantly to preventing COVID-19. Conclusions Thus, even MM patients who respond adequately to vaccination are likely to require more frequent booster doses than the general population.

Background Activating fusions of the NTRK1, NTRK2 and NTRK3 genes are drivers of carcinogenesis and proliferation across a broad range of tumour types in both adult and paediatric patients. Recently, the FDA granted tumour-agnostic approvals of TRK inhibitors, larotrectinib and entrectinib, based on significant and durable responses in multiple primary tumour types. Unfortunately, testing rates in clinical practice remain quite low. Adding plasma next-generation sequencing of circulating tumour DNA (ctDNA) to tissue-based testing increases the detection rate of oncogenic drivers and demonstrates high concordance with tissue genotyping. However, the clinical potential of ctDNA analysis to identify NTRK fusion-positive tumours has been largely unexplored. Methods We retrospectively reviewed a ctDNA database in advanced stage solid tumours for NTRK1 fusions. Results NTRK1 fusion events, with nine unique fusion partners, were identified in 37 patients. Of the cases for which clinical data were available, 44% had tissue testing for NTRK1 fusions; the NTRK1 fusion detected by ctDNA was confirmed in tissue in 88% of cases. Here, we report for the first time that minimally-invasive plasma NGS can detect NTRK fusions with a high positive predictive value. Conclusion Plasma ctDNA represents a rapid, non-invasive screening method for this rare genomic target that may improve identification of patients who can benefit from TRK-targeted therapy and potentially identify subsequent on- and off-target resistance mechanisms.

Multiple myeloma (MM) patients are at higher risk for severe COVID-19. Their mRNA vaccination response against SARS-CoV-2 is unknown. Thus, we analyzed responses to mRNA vaccination against COVID-19 among these patients. Using an ELISA-based assay that detects IgG antibodies to SARS-CoV-2 spike protein, we determined serum antibody levels prior to immunization and 12–21 and 14–21 days following the first and second vaccinations, respectively, with mRNA-1273 (Moderna) or BNT162b2 (Pfizer/BioNTech) among 103 MM patients (96 and 7 with active and smoldering disease, respectively). We stratified patients into clinically relevant responders (>250 IU/mL), partial responders (50–250 IU/mL, which was above pre-COVID-19 background), and nonresponders ( second line of treatment, and among those not in complete remission. Patients who received mRNA-1273 vaccine had higher anti-spike antibody levels than those who were vaccinated with BNT162b2. Thus, most MM patients have impaired responses to mRNA vaccination against COVID-19, and specific clinical and myeloma-related characteristics predict vaccine responsiveness.

Ruxolitinib with lenalidomide and dexamethasone shows anti-myeloma effects in vitro and in vivo. MUC1 leads to lenalidomide resistance in multiple myeloma (MM) cells, and ruxolitinib blocks its expression. Thus, ruxolitinib may restore sensitivity to lenalidomide. A phase I trial was conducted to determine the safety and efficacy of ruxolitinib with lenalidomide and methylprednisolone for patients with relapsed/refractory (RR)MM who had been treated with lenalidomide, steroids and a proteasome inhibitor and showed progressive disease at study entry. A traditional 3 + 3 dose escalation design was used to enroll subjects in four cohorts. Subjects received ruxolitinib twice daily, lenalidomide daily on days 1-21 of a 28 day cycle and methylprednisolone orally every other day. Primary endpoints were safety, clinical benefit rate (CBR) and overall response rate (ORR). Forty-nine patients were enrolled. The median age was 64 years and they had received a median of six prior treatments including lenalidomide and steroids to which 94% were refractory. No dose limiting toxicities occurred. The CBR and ORR were 49% and 36%, respectively. All responding patients were refractory to lenalidomide. Grade 3 or 4 adverse events (AEs) included anemia (17%), decreased lymphocyte count (15%), and hypophosphatemia (10%). Most common serious AEs included sepsis (9.8%) and pneumonia (7.8%). This Phase I trial demonstrates that a JAK inhibitor, ruxolitinib, can overcome refractoriness to lenalidomide and steroids for patients with RRMM. These results represent a promising novel therapeutic approach for treating MM. NCT03110822.

Identifying effective combination regimens is a high priority in multiple myeloma (MM), as most patients eventually become refractory to their current treatments. In this study, we investigated whether the proteasome inhibitor (PI) ixazomib could delay disease progression among patients who failed regimens containing another PI, bortezomib or carfilzomib. This phase 1/2, multicenter, open-label, nonrandomized study enrolled patients who were refractory to a previous regimen containing bortezomib or carfilzomib. Patients continued the other anti-MM drugs in the regimen at the same doses and frequencies. Patients with combination regimens with unknown maximum tolerated dose (MTD) of ixazomib were enrolled in phase 1, with ixazomib starting at 3 mg and then dose escalated to 4 mg. Patients on regimens with a known ixazomib MTD were enrolled in phase 2. Primary endpoints were overall response rate (ORR), clinical benefit rate (CBR), adverse events (AEs), and determination of maximum tolerated dose (MTD). Of the 46 patients enrolled, 39 were evaluable for efficacy. ORR and CBR were 12.8% and 17.9%, respectively. Ixazomib appeared to be well tolerated as a replacement for carfilzomib and bortezomib, with 23.9% of patients experiencing at least one grade ≥3 serious adverse event (SAE) and 37.0% experiencing at least one grade ≥3 AE. The most common grade ≥3 AEs were hyponatremia (8.7%), anemia (8.7%), dyspnea (8.7%), thrombocytopenia (6.5%), dehydration (4.3%), and pneumonia (4.3%). The results indicate that ixazomib is not an effective replacement for bortezomib or carfilzomib for patients with MM who have previously relapsed on other bortezomib/carfilzomib-containing regimens.

Introduction: Amid the current COVID-19 pandemic, the highest mortality rates are among the elderly and immunocompromised. Multiple myeloma (MM) patients are immunocompromised and often are elderly. Not only do MM patients develop more frequent infections, but it is one of the leading causes of death for these patients. This population develops more severe COVID-19 due to various mechanisms that impair their ability to fight infection. This also reduces their ability to generate immunity from vaccination, as has been demonstrated by their diminished responses to vaccines for various respiratory illnesses. It follows that while phase III trial results for mRNA1273 and BNT162b2 COVID-19 vaccines showed an efficacy of 94-95% against even mild infection with this virus, the efficacy has been shown to be lower among MM patients. We recently evaluated patients' antibody responses to vaccination for COVID-19 by measuring anti-spike IgG levels in patient serum from before vaccination (baseline) and two weeks after dose 2 (D2W2) of vaccination with mRNA-1273 or BNT162b2, and found that most MM patients have impaired responses (Stampfer et al., Leukemia 2021). Patients who received mRNA-1273 vaccine had higher antibody levels than those who were vaccinated with BNT162b2, and specific clinical and myeloma-related characteristics predicted vaccine responsiveness. In the current study, we are monitoring anti-spike IgG antibody levels at Dose 2 Week 8 (D2W8) and Dose 2 Week 16 (D2W16) post-mRNA vaccination among these patients and in age-matched healthy controls to investigate antibody decay. Methods: Participants in the trial included MM patients (n=91) at the Berenson Cancer Center, and age-matched healthy controls (n=27). Healthy subjects were not known to be immunocompromised or currently receiving immunosuppressive therapy. Vaccination was done outside of the clinic and subjects provided copies of their CDC-issued COVID-19 vaccination cards to confirm dosing dates. Sera from vaccinated individuals were drawn at baseline (0-60 days prior to first vaccine dose) and at intervals following their second dose (14-21, 56-70, and 112-126 days post-vaccination). Background levels were determined from the clinic's serum bank of healthy subjects drawn pre-April 2019. Using an ELISA-based assay that detects IgG antibodies to SARS-CoV-2 spike protein, we determined Anti-SARS-CoV-2 spike ectodomain serum antibody levels and quantified them in IU/mL based on the WHO International Standard 20/136. Results: We analyzed the patient and control populations, and specifically for those classified as responders from our original published study (D2W2 >50 IU/mL). All controls but only 70% of patients (64/91) were classified as responders, so this responder-specific analysis was only relevant for patients. There was a significant decline in anti-SARS-CoV-2 spike antibodies from D2W2 to D2W8 for both patients and controls; D2W16 results are pending. Median values dropped from 283.1 IU/mL to 90.9 IU/mL among patients, and 893.6 IU/mL to 354.4 IU/mL among controls. Median antibody levels among patients classified as responders dropped from 482.9 IU/mL to 145.5 IU/mL (p Conclusions: The markedly lower anti-spike antibody levels among MM patients compared to healthy controls at week 8 post-vaccination indicate that they remain at high risk for breakthrough infections. Combined with their rapid decline in anti-spike antibody levels over only a 6-week period, this indicates that even MM patients who responded initially to vaccination are likely to require boosters sooner than healthy individuals. This immunocompromised population remains at high risk even following vaccination and should continue to maintain social distancing precautions during periods of high local SARS-CoV-2 transmission. Disclosures No relevant conflicts of interest to declare.

Introduction Ruxolitinib (RUX) is an FDA-approved oral, selective inhibitor of Janus kinase (JAK) 1/2 for the treatment of patients with intermediate or high-risk myelofibrosis and polycythemia vera who have had an inadequate response to or are intolerant of hydroxyurea. Preclinical studies from our laboratory have demonstrated that RUX in combination with dexamethasone shows marked anti-myeloma effects both in vitro and in vivo. Specifically, RUX blocks expression of Mucin 1 whose function is linked to lenalidomide (LEN) resistance, downregulates PD-L1 and PD-L2 expression and reduces tumor stimulatory M2 macrophage polarization in multiple myeloma (MM) bone marrow. Recently published results from a Phase 1 trial for 28 heavily previously treated MM patients administered RUX, LEN and methylprednisolone (MP) demonstrated that the therapy was well tolerated and RUX overcame refractoriness to LEN and steroids (Berenson et al., Clin Cancer Res. 2020). The clinical benefit rate (CBR) and overall response rate (ORR) were 46% and 28%, respectively, and all 12 responding patients were refractory to LEN. We evaluated whether the combination of RUX and steroids without LEN was active for treating similar patients through treatment of a cohort within the current trial using this two-drug combination (NCT03110822). Methods MM patients must have failed > 3 prior regimens and have been exposed to a proteasome inhibitor and LEN. Patients were treated orally (PO) with 15 mg RUX twice daily on days 1-28 of a 28-day cycle and 40 mg MP every other day. The treatment of this two-drug combination was continued until disease progression (PD). Once PD was confirmed, LEN at 10 mg PO daily on days 1-21 of a 28-day cycle was added to the regimen. Primary endpoints were safety, ORR and CBR. Results As of July 15, 2021, 27 patients were enrolled. The median age was 64 years (range, 46-85), and 17 (63%) were male. Patients received a median of 4 (range, 3-11) prior treatments including LEN and steroid-containing regimens to which they were all refractory. Twenty-six patients have completed at least 1 full cycle of therapy; and, thus, were evaluable for efficacy. The ORR and CBR were 35% (n=9) and 39% (n=10), respectively. Notably, all 10 responding patients were refractory to LEN (i.e., progressed while on or within 8 weeks of last dosage). The remaining patients showed stable disease (n=12) or PD (n=4). The median follow-up was 13 months. The median progression-free survival was 4 months (range, 1-21). The median duration of response was 11 months (range, 1-20). Of 13 patients who progressed on the two-drug combination and had LEN added to their regimen, 6 patients responded (3 MR and 3 PR). Nine patients experienced SAEs including sepsis (12%), sepsis with neutropenic fever (4%), thrombocytopenia (4%), hyperglycemia (4%), neutropenia (4%), anemia (4%), acute heart failure (4%), rotator cuff tear (4%), osteomyelitis (4%), aspiration pneumonia (4%), pneumonia and pneumothorax (4%), and deep venous thrombosis (4%). Two patients died (one each from pneumonia and PD). Conclusions This ongoing Phase 1 trial is the first study demonstrating clinical activity of the two-drug combination of the JAK inhibitor RUX with steroids for MM patients. The treatment was well tolerated and shows promising efficacy for treating heavily previously treated MM patients. Because of this, additional studies are being conducted with higher doses of RUX in combination with MP for this patient population. Disclosures Vescio: Janssen: Speakers Bureau; Karyopharm: Speakers Bureau; GlaxoSnithKlein: Speakers Bureau; Amgen: Speakers Bureau. OffLabel Disclosure: Ruxolitinib is being investigated off-label to determine if it shows anti-myeloma activity in combination with steroids for relapsed/refractory myeloma patients.

Background: Anti-CD38 monoclonal antibodies have become a standard treatment option for patients with either relapsed/refractory (RR) or newly diagnosed multiple myeloma (MM). There are two approved drugs in this class, daratumumab and isatuximab. These agents have become a mainstay of myeloma treatment being used in a variety of combination approaches. Although they are widely used, very little has been reported regarding the benefit they provide if they are used again in subsequent lines of therapy among patients that have already been treated with prior anti-CD38 containing regimens. Methods: MM patients that were treated at the Berenson Cancer Center and previously received either daratumumab or isatuximab and were treated with another anti-CD38-containing combination were retrospectively analyzed. Response to each anti-CD38 regimen was evaluated for responses according to the International Myeloma Working Group (IMWG) criteria. We used Kaplan-Meier curves to determine progression free survival (PFS) and overall survival (OS). Results: There were 49 patients identified as having received > 2 anti-CD38 containing regimens. Among these patients, 40, 1, and 8 received only daratumumab, only isatuximab, or a combination of both anti-CD38 regimens. 17, 19, 8, and 41 received an IMiD, PI, a combination of both, or other combinations. Overall, the median PFS was 3.9, 3.2, 2.6, 1.9, and 1.5 months for the first (n=49), second (n=49), third (n=23), fourth (n=15), and fifth (n=8) exposures, respectively. For their first anti-CD38 containing regimen (n=49), the clinical benefit rate (CBR) and overall response rate (ORR) were 44.9% and 38.8%, respectively (7.7% MR, 34.7% PR, and 4.1% CR), and 36.7% showed SD. During their second exposure (n=49), the CBR and ORR were 42.9% and 40.8%, respectively (2.0% MR, 36.7% PR, 12.0% VGPR, and 2.0% CR) and 36.7% showed SD. The PFS among patients who were exposed to a second regimen and discontinued therapy without disease progression to their first anti-CD38 containing regimen was 5.9 months compared with only 3.0 months for those who progressed during their first regimen. OS from the start of anti-CD38 treatment was a median of 36.5 months (range, 1.8-56.5). OS was longer among patients who did not progress on first exposure with a median of 26.2 months (range, 1.8-56.5) compared to patients who progressed during their first regimen (median not yet reached; range, 7.6-54.5). PFS among patients treated with a proteasome inhibitor (PI)-containing combination in their first exposure was 8.7 months versus 4.5 months among patients who were treated with an immunomodulatory agent (IMiD)-containing treatment. Additionally, the PFS among patients treated with a PI versus an IMiD with their second exposure to an anti-CD38 containing regimen was 5.9 and 2.8 months, respectively. Conclusions: Our retrospective study suggests that RRMM patients who are retreated with another anti-CD38 containing regimens can continue to respond after multiple exposures to this drug class. The ORR differs little during the first two exposures to these agents for RRMM patients. To our knowledge, this study provides support for retreatment of RRMM patients with anti-CD38 containing regimens. Additional studies are planned to further investigate this patient population. Disclosures Vescio: Janssen: Speakers Bureau; Karyopharm: Speakers Bureau; GlaxoSnithKlein: Speakers Bureau; Amgen: Speakers Bureau.

Genomic instability, including genetic mutations and chromosomal rearrangements, can lead to cancer development. Aberrant DNA methylation occurs commonly in cancer cells. The aim of this study is to determine the effects of a specific chromosomal lesion the BCR-ABL translocation t(9:22), in establishing DNA methylation profiles in cancer. Materials and methods We compared DNA methylation of 1,505 selected promoter CpGs in chronic myelogenous leukemia (CML), acute lymphoblastic leukemia (ALL) with and without the Philadelphia chromosome t(9:22), CD34+ hematopoietic stem cells transfected with BCR-ABL, and other tumors without BCR-ABL (acute promyelocytic leukemia (APL) and gastrointestinal stromal tumors (GIST). In this study, the DNA methylation profile of CML was more closely related to APL, another myeloid leukemia, than Ph+ ALL. Although DNA methylation profiles were consistent within a specific tumor type, overall DNA methylation profiles were no influenced by BCR-ABL gene translocation in the cancers and tissues studied. We conclude that DNA methylation profiles may reflect the cell of origin in cancers rather than the chromosomal lesions involved in leukemogenesis.

Purpose: Ruxolitinib with lenalidomide and dexamethasone shows antimyeloma effects in vitro and in vivo. MUC1 leads to lenalidomide resistance in multiple myeloma cells, and ruxolitinib blocks its expression. Thus, ruxolitinib may restore sensitivity to lenalidomide. Therefore, a phase I trial was conducted to determine the safety and efficacy of ruxolitinib with lenalidomide and methylprednisolone for patients with relapsed/refractory multiple myeloma (RRMM) who had been treated with lenalidomide/steroids and a proteasome inhibitor and showed progressive disease at study entry. Patients and Methods: A traditional 3+3 dose escalation design was used to enroll subjects in four cohorts with planned total enrollment of 28 patients. Subjects received ruxolitinib twice daily, lenalidomide daily on days 1–21 of a 28-day cycle, and methylprednisolone orally every other day. Primary endpoints were safety, clinical benefit rate (CBR), and overall response rate (ORR). Results: Twenty-eight patients were enrolled. The median age was 67 years and received a median of six prior treatments including lenalidomide and steroids to which 93% were refractory. No dose-limiting toxicities occurred. The CBR and ORR were 46% and 38%, respectively. All 12 responding patients were refractory to lenalidomide. Grade 3 or grade 4 adverse events (AE) included anemia (18%), thrombocytopenia (14%), and lymphopenia (14%). Most common serious AEs included sepsis (11%) and pneumonia (11%). Conclusions: This phase I trial demonstrates that a JAK inhibitor, ruxolitinib, can overcome refractoriness to lenalidomide and steroids for patients with RRMM. These results represent a promising novel therapeutic approach for treating multiple myeloma (ClinicalTrials.gov number, NCT03110822).

New classes of drugs including the proteasome inhibitors (PI) bortezomib and, more recently, carfilzomib and the immunomodulatory agent lenalidomide have shown improved outcomes for multiple myeloma (MM) patients during the past decade. However, most of the studies reporting outcomes for patients receiving these drugs have relied on older data sets derived from large institutions that included patients not receiving their treatment at those facilities and represented only those eligible for clinical trials or were from sites where treatment options were limited. We have analyzed data from 258 MM patients who have received treatment with at least one of three agents: bortezomib, carfilzomib, and lenalidomide in a single clinic specializing in MM with respect to their responses and other outcomes to treatment regimens including these agents. Response rates were similar between these three drugs when used for the first time and again during subsequent treatment regimens. As expected, the clinical benefit rates (CBRs) were better for patients receiving their first treatment when compared to their use in subsequent treatment regimens. The CBRs were similar during their 2nd, 3rd, and 4th treatments containing these agents. Many patients refractory to these agents showed responses to regimens containing these same drugs when used in different combinations. In addition, patients refractory to one PI often responded to the other PI. The results of this study demonstrate that novel agents can be used repeatedly in novel combinations with significant clinical benefit for patients with MM.

Despite recent advances made in its treatment, multiple myeloma (MM) remains an incurable B cell malignancy. Thus, the objective for treating these patients is to prolong overall survival (OS) and preserve patients’ quality of life. We have analyzed data from 264 consecutive MM patients who had their initial visit between July 1, 2004 and December 1, 2014 and have received treatment in a single clinic specializing in MM. We determined their progression-free survival (PFS, OS, and 5-year OS). The PFS for frontline (n = 165 treatments), salvage (n = 980), and all treatments (n = 1145) were 13.9, 4.6, and 5.5 months, respectively. The median OS of all patients was 98 months with a 5-year survival of 74%. The results of this study show a marked improvement in OS for unselected MM patients compared with historical data. There were no significant differences in OS between patients with different International Staging System (ISS) stages. Younger patients (

Introduction Ruxolitinib (RUX) is an FDA-approved oral, selective inhibitor of Janus kinase (JAK) 1/2 for the treatment of patients with intermediate or high-risk myelofibrosis and polycythemia vera who have had an inadequate response to or are intolerant of hydroxyurea. Preclinical studies from our laboratory have demonstrated that RUX in combination with dexamethasone shows marked anti-myeloma effects both in vitro and in vivo. Specifically, RUX blocks expression of Mucin 1 whose function is linked to lenalidomide (LEN) resistance and down-regulates PD-L1 and PD-L2 expression and reduces tumor stimulatory M2 macrophage polarization in multiple myeloma (MM) bone marrow. Recently published results from a Phase 1 trial for 28 heavily previously treated MM patients administered RUX, LEN and methylprednisolone (MP) demonstrated that the therapy was well tolerated and RUX overcame refractoriness to LEN and steroids (Berenson et al., Clin Cancer Res. 2020). The clinical and overall response rates were 46% and 28%, respectively, and all 12 responding patients were refractory to LEN. To further evaluate whether RUX and steroids without LEN demonstrate clinical activity and its tolerability in the same patient population, an ongoing Phase I trial was expanded to also include a cohort of patients treated with this two-drug combination (NCT03110822). Methods MM patients must have failed > 3 prior regimens and have been exposed to a proteasome inhibitor and LEN. Patients were treated orally (PO) with 15 mg RUX twice daily on days 1-28 of a 28-day cycle and 40 mg MP every other day. The treatment of this two-drug combination was continued until disease progression (PD). Once PD was confirmed, LEN at 10 mg PO daily on days 1-21 of a 28-day cycle was added to the regimen. Primary endpoints were safety, clinical benefit rate (CBR) and overall response rate (ORR). Results As of July 25, 2020, 16 of the planned 29 patients have been enrolled, and 15 patients have completed at least 1 full cycle of therapy; and, thus, were evaluable for efficacy. The median age was 64 years (range, 46-77), and 9 (60%) were male. Patients received a median of 4 (range, 3-10) prior treatments including LEN and steroids to which they were all refractory. Of the 15 evaluable patients treated with RUX and steroids, the CBR and ORR were 53% (n=8) and 33% (n=5), respectively. With a median follow-up of 4.7 months, the median duration of response was 3.5 months (range, 1-11+). Five and 2 patients showed stable disease and progressive disease, respectively. Notably, all 8 responding patients were refractory to LEN (i.e. progressed while on or within 8 weeks of last dosage). Of 6 patients who had progressed on the two-drug combination and had LEN added to their regimen, 3 patients responded (2 MR and 1 PR). Four patients experienced SAEs including sepsis (7%), group B strep sepsis with neutropenic fever and nausea (7%), pneumonia and pneumothorax (7%), thrombocytopenia (7%), and hyperglycemia (7%). Two patients died (one each from pneumonia and progressive disease). Conclusions This ongoing phase 1 trial is the first study reporting clinical activity of the JAK inhibitor RUX with steroids for MM. These results demonstrate that RUX with only steroids is also well tolerated and shows promising efficacy for treating heavily previously treated MM patients. Because of this, additional studies are being conducted with higher doses of RUX in combination with MP for this patient population. Disclosures Berenson: Takeda: Consultancy, Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; Incyte: Honoraria, Patents & Royalties: OncoTracker, Speakers Bureau; Amgen: Consultancy, Honoraria, Speakers Bureau. Swift:Jassen: Consultancy, Honoraria, Speakers Bureau; bristol myers squibb: Consultancy, Honoraria, Speakers Bureau; Amgent: Consultancy, Honoraria, Speakers Bureau. Boccia:Incyte: Consultancy, Honoraria, Research Funding, Speakers Bureau; Daiichi Sankyo: Consultancy, Honoraria, Research Funding, Speakers Bureau; Amgen: Consultancy, Honoraria, Research Funding, Speakers Bureau; AstraZeneca: Consultancy, Honoraria, Research Funding, Speakers Bureau; Genentech: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Rigel: Consultancy, Honoraria, Research Funding, Speakers Bureau. OffLabel Disclosure: Ruxolitinib and steroids only for treatment patients with Multiple Myeloma

Summary Immunomodulatory drugs including thalidomide, lenalidomide (LEN) and pomalidomide (POM), are effective for treating multiple myeloma (MM). POM has shown enhanced efficacy with dexamethasone (DEX). Pegylated liposomal doxorubicin (PLD) with bortezomib is US Food and Drug Administration-approved for treating MM. PLD with LEN or thalidomide has shown efficacy for MM patients. LEN with DEX, PLD and bortezomib achieves high response rates. We evaluated the combination of POM with DEX 40 mg and PLD 5 mg/m2 with the latter two drugs administered on days 1, 4, 8 and 11 on a 28-day cycle for the treatment of relapsed/refractory MM patients. During Phase 1, the maximum tolerated dose of POM was 4 mg, and was used in Phase 2, which also required patients to be refractory to LEN. However, neutropenia ≥ grade 3 was observed in 10/17 (59%) patients, and the dose was lowered to 3 mg. Median PFS was 5·4 months (range, 0·3–29·0 + months). Overall response rates for patients in Phase 2 were 39% and 31% among subjects receiving POM at 3 mg and 4 mg, respectively, and clinical benefit rates were 51% and 44%, respectively. POM, PLD and DEX is a treatment option for relapsed/refractory MM patients including those who are refractory to LEN.

Introduction Ruxolitinib (RUX) is an FDA-approved oral, selective inhibitor of Janus kinase (JAK) 1/2 for the treatment of patients with polycythemia vera who have had an inadequate response to or are intolerant of hydroxyurea or intermediate or high-risk myelofibrosis (MF). Preclinical studies from our laboratory have demonstrated that RUX in combination with the immunomodulatory agent lenalidomide (LEN) and dexamethasone shows marked anti-myeloma effects both in vitro and in vivo. Furthermore, MUC1 (Mucin 1) is responsible for LEN resistance in MM cells, and RUX blocks its expression in multiple myeloma (MM) cells. Thus, RUX may restore sensitivity to LEN. RUX also downregulates PD-L1 and PD-L2 expression on MM cells and reduces tumor stimulatory M2 macrophage polarization in MM bone marrow. Therefore, a phase 1 trial was conducted to determine the safety and efficacy of RUX in combination with LEN and the steroid methylprednisolone (MP) for relapsed/refractory (RR) MM patients who had previously been treated with LEN/steroids and a proteasome inhibitor (PI) and showed progressive disease at study entry. Methods A traditional 3+3 dose escalation design was used to enroll subjects in four cohorts with planned total enrollment to be 49 patients. Subjects received RUX twice daily continuously, LEN daily on d1-21 of a 28-d cycle and MP orally every other day. In DL0, patients received RUX 5 mg, LEN 5 mg, and MP 40 mg. In DL+1 and +2, both doses of LEN and MP remained unchanged and RUX was escalated to 10 and 15 mg, respectively. DL+3 escalated LEN to 10 mg with MP unchanged and RUX at 15 mg. Primary endpoints were safety, clinical benefit rate (CBR) and overall response rate (ORR). Results As of March 1, 2019, 43 patients were enrolled, and 40 were evaluable for efficacy. The median age was 65 years (range, 46-81), and 25 (58%) were male. Patients received a median of 6 prior treatments including LEN and steroids to which they were all refractory. No DLTs occurred, and DL+3 was expanded. Among all 40 evaluable patients, the CBR and ORR were 47% and 37%, respectively (1 CR, 4 VGPR, 10 PR and 4 MR), and 16 and 5 patients showed SD and PD. Notably, all 19 patients achieving > MR were refractory to LEN (i.e. progressed while on or within 8 weeks of last dosage). The median PFS for all evaluable patients was 4 months. G3/4 AEs included anemia (16%), sepsis (14%), lymphocytopenia (14%), pneumonia (12%), neutropenia (12%) and hypokalaemia (12%). Most common SAEs included sepsis (14%) and pneumonia (12%). Conclusions This phase 1 trial demonstrates for the first time that a JAK inhibitor, RUX, can overcome refractoriness to LEN and steroids for RRMM patients. This all oral regimen was well tolerated, and reversible Grade 3/4 cytopenias occurred in only a small minority of patients. These promising results are leading to expansion of the current clinical trial to 78 patients, and represent a potential novel therapeutic approach for treating MM. Disclosures Berenson: Takeda: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Amag: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Amag: Consultancy, Speakers Bureau; Sanofi: Consultancy; Amgen: Consultancy, Speakers Bureau; Sanofi: Consultancy; OncoTracker: Equity Ownership, Other: Officer; OncoTracker: Equity Ownership, Other: Officer; Takeda: Consultancy, Speakers Bureau; Incyte Corporation.: Consultancy, Research Funding; Incyte Corporation.: Consultancy, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding. Swift:Bristol Mayers Squib: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Jansen: Consultancy, Honoraria. Eades:Celgene: Other: Stock. Boccia:Genentech: Speakers Bureau; DSI: Speakers Bureau; AMAG: Consultancy; AstraZeneca: Speakers Bureau; Amgen: Speakers Bureau; Celgene: Speakers Bureau. OffLabel Disclosure: The goal of this clinical trial is to establish ruxolitinib in combination with lenalidomide and methylprednisolone as the therapy for relapse/refractory multiple myeloma patients.

8048 Background: Preclinical studies from our laboratory have demonstrated that ruxolitinib (RUX) in combination with lenalidomide (LEN) and dexamethasone shows marked anti-myeloma effects both in vitro and in vivo. Furthermore, MUC1 is responsible for LEN resistance in MM cells, and RUX blocks its expression in MM cells. Thus, RUX may restore sensitivity to LEN. Therefore, a phase 1 trial was conducted to determine the safety and efficacy of RUX in combination with LEN and methylprednisolone (MP) for relapsed/refractory (RR) MM patients (pts) who had previously been treated with LEN/steroids and a proteasome inhibitor (PI) and showed progressive disease at study entry. Methods: A traditional 3+3 dose escalation design was used to enroll subjects in four cohorts with planned total enrollment to be 49 pts. Subjects received RUX twice daily continuously, LEN daily on d1-21 of a 28-d cycle and MP orally every other day. In DL0, pts received RUX 5 mg, LEN 5 mg, and MP 40 mg. In DL+1 and +2, both doses of LEN and MP remained unchanged and RUX was escalated to 10 and 15 mg, respectively. DL+3 escalated LEN to 10 mg with MP unchanged and RUX at 15 mg. Primary endpoints were safety, clinical benefit rate (CBR) and overall response rate (ORR). Results: As of September 1, 2018, 36 pts were enrolled, and 32 were evaluable for efficacy. The median age was 66 years (range, 46-81), and 21 (58%) were male. Pts received a median of 6 prior treatments including LEN and steroids to which they were all refractory and a proteasome inhibitor. No DLTs occurred, and DL+3 was expanded. Among evaluable pts, the CBR and ORR were 47% and 41%, respectively (1 CR, 2 VGPR, 10 PR and 2 MR), and 14 and 3 pts showed SD and PD. All 15 responding pts were refractory to LEN. G3 AEs included anemia (17%), neutropenia (14%), sepsis (14%), lymphocytopenia (11%), thrombocytopenia (11%), and pneumonia (11%). Most common SAEs included sepsis (14%) and pneumonia (11%). Conclusions: This Ph 1 trial demonstrates for the first time that a JAK inhibitor, RUX, can overcome refractoriness to LEN and steroids for RR MM pts. These promising results are leading to expansion of the current clinical trial to 78 pts, and represents a novel therapeutic approach for treating MM. Clinical trial information: NCT03110822.

SUMMARY We report a case of a never-smoker male originally diagnosed with stage IIIb non-small-cell lung cancer that then presented with metastatic disease (EGFR/KRAS wild-type and ALK-positive) confined to the omentum 6 years later. Early metastasis to a variety of internal organs is common in the course of disease. Gastrointestinal metastasis of non-small-cell lung cancer is relatively rare and is typically found in the gastric or small bowel regions. Metastasis to the omentum is commonly seen with certain gynecologic cancer, but has not yet been well documented in pulmonary malignancies. Increasing use of molecular markers has altered the diagnosis, management and possible long-term prognosis of various subcategories of lung cancer. ALK-associated mutations have been found in a small minority of patients and its presence requires the use of novel therapeutics and, possibly, more prolonged surveillance for late metastasis.

To determine the effects of a specific chromosomal lesion, the BCR-ABL translocation t(9:22), in establishing DNA methylation profiles in cancer, we compared DNA methylation of 1,505 selected promoter CpGs in Chronic Myelogenous Leukemia (CML), Acute Lymphoblastic Leukemia (ALL) with and without the Philadelphia chromosome t(9:22), CD34 + hematopoietic stem cells transfected with BCR-ABL, and other tumors without BCR- ABL (Acute Promyelocytic Leukemia (APL) and Gastrointestinal Stromal Tumors (GIST)). In this study, the DNA methylation profile of CML was more closely related to APL, another myeloid leukemia, than Ph + ALL. Although DNA methylation profiles were consistent within a specific tumor type, overall DNA methylation profiles were not influenced by BCR-ABL gene translocation in the cancers and tissues studied. We conclude that DNA methylation profiles may reflect the cell of origin in cancers rather than the chromosomal lesions involved in leukemogenesis.

Neuropathy is an important complication that may limit treatment options for patients with multiple myeloma. Previous studies have focused on treatment efficacy and have shown that retreatment with bortezomib (BTZ) is an effective treatment option. The goal of this study was to focus on the clinical manifestations of peripheral neuropathy (PN) and to retrospectively compare the incidence and severity of PN between the initial BTZ regimen and upon retreatment. Furthermore, this study evaluated how certain factors affect BIPN, which will help determine what conditions should be considered prior to retreatment. Charts were reviewed from 93 patients who were retreated with a BTZ-containing regimen after previously being treated with this drug. Among the patients who developed PN, most patients in the study had low-grade neuropathy during the initial BTZ treatment (n = 52, 68%). The results showed no evidence of cumulative toxicity, and there was no significant difference in the incidence and severity of PN upon retreatment. Factors such as the presence of baseline PN, number of prior treatments, dose of BTZ, and comorbidities did not increase the severity of PN upon retreatment. The lapse of time between the two regimens also did not affect the severity of PN. The results suggest that retreatment with BTZ may be a feasible option, without additional risks of PN, for MM patients even with peripheral neuropathy during their initial treatment with this drug.

Introduction Ruxolitinib (RUX) is an FDA-approved oral, selective inhibitor of Janus kinase (JAK) 1/2 for the treatment of patients with polycythemia vera who have had an inadequate response to or are intolerant of hydroxyurea or intermediate or high-risk myelofibrosis (MF). Preclinical studies from our laboratory have demonstrated that RUX in combination with the immunomodulatory agent lenalidomide (LEN) and dexamethasone shows marked anti-myeloma effects both in vitro and in vivo. Furthermore, MUC1 is responsible for LEN resistance in MM cells, and RUX blocks its expression in multiple myeloma (MM) cells. Thus, RUX may restore sensitivity to LEN. RUX also downregulates PD-L1 and PD-L2 expression on MM cells and reduces tumor stimulatory M2 macrophage polarization in MM bone marrow. Therefore, a phase 1 trial was conducted to determine the safety and efficacy of RUX in combination with LEN and the steroid methylprednisolone (MP) for relapsed/refractory (RR) MM patients who had previously been treated with LEN/steroids and a proteasome inhibitor (PI) and showed progressive disease at study entry. Methods A traditional 3+3 dose escalation design was used to enroll subjects in four cohorts with expansion once a MTD was determined. Subjects received RUX twice daily continuously, LEN daily on d1-21 of a 28-d cycle and MP orally every other day. In DL0, patients received RUX 5 mg, LEN 5 mg, and MP 40 mg. In DL+1 and +2, both doses of LEN and MP remained unchanged and RUX was escalated to 10 and 15 mg, respectively. DL+3 escalated LEN to 10 mg with MP unchanged and RUX at 15 mg. Primary endpoints were safety, clinical benefit rate (CBR) and overall response rate (ORR). Results Thirty-four patients have been enrolled, and 31 were evaluable for response as of July 13, 2018. The median age was 68 years (range, 49-81), and 20 (59%) were male. Patients received a median of 6 prior treatments including lenalidomide and a PI. No DLTs occurred, and DL+3 was expanded (n=25). Among all 31 evaluable patients, the CBR and ORR were 48% and 39%, respectively (1 CR, 9 PR, 2 VGPR, and 3 MR), and 11 and 5 showed SD and PD, respectively. Notably, all 15 patients achieving ≥ MR were refractory to LEN (i.e. progressed while on or within 8 weeks of last dosage). The median PFS for all evaluable patients was 7.4 months. Grade 3/4 adverse events included transient thrombocytopenia (11.1%), anemia (8.8%), neutropenia (6.7%), hypoxia (4.4%) and gastrointestinal bleeding (4.4%). Common serious adverse events included abdominal pain (6.7%), gastrointestinal bleeding (6.7%) and hypoxia (6.7 %). Conclusions This phase 1 trial demonstrates for the first time that a JAK inhibitor, RUX, can overcome refractoriness to LEN and steroids for RRMM patients. This all oral regimen was well tolerated, and reversible Grade 3/4 cytopenias occurred in only a small minority of patients. These promising results are leading to other clinical trials using immunomodulatory agents, steroids and RUX, for treating RRMM patients, and this may represent a new therapeutic approach for these patients. Disclosures Berenson: BMS: Consultancy, Honoraria, Speakers Bureau; Amgen: Consultancy, Honoraria; Takeda: Honoraria, Speakers Bureau; Daiichi: Speakers Bureau; Celgene: Honoraria, Speakers Bureau; Janssen: Honoraria, Speakers Bureau.

8005Background: Preclinical studies from our laboratory have demonstrated that ruxolitinib (RUX) in combination with lenalidomide (LEN) and dexamethasone shows marked anti-myeloma effects both in vitro and in vivo. Furthermore, MUC1 is responsible for LEN resistance in MM cells, and RUX blocks its expression in MM cells. Thus, RUX may restore sensitivity to LEN. Therefore, a phase 1 trial was conducted to determine the safety and efficacy of RUX in combination with LEN and methylprednisolone (MP) for relapsed/refractory (RR)MM patients (pts) who had previously been treated with LEN/steroids and a PI and showed progressive disease at study entry. Methods: A traditional 3+3 dose escalation design was used to enroll subjects in four cohorts with planned total enrollment to be 28 pts. Subjects received RUX twice daily continuously, LEN daily on d1-21 of a 28-d cycle and MP orally every other day. In DL0, pts received RUX 5 mg, LEN 5 mg, and MP 40 mg. In DL+1 and +2, both doses of LEN and MP remained unchanged...

8013 Background: The proteasome inhibitor (PI) ixazomib (Ixz) is the first orally administered PI approved for treating multiple myeloma (MM). It has shown clinical activity as a single agent and when used in other combinations. In this phase 1/2 trial, we evaluated Ixz as a replacement therapy for bortezomib or carfilzomib for MM patients who were refractory to a bortezomib- or carfilzomib-containing combination regimen. Methods: This was a phase 1/2, intra-patient, multicenter, open-label trial evaluating the replacement of ixazomib for bortezomib or carfilzomib for MM patients who were refractory in combination with the other agents that the patients had received and failed. Patients received Ixz on days 1, 8 and 15 on a 28-day schedule and the other drugs were administered using the same doses and schedules as they were receiving during their prior regimen. If the Ixz maximum tolerated dose (MTD) for a particular combination regimen was previously determined, then patients were enrolled directly into Phase 2 (PhII). If not, MTD was determined during the Phase 1 (PhI) portion of the trial. Results: To date, a total of 40 patients have been enrolled; 37 patients (21 were enrolled in PhI and 16 in PhII) had completed at least one cycle of this treatment. Patients received a median of 5 prior treatments (range, 1-22). The median follow-up time for all patients was 1.6 months (range, 0.1-10.7 months), whereas that of PhII was 2.2 months (range, 0.2-10.7 months). There was no clinical benefit (CBR; 0%) nor any overall response rate (ORR; 0%) for patients receiving Ixz 3 mg (PhI). Nine patients (43%) showed stable disease (SD) while 12 (57%) exhibited disease progression (PD). In PhII (4mg Ixz) portion of the trial, ORR and CBR were both 18.7% with 16 (43.2%) patients showing SD, and 18 (48.6%) patients displaying PD. Common ≥ Gr3 adverse events were anemia (11%), thrombocytopenia (5.4%), hyponatremia (5.4%), dehydration (5.4%) and neutropenia (2.7%). Conclusions: Replacement of bortezomib or carfilzomib with Ixz infrequently leads to responses among RRMM patient who have progressed while on proteasome inhibitor -containing combination regimens. Clinical trial information: NCT02206425.

e19528 Background: Pomalidomide (POM) is a third –generation immunomodulatory drug shown to be safe and effective for the treatment of relapsed/refractory multiple myeloma (RRMM) for patients (pts) previously treated with bortezomib and lenalidomide (LEN) and in combination with dexamethasone it has been shown to overcome resistance in RRMM. In this phase 2 trial, we are evaluating the efficacy, safety and tolerability of POM as a replacement therapy for LEN for pts who have progressed receiving a LEN combination regimen. Methods: This is a phase 2, multicenter, open-label and non-randomized study. Pts who have failed a combination regimen containing LEN were treated with POM along with all of the other drugs previously used in the regimen. POM administered orally (dose is determined based on the previous regimen) on days 1-21 of a 28-day cycle, whereas other drugs are administered using the same schedule(s), dose(s) and drug combination as the last LEN-containing regimen that the patient received and failed. The planned enrollment on the study will be 45 pts. Results: To date, a total of 29 pts have been enrolled, 25 pts are evaluable and 12 pts have discontinued treatment. Of the evaluable pts, 9 (36%) and 16 (64%) received 3mg and 4mg of POM, respectively. The median age of all pts was 72 years (range, 52-81), and 17 (68%) were males. Pts have received a median of 3 prior treatments (range, 1-7). The median follow-up time for all pts is 3.1 months (range, 0.2-8.1). Amongst evaluable pts, 5 (23.8%) pts achieved at least a minimal response, 10 (47.6%) pts showed stable disease while 6 (28.6%) pts exhibited disease progression. At the time of data cutoff, only 17 pts have completed more than 1 full cycle of treatment; and, thus, the overall response and clinical benefit rates are fairly low (14.3% and 23.8%, respectively) but expected to improve with further follow up. The median PFS for the cohort was 7.6 months. Common ≥ Gr3 adverse events were neutropenia (8%), hypomania (4%) and leukopenia (4%). Conclusions: We show thatPOM appears to be promising replacement therapy for LEN in RRMM pts who have progressed within receiving a LEN combination regimen.

In this open-label, intra-patient phase I/II trial, bortezomib was replaced with carfilzomib (escalated from 20 to 45 mg/m(2) on days 1, 2, 8, 9, 15 and 16 of a 28-day cycle) for multiple myeloma (MM) patients who progressed while on or within 12 weeks of receiving a bortezomib-containing combination regimen. Study objectives included determination of the maximum tolerated dose (MTD), overall response rate (ORR), clinical benefit rate (CBR), time to progression, time to response, duration of response, progression-free survival and overall survival (OS). Of 38 registered patients, 37 were treated and evaluable for efficacy and safety. Thirty-one carfilzomib-based regimens using 14 different drug combinations were tested. One regimen (carfilzomib (45 mg/m(2)), ascorbic acid (1000 mg) and cyclophosphamide (2.2 mg/kg)) reached MTD. ORR and CBR were 43.2 and 62.2%, respectively. Median progression-free survival, time to progression and OS were 8.3, 9.9 and 15.8 months, respectively. Hematologic adverse events (AEs; ⩾grade 3) included lymphopenia (35.1%), thrombocytopenia (24.3%), anemia (10.8%) and neutropenia (10.8%). Nonhematologic AEs (⩾grade 3) included fever (5.4%) and hypokalemia (5.4%). These results demonstrate that replacing bortezomib with carfilzomib is safe and can be effective for MM patients failing bortezomib-containing combination regimens. This trial was registered at http://www.clinicaltrials.gov (#NCT01365559).

5035Background: Genomic profiling of metastatic PCa tissue is challenging and circulating tumor cells may not recapitulate entire tumor biology. Cell-free ctDNA is detectable in most patients (pts)...

Nearly all patients with multiple myeloma will eventually relapse; and, thus, it is critical to identify new treatments that increase therapeutic options for these patients. This review highlights the newest approaches with already approved drugs for treating this common B-cell malignancy.Most patients with multiple myeloma in both the frontline and relapsed/refractory settings are now treated with a combination of dexamethasone with the proteasome inhibitor bortezomib and/or an immunomodulatory agent thalidomide or lenalidomide. However, alkylating agents including melphalan, cyclophosphamide and most recently bendamustine as well as anthracyclines, especially the pegylated liposomal doxorubicin, have shown high response rates and prolonged remissions when combined with these agents. There are emerging data showing the importance of maintenance therapy especially with lenalidomide. Because of the marked improvement in survival of multiple myeloma during the past decade, there has been a renewed emphasis on developing therapies that are not only effective but also well tolerated. Alternative dosing, scheduling and routes of administration of already approved drugs have proven effective in accomplishing these goals.The availability of drugs with different mechanisms that produce anti-multiple myeloma effects and also show synergistic effects has paved the way for more effective and safer combinations and led to multiple myeloma patients living longer with improved quality of lives.

Division of Hematology and Oncology, Department of Medicine, David Geffen School of Medicine at UCLA,Los Angeles, CaliforniaData on Epstein–Barr virus-related hemophago-cytic lymphohistiocytosis (EBV-HLH) in adultsin the United States remain very limited. A clus-ter of four cases of EBV-HLH was observed in a4-month period at a tertiary center in LosAngeles County (LA County) and the clinicaland molecular characteristics identified in thesecases are being described. EBV typing, immu-nophenotypic and molecular genetic studieswere performed. Diagnostic criteria that may beused to identify EBV as a cause of HLH in adultsare also being suggested. Finally, the crude in-cidence rate for HLH in LA County was deter-mined and was compared to the worldwidecrude incidence rate for HLH. The cases eachoccurred in young male adult residents ofCalifornia and were associated with evidenceof EBV reactivation and ferritin levels of>20,000 mg/L. A higher rate of cases of EBV-HLH in 2010 was found at UCLA Medical Centerthan for 2007–2009 (4.9/10,000 hospitaldischarges vs. 0.14/10,000 hospital discharges,respectively; P ¼ 0.0017). The cases were asso-ciated with EBV type 1, and the insertion of thecodon CTC (leucine) was found in numerous ofthe EBNA-2 gene sequences. The annualincidence of secondary, non-familial HLH wasestimated to be 0.9 cases per million persons>15 years of age in LA County. Although EBV-HLH is a rare disease, the incidence in adultsin Western countries may be underestimated.J. Med. Virol. 84:777–785, 2012.

Hemophagocytic lymphohistiocytosis (HLH) is a heterogeneous disorder characterized by excessive activation and proliferation of nonmalignant histiocytes, which are commonly found in bone marrow, lymph nodes, spleen and liver in affected patients. Here, we report the presence of glomerular macrophages, including one showing erythrophagocytosis, on renal biopsy in a 25-year-old patient with clinical presentation and laboratory changes consistent with HLH. The clinical course was marked by persistent fever for 2 months, pleural and pericardial effusion, splenomegaly, lymphadenopathy, pancytopenia, cardiac arrhythmias, multiple organ dysfunction, and proteinuria, with demise after a 2-month hospitalization. Positive assay for Epstein-Barr virus (EBV), marked hyperferritinemia, hypofibrinogenemia, hypertriglyceridemia, elevated anti-nuclear antibody, proteinuria, and decreased circulating NK cells by flow cytometry were compatible with the diagnosis of HLH. We suggest that the glomerular hemophagocytic macrophages, which have not heretofore been described in the kidney of a patient with HLH, may have contributed to renal dysfunction manifest as proteinuria.

Background Immunomodulatory drugs (IMiD), such as thalidomide and lenalidomide (LEN) and its newest derivative pomalidomide (POM), have shown great promise for the treatment of multiple myeloma (MM) patients (pts). POM has in vitro anti-MM potency and has shown efficacy for the treatment of relapsed/refractory (RR) MM pts. POM with dexamethasone (DEX) induces responses even for MM pts who are refractory to bortezomib (BORT) and LEN (Richardson et al, 2012). Pegylated liposomal doxorubicin (PLD) with BORT is FDA-approved for the treatment of MM pts who have received one prior therapy not containing BORT. The combination of PLD and LEN or thalidomide has shown efficacy for both RR and frontline MM pts (Offidani et al, 2006; 2007). We have also demonstrated that both the efficacy and tolerability of LEN in combination with DEX, PLD and BORT (DVD-R) may be improved by changing the doses and schedules of these drugs (Berenson et al, 2012). Based on these results, we hypothesized that the combination of POM, DEX and PLD would be effective for the treatment of RRMM pts. Thus, we conducted the first study investigating the safety and efficacy of POM in combination with intravenous (IV) DEX and PLD as a phase 1/2 trial using a modified dose, schedule and longer 28-day cycles for pts with RRMM. Methods The phase 1 portion enrolled MM pts w/progressive disease whereas those enrolled in phase 2 also had to be refractory to LEN (single-agent or in combination), as demonstrated by progressive disease while receiving their last LEN-containing regimen or relapsed within 8 weeks of their last dose of this IMiD. Pts who previously received POM treatment were ineligible. POM was administered orally on days 1-21 of a 28-day cycle, while DEX (40 mg) and PLD (5mg/m2) were both infused on days 1, 4, 8, and 11. During phase 1 enrollment, three cohorts were enrolled at 2, 3 and 4 mg doses of POM, and DEX and PLD were both administered at fixed doses. Phase 2 enrollment commenced once the MTD was established from the phase 1 portion of the study. Results As of August 1, 2015, 70 pts were screened, 68 were enrolled in the trial (with the pre-planned enrollment goal reached) and had received study drug, and a total of 50 pts were evaluable for safety and efficacy. Among all enrolled pts, 60pts discontinued treatment and 8 remain active. Pts had received a median of 4 prior treatments (range 1-18). Median number of cycles for all pts was 4 (range 1-8), with a median follow-up time of 5.5 months (range 0-22). During the phase 1 portion of the trial, the maximum tolerated dose (MTD) of POM was established at 4 mg. Enrollment of pts into the phase 2 portion of the trial began at the MTD. However, neutropenia ≥ grade 3 was observed at this dose in 10/17 (58.8%) phase 2 pts; and, as a result, the protocol was amended so that the MTD was lowered to 3 mg for all pts subsequently enrolled. Among the 57 pts enrolled in phase 2, 53% percent were refractory to LEN and steroids with or without other agents and 31% had previously received PLD. A median of 4 cycles (range, 1 to 8) were administered among the pts enrolled in phase 2. Fifty-five pts were evaluable for response as 2 pts are active but have not yet had any post-baseline disease assessment. Among all evaluable pts (n=55) enrolled in phase 2, the overall response rate (ORR) was 33% (CR= 5%, VGPR= 5% and PR=23%) and clinical benefit rate (CBR) was 47% with 11 pts (26%) showing stable disease and 5 pts (12%) demonstrating progressive disease. For all evaluable pts enrolled in phase 2, the median follow-up time was 3.6 months (range 0-12 months) and the median PFS was 4.2 months (range 0.3- 29.0+). ORR and CBR for pts in the phase 2 were higher among pts receiving POM at 3 mg (36% and 51%, respectively) than among pts receiving POM at 4 mg (25% and 37%, respectively). Pts receiving the 4 mg dose of POM experienced more toxicities resulting in discontinuations, which likely explains the lower ORR and CBR observed among pts receiving this POM dose. Common ≥ grade 3 adverse events observed throughout the trial were neutropenia (49 pts; 72.0%), lymphopenia (36 pts; 52.9%), leukopenia (33 pts; 48.5%), hyponatremia (27 pts; 39.7%), and hypokalemia (26pts; 38.2%). One pt died of grade 5 sepsis. Conclusions This phase 1/2 trial is the first evaluating POM with PLD and DEX and demonstrates that the combination of POM at 3 mg, PLD and DEX using a modified 28-day cycle schedule is safe and effective for the treatment of MM pts refractory to LEN. Disclosures No relevant conflicts of interest to declare.

8591 Background: Treatment with POM and DEX has shown efficacy in RRMM patients (pts), even in those refractory to lenalidomide (LEN). In this phase (Ph) 1/2 trial, we investigated the POM-DEX-PLD ...

e19586^ Background: The POM-DEX combination is an effective treatment for RRMM patients (pts), even those refractory to lenalidomide (LEN). We investigated the combination of POM-DEX-PLD using a mo...

Background Thalidomide and its immunomodulatory drug (IMiD) derivatives such as lenalidomide (LEN) have shown great promise as treatment options for multiple myeloma (MM) patients (pts). Pomalidomide (POM) is a newer IMiD with high in vitro anti-MM potency that has shown promise as an effective treatment option for relapsed/refractory (R/R) MM pts. Recent data has shown POM to be effective in combination with dexamethasone (DEX), even for patients who are refractory to bortezomib and lenalidomide (Richardson et al, 2012). It has been demonstrated that the addition of pegylated liposomal doxorubicin (PLD) to lenalidomide and thalidomide is effective for both R/R and frontline MM pts (Offidani et al, 2006; 2007). Our recent trial evaluating lenalidomide in combination with dexamethasone, PLD, and bortezomib (DVD-R) showed that both efficacy and tolerability may be improved by changing the doses and schedules of these drugs (Berenson et al, 2012). These results imply that the combination of POM, DEX and PLD may be an effective regimen for treating R/R MM pts. We conducted a phase 1/2 trial investigating the safety and efficacy of POM in combination with IV DEX and PLD using our modified dose, schedule, and longer 28-day cycle for pts with R/R MM. Methods For enrollment into the phase 1 portion of the study, eligible pts had to show progressive MM at the time of enrollment. For participation in the phase 2 portion, pts had to be refractory to LEN (singe-agent or in combination) demonstrated by progressive disease while receiving this IMiD or relapsed within 8 weeks of their last dose. Pts who received previous POM treatment were ineligible. During the phase 1 part of the trial, POM was administered orally at 2, 3, or 4 mg daily in 3 successive cohorts of 3 pts each on days 1-21 of each 28-day cycle. DEX (40 mg intravenously over 30 min) and PLD (5 mg/m2infused over 30-90 min) both were given on days 1, 4, 8, and 11 of each cycle. POM doses were escalated per cohort until maximum tolerated dose (MTD) was reached. Phase 2 patients were all enrolled at what was established as the MTD. Results As of July 9, 2013, 33 pts were registered: 11 and 22 enrolled in the phase 1 and 2 portions, respectively. Fifteen patients have discontinued treatment thus far and 18 remain active. Pts had received a median of 5 prior treatments (range, 1-18) with a median of 1 prior PLD regimen (range, 0-2) and 1 prior IMiD regimen (range, 0-4). Pts have completed a median of 4 cycles (range: 0-8) with a median of 3.2 months of follow up (range: 0-9.7). During the phase 1 portion, no dose-limiting toxicities were identified, so the highest administered dose of 4 mg was initially established as the MTD. However, neutropenia ≥ G3 was observed in 10/17 phase 2 pts (58.8%) at this dose; and, as a result, the protocol was amended so that the MTD was lowered to 3 mg for all phase 2 pts beyond the 29thpt (n = 4). Thirty-two pts have received study drug. Twenty-nine pts were evaluable for response at data cutoff as 3 were active but had not yet had any post-baseline disease assessments. Of the 29 considered evaluable, overall response rate and clinical benefit rates were 34.5% and 48.3%, respectively, Six pts (20.7%) showed stable disease while 8 (27.6%) pts exhibited progressive disease. There was ≥ grade 3 (G3) neutropenia in 12 (37.5%) pts. Two pts experienced this toxicity during the phase 1 portion (3 mg and 4 mg doses- 1 each) whereas the other 10 pts experienced this side effect at the 4 mg dose during phase 2 enrollment. Following the lowering of the MTD to 3 mg due to this issue, neutropenia (≥ G3) has not been observed to date. Other treatment-emergent AEs ≥ G3 occurring in more than 1 pt were leukopenia in 12 (37.5%), lymphopenia in 8 (25%), anemia in 3 (9.4%), hyponatremia in 3 (9.4%), thrombocytopenia in 2 (6.3%), and duodenal ulcers in 1 (3.4%) pts. One pt died during treatment due to sepsis. Conclusions Results from this phase 1/2 trial demonstrate that the combination of pomalidomide with dexamethasone and PLD using a 28-day cycle shows efficacy for MM pts with progressive disease who are refractory to lenalidomide. Notably, less neutropenia has been observed at the 3 mg dose of pomalidomide, with 4 mg POM dosing associated with a high incidence of ≥ G3 neutropenia when used in this three-drug combination. Disclosures: Berenson: Celgene: Honoraria, Research Funding, Speakers Bureau. Swift:Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Vescio:Celgene: Speakers Bureau.

8599 Background: Recent data has shown that single-agent CFZ can produce responses among MM pts refractory to previous treatment regimens including those containing BTZ. We conducted an intrapatient phase I/II trial investigating the safety and efficacy of CFZ as a replacement for BTZ in BTZEcontaining regimens to which pts have progressed. Methods: Eligible pts progressed while receiving their most recent BTZEcontaining regimen after at least 4 doses of BTZ at ≥1.0 mg/m² in ≤4 weeks per cycle. CFZ replaced BTZ in each regimen via intravenous administration over 30 min on days 1, 2, 8, 9, 15, and 16 of each cycle. Treatment continued using the same dose(s) and schedule(s) of each drug administered in the BTZEcontaining regimen. CFZ doses were escalated on each of the first 4 cycles from 20 to 27, 36, and 45 mg/m² or until a maximum tolerated dose (MTD) was reached for that regimen. Results: Of 37 enrolled pts, 33 were evaluable for efficacy and 37 for safety. Pts received a median of 4 prior treatments (range, 1-23) and 2 different BTZ-containing regimens (range, 1-13). Pts were treated with CFZ and a variety of different combinations of agents, including: bendamustine, clarithromycin, cyclophosphamide, dexamethasone, melphalan, methylprednisolone, pegylated liposomal doxorubicin, thalidomide, lenalidomide, and ascorbic acid. Pts have completed a median of 3 cycles with 12 pts going on to maintenance. One of 14 combinations, CFZ + ascorbic acid + cyclophosphamide, has reached a MTD at 45 mg/m2. Clinical benefit was seen in 23 (70%) evaluable pts (complete response = 6%; very good partial response = 18%; partial response = 21%; minor response = 24%) with another 18% showing stable disease. The median time to progression is 8.8 mo. (95% CI: 6.4-11.1 mo.) with 21 pts progressing overall and 9 progressing on study treatment. The most common ≥ G3 adverse events were lymphopenia (35% of pts), thrombocytopenia (19%), neutropenia (11%), and anemia (8%). Conclusions: These results suggest that replacement of BTZ with CFZ in a BTZ-containing combination regimen to which a MM patient is failing often leads to responses and is well-tolerated. Clinical trial information: NCT01365559.

8598 Background: POM is a newer IMiD immunomodulatory compound with high in vitro potency that has shown promise in combination with DEX as an effective treatment option for R/R MM patients (pts), even those refractory to bortezomib and lenalidomide (LEN). We conducted a phase I/II trial investigating the safety and efficacy of POM in combination with IV DEX and PLD using a modified dose and longer 28-day schedule in R/R MM pts. Methods: Phase I pts had progressive MM at the time of enrollment that was R/R to at least one anti-MM regimen. Phase II pts had to be refractory to LEN (singe-agent or in combination) demonstrated by progressive disease while receiving LEN or relapse within 8 weeks of its last dose. During phase I, POM was administered orally at 2, 3, or 4 mg daily in 3 successive cohorts of 3 pts each on days 1-21 of each 28-day cycle. DEX was administered IV at 40 mg over 30 min and PLD was administered at 5 mg/m2as an IV infusion over 30-90 min on days 1, 4, 8, and 11 of each cycle. POM doses were escalated until maximum tolerated dose (MTD) was reached. Once MTD was reached, all subsequent pts were enrolled at that dose. Results: To date, 27 pts have been enrolled, with 18 evaluable for efficacy and 19 for safety. Pts received a median of 5 prior treatments (range, 1-18) with a median of 1 prior PLD regimens (range, 0-2). Pts have completed a median of 1 cycle (range: 0-8) with a median of 1.4 months of follow up (range: 0-7.1). No DLTs were seen during phase I, which established the MTD at 4 mg. Phase II has enrolled 16 pts at 4 mg so far. Clinical benefit was seen in 7 (39%) of evaluable pts (partial response = 22%; minor response = 17%) with another 44% showing stable disease. Common ≥ G3 adverse events included leukopenia (32% of pts), neutropenia (32%), lymphopenia (26%), and hyponatremia (16%). Neutropenia at ≥ G3 occurred in more than half of patients on the phase II trial (4 mg POM). Conclusions: The combination of pomalidomide with dexamethasone and PLD on a 28-day cycle shows efficacy for R/R MM pts. However, because of excessive ≥ G3 neutropenia, POM is being reduced to 3 mg for newly enrolled patients. Clinical trial information: NCT01541332.

Abstract 2979 Background: Thalidomide and its immunomodulatory (IMiD) derivatives such as lenalidomide have shown great promise as a treatment option for multiple myeloma (MM) patients. Pomalidomide is a newer IMiD with high in vitro potency that has shown promise as an effective treatment option for relapsed/refractory (R/R) MM patients. Recent data has shown pomalidomide to be effective in combination with dexamethasone, even for patients who are refractory to bortezomib and lenalidomide. It has been demonstrated that the addition of pegylated liposomal doxorubicin (PLD) to lenalidomide and thalidomide is effective for both R/R and frontline MM patients. Our recent trial evaluating lenalidomide in combination with dexamethasone, PLD, and bortezomib (DVD-R) showed that both efficacy and tolerability may be improved by changing the dose and schedule of these drugs. These data imply that the combination of pomalidomide, dexamethasone, and PLD for R/R MM patients may be an effective regimen. We conducted a phase 1/2 trial investigating the safety and efficacy of pomalidomide in combination with IV dexamethasone and (PLD) using a modified dose and longer 28-day schedule for patients with R/R MM. The combination of PLD, dexamethasone and lenalidomide without bortezomib has not been previously evaluated. Methods: For enrollment into the phase 1 dose-escalation portion of the study, eligible pts had to have progressive MM at the time of enrollment that has relapsed following stabilization to at least one anti-myeloma regimen or is refractory defined as progressed while receiving anti-myeloma treatment. For enrollment into the planned phase 2 portion of the study, eligible pts have to be refractory to lenalidomide (singe-agent or in combination) demonstrated by progressive disease while receiving lenalidomide or relapse within 8 weeks of the last dose of lenalidomide. Patients who have received previous pomalidomide treatment were not eligible. Patients must not have received chemotherapy, corticosteroids, immunotherapy, antibody therapy, or treatment with thalidomide, lenalidomide, or bortezomib within 3 weeks of receiving study drug, nor extensive radiation therapy within 4 weeks of receiving study drug. During the phase 1 part of the trial, pomalidomide was administered orally at 2, 3, or 4 mg daily in 3 successive cohorts of 3 patients each on days 1–21 of each 28-day cycle. Dexamethasone was administered intravenously at 40 mg over 30 min on days 1, 4, 8, and 11 of each cycle. PLD was administered at 5 mg/m2 as an IV infusion over 30–90 min on days 1, 4, 8, and 11 of each cycle. Pomalidomide doses were escalated until maximum tolerated dose (MTD) was reached. Once MTD was reached, all subsequent patients will be enrolled at that dose. Results: Ten of 40 planned patients have been enrolled to date, all during the phase 1 portion of the trial. Pts received a median of 4 prior treatments (range, 1–8) with a median of 1 prior PLD regimens (range, 0–1). Pts have completed a median of 1 cycle (range: 0–12) with a median of 1.2 months of follow up (range: 0.2–3.6). To date, the trial has enrolled all three cohorts in phase 1 (1 patient in cohort 1 chose to withdraw during Cycle 1 and was replaced). MTD has not yet been reached with no dose limiting toxicities (DLTs) in the first 2 cohorts. The 3 patients in cohort 3 are currently in Cycle 1. Seven patients are currently evaluable for efficacy and the 3 patients in cohort 3 have not yet had response assessment. Best response for evaluable patients is as follows: 3 patients have shown partial response, 2 patients have shown minor response, 1 is exhibiting stable disease (SD) but shows a decreasing monoclonal protein, and 1 has progressed. The incidence of reported adverse events (AEs) so far is low. Only 1 grade 3 or 4 (G3, G4) AE has been observed. The most common AEs were lymphopenia, which occurred in 6 pts (G1: 3; G2: 2; G3: 1), elevated urea nitrogen occurred in 4 pts (all G1), neutropenia occurred in 4 pts (all G1), and leukopenia occurred in 4 pts (all G1). Conclusions: The combination of pomalidomide with dexamethasone and PLD on a 28-day cycle may be an effective treatment option with acceptable tolerability for relapsed/refractory MM patients. Disclosures: Berenson: Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau.

Abstract 4063 Background: Carfilzomib has shown promise in treating both newly diagnosed and relapsed/refractory MM patients. Recent data has suggested that single agent carfilzomib can produce a response for MM pts refractory to previous treatment regimens, including prior bortezomib-containing regimens. We conducted an intrapatient Phase 1/2 trial investigating the safety and efficacy of carfilzomib as a replacement for bortezomib in bortezomib-containing regimens to which pts have progressed. Methods: Eligible pts had to have progressed while receiving their most recent bortezomib-containing regimen after at least 4 doses of bortezomib at ≥ 1.0 mg/m2 in ≤ 4 weeks per cycle. Patients treated with bortezomib-containing combination regimens, including alkylating agents, anthracyclines, glucocorticosteroids, and/or immunomodulatory agents were eligible. Carfilzomib replaced bortezomib in each regimen via intravenous administration over 30 min on days 1, 2, 8, 9, 15, and 16 of each 28 day cycle. For each pt, carfilzomib doses were escalated on each of the first 4 cycles from 20 to 27, 36, and 45 mg/m2 or until maximum tolerated dose (MTD) was reached for that regimen. Aside from carfilzomib replacing bortezomib, regimens utilized the same dose(s) and schedule(s) of each drug administered in the most recent bortezomib-containing regimen to which the patient was refractory. Results: The trial has currently enrolled 32 of 45 planned patients. Pts received a median of 6 prior treatments (range, 1–18) and 2 different bortezomib-containing regimens (range, 1–13). Pts were treated with carfilzomib and 13 different combination regimens at varying doses and schedules. Agents in these combination regimens varied per patient, potentially including any of the following: ascorbic acid, bendamustine, clarithromycin, cyclophosphamide, dexamethasone, lenalidomide, melphalan, methylprednisolone, pegylated liposomal doxorubicin, and/or thalidomide. Six patients received a regimen containing IMiDs (thalidomide or lenalidomide). Pts have completed a median of 3 cycles (range: 0–12) with a median of 5.9 months of follow up (range: 0.4–14.1). To date, 1 regimen, carfilzomib + ascorbic acid + cyclophosphamide, has reached MTD with the maximum dose of carfilzomib (45 mg/m2) without any DLTs. Efficacy data stems from 24 of 32 enrolled pts who have completed at least 1 cycle of treatment. Clinical benefit was seen in 18 (75%) pts (complete response = 8.3%; very good partial response = 16.7%; partial response = 29.2%; minor response = 20.8%) with another 16.7% showing stable disease. Notably, response was achieved for each type of agent included in the trial across a variety of specific treatment combinations. Only 3 pts have progressed while on study, one with progressive disease after an initial response. Kaplan-Meier analysis reveals the median progression-free survival (PFS) at this point in the study to be 10 months (95% confidence interval: 8.6–11.2). The median time to progression (TTP) and duration of response (DOR) have not yet been reached. Safety data stems from all 32 enrolled pts. The most common grade 3/4 hematologic adverse events include: thrombocytopenia, occurring in 16 pts (G3: 5; G4: 2); lymphopenia occurring in 12 pts (G3: 6; G4: 1) and fever occurring in 2 pts (G3). The most common grade 3/4 non-hematologic adverse events include: fever, occurring in 8 pts (G3: 2); urinary tract infection, occurring in 3 pts (G3: 1); sepsis occurring in 1 pt (G4); pneumonia occurring in 2 pts (G3: 1); tumor lysis syndrome occurring in 1 pt (G3). Ten pts experienced a serious adverse event on study. Conclusions: These early results suggest that carfilzomib is an effective and tolerable replacement for bortezomib among pts who are refractory to bortezomib-containing combination regimens. These results appear to be applicable to a variety of combination regimens consisting of a wide array of therapeutic agents. Disclosures: Berenson: Onyx: Consultancy, Honoraria, Research Funding, Speakers Bureau.

DNA methylation changes are a common finding in leukemia, and hypermethylation of CpG island promoters is associated with aberrant gene silencing. Some abnormal cancer related methylation changes have been associated with clinical phenotype including pathologic features, prognosis, and treatment response. However, other DNA methylation changes do not appear to have phenotypic consequences and may reflect a stochastic event or a downstream event of tumorogenesis, such as the CpG island methylator phenotype (CIMP). In order to obtain a better understanding of the DNA methylation changes found in leukemia we analyzed 18 acute promyelocytic leukemia (APL) and 36 chronic myeloid leukemia (CML) patients. We specifically chose to study APL and CML as these leukemia are initiated by specific genetic translocation events, t(15:17) and t(9:22) respectively. To measure the DNA methylation status, we used the GoldenGate Assay for Methylation and BeadArray technology from Illumina, Inc. The Standard Methylation Cancer Panel I from Illumina interrogates 1505 CpG sites, selected from 807 genes (231 genes contain one CpG site per gene, 463 genes contain two CpG sites and 114 genes have three or more CpG sites). In our study we found 142 and 269 genes that were hypermethylated in CML and APL. 31 genes were uniquely hypermethylated in CML, 158 genes were hypermethylated only in APL, and 111 genes were hypermethylated in both leukemias. There was a unique pattern of hypermethylated genes in each cancer; such there was a high concordance of hypermethylated genes within each leukemia type. These data suggest that the epigenetic events were a result of the genetic translocation BCR/ABL or PML/RARα (associated with chromosomal aberrations t(9:22) or t(15:17)) that initiates these leukemias. Analysis of the number of hypermethylated genes in these two leukemias showed a bimodal distribution suggestive of CIMP, however, closer examination showed that this bimodal distribution could be attributed to the two different types of leukemia. APL patients had mean of 280 genes hypermethylated while CML patients only had a mean of 193 genes hypermethylated. APL had a stronger methylator phenotype than CML for the loci studied, which underscores the possible relationship of CIMP to a genetic phenotype. Subset analysis of our CML samples by chronic phase (23 patients), accelerated phase (5 patients), and blast crisis (8 patients) revealed 42 genes that became hypermethylated with progression of CML. It is possible that hypermethylation of these genes are clinically important in the leukemia phenotype, and maybe targets for epigenetic therapy. We examined the DNA methylation changes induced by the DNA methylation inhibitor, azacitidine, in a patient with blast crisis CML and refractory to imatinib mesylate therapy. Azacitidine could reverse the aberrant hypermethylation associated with progression of CML to blast crisis and supports the use of this drug as an epigenetic therapy. Our data show that the majority of DNA hypermethylation events in leukemia are dependent on genetic events, but there is a subset of DNA hypermethylation events that are involved in the progression of leukemia and may be therapeutically reversed by DNA methylation inhibitors.

Aberrant promoter DNA methylation is found in many cancers and is associated with aberrant gene silencing. Some abnormal cancer related methylation changes have been associated with a number of clinical features including pathologic features, prognosis, and treatment response. However, other DNA methylation changes do not appear to have phenotypic consequences and may reflect a stochastic event or a downstream event of tumorogenesis. In order to obtain a better understanding of DNA methylation changes in cancer we chose to study three very homogenous cancers: CML, APL and GIST. CML is a leukemia characterized by the Philadelphia chromosome, t(9:22), and is very sensitive to imatinib mesylate. APL is another leukemia characterized by a specific chromosomal translocation, t(15:17), and a high response to all-trans-retinoic acid. Lastly, GIST is a sarcoma with a high frequency of c-kit mutations and sensitivity to imatinib mesylate. In this study, we used bisulfite PCR/Pyrosequencing to accurately quantitate the absolute amount of DNA methylation of 15 genes (MDR, ID4, PROX1, ER, Jun B, p73, p15, ABL1, THBS1, p16, RASSF1A, RUNX3, SOCS1, RARA, and PML). We analyzed the blood and bone marrow of 58 patients with CML (chronic phase n=32, accelerated phase n=11, blast crisis n=13), and 23 patients with APL. We also analyzed tumors from 9 GIST patients. We show clearly that DNA methylation changes are not random events, but related to the biology of the cancer. We have identified at least four specific types of aberrant DNA methylation. Cancer specific hypermethylation of genes related to clinical phenotype. ID4 and PROX1 were hypermethylated in APL and CML, but not GIST. Methylation of these genes in CML was strongly associated with the development of blast crisis (P=0.0005), and the methylation of these two genes was also associated with an increase in the percentage of bone marrow blasts in APL (p In conclusion, ID4 and PROX1 hypermethylation is a marker of disease progression in leukemia such as blast crisis in CML and higher percentage of bone marrow blasts in APL. Methylation of ABL and RARalpha are specific to CML and APL, respectively, and is likely related to specific chromosomal translocations. There are genes that are subject to small increases or decreases in DNA methylation that do not correlate with any obvious biology or clinical features. Finally, there are genes that are clearly resistant to DNA methylation changes. Our study shows that DNA methylation changes are clearly non-random events, and suggests there are several mechanisms that drive DNA methylation changes.