Partial Clinical Responses Research Articles

Novel PAX3::MAML3 Fusion Identified in Alveolar Rhabdomyosarcoma, Using DNA Methylation Profiling to Expand the Genetic Spectrum of “Fusion-Positive” Cases

Alveolar rhabdomyosarcoma (ARMS) with FOXO1 gene rearrangements is an aggressive pediatric rhabdomyosarcoma subtype that is prognostically distinct from embryonal rhabdomyosarcoma and fusion-negative ARMS. Here, we report 2 cases of ARMS with PAX3::MAML3 fusions. The tumors arose in an infant and an adolescent as stage IV metastatic disease (by Children’s Oncology Group staging system). Histologically, both cases were small round blue cell tumors arranged in vague nests and solid sheets that were diffusely positive for desmin and myogenin. By methylation profiling and unsupervised clustering analysis, the tumors clustered with ARMS with classic FOXO1 rearrangements and ARMS with variant PAX3::NCOA1/INO80D fusions, but not with biphenotypic sinonasal sarcoma (BSNS) with PAX3::MAML3/NCOA2/FOXO1/YAP1 fusions nor with other small round blue cell tumors, including embryonal rhabdomyosarcoma. The differentially methylated genes between ARMS and BSNS were highly enriched in genes involved in myogenesis, and 21% of these genes overlap with target genes of the PAX3::FOXO1 fusion transcription factor. On follow-up after initiation of vincristine/actinomycin/cyclophosphamide chemotherapy, the tumors showed partial and complete clinical responses, consistent with typical upfront chemotherapy responsiveness of ARMS with the classic FOXO1 rearrangement. We conclude that PAX3::MAML3 is a novel variant fusion of ARMS, which displays a methylation signature distinct from BSNS despite sharing similar PAX3 fusions. These findings highlight the utility of methylation profiling in classifying ARMS with noncanonical fusions.

IgG4-related disease and B-cell malignancy due to an IKZF1 gain-of-function variant

BackgroundMonoallelic loss-of-function IKZF1 (IKAROS) variants cause B-cell deficiency or combined immunodeficiency, whereas monoallelic gain-of-function IKZF1 variants have recently been reported to cause hypergammaglobulinemia, abnormal plasma cell differentiation, autoimmune and allergic manifestations, and infections. MethodsWe studied seven relatives with autoimmune/inflammatory diseases and lymphoproliferative diseases. We analysed biopsy results and performed whole-exome sequencing and immunological studies. ResultsDisease onset occurred at a mean age of 25.2 years (range: 10-64, years). Six patients suffered from autoimmune/inflammatory diseases, four had confirmed immunoglobulin G4-related disease (IgG4-RD), and five developed B-cell malignancies: lymphoma in four and multiple myeloma in the remaining patient. Patients without immunosuppression were not particularly prone to infectious diseases. Three patients suffered from life-threatening COVID-19 pneumonia, one of whom had autoantibodies neutralizing interferon (IFN)-α. The recently described IKZF1 gain-of-function p.R183H variant was found in the five affected relatives tested and in a six-year-old asymptomatic girl. Immunological analysis revealed hypergammaglobulinemia and high frequencies of certain lymphocyte subsets (exhausted B cells, effector memory CD4 T cells, TEMRA and CD28-CD57+ CD4+ and CD8+ T cells, Th2 and Tfh2 cells) attesting to immune dysregulation. Partial clinical responses to rituximab and corticosteroids were observed, and treatment with lenalidomide, which promotes IKAROS degradation, was initiated in three patients. ConclusionHeterozygosity for gain-of-function IKZF1 variants underlies autoimmunity/inflammatory diseases, IgG4-RD and B-cell malignancies, the onset of which may occur in adulthood. Clinical and immunological data are similar to those for patients with unexplained IgG4-RD. Patients may therefore benefit from treatments inhibiting pathways displaying IKAROS-mediated overactivity.

Long Term Outcomes in Idiopathic Inflammatory Myositis: An Observational Epidemiologic Study over 15 Years.

We report a longitudinal observational cohort of idiopathic inflammatory myositis (IIM) focusing on the long-term clinical outcome and associated parameters. IIM patients were classified as per Bohan and Peter criteria. In those with ≥ 24 months of follow-up; the treatment response, functional outcomes, and damage at last follow-up were recorded. Complete clinical response and clinical remission as defined by Oddis et al., was used to define outcomes at last follow-up. The cohort consists of 175 patients, mean age 40.9 (+12.6) years, M:F 1:3.3; and the major subsets were dermatomyositis (44.6%), overlap myositis (25.7%), antisynthetase syndrome (6.3%), polymyositis (14.3%), and juvenile DM/OM (8.6%). Ninety-four patients have followed up for 24 months or more, with the median (IQR) of 65(35,100.7) months. Of them, 74.1% and 11.8% had complete and partial clinical responses respectively at the last follow-up. In our cohort 40.2% were off-steroids and 13.8% were in clinical remission at the last follow-up. Complete clinical response was associated with better functional outcomes and lesser damage as determined by HAQ-DI of 0[OR10.9; 95%CI (3.3,160)], MRS [OR 3.2; 95%CI (1.4,7.3)] and lesser MDI [OR 1.7; 95% CI (1.1,2.7)] respectively as compared to partial response (unadjusted analysis). Baseline parameters and IIM subsets did not significantly influence the functional outcome and damage. The mortality rate in our cohort is 24/175 (13.7%), the disease-specific mortality rate being 9.1%. Large majority of deaths were early, associated with active disease. We report good long-term outcomes in all major myositis subsets. Partial clinical response to treatment is associated with worse functional outcomes and damage accrual. Death occurs early in association with active disease.

Efficacy of the Allosteric MEK Inhibitor Trametinib in Relapsed and Refractory Juvenile Myelomonocytic Leukemia: A Report from the Children's Oncology Group

Background: Juvenile myelomonocytic leukemia (JMML) is an aggressive myeloproliferative neoplasm of infants and toddlers. Upfront therapies typically include high-dose cytarabine or azacitidine, but the only definitive treatment is hematopoietic stem cell transplantation (HSCT). While HSCT cures ~50% of patients, the prognosis is dismal for those who relapse. In the absence of a second HSCT, patients who relapse have a 2-year overall survival of ~10%. JMML is initiated by germline and somatic driver mutations in NF1, KRAS, NRAS, PTPN11, and CBL. These mutations converge on Ras signaling, leading to elevated levels of active Ras-GTP in specific cell lineages. Genetically engineered mouse (GEM) models accurately model key molecular, biologic, and biochemical features of JMML. Preclinical trials of the allosteric MEK inhibitors in Kras and Nf1 mutant mice demonstrated dramatic phenotypic responses with reduction in white blood cell counts, resolution of splenomegaly, and reversion to normal erythropoiesis. Based on the promising efficacy signal in GEM models, we evaluated trametinib, an orally bioavailable allosteric inhibitor of MEK1/2, in a prospective clinical trial in children with relapsed or refractory JMML to determine the overall response rate to trametinib. Results: Ten infants and children with JMML (median age 23.6 months) were enrolled and all were evaluable for safety and efficacy. Patients received age-adjusted dosing of trametinib for 28-day cycles and could remain on study for up to 12 cycles in the absence of disease progression or toxicity. A clonal Ras pathway mutation was confirmed in the blood and/or bone marrow of all patients. The objective response rate was 50% (two complete and three partial clinical responses). Four patients proceeded to HSCT after receiving protocol therapy and remain alive in complete remission with undetectable levels of the Ras pathway mutation identified at enrollment. Three additional patients completed 12 cycles of trametinib and continue to receive off-protocol therapy 6-24 months later with no change in the variant allele frequency of the underlying Ras pathway mutation. The remaining three patients had progressive disease with two demonstrating molecular evolution by the end of cycle 2. Paired pre- and post-trametinib RNASeq and proteomic analyses confirmed on-target biochemical effects of trametinib with down-regulation of both Ras/MAPK pathway related gene expression and MEK1/2 kinase activity, respectively. To gain deeper insight into how trametinib might affect distinct populations of hematopoietic cells, we generated single cell RNASeq data before and after treatment. The most prominent finding was a reduction in the proportions of classical and non-classical monocytes and broad downregulation of immune-related pathways in all cell populations. However, the downregulation of MAPK signaling genes was confined to specific cell types including, macrophages, classical monocytes, and granulocyte-monocyte progenitors, which uniquely displayed downregulated KRAS-related signatures following treatment. High DNA methylation and the presence of >1 mutation at enrollment correlated with lack of response to trametinib. Conclusions: We conducted an open label, phase 2 trial of trametinib in children with relapsed or refractory JMML. This is the first completed study of a MEK inhibitor in any hematologic malignancy in children. The trial met its primary objective and demonstrated a 50% objective response rate with 70% of patients bridging to a successful HSCT or completing the maximum 12 cycles permitted on study. The three patients who completed 12 cycles continue to receive trametinib off study for as long as 2 years. Although limited by a small number of patients, the long-term survival, and correlative molecular analyses from this trial raise provocative questions about whether certain patients with JMML might be spared the long-term adverse health risks of genotoxic HSCT conditioning regimens based on favorable molecular characteristics at diagnosis. This hypothesis will now be tested in a national, risk-stratified therapeutic trial (NCT05849662) of trametinib in combination with azacitidine in newly diagnosed patients. The authors would like to acknowledge funding and support from: NCTN Operations Center Grant (U10CA180886), NCTN Statistics & Data Center Grant (U10CA180899), and the St. Baldrick's Foundation.

Effective treatment with the selective cytokine inhibitor BNZ-1 reveals the cytokine dependency of T-LGL leukemia

AbstractT-cell large granular lymphocytic leukemia (T-LGLL) is a clonal proliferation of cytotoxic T lymphocytes that can result in severe neutropenia, anemia, and bone marrow failure. Strong evidence from patients and mouse models demonstrate the critical role of interleukin-15 (IL-15) in T-LGLL pathogenesis. BNZ-1 is a pegylated peptide that selectively inhibits the binding of IL-15 and other γc cytokines to their cellular receptor complex, which has demonstrated efficacy in ex vivo T-LGLL cells and transgenic mice in preclinical studies. We conducted a phase 1/2 trial of BNZ-1 in patients with T-LGLL who had hematocytopenias (anemia or neutropenia) and required therapy. Clinical responses were assessed using hematologic parameters (improvement in hematocytopenias) based on response criteria from the Eastern Cooperative Oncology Group 5998 T-LGLL trial. BNZ-1 demonstrated clinical partial responses in 20% of patients with T-LGLL with minimal toxicity and the maximum tolerated dose was not reached. Furthermore, T-LGL leukemic cells showed significantly increased apoptosis in response to BNZ-1 treatment as early as day 2, including in clinical nonresponders, with changes that remained statistically different from baseline throughout treatment (P < .005). We report first-in-human proof that T-LGL leukemic cells are dependent on IL-15 and that intervention with IL-15 inhibition with BNZ-1 in patients with T-LGLL shows therapeutic effects, which carries important implications for the understanding of the pathogenesis of this disease. This trial was registered at www.clinicaltrials.gov as #NCT03239392.

Efficacy of direct-acting antivirals in patients with hepatitis C virus-associated cryoglobulinemic vasculitis: Results of a long-term follow-up

Objective – to evaluate the long-term outcomes of HCV eradication with direct-acting antivirals (DAAs) in patients with hepatitis C-associated cryoglobulinemic vasculitis (HCV-CV)Materials and methods. We retrospectively assessed 48 patients with HCV-CV treated with DAAs. The activity of HCV-CV was assessed by using Birmingham Vasculitis Activity Score version 3 (BVAS v. 3). In patients with HCV-CV the rate of sustained virologic (defined as undetectable HCV-RNA levels 12 weeks after treatment cessation) and immunological (defined as absence of circulating cryoglobulins, rheumatoid factor and normal C4 level) response; and the rate of complete (defined by a BVAS v. 3 score of 0) and partial (defined as BVAS v. 3 score &lt;50% of the baseline score) clinical response were evaluated. Immunosupressants were given before or after DAAs therapy if clinically needed.Results. Median time of follow-up from treatment cessation were 26,5 (11,5–62,3) months. All 48 (100%) patients achieved sustained virologic response. Elimination of cryoglobulins were reported in 20 (41,7%) patients, complete immunological response-in 4 (8,3%) cases. Complete and partial clinical responses were observed in 13 (27,1%) and 19 (39,6%) patients, respectively. BVAS v. 3 score &lt;4 at baseline was independently associated with complete clinical response (ОR=7,58; 95% CI: 1,42–40,48; р=0,018). 3 (6,3%) patients demonstrated HCV-CV relapse.Conclusion. Patients with HCV-CV require a long-term follow-up period even after reaching the SVR. The use of BVAS v. 3 score before the DAAs therapy can facilitate the planning of therapeutic approach, particularly, when identifying the patients in whom the immunosupressive therapy should be considered after viral eradication.

Severe acute respiratory syndrome coronavirus-2 Alpha variant (B.1.1.7), original wild-type severe acute respiratory syndrome coronavirus 2, and cytomegalovirus co-infection in a young adult with acute lymphoblastic leukemia, case report, and review of the possible cytomegalovirus reactivation mechanisms

BackgroundLike other viral infections, severe acute respiratory syndrome coronavirus-2 infection could affect different human body systems, including host immune responses. Three years after its pandemic, we learn more about this novel coronavirus. As we expected, different co-infections with various organisms, such as viruses, bacteria, and even fungi, have been reported. However, concurrent infection with two severe acute respiratory syndrome coronavirus-2 strains and cytomegalovirus is extremely unusual. We have only a rudimentary understanding of such co-infections and their long-term consequences for patients with cancer.Case presentationAn 18-year-old young Iranian adult with acute lymphoblastic leukemia presented with abdominal pain, diarrhea, nausea, and vomiting following a recent history of severe acute respiratory syndrome coronavirus-2 infection. The patient never experienced respiratory symptoms, and the chest imaging study was normal on admission. His primary laboratory investigation revealed prerenal azotemia and severe abnormal liver function tests (blood urea nitrogen 32 mg/dL, creatinine 1.75 mg/dL, prothrombin time 66 s, partial thromboplastin time 44.5 s, international normalized ratio 5.14, total bilirubin 2.9 mg/dL, and direct bilirubin 2.59 mg/dL). Cytomegalovirus disease was diagnosed by polymerase chain reaction in his blood and stool samples. The patient’s gastrointestinal signs and symptoms improved shortly after receiving intravenous ganciclovir treatment. His gastrointestinal symptoms continued intermittently for weeks despite maintenance valganciclovir prescription, necessitating frequent hospitalizations. The patient was complicated by the recurrence of gastrointestinal symptoms during the sixth hospitalization, even though he had no respiratory symptoms, and the nasopharyngeal test revealed severe acute respiratory syndrome coronavirus-2 Wuhan strain for the first time. Remdesivir and valganciclovir were administrated due to persistent enteritis and evidence of intestinal tissue invasion by severe acute respiratory syndrome coronavirus 2 and cytomegalovirus on multiple intestinal biopsies, which led to partial clinical responses. Cytomegalovirus and severe acute respiratory syndrome coronavirus-2 fecal shedding continued for more than 6 months despite repeated antiviral therapy, and the Wuhan and Alpha strains were also detected in his nasopharyngeal samples through repeated sampling (confirmed by four nasopharyngeal sampling and multiple stool specimens and several intestinal biopsies). Finally, during the Delta-variant (B.1.617.2) outbreak in Iran, the patient was admitted again with febrile neutropenia and decreased level of consciousness, necessitating respiratory support and mechanical ventilation. During the Delta-variant peak, the patient’s nasopharyngeal sample once more tested positive for severe acute respiratory syndrome coronavirus 2. The patient died a few days later from cardiopulmonary arrest.ConclusionThe coronavirus disease 2019 pandemic has encountered patients with cancer with critical diagnostic and treatment challenges. Patients who are immunocompromised may co-infect with multiple severe acute respiratory syndrome coronavirus-2 strains and cytomegalovirus, and even with timely diagnosis and treatment, the prognosis may be poor.

Modulation of myeloid and T cells in vivo by Bruton’s tyrosine kinase inhibitor ibrutinib in patients with metastatic pancreatic ductal adenocarcinoma

BackgroundIn preclinical studies of pancreatic ductal adenocarcinoma (PDAC), ibrutinib improved the antitumor efficacy of the standard of care chemotherapy. This led to a phase 1b clinical trial to determine the...

MEK Inhibition Demonstrates Activity in Relapsed, Refractory Patients with Juvenile Myelomonocytic Leukemia: Results from COG Study ADVL1521

▪Background: Juvenile myelomonocytic leukemia (JMML) is a hematologic malignancy of infants and toddlers with both myelodysplastic and myeloproliferative features. The prognosis for patients (pts) with relapsed or refractory (r/r) JMML is poor and hematopoietic stem cell transplant (HCT) is the only curative therapy. The molecular hallmark of JMML is activation of the Ras/MAPK pathway. In preclinical studies, MEK inhibition was shown to be effective at reducing spleen sizes, restoring normal hematopoiesis, and extending survival compared to placebo in several genetically engineered mouse models of JMML. Trametinib is an orally bioavailable MEK1/2 inhibitor and is approved for treatment of several malignancies in adults. This Children's Oncology Group study (ADVL1521, NCT03190915) is the first clinical trial for pts with r/r JMML conducted in the United States. Pts are eligible if they have persistent clinical or molecular evidence of JMML after 1 cycle of high dose cytarabine, 2 cycles of a hypomethylating agent or HCT. Pts receive daily trametinib for up to 12 cycles (28 days) in the absence of disease progression or dose-limiting toxicity (DLT). Dosing is age-based with pts less than 6 years of age receiving 0.032mg/kg/day and those 6 years or older receiving 0.025mg/kg/day. An oral suspension is available for pts unable to swallow tablets. Using a Simon 2-stage design (10 pts in each stage), trametinib would be deemed effective if 3 or more pts achieved an objective response.Results: From 2018-2021, 9 pts were enrolled; all 9 were eligible and evaluable for toxicity and response. Each pt had a detectable Ras mutation at the time of enrollment and was monitored for response using clinical and molecular criteria developed by an international consensus panel (Niemeyer et al, 2015). Five pts were less than 2 years of age. Three patients had relapsed post-HCT prior to enrolling and 6 patients were refractory to a median of 1.5 prior therapies (range 1-3). Four pts had an objective response (1 clinical complete response (cCR), 3 clinical partial responses, (cPR); 2 pts had stable disease and 3 had progressive disease (Table 1). Both pts with stable disease completed the maximum 12 cycles permitted on study. Two pts who achieved a cPR proceeded to HCT. One patient who achieved a cCR remains on study. No molecular responses were achieved. There were no dose-limiting toxicities; one pt had grade 4 thrombocytopenia probably related to trametinib. Of the 8 patients who consented to correlative studies, 7 had DNA methylation testing, 6 had kinome profiling, and 5 had RNASeq testing performed on both pre- and post-trametinib paired samples. DNA methylation testing revealed stable intrapatient methylation signatures across diagnostic, relapse and post-trametinib timepoints using the international consensus criteria (Schönung et al, 2020). Integrated kinome and RNASeq analysis revealed downregulation of proteins and genes involved in Ras/MAPK signaling.Conclusions: In the first clinical trial for r/r JMML patients in the United States, 4 objective responses were observed among the initial 9 patients meeting the pre-defined criteria to deem trametinib effective. While clinical responses including resolution of splenomegaly, resolution of monocytosis and increased platelets counts were observed, no molecular responses were noted. The treatment of r/r JMML has historically depended on HCT. Recently, azacitidine has shown promise in treating r/r JMML. This trial demonstrates that trametinib is active in r/r JMML and has a favorable side effect profile. Ongoing analysis of extensive correlative testing results have revealed potential mechanisms of response and resistance to MEK inhibition. Future studies will focus on children with newly diagnosed JMML and combine trametinib with azacitidine with or without HCT. [Display omitted] DisclosuresLoh: MediSix therapeutics: Membership on an entity's Board of Directors or advisory committees. Barkauskas: Genentech: Current Employment.

Clinical, Radiometabolic and Immunologic Effects of Olaparib in Locally Advanced Triple Negative Breast Cancer: The OLTRE Window of Opportunity Trial.

IntroductionOlaparib is effective in metastatic triple negative breast cancer (TNBC) carrying germline mutations in DNA damage repair (DDR) genes BRCA1/2 (gBRCA-mut). The OLTRE window-of-opportunity trial preliminarily investigated potential pathologic, radiometabolic and immune biomarkers of early-response to olaparib in gBRCA-wild-type (wt) TNBC and, as proof-of-concept in gBRCA-mut HER2-negative BC.MethodsPatients received olaparib for 3 weeks (3w) before standard neoadjuvant chemotherapy and underwent multiple FDG18-PET/CT scan (basal, after olaparib), clinical assessments (basal, every 3w), tumor biopsies and blood samplings (baseline, after olaparib). Clinical and radiometabolic responses were evaluated according to RECIST1.1 and PERCIST criteria.Results27 patients with gBRCA-wt TNBC and 8 with gBRCA-mut BC (6 TNBC, 2 HR+/HER2-negative) were enrolled. Three (11.1%) patients showed mutations in non-BRCA1/2 DDR genes and 4 (14.8%) in other genes. 3w olaparib induced 16/35 and 15/27 partial clinical and radiometabolic responses, including in 40.7% and 50.0% gBRCA-wt patients. gBRCA-mut tumors presented numerically higher tumor-infiltrating lymphocytes (TILs) levels and PD-L1 positive tumors. Clinical responders experienced a reduction in T-regs/T-eff ratio (p=0.05), B and NK lymphocytes (p=0.003 both), with an average increase in T-helpers rate (p<0.001) and CD4/CD8 ratio (p=0.02). Ki67% and TILs did not vary significantly (p=0.67 and p=0.77). A numerical increase in PD-L1 positive cases after olaparib was observed, though non-significant (p=0.134). No differences were observed according to gBRCA status and type of response.ConclusionsEarly-stage TNBC might be a target population for olaparib, irrespective of gBRCA mutations. Future trials should combine TILs, PD-L1 and gBRCA status to better identify candidates for escalated/de-escalated treatment strategies including olaparib.

CANOMAD: a neurological monoclonal gammopathy of clinical significance that benefits from B-cell–targeted therapies

AbstractCANOMAD (chronic ataxic neuropathy, ophthalmoplegia, immunoglobulin M [IgM] paraprotein, cold agglutinins, and disialosyl antibodies) is a rare syndrome characterized by chronic neuropathy with sensory ataxia, ocular, and/or bulbar motor weakness in the presence of a monoclonal IgM reacting against gangliosides containing disialosyl epitopes. Data regarding associated hematologic malignancies and effective therapies in CANOMAD are scarce. We conducted a French multicenter retrospective study that included 45 patients with serum IgM antibodies reacting against disialosyl epitopes in the context of evocating neurologic symptoms. The main clinical features were sensitive symptoms (ataxia, paresthesia, hypoesthesia; n = 45, 100%), motor weakness (n = 18, 40%), ophthalmoplegia (n = 20, 45%), and bulbar symptoms (n = 6, 13%). Forty-five percent of the cohort had moderate to severe disability (modified Rankin score, 3-5). Cold agglutinins were identified in 15 (34%) patients. Electrophysiologic studies showed a demyelinating or axonal pattern in, respectively, 60% and 27% of cases. All patients had serum monoclonal IgM gammopathy (median, 2.6 g/L; range, 0.1-40 g/L). Overt hematologic malignancies were diagnosed in 16 patients (36%), with the most frequent being Waldenström macroglobulinemia (n = 9, 20%). Forty-one patients (91%) required treatment of CANOMAD. Intravenous immunoglobulins (IVIg) and rituximab-based regimens were the most effective therapies with, respectively, 53% and 52% of partial or better clinical responses. Corticosteroids and immunosuppressive drugs were largely ineffective. Although more studies are warranted to better define the optimal therapeutic sequence, IVIg should be proposed as the standard of care for first-line treatment and rituximab-based regimens for second-line treatment. These compiled data argue for CANOMAD to be included in neurologic monoclonal gammopathy of clinical significance.

Biomarkers of minimal residual disease in rituximab-treated patients with mixed cryoglobulinemia.

Hepatitis C virus (HCV) represents the major risk factor for mixed cryoglobulinemia (MC), a small-vessel vasculitis that may evolve into an overt B-cell non-Hodgkin's lymphoma. Here, we aimed to identify a biomarker signature for the early diagnosis of minimal residual disease (MRD). We assessed free light chains (FLCs), IgM k,and IgM λ heavy/light chain (HLC) pairs, and vascular endothelial growth factor (VEGF) in sera from 34 patients with MC vasculitis (32 HCV- and 2 HBV-related), treated with low-dose rituximab (RTX). FLCs and IgM HLCs were measured by turbidimetric assay; VEGF by an enzyme-linked immunosorbent assay. After RTX, the positive (complete+partial) clinical and laboratory responses were of 85.29% and 50%, respectively; in contrast, the mean levels of FLCs, IgM HLCs, and VEGF were substantially unaffected in most patients and still above the normal range. In those achieving a reduction of FLCs and IgM k and λ chains values within the range of normality, we found that post-treatment free λ chains and IgM k values correlated with clinical and laboratory response. Our results suggest that high levels of FLCs, IgM HLCs, and VEGF could represent the signature of "dormant" B cell clones' activity that could be very useful to identify MRD indicative of possible relapse or worsening outcome.

Inhibition of Unc-51-like Kinase 1 (ULK1) with Novel Small Molecular Inhibitor MRT68921 Preferentially Induces Apoptosis and Autophagy in FLT3-ITD- Mutated Acute Myeloid Leukemia

In normal karyotype acute myeloid leukemia (AML), FLT3-ITD mutation is associated with dismal prognosis with early relapse even after allogeneic stem cell transplantation. Unfortunately, to date small-molecule inhibitors of FLT3 have resulted in only partial and transient clinical responses with residual leukemic blasts acquiring resistance to FLT3 inhibitors. Therefore, elucidation of novel molecular targets should be necessary for effective eradication of FLT3-ITD AML cells. Evidences are accumulating on the functional roles of autophagy in the initiation and maintenance of AML as well as the development of drug resistance. Unc-51-like kinase 1 (ULK1) is a conserved serine-threonine kinase that plays a central role in the initiation of autophagy. Our group demonstrated that ULK1 is potentially involved in the development of resistance of AML leukemia stem cells to BET inhibitor, JQ1. The fact that ULK1 is the only conserved serine/threonine kinase in the autophagy cascade makes it a very attractive target for therapeutic development. However, the role of Ulk1 in FLT3-ITD AML remains unclear. In this study, we observed that MRT68921, a potent inhibitor of both ULK1 and ULK2, induced apoptotic cell death in FLT3-ITD-mutated AML cell lines (MV4-11, Molm13, U937/FLT3-ITD-muated) in a dose-dependent manner. However, apoptosis-inducing effect of MRT68921 was significantly lower in FLT3-WT AML (HL-60, U937). Cell death was accompanied with cleavage of caspases and PARP, which were partially blocked with caspase inhibitor z-VAD-fmk, indicating the caspase-dependent mechanism exists. MRT68921 treatment led to a notable decrease in the levels of phosphorylated (p) ATG13 (Ser 318) as well as total ULK1 and p-ULK1 (Ser 555). Interestingly, MRT68921 induced LC3-II lipidation, autophagosome, and GFP/LC3 punta formation, indicating autophagy was paradoxically activated in FLT3-ITD-mutated AML cells. AMPKa phosphorylation (T712) was increased in MTR68921-responsive cells. In contrast, autophagy induction was negligible to modest in FLT3-WT AML cells. Treatment of FLT3-ITD cells with autophagy inhibitors, 3-MA, bafilomycin A1, and hydroxychloroquine, markedly enhanced the MRT68921-induced apoptosis, strongly suggesting that prosurvival autophagy activation occurred with MRT68921 in FLT3-ITD cells. Reduction in the levels of total FLT3 and p-FLT3 protein were observed concurrently with downregulation of p-STAT5 in FLT3-ITD cells. Endoplasmic reticulum stress-associated proteins, p-PERK and p-eIF2a were also downregulated with MRT68921 in FLT3-ITD cells. Taken together, targeting the ULK1 pathway could be an effective therapeutic strategy for combating FLT3-ITD AML. Inhibition of prosurvival autophagy pathway could enhance the anti-leukemia effects of MRT68921. DisclosuresNo relevant conflicts of interest to declare.

Monoclonal antibody therapy in cancer: When two is better (and considerably more expensive) than one.

It is 20years since the US Food and Drug Administration approved the first successful monoclonal anticancer antibody, trastuzumab. The therapeutic utility of monoclonal antibodies in cancer is often limited by partial clinical responses and the development of tumour resistance. An expanding strategy, to be reviewed here, to overcome the limited response and resistance to monotherapy utilizes concurrent treatment with two synergistic monoclonal antibodies. Key examples include two monoclonal antibodies, each engaging a distinct site of human epidermal growth factor receptor 2 (HER2), in the treatment of breast cancer and a combination of antibodies to two distinct T-cell antigens for the treatment of melanoma. Here, we provide an overview of the rationale and evidence for using selected monoclonal antibodies in combination for treating some cancers, along with potential hazards, especially autoimmune-related toxicities. Thorough research, the development of panels of biomarkers and individualization of therapy will be necessary to optimize the use of these combinations and minimize the substantial risk of overstimulating the immune system.

Abstract 5016: High-throughput patient-derived 3-dimensional organoid cultures as personalized models to assess drug response and post-treatment residual disease

Abstract Cell lines grown in 2-dimensional (2D) plastic surfaces have historically been the main models to identify cancer cell vulnerabilities. In contrast, malignant tumors in vivo grow as 3-dimensional (3D) cellular masses and manifest remarkably different drug-response patterns compared to cell lines. We have shown that breast cancer (BrCa) cells in 3D extracellular matrices acquire distinct phenotypic properties, compared to the 2D counterparts. Notably, serum-free culture medium supplemented with defined cocktails of developmental morphogens was sufficient to support the growth of BrCa cell lines in 3D, but not in 2D conditions; indicating a key role of 3D architecture in in vivo growth regulation. CRISPR/Cas9-based gene editing screens in BrCa cells indicated that distinct gene subset govern the growth in 3D vs. 2D conditions. Using similar culture conditions, we expanded several molecularly annotated BrCa patient-derived 3D Organoids (3D PDOs), adapted them into a high-throughput miniaturized drug screening platform, and interrogated them against a panel of ~500 target-annotated kinase inhibitors and FDA-approved anti-cancer agents. This pharmacological screen revealed high sensitivity to distinct classes of agents (eg. PI3K inhibitors), but also variable responses to others (eg. EGFR inhibitors); and provided insight into potentially driver vs. passenger mutations in the respective patients. Co-culture of PDOs with non-malignant stromal cells from bone metastatic niche reduced sensitivity to some classes of drugs (eg. PLK inhibitors). Importantly, long-term (up to 3-4 weeks) treatment of 3D organoids did not eradicate the cells in culture (in contrast to conventional 2D assays), but generated a cell subpopulation which remained viable for the duration of the experiment, reminiscent of the residual disease after initial clinical partial responses to a drug. This residual disease phenotype was also confirmed in vivo, when the respective PD-Xenografts were treated with the same agents. The majority of transcripts with differential expression in residual PDX tumor cells were also concordantly up- or down-regulated in the respective 3D PDO model. These transcripts included canonical regulators of cell cycle, chromatin signaling and epigenetic mechanisms, cytoskeletal function and metabolic pathways; as well as other transcripts not previously linked to drug resistance in conventional 2D models. To our knowledge this is the first description of an in vitro personalized cancer model that simulates both molecularly and phenotypically the post-treatment residual disease in vivo. Our high-throughput functional platform can potentially complement genomic approaches in personalized medicine and help discover novel drugs that overcome tumor drug resistance. Citation Format: Eugen Dhimolea, Ricardo De Matos Simoes, Yoko DeRose, Pallavi Awate, Xiang Weng, Huihui Tang, Aedin Culhane, Alana Welm, Constantine Mitsiades. High-throughput patient-derived 3-dimensional organoid cultures as personalized models to assess drug response and post-treatment residual disease [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5016. doi:10.1158/1538-7445.AM2017-5016

Presynaptic Dopamine Synthesis Capacity in Schizophrenia and Striatal Blood Flow Change During Antipsychotic Treatment and Medication-Free Conditions.

Standard-of-care biological treatment of schizophrenia remains dependent upon antipsychotic medications, which demonstrate D2 receptor affinity and elicit variable, partial clinical responses via neural mechanisms that are not entirely understood. In the striatum, where D2 receptors are abundant, antipsychotic medications may affect neural function in studies of animals, healthy volunteers, and patients, yet the relevance of this to pharmacotherapeutic actions remains unresolved. In this same brain region, some individuals with schizophrenia may demonstrate phenotypes consistent with exaggerated dopaminergic signaling, including alterations in dopamine synthesis capacity; however, the hypothesis that dopamine system characteristics underlie variance in medication-induced regional blood flow changes has not been directly tested. We therefore studied a cohort of 30 individuals with schizophrenia using longitudinal, multi-session [15O]-water and [18F]-FDOPA positron emission tomography to determine striatal blood flow during active atypical antipsychotic medication treatment and after at least 3 weeks of placebo treatment, along with presynaptic dopamine synthesis capacity (ie, DOPA decarboxylase activity). Regional striatal blood flow was significantly higher during active treatment than during the placebo condition. Furthermore, medication-related increases in ventral striatal blood flow were associated with more robust amelioration of excited factor symptoms during active medication and with higher dopamine synthesis capacity. These data indicate that atypical medications enact measureable physiological alterations in limbic striatal circuitry that vary as a function of dopaminergic tone and may have relevance to aspects of therapeutic responses.

Abstract B40: Intratumor heterogeneity in lung adenocarcinoma: Beyond genetics

Abstract Background: For decades, pathologists have been describing new anatomopathological entities in lung carcinomas emphasizing the great heterogeneity of intratumor cell growth patterns. Modern genomic studies have identified a vast number of genetic alterations throughout the whole tumor and metastasis. In lung, oncogenic drivers such as EGFR, ALK, ROS, and RAS are mutated or translocated in a significant number of adenocarcinomas. Because genetic alteration can be heterogeneous, we studied DNA mutations and RNA expression in three different areas of the tumors, as well as protein expression of receptors and signaling factors in whole lung carcinomas sections. Material and Methods: In 130 lung adenocarcinomas the histopathological features were assessed as well as the expression of ALK, HER2, ki67, pMAPK, mtor, 4E-BP1, p4E-BP1, eIF4E, peIF4E, and Epithelial-Mesenchymal Transition (EMT) factors (N-cadherin, YB1, pYB1) by specific antibodies in whole paraffin sections. Homogeneous expression was considered when &amp;gt;80% tumor cells displayed a strong positivity. In 30 adenocarcinomas, molecular studies were run in three different histological cores with acinar, papilar and micropapilar cell growth patterns. DNA extraction for detection of mutations of EGFR and KRAS and RNA extraction, and gene expression of 110 genes was screened using NanoString technology. Levels of expression were evaluated semi quantitatively as percentage and intensity of stained tumor cells (histo-score [H score]) and correlations were analyzed using Kruskal-Wallis and Kaplan-Meier (log Rank) statistical tests. Results: The predominant histological subtypes were acinar, papillary and solid, with the latter associated with higher histological grade and more necrosis. p4E-BP1 and peIF4E displayed a homogenous and diffuse protein expression in about 80% of tumors. High expression of eIF4E, 4EBP1 and YB1 was associated with higher recurrence rate, histological grade and metastases. Regarding RNA expression, 90% of cases showed intratumoral heterogeneity in some genes. EGFR mutations show a heterogeneous distribution in about 20% cases while KRAS mutations were positive in all the areas of the tumors. Conclusions: In lung adenocarcinomas, there is intratumor heterogeneity of mRNA expression in most cases, of EGFR mutations in a significant number of cases and a striking diversity in protein expression. With this data, several conclusions can be drawn: first, the importance to study several areas of the tumors to assess tumor heterogeneity both at molecular and protein levels; second, the patchy expression of signaling factors, probably due to local factors such as hypoxia, may explain partial clinical responses, third, in most cases peIF4E displayed a homogeneous expression supporting its role as a therapeutical target and fourth, pathologists need to integrate the molecular data, protein and histopathological data and apply systems biology and topologic approaches to highlight the more relevant prognostic information and therapeutical options for each tumor and patient. Citation Format: Santiago Ramon y Cajal. Intratumor heterogeneity in lung adenocarcinoma: Beyond genetics. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr B40.

Clinical Response Rates From Interleukin-2 Therapy for Metastatic Melanoma Over 30 Years' Experience: A Meta-Analysis of 3312 Patients.

Interleukin-2 (IL-2), initially used in 1986, can induce clinical regression-complete responses (CR) and partial responses (PR) of metastatic malignant melanoma. IL-2 has been used alone or in combination, and in different dosage schedules, as an immunotherapeutic agent for melanoma treatment. This meta-analysis aimed to document and evaluate the spectrum of reported clinical response rates from the combined experience of almost 30 years of IL-2 clinical usage. Clinical trials using IL-2 for metastatic melanoma therapy that reported: dosage, combinations, study details, definitions and clinical CR, PR, and overall response (OR) rates were included. A meta-analysis was conducted using the preferred reporting items for systematic reviews and meta-analyses (PRISMA) guidelines. In total, 34 studies met inclusion criteria, with 41 separate treatment arms. For all IL-2 treatment modalities collectively, the CR rate was 4.0% [95% confidence interval (CI), 2.8-5.3], PR 12.5% (95% CI, 10.1-15.0), and OR 19.7% (95% CI, 15.9-23.5). CR pre-1994 was 2.7% versus 6.1% post-1994. High and intermediate-IL-2 dosage showed no CR difference, while low-dose IL-2 showed a nonstatistical trend toward an increased CR rate. The highest CR rate resulted from IL-2 combined with vaccine at 5.0%. The meta-analysis showed that IL-2 immunotherapy for advanced metastatic melanoma delivered a CR rate of 4% (range, 0-23%) across nearly 30 years of clinical studies, with gradual improvement over time. The significance is that, contrary to popular belief, the data demonstrated that CR rates were similar for intermediate versus high-IL-2 dosing.

Receptor tyrosine kinase Axl is required for resistance of leukemic cells to FLT3-targeted therapy in acute myeloid leukemia.

In acute myeloid leukemia (AML), about 25-30% of patients harbor a constitutively active receptor tyrosine kinase (RTK) FLT3 encoded by a FLT3 allele harboring internal tandem duplication (FLT3-ITD) mutation. The presence of FLT3-ITD correlates with poor prognosis in AML and it makes FLT3 an attractive therapeutic target in AML. Unfortunately, to date small-molecule inhibitors of FLT3 have resulted in only partial and transient clinical responses with residual leukemic blasts resistant to FLT3 inhibitors detected in blood or bone marrow. In this study, we investigated whether the RTK Axl is responsible for resistance of FLT3-ITD(+) AML cells to PKC412 and AC220, FLT3 inhibitors currently under clinical trials for FLT3-ITD(+) AML patients. Upon treatment with PKC412 or AC220, phosphorylation of Axl was significantly enhanced in the FLT3-ITD(+) MV4-11 AML cell line and in primary blasts from a FLT3-ITD(+) AML patient. Consistently, a PKC412-resistant AML cell line and PKC412-resistant primary blasts from FLT3-ITD(+) AML patients had significantly higher levels of constitutively phosphorylated Axl and total Axl when compared with a PKC412-sensitive AML cell line and PKC412-sensitive primary blasts from FLT3-ITD(+) AML patients. We also found that resistance of AML cells against the FLT3 inhibitor PKC412 and AC220 was substantially diminished by the inhibition of Axl via a small-molecule inhibitor TP-0903, a soluble receptor Axl fusion protein Axl-Fc or knockdown of Axl gene expression by shRNA. Collectively, our study suggests that Axl is required for resistance of FLT3-ITD(+) AML cells against the FLT3 inhibitor PKC412 and AC220, and that inhibition of Axl activation may overcome resistance to FLT3-targeted therapy in FLT3-ITD(+) AML.

The Receptor Tyrosine Kinase Axl Is Required for Resistance to FLT3-Targeted Therapy in Acute Myeloid Leukemia

In acute myeloid leukemia (AML), about 25 to 30% of patients harbor a constitutively active receptor tyrosine kinase (RTK) FLT3 encoded by an FLT3 allele harboring internal tandem duplication (FLT3-ITD) mutation. Clinical studies have shown that the presence of FLT3-ITD correlates with poor prognosis in AML. Therefore, these features make FLT3-ITD an attractive therapeutic target in AML and several tyrosine kinase inhibitors (TKIs) targeting constitutively activated FLT3 have been developed and tested in clinical trials. Unfortunately, to date these small molecule inhibitors have resulted in only partial and transient clinical responses with residual leukemic blasts resistant to the TKIs detected in blood or bone marrow. This poses a serious obstacle to improving clinical outcome of FLT3-ITD AML patients. Thus understanding the molecular basis of resistance may ultimately help to optimize FLT3-ITD targeting to achieve long-term survival of these AML patients. Axl is a member of the RTK Axl family and is over-expressed and/or aberrantly activated in many types of cancers including AML. We have previously shown that Axl is involved in leukemia growth and survival in FLT3-ITD AML (Park et al, Blood 121, 2064-73, 2013). Thus, targeting Axl may represent a novel therapeutic strategy for the treatment of FLT3-ITD AML. In this study, we investigated whether Axl is responsible for resistance of leukemic cells to PKC412, a TKI that is undergoing clinical investigation in FLT3-ITD AML patients. Upon treatment of FLT3 inhibitor PKC412, phosphorylation of Axl was significantly enhanced in the FLT3-ITD MV4-11 AML cell line and primary blasts from an FLT3-ITD AML patient. When primary blasts from FLT3-ITD AML patients were treated with PKC412, the majority of AML cells undergoing apoptosis (Annexin V-positive) harbored little phosphorylated Axl. On the other hand, most AML cells resistant to PKC412 (Annexin V-negative) remained alive and showed a significantly higher level of phosphorylated Axl (75.2 ± 9.3% Axl phosphorylation for Annexin V-negative vs. 8.4 ± 1.7% for Annexin V-positive, p < 0.01). These results suggest that increased Axl phosphorylation following treatment of PKC412 may give a survival advantage to a subset of AML cells treated with the FLT3 inhibitor PKC412. It also suggests that Axl phosphorylation may represent at least part of the molecular mechanism explaining resistance of FLT3-ITD AML cells to FLT3-targeted therapy. To further investigate this, we analyzed a PKC412-resistant AML cell line, FLT3-ITD MOLM13-R-PKC412, which was generated from its parental PKC412-sensitive cell line, FLT3-ITD MOLM13. The PKC412-resistant MOLM13-R-PKC412 AML cell line possessed a significantly higher level of phosphorylated Axl and total Axl when compared to PKC412-sensitive MOLM13. An Axl-specific TKI, TP-0903(Tolero Pharmaceuticals), was able to inhibit phosphorylation of Axl and to re-sensitize MOLM13-R-PKC412 cells to PKC412 (82.2 ± 5.6% cell viability for PKC412 only vs. 9.5 ± 2.3% for PKC412 + TP-0903, p < 0.01). When the PKC412-resistant MOLM13-R-PKC412 AML cells were treated with Axl-Fc, which is a soluble Axl chimeric protein to sequester ligands for Axl, resistance of MOLM13-R-PKC412 AML cells to PKC412 was abrogated (77.4 ± 8.2% cell viability for PKC412 vs. 12.5 ± 2.1% for PKC412 + Axl-Fc, p < 0.01). In addition, when MOLM13-R-PKC412 AML cells were transfected with lentivirus encoding shRNA targeting Axl, sensitivity to PKC412 was restored (81.6 ± 10.5% cell viability for control shRNA vs. 18.3 ± 2.4% for Axl shRNA, p < 0.01). While phosphorylated Axl and total Axl were easily detectable in primary AML blasts intrinsically resistant to PKC412 (0.33 and 0.75 for median phospho-Axl and total Axl level, respectively), we observed little or no phosphorylated Axl or total Axl protein in AML patient blasts intrinsically sensitive to PKC412 (0.03 and 0.08 for median phospho-Axl and Axl, respectively). Collectively, these results indicate that Axl is specifically required for resistance of FLT3-ITD AML cells against the FLT3 inhibitor PKC412, and that Axl should be explored as a potential therapeutic target to overcome resistance to FLT3-targeted therapy in FLT3-ITD AML. We provide a rationale for testing an Axl-specific inhibitor, such as TP-0903, alone or in combination with FLT3 inhibitors in clinical trials for FLT3-ITD AML patients. DisclosuresWarner:Tolero Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties. Bearss:Tolero Pharmaceuticals: Employment.