Citations

3535 Background: Peripheral blood allows for a simple non-invasive method for isolating various cancer associated circulating stromal cells (CStCs) which may predict for cancer presence. Cancer Associated Macrophage-Like cells (CAMLs), a specific CStC, are phagocytic myeloid cells that derive from an immunological response to cancer and emanate from primary tumors. Using a filtration platform we screened the peripheral blood of untreated newly diagnosed cancer patients (n = 308) for CAMLs. In parallel, we screened patients with newly diagnosed non-malignant diseases, i.e. lupus, benign cysts, etc. (n = 39), and healthy control samples (n = 76). We found that CAMLs are highly prevalent (87%) in the blood of cancer patients, but uncommon in non-malignant conditions (20%) & absent in healthy individuals (0%). Methods: Anonymized peripheral blood were taken from 308 cancer patients after confirmation of invasive malignancy [stage I (n = 76), stage II (n = 73), stage III (n = 72), stage IV (n = 65) and unstaged non-metastatic (n = 22)] with pathologically confirmed lung (n = 65), pancreas (n = 53), breast (n = 52), prostate (n = 40), esophageal (n = 30), renal cell (n = 18), hepatocellular (n = 15), neuroblastoma (n = 10), melanoma (n = 8), and other (n = 17). Further, anonymized blood was taken from patients with untreated non-malignant conditions including benign breast masses (n = 19), lupus (n = 11), liver cirrhosis (n = 5), benign prostatic hyperplasia (BPH) (n = 3), and viral infection (n = 1); or from healthy control volunteers (n = 76). CAMLs were isolated from whole peripheral blood by the CellSieve™ microfiltration technique and defined as enlarged, multinuclear cells with cytokeratin and/or CD45/CD14 positive. Results: CAMLs were found in 87% of all cancer patients regardless of stage, ~5.4 CAMLs/7.5mL blood. Specifically, CAMLs were found in 80% of Stage I, 90% Stage II, 89% Stage III, and 97% Stage IV patients. No CAMLs were found in any healthy controls, but were found in 26% of benign breast masses, 18% of lupus, 0% of BPH and 0% of cirrhosis. In total, CAML sensitivity in cancer vs healthy was 87% (CI95% 82-90%), specificity = 100% (CI95% 95-100%), PPV = 100% (CI95% 100%), NPV = 67% (CI95% 58-71%). CAML sensitivity in cancer vs benign was 87% (CI95% 82-90%), specificity = 80% (CI95% 64-91%), PPV = 97% (CI95% 95-98%), NPV = 43% (CI95% 35-51%). Conclusions: CAMLs, a Circulating Stromal Cell subtype, is a sensitive blood based biomarker found in all stages of cancer that is rare in non-malignant conditions and absent in healthy individuals.

Purpose. Osteosarcoma is a rare cancer and a third of patients who have completed primary treatment will develop osteosarcoma recurrence. The Src pathway has been implicated in the metastatic behavior of osteosarcoma; about 95% of samples examined express Src or have evidence of downstream activation of this pathway. Saracatinib (AZD0530) is a potent and selective Src kinase inhibitor that was evaluated in adults in Phase 1 studies. The primary goal of this study was to determine if treatment with saracatinib could increase progression-free survival (PFS) for patients who have undergone complete resection of osteosarcoma lung metastases in a double-blinded, placebo-controlled trial. Patients and Methods. Subjects with recurrent osteosarcoma localized to lung and who had complete surgical removal of all lung nodules were randomized within six weeks after complete surgical resection. Saracatinib, or placebo, was administered at a dose of 175 mg orally, once daily, for up to thirteen 28-day cycles. Results. Thirty-seven subjects were included in the analyses; 18 subjects were randomized to receive saracatinib and 19 to receive placebo. Intent-to-treat analysis demonstrated a median PFS of 19.4 months in the saracatinib treatment group and 8.6 months in the placebo treatment group (p=0.47). Median OS was not reached in either arm. Conclusions. Although saracatinib was well tolerated in this patient population, there was no apparent impact of the drug in this double-blinded, placebo-controlled trial on OS, and Src inhibition alone may not be sufficient to suppress metastatic progression in osteosarcoma. There is a suggestion of potential clinical benefit as evidenced by longer PFS in patients randomized to saracatinib based on limited numbers of patients treated.

TPS10591 Background: Osteosarcoma is a rare cancer and 33% of patients who have completed primary treatment will recur. The Src pathway has been implicated in the metastatic behavior of several tumors including osteosarcoma where 95% of samples express Src or have evidence of downstream activation of this pathway. Saracatinib (AZD0530) is a potent and selective Src kinase inhibitor. The recommended phase II dose in adults was found to be 175mg daily. The primary goal of this study is to determine if treatment with Saracatinib can increase progression free survival (PFS) for patients who have undergone complete resection of metastatic osteosarcoma nodules in the lung. Secondary goals are evaluation of overall survival, time to treatment failure, and evaluation of several biological correlatives. Methods: This is a multi-institutional, phase II.5, placebo-controlled study with an accrual goals of 88 randomized patients. Patients between 15 and 75 years, with histological confirmation of recurrent osteosarcoma, localized to the lung, who have potential for complete surgical resection, are eligible for enrollment. After complete resection, patients are randomized to treatment with saracatinib or placebo, of a daily oral dose of 175 mg, continuously for up to 1 year or until progression. Patients who recur in the lung while on-study and who are amenable to complete surgical resection will be un-blinded. Those patients who received placebo may have the option to undergo surgical resection. If fully resected, they will be offered therapy with saracatinib under the same treatment guidelines as above. As of January 2013, 38 patients have enrolled and 32 patients met the criteria to be randomized and began oral therapy with either saracatinib or placebo. An interim analysis is planned after 40 patients have been randomized. Clinical trial information: NCT00752206.

Abstract Multiple myeloma (MM) is a tumor of terminally differentiated plasma cells that underwent affinity maturation and class switch recombination processes in germinal centers before homing to the bone marrow (BM), where they secrete large amounts of monoclonal immunoglobulins, clinically detected as an M-spike by serum protein electrophoresis (SPEP). Clinical manifestations of MM also include renal impairment, anemia, and bone disease, the latter resulting from abnormal osteoclast activation, which highlights the complex crosstalk between MM cells and their BM microenvironment. Chromosomal translocations into the immunoglobulin loci are the primary oncogenic events in ~50% of MM cases and are thought to occur as a result of errors during somatic hypermutation or class switch recombination. The other half of MM is characterized by trisomies of odd-numbered chromosomes. Both of these genomic abnormalities are present in the premalignant phase of MM called MGUS, which progresses to MM at a rate of 1% per year. Progression events include mutations of genes in the RAS/MAPK pathways; chromosomal rearrangements of the MYC locus; and mutations, amplifications, or biallelic deletions of genes in the NFkB pathway. Among the latter, the most common are deletions of TRAF3 or cIAP1 and -2 (BIRC2 and -3) and amplification or translocations of NIK (Map3k14), all resulting in NIK stabilization, phosphorylation of IKKα, processing of p100 to p52, NFKB2 nuclear translocation, and activation of the noncanonical NFKB pathway. This can be induced in MM cell lines in vitro by treatment with SMAC mimetics or IAP antagonists (IAPa), which induce cIAP1 auto-ubiquitination and degradation. We postulated that the resulting constitutive activation of the noncanonical NFKB pathway would render MM cells independent on the BM-localized survival signals provided by BAFF and APRIL, leading to extramedullary dissemination. We tested this hypothesis in vivo by exposing Vk*MYC mice with BM-localized MM to the IAP antagonist LCL161 and monitoring disease progression. In the immunocompetent Vk*MYC transgenic mouse model MYC expression is sporadically induced in germinal center cells by AID-dependent somatic hypermutation, leading to progressive accumulation of monoclonal plasma cells restricted to the BM that can be monitored over time by SPEP and M-spike quantization, as is done clinically in MM patients. Through a very rigorous testing of drugs whose clinical activity in MM has been assessed in clinical trials, we have also validated Vk*MYC mice as a faithful model to predict drug activity in MM, with a positive predictive value of 73% and a negative predictive value of 92%. To our surprise, treatment of Vk*MYC mice with the IAPa LCL161 did not promote MM extramedullary dissemination as we predicted, but rather induced a significant reduction in M-spike levels, indicative of reduction of tumor burden. Based on these preclinical data, we conducted a phase 2 clinical trial of LCL161 in combination with cyclophosphamide in relapsed/refractory MM patients. Results from 25 patients enrolled in the study demonstrated a response in five patients (1CR, 1 VGPR, 2PR, 1MR) and a median PFS of 10 months. Consistent with in vitro data on MM cell lines, LCL161 treatment did not promote direct apoptosis of Vk*MYC cells but phagocytosis of live cells, detectable by intravital microscopy within four hours after treatment. While LCL161 treatment of BM-derived macrophages (MΦ) in vitro promoted the acquisition of an inflammatory phenotype, with increased expression of IL6, IL12, and CD86 that resembled activation with CD40 agonists, it did not increase their phagocytic ability. In contrast, exposure of MΦ to LCL161 treated tumor cells or to the conditioned medium collected after LCL161 treatment enhanced phagocytosis. We therefore performed gene expression analysis on MM cells collected from Vk*MYC mice and MM patients before and after LCL161 treatment and identified marked upregulation of NFKB target genes, as expected, but also a type-I interferon (IFN) signature. Interestingly, the presence of blocking antibody to the IFN receptor subunit 1 (aIFNAR1) during LCL161 treatment of tumor cells inhibited their phagocytosis in vitro, and pretreatment of Vk*MYC mice with aIFNAR antibody abrogated LCL161 activity in vivo. Pretreatment of Vk*MYC mice with liposomal clodronate, known to deplete phagocytic cells, also abrogated LCL161 activity, indicating that MΦ-dependent phagocytosis, stimulated by the LCL161 induced tumor cell autonomous IFN response, is required for the anti-MM effects of LCL161. LCL161 has been shown to promote T-cell function in vitro, but in our hand LCL161 retained full anti-MM activity in T-cell deficient mice. However, while ~15% of immunocompetent mice were cured by just two weeks of LCL161 treatment, none of the T-cell deficient mice were, indicating a role for T cells in preventing disease recurrence. Consistently, rechallenge of cured mice with specific MM tumors induced expansion of IFNγ-producing T cells, indicative of reactivation of immunologic memory. We were surprised by the hyporesponsiveness of T cells to LCL161 in vivo and attributed it to the high levels of expression of the coinhibitory molecule PD1 we noted on both CD4 and CD8 cells irrespective of treatment. We also detected high levels of PDL1 on most CD11b+ myeloid cells as well as on a fraction of MM cells, which increased following LCL161 treatment. We therefore sought to combine LCL161 with an antibody against PD1. Remarkably, the combination of LCL161 with aPD1 was curative in all the mice that completed the two-week treatment, and we are in the process of translating this discovery to the clinic by opening a new trial of LCL161 in combination with aPD1. Citation Format: Marta Chesi, David Fooksman, Peter Leif Bergsagel. Immunomodulatory therapy of multiple myeloma with IAP antagonists [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr IA21.

BackgroundCurative therapy for ES requires both chemotherapy and local control of primary tumor. There is no universally accepted standard approach to local control modalities. This survey was conducted to determine practice patterns and factors influencing the choice to offer various local control modalities to patients with ES of the spine and pelvis.MethodsThe survey consisted of four scenarios involving a 15‐year‐old girl who presented with Ewing sarcoma of thoracic vertebra, sacrum, iliac wing, and acetabulum with or without neurologic compromise. The questionnaire was sent to oncologists, orthopedic surgeons, and radiation oncologists, asking their recommendations for local control modality.ResultsAmong 94 respondents, radiotherapy was most frequently chosen for sacral tumors (68.1%) and T10 vertebral tumors (46.2%) whereas surgery was preferred for iliac wing pelvic tumors (45.7%) and acetabular tumors (43.6%). Orthopedic surgeons were significantly more likely to offer surgery than radiation oncologists (OR 3.07, 95%CI 1.37‐6.88, P = 0.007). Providers outside North America were more likely to offer combined surgery plus radiotherapy (OR 10.58, 95%CI 5.41‐20.70, P < 0.001).ConclusionConsiderable heterogeneity exists in local control modalities for Ewing sarcoma of the spine and pelvis. Specialty and location of practice may influence treatment recommendations.

Abstract Genetic lesions such as BCR–ABL1, E2A–PBX1, and MLL rearrangements (MLLr) are associated with unfavorable outcomes in adult B-cell precursor acute lymphoblastic leukemia (B-ALL). Leukemia oncoproteins may directly or indirectly disrupt cytosine methylation patterning to mediate the malignant phenotype. We postulated that DNA methylation signatures in these aggressive B-ALLs would point toward disease mechanisms and useful biomarkers and therapeutic targets. We therefore conducted DNA methylation and gene expression profiling on a cohort of 215 adult patients with B-ALL enrolled in a single phase III clinical trial (ECOG E2993) and normal control B cells. In BCR–ABL1-positive B-ALLs, aberrant cytosine methylation patterning centered around a cytokine network defined by hypomethylation and overexpression of IL2RA(CD25). The E2993 trial clinical data showed that CD25 expression was strongly associated with a poor outcome in patients with ALL regardless of BCR–ABL1 status, suggesting CD25 as a novel prognostic biomarker for risk stratification in B-ALLs. In E2A–PBX1-positive B-ALLs, aberrant DNA methylation patterning was strongly associated with direct fusion protein binding as shown by the E2A–PBX1 chromatin immunoprecipitation (ChIP) sequencing (ChIP-seq), suggesting that E2A–PBX1 fusion protein directly remodels the epigenome to impose an aggressive B-ALL phenotype. MLLr B-ALL featured prominent cytosine hypomethylation, which was linked with MLL fusion protein binding, H3K79 dimethylation, and transcriptional upregulation, affecting a set of known and newly identified MLL fusion direct targets with oncogenic activity such as FLT3 and BCL6. Notably, BCL6 blockade or loss of function suppressed proliferation and survival of MLLr leukemia cells, suggesting BCL6-targeted therapy as a new therapeutic strategy for MLLr B-ALLs. Significance: We conducted the first integrative epigenomic study in adult B-ALLs, as a correlative study to the ECOG E2993 phase III clinical trial. This study links for the first time the direct actions of oncogenic fusion proteins with disruption of epigenetic regulation mediated by cytosine methylation. We identify a novel clinically actionable biomarker in B-ALLs: IL2RA(CD25), which is linked with BCR–ABL1 and an inflammatory signaling network associated with chemotherapy resistance. We show that BCL6 is a novel MLL fusion protein target that is required to maintain the proliferation and survival of primary human adult MLLr cells and provide the basis for a clinical trial with BCL6 inhibitors for patients with MLLr. Cancer Discov; 2(11); 1004–23. ©2012 AACR. Read the Commentary on this article by Cimmino and Aifantis, p. 976. This article is highlighted in the In This Issue feature, p. 961