CD74 is expressed in a subset of pediatric acute myeloid leukemia patients and is a promising target for therapy: a report from the Children's Oncology Group.

As curative therapies for pediatric AML remain elusive, identifying potential new treatment targets is vital. We assessed the cell surface expression of CD74, also known as the MHC-II invariant chain, by multidimensional flow cytometry in 973 patients enrolled in the Children's Oncology Group AAML1031 clinical trial. 38% of pediatric AML patients expressed CD74 at any level and a comparison to normal hematopoietic cells revealed a subset with increased expression relative to normal myeloid progenitor cells. Pediatric AML patients expressing high intensity CD74 typically had an immature immunophenotype and an increased frequency of lymphoid antigen expression. Increased CD74 expression was associated with older patients with lower WBC and peripheral blood blast counts, and was enriched for t(8;21), trisomy 8, and CEBPA mutations. Overall, high CD74 expression was associated with low-risk status, however 26% of patients were allocated to high-risk protocol status and 5-year event free survival was 53%, indicating that a significant number of high expressing patients had poor outcomes. In vitro pre-clinical studies indicate that anti-CD74 therapy demonstrates efficacy against AML cells but has little impact on normal CD34+ cells. Together, we demonstrate that CD74 is expressed on a subset of pediatric AMLs at increased levels compared to normal hematopoietic cells and is a promising target for therapy in expressing patients. Given that nearly half of patients expressing CD74 at high levels experience an adverse event within 5 years, and the availability of CD74 targeting drugs, this represents a promising line of therapy worthy of additional investigation.

Cytokine release syndrome after bronchoalveolar lavage.

BACKGROUND: Immunosuppressed bone marrow transplant patients with pulmonary infiltrates routinely undergo bronchoscopy with bronchoalveolar lavage (BAL) to investigate potential etiologies. Cytokine release syndrome after BAL is unreported in the literature in general and in this patient population. CASE PRESENTATION: We report on an allogeneic bone marrow transplant patient with non-infectious organizing pneumonia of the lungs who developed delayed and rapidly progressive shock and hypoxia post-procedure over the course of 12 h resulting in intensive care unit admission for supportive care. BAL was characterized by a marked lymphocytic, cytotoxic T cell infiltrate on pathology and flow cytometry without clear evidence of infection. The patient's clinical status improved quickly only after the initiation of high dose intravenous steroids and returned to baseline as an outpatient. CONCLUSION: The patient's clinical data and course suggest a cytotoxic T cell response from the lung and BAL as the etiology. With an increasing number of cellular therapies for cancer entering the clinic, the potential for unusual but morbid complications from routine bronchoscopy should be considered.

Donor bone marrow-derived macrophage engraftment into the central nervous system of patients following allogeneic transplantation.

Hematopoietic stem cell transplantation is a well-known treatment for hematologic malignancies, wherein nascent stem cells provide regenerating marrow and immunotherapy against the tumor. The progeny of hematopoietic stem cells also populate a wide spectrum of tissues, including the brain, as bone marrow-derived macrophages similar to microglial cells. We developed a sensitive and novel combined immunohistochemistry (IHC) and XY fluorescence in situ hybridization assay to detect, quantify, and characterize donor cells in the cerebral cortices of 19 female patients who underwent allogeneic stem cell transplantation. We showed that the number of male donor cells ranged from 0.14% to 3.0% of the total cells or from 1.2% to 25% of microglial cells. Using tyramide-based fluorescent IHC, we found that at least 80% of the donor cells expressed the microglial marker ionized calcium-binding adapter molecule-1, consistent with bone marrow-derived macrophages. The percentage of donor cells was related to pretransplantation conditioning; donor cells from radiation-based myeloablative cases averaged 8.1% of microglial cells, whereas those from nonmyeloablative cases averaged only 1.3%. The number of donor cells in patients conditioned with busulfan- or treosulfan-based myeloablation was similar to that in total body irradiation-based conditioning; donor cells averaged 6.8% of the microglial cells. Notably, patients who received multiple transplantations and those with the longest posttransplantation survival had the highest level of donor engraftment, with donor cells averaging 16.3% of the microglial cells. Our work represents the largest study characterizing bone marrow-derived macrophages in patients after transplantation. The efficiency of engraftment observed in our study warrants future research on microglial replacement as a therapeutic option for disorders of the central nervous system.

Therapeutic targeting of PRAME with mTCRCAR T cells in acute myeloid leukemia.

Preferentially Expressed Antigen in Melanoma (PRAME), a cancer-testis antigen, provides an ideal target for immunotherapy in acute myeloid leukemia (AML). We have shown expression of PRAME in a significant subset of childhood and adult AML and lack of expression in normal hematopoiesis. Although an intracellular antigen, we developed a novel approach to target PRAME using a chimeric antigen receptor (CAR) construct encoding a targeting domain based on T-cell receptor (TCR) mimic antibodies that target the peptide-HLA complex. We used the antibody sequence from a previously designed TCR mimic (mTCR) antibody, Pr20, that recognizes the PRAME ALY peptide in complex with HLA-A∗02 and verified expression of PRAME in AML cell lines and primary AML blasts. Using the Pr20 antibody sequence, we developed CAR T cells (PRAME mTCRCAR T) to be tested against primary samples from patients with AML and AML cell lines that express the PRAME antigen in the context of HLA-A2 expression. In contrast to appropriate controls, PRAME mTCRCAR T cells demonstrate target-specific and HLA-mediated in vitro activity in OCI-AML2 and THP-1 cell lines, HLA-A2 cell lines expressing the PRAME antigen, and against primary AML patient samples. In vivo cell-derived xenograft models treated with PRAME mTCRCAR T cells demonstrated potent leukemia clearance and improved survival compared with unmodified T-cell controls. Furthermore, the cytolytic activity of PRAME mTCRCAR T cells was enhanced by treating the target cells with interferon gamma, which increases PRAME antigen expression. These results demonstrate the feasibility and efficacy of targeting PRAME with novel PRAME mTCRCAR T cells.

Oncogenic role of a developmentally regulated NTRK2 splice variant.

Temporally regulated alternative splicing choices are vital for proper development, yet the wrong splice choice may be detrimental. Here, we highlight a previously unidentified role for the neurotrophin receptor splice variant TrkB.T1 in neurodevelopment, embryogenesis, transformation, and oncogenesis across multiple tumor types in humans and mice. TrkB.T1 is the predominant NTRK2 isoform across embryonic organogenesis, and forced overexpression of this embryonic pattern causes multiple solid and nonsolid tumors in mice in the context of tumor suppressor loss. TrkB.T1 also emerges as the predominant NTRK isoform expressed in a wide range of adult and pediatric tumors, including those harboring tropomyosin receptor kinase fusions. Affinity purification-mass spectrometry proteomic analysis reveals distinct interactors with known developmental and oncogenic signaling pathways such as Wnt, transforming growth factor-ß, Sonic Hedgehog, and Ras. From alterations in splicing factors to changes in gene expression, the discovery of isoform specific oncogenes with embryonic ancestry has the potential to shape the way we think about developmental systems and oncology.

CBFA2T3-GLIS2 model of pediatric acute megakaryoblastic leukemia identifies FOLR1 as a CAR T cell target.

The CBFA2T3-GLIS2 (C/G) fusion is a product of a cryptic translocation primarily seen in infants and early childhood and is associated with dismal outcome. Here, we demonstrate that the expression of the C/G oncogenic fusion protein promotes the transformation of human cord blood hematopoietic stem and progenitor cells (CB HSPCs) in an endothelial cell coculture system that recapitulates the transcriptome, morphology, and immunophenotype of C/G acute myeloid leukemia (AML) and induces highly aggressive leukemia in xenograft models. Interrogating the transcriptome of C/G-CB cells and primary C/G AML identified a library of C/G-fusion-specific genes that are potential targets for therapy. We developed chimeric antigen receptor (CAR) T cells directed against one of the targets, folate receptor α (FOLR1), and demonstrated their preclinical efficacy against C/G AML using in vitro and xenograft models. FOLR1 is also expressed in renal and pulmonary epithelium, raising concerns for toxicity that must be addressed for the clinical application of this therapy. Our findings underscore the role of the endothelial niche in promoting leukemic transformation of C/G-transduced CB HSPCs. Furthermore, this work has broad implications for studies of leukemogenesis applicable to a variety of oncogenic fusion-driven pediatric leukemias, providing a robust and tractable model system to characterize the molecular mechanisms of leukemogenesis and identify biomarkers for disease diagnosis and targets for therapy.

Accurate detection of subclonal variants in paired diagnosis-relapse acute myeloid leukemia samples by next generation Duplex Sequencing.

Mutations characterize diverse human cancers; there is a positive correlation between elevated mutation frequency and tumor progression. One exception is acute myeloid leukemia (AML), which has few clonal single nucleotide mutations. We used highly sensitive and accurate Duplex Sequencing (DS) to show now that AML, in addition, has an extensive repertoire of variants with low allele frequencies, < 1%, which is below the accurate detection limit of most other sequencing methodologies. The subclonal variants are unique to each individual and change in composition, frequency, and sequence context from diagnosis to relapse. Their functional significance is apparent by the observation that many are known variants and cluster within functionally important protein domains. Subclones provide a reservoir of variants that could expand and contribute to the development of drug resistance and relapse. In accord, we accurately identified subclonal variants in AML driver genes NRAS and RUNX1 at allele frequencies between 0.1% and 0.3% at diagnosis, which expanded to comprise a major fraction (14-53%) of the blast population at relapse. Early and accurate detection of subclonal variants with low allele frequency thus offers the opportunity for early intervention, prior to detection of clinical relapse, to improve disease outcome and enhance patient survival.

FOXM1 drives HPV+ HNSCC sensitivity to WEE1 inhibition.

Head and neck squamous cell carcinoma (HNSCC) associated with high-risk human papilloma virus (HPV) infection is a growing clinical problem. The WEE1 kinase inhibitor AZD1775 (WEE1i) overrides cell cycle checkpoints and is being studied in HNSCC regimens. We show that the HPV16 E6/E7 oncoproteins sensitize HNSCC cells to single-agent WEE1i treatment through activation of a FOXM1-CDK1 circuit that drives mitotic gene expression and DNA damage. An isogenic cell system indicated that E6 largely accounts for these phenotypes in ways that extend beyond p53 inactivation. A targeted genomic analysis implicated FOXM1 signaling downstream of E6/E7 expression and analyses of primary tumors and The Cancer Genome Atlas (TCGA) data revealed an activated FOXM1-directed promitotic transcriptional signature in HPV+ versus HPV- HNSCCs. Finally, we demonstrate the causality of FOXM1 in driving WEE1i sensitivity. These data suggest that elevated basal FOXM1 activity predisposes HPV+ HNSCC to WEE1i-induced toxicity and provide mechanistic insights into WEE1i and HPV+ HNSCC therapies.

Targeted Sequencing Improves DIPSS-Plus Prognostic Scoring in Myelofibrosis Patients Undergoing Allogeneic Transplantation.

Primary myelofibrosis (MF) and secondary MF developing after polycythemia vera or essential thrombocythemia are clonal disorders of hematopoiesis. Currently the sole therapy offering the potential of cure is hematopoietic cell transplantation (HCT). Several risk classification systems including clinical, hematologic, and mutational parameters have been proposed. We analyzed the mutational landscape in addition to the Dynamic International Prognostic Scoring System (DIPSS)-plus in 55 patients with MF to determine the combined impact on post-HCT outcome. Mutations, analyzed in 75 genes, were most common in JAK2, CALR, ASXL1, TET2, GATA2, EZH2, U2AF1, and ETV6. Patients with ≥3 mutations in addition to JAK2 or CALR mutations had a higher post-transplantation relapse rate and nonrelapse mortality than patients with fewer mutations, independent of DIPSS-plus risk. The presence of higher numbers of mutations identified patients at the greatest risk of relapse within the highest overall risk group as determined by DIPSS-plus. These findings are consistent with molecular risk classifications for patients who do not undergo HCT and support the proposed transplantation risk classification incorporating mutational information.

Comprehensive Transcriptome Profiling of Cryptic CBFA2T3-GLIS2 Fusion-Positive AML Defines Novel Therapeutic Options: A COG and TARGET Pediatric AML Study.

PURPOSE: A cryptic inv(16)(p13.3q24.3) encoding the CBFA2T3-GLIS2 fusion is associated with poor outcome in infants with acute megakaryocytic leukemia. We aimed to broaden our understanding of the pathogenesis of this fusion through transcriptome profiling. EXPERIMENTAL DESIGN: Available RNA from children and young adults with de novo acute myeloid leukemia (AML; N = 1,049) underwent transcriptome sequencing (mRNA and miRNA). Transcriptome profiles for those with the CBFA2T3-GLIS2 fusion (N = 24) and without (N = 1,025) were contrasted to define fusion-specific miRNAs, genes, and pathways. Clinical annotations defined distinct fusion-associated disease characteristics and outcomes. RESULTS: The CBFA2T3-GLIS2 fusion was restricted to infants <3 years old (P < 0.001), and the presence of this fusion was highly associated with adverse outcome (P < 0.001) across all morphologic classifications. Further, there was a striking paucity of recurrent cooperating mutations, and transduction of cord blood stem cells with this fusion was sufficient for malignant transformation. CBFA2T3-GLIS2 positive cases displayed marked upregulation of genes with cell membrane/extracellular matrix localization potential, including NCAM1 and GABRE. Additionally, miRNA profiling revealed significant overexpression of mature miR-224 and miR-452, which are intronic miRNAs transcribed from the GABRE locus. Gene-set enrichment identified dysregulated Hippo, TGFß, and hedgehog signaling, as well as NCAM1 (CD56) interaction pathways. Therapeutic targeting of fusion-positive leukemic cells with CD56-directed antibody-drug conjugate caused significant cytotoxicity in leukemic blasts. CONCLUSIONS: The CBFA2T3-GLIS2 fusion defines a highly refractory entity limited to infants that appears to be sufficient for malignant transformation. Transcriptome profiling elucidated several highly targetable genes and pathways, including the identification of CD56, providing a highly plausible target for therapeutic intervention.

A high-resolution landscape of mutations in the BCL6 super-enhancer in normal human B cells.

The super-enhancers (SEs) of lineage-specific genes in B cells are off-target sites of somatic hypermutation. However, the inability to detect sufficient numbers of mutations in normal human B cells has precluded the generation of a high-resolution mutational landscape of SEs. Here we captured and sequenced 12 B cell SEs at single-nucleotide resolution from 10 healthy individuals across diverse ethnicities. We detected a total of approximately 9,000 subclonal mutations (allele frequencies <0.1%); of these, approximately 8,000 are present in the BCL6 SE alone. Within the BCL6 SE, we identified 3 regions of clustered mutations in which the mutation frequency is ∼7 × 10-4 Mutational spectra show a predominance of C > T/G > A and A > G/T > C substitutions, consistent with the activities of activation-induced-cytidine deaminase (AID) and the A-T mutator, DNA polymerase η, respectively, in mutagenesis in normal B cells. Analyses of mutational signatures further corroborate the participation of these factors in this process. Single base substitution signatures SBS85, SBS37, and SBS39 were found in the BCL6 SE. While SBS85 is a denoted signature of AID in lymphoid cells, the etiologies of SBS37 and SBS39 are unknown. Our analysis suggests the contribution of error-prone DNA polymerases to the latter signatures. The high-resolution mutation landscape has enabled accurate profiling of subclonal mutations in B cell SEs in normal individuals. By virtue of the fact that subclonal SE mutations are clonally expanded in B cell lymphomas, our studies also offer the potential for early detection of neoplastic alterations.

Insertional mutagenesis using the Sleeping Beauty transposon system identifies drivers of erythroleukemia in mice.

Insertional mutagenesis is a powerful means of identifying cancer drivers in animal models. We used the Sleeping Beauty (SB) transposon/transposase system to identify activated oncogenes in hematologic cancers in wild-type mice and mice that express a stabilized cyclin E protein (termed cyclin ET74AT393A). Cyclin E governs cell division and is misregulated in human cancers. Cyclin ET74AT393A mice develop ineffective erythropoiesis that resembles early-stage human myelodysplastic syndrome, and we sought to identify oncogenes that might cooperate with cyclin E hyperactivity in leukemogenesis. SB activation in hematopoietic precursors caused T-cell leukemia/lymphomas (T-ALL) and pure red blood cell erythroleukemias (EL). Analysis of >12,000 SB integration sites revealed markedly different oncogene activations in EL and T-ALL: Notch1 and Ikaros were most common in T-ALL, whereas ETS transcription factors (Erg and Ets1) were targeted in most ELs. Cyclin E status did not impact leukemogenesis or oncogene activations. Whereas most SB insertions were lost during culture of EL cell lines, Erg insertions were retained, indicating Erg's key role in these neoplasms. Surprisingly, cyclin ET74AT393A conferred growth factor independence and altered Erg-dependent differentiation in EL cell lines. These studies provide new molecular insights into erythroid leukemia and suggest potential therapeutic targets for human leukemia.

Whole-genome sequencing of esophageal adenocarcinoma in Chinese patients reveals distinct mutational signatures and genomic alterations.

While the incidence of esophageal adenocarcinoma (EAC) has risen drastically in Western countries over the last 40 years, a similar trend has not been observed for EAC in China. Here, we analyzed mutational spectrum, copy number alterations, and structural variants from whole-genome sequencing of 10 Chinese EAC tumor samples and their matched normal samples, and compared them to previously reported EAC tumor specimens from Western countries. The mutational burden in Chinese EAC was significantly lower than that found in EAC from Western countries. The hallmark A>C mutational signature observed at high frequency in EAC from Western countries, which has been linked to acid reflux, is completely absent in Chinese samples. Furthermore, none of the Chinese samples showed evidence of chromothripsis and genome doubling that are often found in EAC from Western countries. In summary, Chinese EAC tumor samples had distinct genomic profiles and signatures, suggesting that EAC in Chinese individuals may arise from a different etiological pathway.

Pilot study of lithium to restore intestinal barrier function in severe graft-versus-host disease.

Severe intestinal graft-vs-host disease (GVHD) after allogeneic hematopoietic cell transplantation (HCT) causes mucosal ulceration and induces innate and adaptive immune responses that amplify and perpetuate GVHD and the associated barrier dysfunction. Pharmacological agents to target mucosal barrier dysfunction in GVHD are needed. We hypothesized that induction of Wnt signaling by lithium, an inhibitor of glycogen synthase kinase (GSK3), would potentiate intestinal crypt proliferation and mucosal repair and that inhibition of GSK3 in inflammatory cells would attenuate the deregulated inflammatory response to mucosal injury. We conducted an observational pilot study to provide data for the potential design of a randomized study of lithium. Twenty patients with steroid refractory intestinal GVHD meeting enrollment criteria were given oral lithium carbonate. GVHD was otherwise treated per current practice, including 2 mg/kg per day of prednisone equivalent. Seventeen patients had extensive mucosal denudation (extreme endoscopic grade 3) in the duodenum or colon. We observed that 8 of 12 patients (67%) had a complete remission (CR) of GVHD and survived more than 1 year (median 5 years) when lithium administration was started promptly within 3 days of endoscopic diagnosis of denuded mucosa. When lithium was started promptly and less than 7 days from salvage therapy for refractory GVHD, 8 of 10 patients (80%) had a CR and survived more than 1 year. In perspective, a review of 1447 consecutive adult HCT patients in the preceding 6 years at our cancer center showed 0% one-year survival in 27 patients with stage 3-4 intestinal GVHD and grade 3 endoscopic appearance in the duodenum or colon. Toxicities included fatigue, somnolence, confusion or blunted affect in 50% of the patients. The favorable outcomes in patients who received prompt lithium therapy appear to support the future conduct of a randomized study of lithium for management of severe GVHD with extensive mucosal injury. TRIAL REGISTRATION: ClinicalTrials.gov NCT00408681.

Outcomes of acute leukemia patients transplanted with naive T cell-depleted stem cell grafts.

BACKGROUND: Graft-versus-host disease (GVHD) is a major cause of morbidity and mortality following allogeneic hematopoietic stem cell transplantation (HCT). In mice, naive T cells (TN) cause more severe GVHD than memory T cells (TM). We hypothesized that selective depletion of TN from human allogeneic peripheral blood stem cell (PBSC) grafts would reduce GVHD and provide sufficient numbers of hematopoietic stem cells and TM to permit hematopoietic engraftment and the transfer of pathogen-specific T cells from donor to recipient, respectively. METHODS: In a single-arm clinical trial, we transplanted 35 patients with high-risk leukemia with TN-depleted PBSC grafts following conditioning with total body irradiation, thiotepa, and fludarabine. GVHD prophylactic management was with tacrolimus immunosuppression alone. Subjects received CD34-selected PBSCs and a defined dose of TM purged of CD45RA+ TN. Primary and secondary objectives included engraftment, acute and chronic GVHD, and immune reconstitution. RESULTS: All recipients of TN-depleted PBSCs engrafted. The incidence of acute GVHD was not reduced; however, GVHD in these patients was universally corticosteroid responsive. Chronic GVHD was remarkably infrequent (9%; median follow-up 932 days) compared with historical rates of approximately 50% with T cell-replete grafts. TM in the graft resulted in rapid T cell recovery and transfer of protective virus-specific immunity. Excessive rates of infection or relapse did not occur and overall survival was 78% at 2 years. CONCLUSION: Depletion of TN from stem cell allografts reduces the incidence of chronic GVHD, while preserving the transfer of functional T cell memory. TRIAL REGISTRATION: ClinicalTrials.gov (NCT 00914940).

Germline ETV6 mutations in familial thrombocytopenia and hematologic malignancy.

We report germline missense mutations in ETV6 segregating with the dominant transmission of thrombocytopenia and hematologic malignancy in three unrelated kindreds, defining a new hereditary syndrome featuring thrombocytopenia with susceptibility to diverse hematologic neoplasms. Two variants, p.Arg369Gln and p.Arg399Cys, reside in the highly conserved ETS DNA-binding domain. The third variant, p.Pro214Leu, lies within the internal linker domain, which regulates DNA binding. These three amino acid sites correspond to hotspots for recurrent somatic mutation in malignancies. Functional studies show that the mutations abrogate DNA binding, alter subcellular localization, decrease transcriptional repression in a dominant-negative fashion and impair hematopoiesis. These familial genetic studies identify a central role for ETV6 in hematopoiesis and malignant transformation. The identification of germline predisposition to cytopenias and cancer informs the diagnosis and medical management of at-risk individuals.

Disruption of IKAROS activity in primitive chronic-phase CML cells mimics myeloid disease progression.

Without effective therapy, chronic-phase chronic myeloid leukemia (CP-CML) evolves into an acute leukemia (blast crisis [BC]) that displays either myeloid or B-lymphoid characteristics. This transition is often preceded by a clinically recognized, but biologically poorly characterized, accelerated phase (AP). Here, we report that IKAROS protein is absent or reduced in bone marrow blasts from most CML patients with advanced myeloid disease (AP or BC). This contrasts with primitive CP-CML cells and BCR-ABL1-negative acute myeloid leukemia blasts, which express readily detectable IKAROS. To investigate whether loss of IKAROS contributes to myeloid disease progression in CP-CML, we examined the effects of forced expression of a dominant-negative isoform of IKAROS (IK6) in CP-CML patients' CD34(+) cells. We confirmed that IK6 disrupts IKAROS activity in transduced CP-CML cells and showed that it confers on them features of AP-CML, including a prolonged increased output in vitro and in xenografted mice of primitive cells with an enhanced ability to differentiate into basophils. Expression of IK6 in CD34(+) CP-CML cells also led to activation of signal transducer and activator of transcription 5 and transcriptional repression of its negative regulators. These findings implicate loss of IKAROS as a frequent step and potential diagnostic harbinger of progressive myeloid disease in CML patients.