Jennifer J.D. Morrissette

Disruption of TET2 promotes the therapeutic efficacy of CD19-targeted T cells

Cancer immunotherapy based on genetically redirecting T cells has been used successfully to treat B cell malignancies1–3. In this strategy, the T cell genome is modified by integration of viral vectors or transposons encoding chimaeric antigen receptors (CARs) that direct tumour cell killing. However, this approach is often limited by the extent of expansion and persistence of CAR T cells4,5. Here we report mechanistic insights from studies of a patient with chronic lymphocytic leukaemia treated with CAR T cells targeting the CD19 protein. Following infusion of CAR T cells, anti-tumour activity was evident in the peripheral blood, lymph nodes and bone marrow; this activity was accompanied by complete remission. Unexpectedly, at the peak of the response, 94% of CAR T cells originated from a single clone in which lentiviral vector-mediated insertion of the CAR transgene disrupted the methylcytosine dioxygenase TET2 gene. Further analysis revealed a hypomorphic mutation in this patient’s second TET2 allele. TET2-disrupted CAR T cells exhibited an epigenetic profile consistent with altered T cell differentiation and, at the peak of expansion, displayed a central memory phenotype. Experimental knockdown of TET2 recapitulated the potency-enhancing effect of TET2 dysfunction in this patient’s CAR T cells. These findings suggest that the progeny of a single CAR T cell induced leukaemia remission and that TET2 modification may be useful for improving immunotherapies.Genetically engineered T cells that induced remission in a patient with chronic lymphocytic leukaemia were found to have disruption of the TET2 gene, which caused T cell changes that potentiated their anti-tumour effects.

Co‐targeting BET and MEK as salvage therapy for MAPK and checkpoint inhibitor‐resistant melanoma

Despite novel therapies for melanoma, drug resistance remains a significant hurdle to achieving optimal responses. NRAS‐mutant melanoma is an archetype of therapeutic challenges in the field, which we used to test drug combinations to avert drug resistance. We show that BET proteins are overexpressed in NRAS‐mutant melanoma and that high levels of the BET family member BRD4 are associated with poor patient survival. Combining BET and MEK inhibitors synergistically curbed the growth of NRAS‐mutant melanoma and prolonged the survival of mice bearing tumors refractory to MAPK inhibitors and immunotherapy. Transcriptomic and proteomic analysis revealed that combining BET and MEK inhibitors mitigates a MAPK and checkpoint inhibitor resistance transcriptional signature, downregulates the transcription factor TCF19, and induces apoptosis. Our studies demonstrate that co‐targeting MEK and BET can offset therapy resistance, offering a salvage strategy for melanomas with no other therapeutic options, and possibly other treatment‐resistant tumor types.

In vivo evaluation of EGFRvIII mutation in primary glioblastoma patients via complex multiparametric MRI signature

Background Epidermal growth factor receptor variant III (EGFRvIII) is a driver mutation and potential therapeutic target in glioblastoma. Non-invasive in vivo EGFRvIII determination, using clinically acquired multiparametric MRI sequences, could assist in assessing spatial heterogeneity related to EGFRvIII, currently not captured via single-specimen analyses. We hypothesize that integration of subtle, yet distinctive, quantitative imaging/radiomic patterns using machine learning may lead to non-invasively determining molecular characteristics, and particularly the EGFRvIII mutation. Methods We integrated diverse imaging features, including the tumors spatial distribution pattern, via support vector machines, to construct an imaging signature of EGFRvIII. This signature was evaluated in independent discovery (n = 75) and replication (n = 54) cohorts of de novo glioblastoma, and compared with the EGFRvIII status obtained through an assay based on next-generation sequencing. Results The cross-validated accuracy of the EGFRvIII signature in classifying the mutation status in individual patients of the independent discovery and replication cohorts was 85.3% (specificity = 86.3%, sensitivity = 83.3%, area under the curve [AUC] = 0.85) and 87% (specificity = 90%, sensitivity = 78.6%, AUC = 0.86), respectively. The signature was consistent with EGFRvIII+ tumors having increased neovascularization and cell density, as well as a distinctive spatial pattern involving relatively more frontal and parietal regions compared with EGFRvIII- tumors. Conclusions An imaging signature of EGFRvIII was found, revealing a complex, yet distinct macroscopic glioblastoma phenotype. By non-invasively capturing the tumor in its entirety, the proposed methodology can assist in evaluating the tumors spatial heterogeneity, hence overcoming common spatial sampling limitations of tissue-based analyses. This signature can preoperatively stratify patients for EGFRvIII-targeted therapies, and potentially monitor dynamic mutational changes during treatment.

Germline duplication of ATG2B and GSKIP genes is not required for the familial myeloid malignancy syndrome associated with the duplication of chromosome 14q32

We read with interest the recent Perspective by Tawana and colleagues [1] discussing the advantages and disadvantages of upfront genetic testing for inherited susceptibility to myeloid neoplasms for all patients with myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). At least 10% of patients with myeloid neoplasms are believed to have a genetic predisposition to MDS/AML [1]. One additional consideration for implementing universal genetic testing for myeloid malignancies is the still incomplete understanding of genotype–phenotype correlations for the more recently described and emerging syndromes. Recently, Saliba et al. [2] identified a novel type of myeloid neoplasm predisposition in four large West Indian families, where a germline tandem duplication of a 700 kilobase (kb) region on chromosome 14q32 conferred a highly penetrant form of a myeloid neoplasm inherited in an autosomal dominant manner. The duplicated region encompassed five protein-coding genes, TCL1A, GSKIP, ATG2B, BDKRB1, and BDKRB2. Extensive correlative studies concluded that malignant predisposition in these families was due to a germline duplication of the GSKIP and ATG2B genes and not the other genes in the duplicated region. Based on these findings, the recommended testing for familial myeloid neoplasms has been expanded to include duplications of the GSKIP and ATG2B genes [1]. Here we report a North American family with an autosomal dominant predisposition to myeloid neoplasms and a duplication of chromosome 14q32 that does not involve the GSKIP and ATG2B genes, suggesting that duplication of GSKIP and ATG2B may not be required for this autosomal dominant myeloid neoplasm predisposition syndrome. We identified a North American family of Caucasian ancestry with three affected generations who presented with myeloid neoplasms similar to the four families reported by Saliba et al. (Fig. 1a, Table 1). The proband (IV-3) developed a JAK2 V617F mutation-positive primary myelofibrosis at the age of 22, and eight years later developed AML. The proband’s father (III-2) was diagnosed with MDS with ringed sideroblasts and multilineage dysplasia at the age of 64 years and rapidly progressed to AML within months of presentation. The proband’s paternal grandmother (II-2) died of acute leukemia at the age of 37 years. Whole exome sequencing (WES) of skin fibroblast DNA from the proband’s father (III-2) detected no genetic lesions associated with known familial myeloid neoplasms. However, single nucleotide polymorphism array (SNP-A) analysis identified a germline duplication on chromosome 14q32. The same chromosome 14q32 duplication was also present in the proband (IV-3) and was absent in the unaffected relatives. The duplicated region partially overlapped the duplication reported by Saliba et al.; however, among the five genes duplicated in the West Indian families, only TCL1A was duplicated in our family. To characterize the duplicated region more precisely, we used whole genome sequencing (WGS) in combination * Daria V. Babushok daria.babushok@uphs.upenn.edu

A KRAS wild type mutational status confers a survival advantage in pancreatic ductal adenocarcinoma

Background The KRAS oncogene is a driver mutation and is present in greater than 90% of pancreatic ductal adenocarcinomas (PDAC). A subset of these tumors, however, do not harbor mutations in KRAS (wild type KRAS). Studies have shown that patients with mutated KRAS have a poorer survival on first-line gemcitabine-based chemotherapy compared to wild type KRAS. In this study, we examined a cohort of patients with PDAC at our institution who were either wild type or mutant for the KRAS gene and assessed for differences in survival and response to different chemotherapeutic regimens. Methods We examined clinical records of patients treated at the Abramson Cancer Center of the University of Pennsylvania from 2013 to 2017. Patients with a pancreatic mass and a histologic diagnosis of pancreatic or pancreaticobiliary adenocarcinoma were identified. Thirty-nine patients with PDAC who underwent tumor sequencing at Penn Medicines Center for Personalized Diagnostics (CPD) were selected for further study. Twelve patients were identified whose tumors were KRAS wild type. Twenty-seven patients with PDAC whose tumors harbored KRAS mutations were selected as controls (KRAS mutant). Results We noted a longer overall survival (OS) among KRAS wild type patients compared to KRAS mutant patients (P=0.026). This was independent of the age at diagnosis, patient gender, stage of diagnosis, tumor morphology, mismatch repair (MMR) status, and chemotherapeutic regimen. Conclusions Similar to previously reported studies, PDAC with a KRAS wild type mutational profile has a better prognosis with a longer OS. This improved prognosis is independent of the protocol utilized in therapy for these patients. Our findings suggest that future clinical trials in pancreatic cancer should take into consideration the presence of KRAS mutations in their pre-planned analysis when assessing the efficacy of a novel therapeutic approach. This may be a crucial factor in trial concepts and outcomes.

ROS1 Rearrangement in a Case of Classic Biphasic Pulmonary Blastoma

Classic biphasic pulmonary blastoma (CBPB) is a rare and aggressive type of non–small cell lung carcinoma (NSCLC) presenting in adults in the fourth to fifth decade. The prognosis is poor and after surgical resection, therapeutic options are often limited. ROS1 is a proto-oncogene receptor tyrosine kinase that has been identified in some types of NSCLC. We report a case of a 36-year-old woman with CBPB, which was subsequently found to have a ROS1 rearrangement. This is the first reported case of a ROS1-rearranged CBPB. This finding has therapeutic implications as these tumors have the potential to be treated with receptor tyrosine kinase inhibitors.

Interim PET/CT Result Is Not Predictive of Survival in Patients With MYC-rearranged Non–Burkitt Aggressive B-cell Lymphoma

Micro‐Abstract: Non–Burkitt B‐cell lymphoma with a rearrangement of MYC is an aggressive lymphoma that can fail initial treatment. We reviewed the data from 28 patients and investigated whether the mid‐treatment positron emission tomography/computed tomography (PET/CT) scan could predict treatment failure. The results of that PET/CT scan did not predict in which patients the initial treatment would fail. Therefore, it should not be used to guide treatment decisions in this population. Background: Patients with a diagnosis of MYC‐rearranged non–Burkitt aggressive B‐cell lymphoma (MYC‐R), including those with double hit lymphoma, are at high risk of developing relapsed/refractory disease, even if treated with intensive front‐line immunochemotherapy. It is common in clinical practice and clinical trials to perform an interim positron emission tomography (PET)/computed tomography (CT) scan (iPET) during front‐line therapy for diffuse large B‐cell lymphoma. However, the utility of the iPET result for MYC‐R patients for predicting outcomes is unclear. Patients and Methods: We performed a single‐center retrospective study with centralized pathologic review and PET/CT image acquisition and interpretation for 28 MYC‐R patients. The patients received front‐line therapy with R‐CHOP (rituximab, cyclophosphamide, hydroxydaunorubicin [doxorubicin], Oncovin [vincristine], prednisone) or intensive immunochemotherapy. Results: Eight patients had iPET‐positive (iPET+) and 20 patients had iPET‐negative (iPET−) results using the Deauville visual assessment criteria. At a median follow‐up length of 30.4 months, progression‐free survival was 65% and overall survival was 76%, neither of which differed significantly between the iPET− and iPET+ patients. The positive predictive value of iPET for progression at 30 months was 25%, and the negative predictive value was 65%. Conclusion: Although patients with MYC‐R lymphoma have been reported to be at high risk of primary treatment failure, this was not predicted by iPET+ results. Thus, the iPET result should not be used to guide changes in front‐line or consolidative therapy for these patients.

Epidermal Growth Factor Receptor Extracellular Domain Mutations in Glioblastoma Present Opportunities for Clinical Imaging and Therapeutic Development

We explored the clinical and pathological impact of epidermal growth factor receptor (EGFR) extracellular domain missense mutations. Retrospective assessment of 260 de novo glioblastoma patients revealed a significant reduction in overall survival of patients having tumors with EGFR mutations at alanine 289 (EGFRA289D/T/V). Quantitative multi-parametric magnetic resonance imaging analyses indicated increased tumor invasion for EGFRA289D/T/V mutants, corroborated in mice bearing intracranial tumors expressing EGFRA289V and dependent on ERK-mediated expression of matrix metalloproteinase-1. EGFRA289V tumor growth was attenuated with an antibody against a cryptic epitope, based on in silico simulation. The findings of this study indicate a highly invasive phenotype associated with the EGFRA289V mutation in glioblastoma, postulating EGFRA289V as a molecular marker for responsiveness to therapy with EGFR-targeting antibodies.

JAK2 V617F-positive acute myeloid leukaemia (AML): a comparison between de novo AML and secondary AML transformed from an underlying myeloproliferative neoplasm. A study from the Bone Marrow Pathology Group

The JAK2 V617F mutation is characteristic of most Philadelphia chromosome‐negative myeloproliferative neoplasms (MPNs) and occurs rarely in de novo acute myeloid leukaemia (AML). We sought to characterize AMLs that harbour this mutation and distinguish those that arise de novo (AML‐DN) from those that reflect transformation of an underlying MPN (AML‐MPN). Forty‐five patients with JAK2 V617F‐mutated AML were identified; 15 were AML‐DN and 30 were AML‐MPN. AML‐MPN cases were more likely to have splenomegaly (P = 0·02), MPN‐like megakaryocytes and higher mean JAK2 V617F VAF at diagnosis (P = 0·04). Mutations involving TET2 were exclusively identified in AML‐DN patients. Mutations of genes affecting DNA methylation were more common in AML‐DN (P < 0·01). A complex karyotype was more frequent in AML‐MPN cases than in AML‐DN (P < 0·01), with AML‐DN more likely to display a normal karyotype (P = 0·02). Bone marrow histology after recovery from induction chemotherapy in AML‐DN cases revealed no morphological evidence of any previously occult MPNs, while this was evident in most of the AML‐MPN specimens (P < 0·01). These findings in this largest study of JAK2 V617F‐mutated AMLs indicate that AML‐DN is distinct from AML‐MPN.