Cyrus V. Hedvat

The Common Feature of Leukemia-Associated IDH1 and IDH2 Mutations Is a Neomorphic Enzyme Activity Converting α-Ketoglutarate to 2-Hydroxyglutarate

The somatic mutations in cytosolic isocitrate dehydrogenase 1 (IDH1) observed in gliomas can lead to the production of 2-hydroxyglutarate (2HG). Here, we report that tumor 2HG is elevated in a high percentage of patients with cytogenetically normal acute myeloid leukemia (AML). Surprisingly, less than half of cases with elevated 2HG possessed IDH1 mutations. The remaining cases with elevated 2HG had mutations in IDH2, the mitochondrial homolog of IDH1. These data demonstrate that a shared feature of all cancer-associated IDH mutations is production of the oncometabolite 2HG. Furthermore, AML patients with IDH mutations display a significantly reduced number of other well characterized AML-associated mutations and/or associated chromosomal abnormalities, potentially implicating IDH mutation in a distinct mechanism of AML pathogenesis.

CD19-targeted T cells rapidly induce molecular remissions in adults with chemotherapy-refractory acute lymphoblastic leukemia.

Five adults with chemotherapy-refractory B-ALL were induced into molecular remissions after treatment with CD19 CAR-targeted T cells. CARving a Niche for Cancer Immunotherapy Acute lymphoblastic leukemia (ALL) is a cancer of the white blood cells that fend off infection. It’s most common in children but—as with many diseases that primarily affect children—has a much worse prognosis when it affects adults. Adults with relapsed disease have a very low chance of survival, and new therapies are desperately needed. Now, Brentjens et al. test T cells engineered to target CD19, which is expressed on both healthy B lymphocytes and B-ALL cells, in five chemotherapy-refractory adult B-ALL patients. Here, the authors treat patients with the patients’ own T cells altered to express not only CD19 but also a fusion of the costimulatory molecule CD28 with CD3ζ chain—so-called “second-generation chimeric antigen receptor (CAR) T cells.” All patients treated with these cells achieved tumor eradication and complete remission. These CAR T cells were well tolerated, although there was substantial cytokine release in some patients that correlated to tumor burden. These patients were treated with steroid therapy. Long-term follow-up in four of these patients was not possible because the CAR T cell therapy allowed these patients to be eligible for subsequent hematopoietic stem cell transplant (HSCT), which resulted in restored hematopoiesis. The remaining patient experienced a relapse of CD19+ cells that coincided with the lack of persistence of the CAR T cells from circulation. These data suggest that subsequent transfusions should be considered for patients unable to undergo HSCT. Adults with relapsed B cell acute lymphoblastic leukemia (B-ALL) have a dismal prognosis. Only those patients able to achieve a second remission with no minimal residual disease (MRD) have a hope for long-term survival in the context of a subsequent allogeneic hematopoietic stem cell transplantation (allo-HSCT). We have treated five relapsed B-ALL subjects with autologous T cells expressing a CD19-specific CD28/CD3ζ second-generation dual-signaling chimeric antigen receptor (CAR) termed 19-28z. All patients with persistent morphological disease or MRD+ disease upon T cell infusion demonstrated rapid tumor eradication and achieved MRD− complete remissions as assessed by deep sequencing polymerase chain reaction. Therapy was well tolerated, although significant cytokine elevations, specifically observed in those patients with morphologic evidence of disease at the time of treatment, required lymphotoxic steroid therapy to ameliorate cytokine-mediated toxicities. Indeed, cytokine elevations directly correlated to tumor burden at the time of CAR-modified T cell infusions. Tumor cells from one patient with relapsed disease after CAR-modified T cell therapy, who was ineligible for additional allo-HSCT or T cell therapy, exhibited persistent expression of CD19 and sensitivity to autologous 19-28z T cell–mediated cytotoxicity, which suggests potential clinical benefit of additional CAR-modified T cell infusions. These results demonstrate the marked antitumor efficacy of 19-28z CAR-modified T cells in patients with relapsed/refractory B-ALL and the reliability of this therapy to induce profound molecular remissions, forming a highly effective bridge to potentially curative therapy with subsequent allo-HSCT.

Development and validation of a clinical cancer genomic profiling test based on massively parallel DNA sequencing

As more clinically relevant cancer genes are identified, comprehensive diagnostic approaches are needed to match patients to therapies, raising the challenge of optimization and analytical validation of assays that interrogate millions of bases of cancer genomes altered by multiple mechanisms. Here we describe a test based on massively parallel DNA sequencing to characterize base substitutions, short insertions and deletions (indels), copy number alterations and selected fusions across 287 cancer-related genes from routine formalin-fixed and paraffin-embedded (FFPE) clinical specimens. We implemented a practical validation strategy with reference samples of pooled cell lines that model key determinants of accuracy, including mutant allele frequency, indel length and amplitude of copy change. Test sensitivity achieved was 95–99% across alteration types, with high specificity (positive predictive value >99%). We confirmed accuracy using 249 FFPE cancer specimens characterized by established assays. Application of the test to 2,221 clinical cases revealed clinically actionable alterations in 76% of tumors, three times the number of actionable alterations detected by current diagnostic tests.

Genetic characterization of TET1, TET2, and TET3 alterations in myeloid malignancies

Disease alleles that activate signal transduction are common in myeloid malignancies; however, there are additional unidentified mutations that contribute to myeloid transformation. Based on the recent identification of TET2 mutations, we evaluated the mutational status of TET1, TET2, and TET3 in myeloproliferative neoplasms (MPNs), chronic myelomonocytic leukemia (CMML), and acute myeloid leukemia (AML). Sequencing of TET2 in 408 paired tumor/normal samples distinguished between 68 somatic mutations and 6 novel single nucleotide polymorphisms and identified TET2 mutations in MPN (27 of 354, 7.6%), CMML (29 of 69, 42%), AML (11 of 91, 12%), and M7 AML (1 of 28, 3.6%) samples. We did not identify somatic TET1 or TET3 mutations or TET2 promoter hypermethylation in MPNs. TET2 mutations did not cluster in genetically defined MPN, CMML, or AML subsets but were associated with decreased overall survival in AML (P = .029). These data indicate that TET2 mutations are observed in different myeloid malignancies and may be important in AML prognosis.

PIK3CA mutation associates with improved outcome in breast cancer.

Purpose: In breast cancer, somatic mutations in the PIK3CA gene are common. The prognostic implication of these activating mutations remains uncertain as moderately sized studies have yielded variable outcomes. Our aim was to determine the prognostic implications of PIK3CA mutations in breast cancer. Experimental Design: Archival formalin-fixed paraffin-embedded primary breast tumors, from 590 patients selected for known vital status with a median follow-up of 12.8 years and a tumor >1 cm, were genotyped for PIK3CA mutations. Mutation rates and associations between mutation site and clinicopathologic characteristics were assessed. Progression-free survival, overall survival, and breast cancer–specific survival were examined using Kaplan-Meier or competing risk methodology. Results: PIK3CA mutation is identified in 32.5% of breast cancers. PIK3CA mutation significantly associates with older age at diagnosis, hormone receptor positivity, HER2 negativity, lower tumor grade and stage, and lymph node negativity. Patients with PIK3CA mutated tumors have significant improvement in overall survival (P = 0.03) and breast cancer–specific survival (P = 0.004). Analysis for PIK3CA mutation site-specific associations reveals that the H1047R kinase domain mutation highly associates with node negativity (P = 0.007), whereas helical domain hotspot mutations associate with older age at diagnosis (P = 0.004). Conclusion: This study defines the positive prognostic significance of PIK3CA mutations. This work is clinically relevant, as it will significantly affect the design of clinical trials planned for phosphatidylinositol 3-kinase–targeted therapy. Future work may define a population of older age breast cancer patients in whom therapy can be minimized. (Clin Cancer Res 2009;15(16):5049–59)

Genetic Analysis of Transforming Events That Convert Chronic Myeloproliferative Neoplasms to Leukemias

The oncogenetic events that transform chronic myeloproliferative neoplasms (MPN) to acute myeloid leukemias (AML) are not well characterized. We investigated the role of several genes implicated in leukemic transformation by mutational analysis of 63 patients with AML secondary to a preexisting MPN (sAML). Frequent mutations were identified in TET2 (26.3%), ASXL1 (19.3%), IDH1 (9.5%), and JAK2 (36.8%) mutations in sAML, and all possible mutational combinations of these genes were also observed. Analysis of 14 patients for which paired samples from MPN and sAML were available showed that TET2 mutations were frequently acquired at leukemic transformation [6 of 14 (43%)]. In contrast, ASXL1 mutations were almost always detected in both the MPN and AML clones from individual patients. One case was also observed where TET2 and ASXL1 mutations were found before the patient acquired a JAK2 mutation or developed clinical evidence of MPN. We conclude that mutations in TET2, ASXL1, and IDH1 are common in sAML derived from a preexisting MPN. Although TET2/ASXL1 mutations may precede acquisition of JAK2 mutations by the MPN clone, mutations in TET2, but not ASXL1, are commonly acquired at the time of leukemic transformation. Our findings argue that the mutational order of events in MPN and sAML varies in different patients, and that TET2 and ASXL1 mutations have distinct roles in MPN pathogenesis and leukemic transformation. Given the presence of sAML that have no preexisting JAK2/TET2/ASXL1/IDH1 mutations, our work indicates the existence of other mutations yet to be identified that are necessary for leukemic transformation.

Adoptive immunotherapy with unselected or EBV-specific T cells for biopsy-proven EBV+ lymphomas after allogeneic hematopoietic cell transplantation.

We evaluated HLA-compatible donor leukocyte infusions (DLIs) and HLA-compatible or HLA-disparate EBV-specific T cells (EBV-CTLs) in 49 hematopoietic cell transplantation recipients with biopsy-proven EBV-lymphoproliferative disease (EBV-LPD). DLIs and EBV-CTLs each induced durable complete or partial remissions in 73% and 68% of treated patients including 74% and 72% of patients surviving ≥ 8 days after infusion, respectively. Reversible acute GVHD occurred in recipients of DLIs (17%) but not EBV-CTLs. The probability of complete response was significantly lower among patients with multiorgan involvement. In responders, DLIs and EBV-CTLs regularly induced exponential increases in EBV-specific CTL precursor (EBV-CTLp) frequencies within 7-14 days, with subsequent clearance of EBV viremia and resolution of disease. In nonresponders, EBV-CTLps did not increase and EBV viremia persisted. Treatment failures were correlated with impaired T-cell recognition of tumor targets. Either donor-derived EBV-CTLs that had been sensitized with autologous BLCLs transformed by EBV strain B95.8 could not lyse spontaneous donor-derived EBV-transformed BLCLs expanded from the patients blood or biopsied tumor or they failed to lyse their targets because they were selectively restricted by HLA alleles not shared by the EBV-LPD. Therefore, either unselected DLIs or EBV-specific CTLs can eradicate both untreated and Rituxan-resistant lymphomatous EBV-LPD, with failures ascribable to impaired T-cell recognition of tumor-associated viral antigens or their presenting HLA alleles.

The ETS protein MEF plays a critical role in perforin gene expression and the development of natural killer and NK-T cells

We utilized gene targeting by homologous recombination to define the role that MEF, a transcriptional activating member of the ETS family of transcription factors, plays in lymphopoiesis. MEF-/- mice have a profound reduction in the number of NK-T and NK cells. Purified MEF-/- NK cells cannot lyse tumor cell targets and secrete only minimal amounts of IFNgamma. Perforin protein expression is severely impaired in MEF-deficient NK cells, likely accounting for the lack of tumor cell cytotoxicity. Promoter studies and chromatin immunoprecipitation analyses demonstrate that MEF and not ETS-1 directly regulates transcription of the perforin gene in NK cells. Our results uncover a specific role of MEF in the development and function of NK cells and in innate immunity.

Effect of Selumetinib vs Chemotherapy on Progression-Free Survival in Uveal Melanoma A Randomized Clinical Trial

IMPORTANCE Uveal melanoma is characterized by mutations in GNAQ and GNA11, resulting in mitogen-activated protein kinase pathway activation. OBJECTIVE To assess the efficacy of selumetinib, a selective, non-adenosine triphosphate competitive inhibitor of MEK1 and MEK2, in uveal melanoma. DESIGN, SETTING, AND PARTICIPANTS Randomized, open-label, phase 2 clinical trial comparing selumetinib vs chemotherapy conducted from August 2010 through December 2013 among 120 patients with metastatic uveal melanoma at 15 academic oncology centers in the United States and Canada. INTERVENTIONS One hundred one patients were randomized in a 1:1 ratio to receive selumetinib, 75 mg orally twice daily on a continual basis (n = 50), or chemotherapy (temozolomide, 150 mg/m2 orally daily for 5 of every 28 days, or dacarbazine, 1000 mg/m2 intravenously every 21 days [investigator choice]; n = 51) until disease progression, death, intolerable adverse effects, or withdrawal of consent. After primary outcome analysis, 19 patients were registered and 18 treated with selumetinib without randomization to complete the planned 120-patient enrollment. Patients in the chemotherapy group could receive selumetinib at the time of radiographic progression. MAIN OUTCOMES AND MEASURES Progression-free survival, the primary end point, was assessed as of April 22, 2013. Additional end points, including overall survival, response rate, and safety/toxicity, were assessed as of December 31, 2013. RESULTS Median progression-free survival among patients randomized to chemotherapy was 7 weeks (95% CI, 4.3-8.4 weeks; median treatment duration, 8 weeks; interquartile range [IQR], 4.3-16 weeks) and among those randomized to selumetinib was 15.9 weeks (95% CI, 8.4-21.1 weeks; median treatment duration, 16.1 weeks; IQR, 8.1-25.3 weeks) (hazard ratio, 0.46; 95% CI, 0.30-0.71; P < .001). Median overall survival time was 9.1 months (95% CI, 6.1-11.1 months) with chemotherapy and 11.8 months (95% CI, 9.8-15.7 months) with selumetinib (hazard ratio, 0.66; 95% CI, 0.41-1.06; P = .09). No objective responses were observed with chemotherapy. Forty-nine percent of patients treated with selumetinib achieved tumor regression, with 14% achieving an objective radiographic response to therapy. Treatment-related adverse events were observed in 97% of patients treated with selumetinib, with 37% requiring at least 1 dose reduction. CONCLUSIONS AND RELEVANCE In this hypothesis-generating study of patients with advanced uveal melanoma, selumetinib compared with chemotherapy resulted in a modestly improved progression-free survival and response rate; however, no improvement in overall survival was observed. Improvement in clinical outcomes was accompanied by a high rate of adverse events. TRIAL REGISTRATION clinicaltrials.gov Identifier: NCT01143402.

Objective quantification of the Ki67 proliferative index in neuroendocrine tumors of the gastroenteropancreatic system: a comparison of digital image analysis with manual methods.

Pathologic grading for prognostic stratification of neuroendocrine tumors (NETs) is critical but presents a challenging interpretive dilemma. Tumor cell proliferative rate is an important factor in the determination of prognosis, and immunohistochemical analysis with Ki67 is becoming more widely used to quantify the proliferative rate. However, Ki67 assessment has limitations due to lack of uniformity and consistency in quantification. These limitations are accentuated in well-differentiated NETs, as differences in the range of 1% to 5% can alter tumor grade, with potential implications for treatment. We therefore performed a concordance study to assess different Ki67 quantification techniques including: (a) digital image analysis (DIA); (b) manual counting (MC) of >2000 cells; and (c) “eyeballed” estimate (EE) of labeling percentage by pathologists (n=18), including individuals experienced in evaluating Ki67 labeling as well as others who had little prior experience assessing Ki67 percentages. Forty-five Ki67 images were selected and analyzed using the 3 methods. On the basis of the recommendations of the World health Organization (WHO) for grading NETs, MC of 2000 cells was used as the “gold standard” reference against which the other techniques were compared. Three images were presented twice, the second being inverted, to assess intraobserver consistency. Statistical analyses were performed to evaluate: (a) the concordance between methods; (b) intraobserver and interobserver consistency; and (c) correlation of NET grades on the basis of Ki67 scores by EE versus the gold standard. Agreement between scores was assessed by intraclass correlation (ICC). DIA and MC were highly concordant (ICC=0.98). The ICC between DIA and the mean EE of all observers was 0.88. However, there was discordance among individual observers on all cases quantified by EE (ICC=0.13). The ICC for intraobserver consistency was 0.39±0.26. With Ki67 in the ranges of <1%, 2% to 3%, and >20%, the mean of Ki67 by EE was, respectively, 93%±2%, 55%±7%, and 55%±15% correct against the gold standard. The &kgr; statistics for EE exhibited low agreement (&kgr;=0.24; 95% confidence interval, 0.23-0.25) for all WHO NET grades. Incorrect assessment by EE resulted in upgrading of all WHO G1 group tumors (n=14); in the WHO G2 group, downgrading of 41% cases occurred (n=11) when Ki67 was <5% (by DIA or MC), and upgrading of 59% cases occurred (n=16) when Ki67 was >5%. We conclude that DIA and MC are the acceptable standards for Ki67 assessment. Given the inherent discordance in determining the grade, the use of an approximate EE of the Ki67-labeling index requires critical reevaluation, especially for NETs with a labeling index straddling the cut-points between grades. Consequently, determination of therapeutic strategies should be guided by an amalgamation of clinicopathologic characteristics, including but not limited to the Ki67 index.