Gautam U. Mehta

Identification of essential genes for cancer immunotherapy.

Somatic gene mutations can alter the vulnerability of cancer cells to T-cell-based immunotherapies. Here we perturbed genes in human melanoma cells to mimic loss-of-function mutations involved in resistance to these therapies, by using a genome-scale CRISPR–Cas9 library that consisted of around 123,000 single-guide RNAs, and profiled genes whose loss in tumour cells impaired the effector function of CD8+ T cells. The genes that were most enriched in the screen have key roles in antigen presentation and interferon-γ signalling, and correlate with cytolytic activity in patient tumours from The Cancer Genome Atlas. Among the genes validated using different cancer cell lines and antigens, we identified multiple loss-of-function mutations in APLNR, encoding the apelin receptor, in patient tumours that were refractory to immunotherapy. We show that APLNR interacts with JAK1, modulating interferon-γ responses in tumours, and that its functional loss reduces the efficacy of adoptive cell transfer and checkpoint blockade immunotherapies in mouse models. Our results link the loss of essential genes for the effector function of CD8+ T cells with the resistance or non-responsiveness of cancer to immunotherapies.

Mitochondrial Membrane Potential Identifies Cells with Enhanced Stemness for Cellular Therapy.

Long-term survival and antitumor immunity of adoptively transferred CD8(+) T cells is dependent on their metabolic fitness, but approaches to isolate therapeutic T cells based on metabolic features are not well established. Here we utilized a lipophilic cationic dye tetramethylrhodamine methyl ester (TMRM) to identify and isolate metabolically robust T cells based on their mitochondrial membrane potential (ΔΨm). Comprehensive metabolomic and gene expression profiling demonstrated global features of improved metabolic fitness in low-ΔΨm-sorted CD8(+) T cells. Transfer of these low-ΔΨm T cells was associated with superior long-term in vivo persistence and an enhanced capacity to eradicate established tumors compared with high-ΔΨm cells. Use of ΔΨm-based sorting to enrich for cells with superior metabolic features was observed in CD8(+), CD4(+) T cell subsets, and long-term hematopoietic stem cells. This metabolism-based approach to cell selection may be broadly applicable to therapies involving the transfer of HSC or lymphocytes for the treatment of viral-associated illnesses and cancer.

Prospective evaluation of radiosurgery for hemangioblastomas in von Hippel-Lindau disease.

To determine the effectiveness of stereotactic radiosurgery (SRS) treatment to central nervous system (CNS) hemangioblastomas in von Hippel-Lindau disease (VHL), we analyzed long-term results in VHL patients treated with SRS. Patients were enrolled in a prospective VHL natural history study, undergoing SRS treatment of CNS hemangioblastomas. Treatment regimens, serial clinical evaluations, and longitudinal imaging data were analyzed. Twenty VHL patients (10 males and 10 females) underwent SRS treatment of 44 CNS hemangioblastomas (39 cerebellar and 5 brainstem). Mean (+/-SD) age at treatment was 37.5 +/- 12.0 years (range: 13-67). Mean follow-up was 8.5 +/- 3.2 years (range: 3.0-17.6 years). All patients were alive at last follow-up. Mean treated tumor volume was 0.5 +/- 0.7 cm(3) (range: 0.01-3.6 cm(3)). Mean prescription dose was 18.9 Gy (range: 12-24 Gy) at the tumor margin. Local control rate at 2, 5, 10, and 15 years after SRS treatment was 91%, 83%, 61%, and 51%, respectively. Univariate analysis did not identify variables associated (P > .05) with worse tumor control at last follow-up. Thirty-three percent of SRS-treated small (<1.0 cm diameter), asymptomatic tumors progressed over a long-term follow-up. There were no long-term adverse radiation effects. Although SRS treatment of hemangioblastomas in VHL has a low risk for adverse radiation effects, it is associated with diminishing control over a long-term follow-up. These results indicate that SRS should not be used to prophylactically treat asymptomatic tumors and should be reserved for the treatment of tumors that are not surgically resectable.

Somatic GNAS Mutation Causes Widespread and Diffuse Pituitary Disease in Acromegalic Patients with McCune-Albright Syndrome

Context: McCune-Albright syndrome (MAS) is caused by sporadic mutations of the GNAS. Patients exhibit features of acromegaly. In most patients, GH-secreting pituitary adenomas have been held responsible for this presentation. However, surgical adenomectomy rarely eliminates excess GH production. Objective: The aim of this study was to elucidate pituitary pathology in patients with MAS and to explain the basis of failure of adenomectomy to eliminate GH hypersecretion. Design and Setting: We conducted a case series at the National Institutes of Health. Intervention(s): Interventions included medical therapy and transsphenoidal surgery. Patients and Main Outcome Measures: We studied clinical and imaging features and the histology and molecular features of the pituitary of four acromegalic MAS patients. Results: We identified widespread and diffuse pituitary gland disease. The primary pathological changes were characterized by hyperplastic and neoplastic change, associated with overrepresentation of somatotroph cells in structurally intact tissue areas. Genetic analysis of multiple microdissected samples of any type of histological area consistently revealed identical GNAS mutations in individual patients. The only patient with remission after surgery received complete hypophysectomy in addition to removal of multiple GH-secreting tumors. Conclusions: These findings indicate developmental effects of GNAS mutation on the entire anterior pituitary gland. The pituitary of individual cases contains a spectrum of changes with regions of normal appearing gland, hyperplasia, and areas of fully developed adenoma formation, as well as transitional stages between these entities. The primary change underlying acromegaly in MAS patients is somatotroph hyperplasia involving the entire pituitary gland, with or without development of somatotroph adenoma. Thus, successful clinical management, whether it is medical, surgical, or via irradiation, must target the entire pituitary, not just the adenomas evident on imaging.

Prevention of intraoperative cerebrospinal fluid leaks by lumbar cerebrospinal fluid drainage during surgery for pituitary macroadenomas

OBJECT Cerebrospinal fluid leakage is a major complication of transsphenoidal surgery. An intraoperative CSF leak, which occurs in up to 50% of pituitary tumor cases, is the only modifiable risk factor for postoperative leaks. Although several techniques have been described for surgical repair when an intraoperative leak is noted, none has been proposed to prevent an intraoperative CSF leak. The authors postulated that intraoperative CSF drainage would diminish tension on the arachnoid, decrease the rate of intraoperative CSF leakage during surgery for larger tumors, and reduce the need for surgical repair of CSF leaks. METHODS The results of 114 transsphenoidal operations for pituitary macroadenoma performed without intraoperative CSF drainage were compared with the findings from 44 cases in which a lumbar subarachnoid catheter was placed before surgery to drain CSF at the time of dural exposure and tumor removal. RESULTS Cerebrospinal fluid drainage reduced the rate of intraoperative CSF leakage from 41% to 5% (p < 0.001). This reduction occurred in macroadenomas with (from 57% to 5%, p < 0.001) and those without suprasellar extension (from 29% to 0%, p = 0.31). The rate of postoperative CSF leakage was similar (5% vs 5%), despite the fact that intraoperative CSF drainage reduced the need for operative repair (from 32% to 5%, p < 0.001). There were no significant catheter-related complications. CONCLUSIONS Cerebrospinal fluid drainage during transsphenoidal surgery for macroadenomas reduces the rate of intraoperative CSF leaks. This preventative measure obviated the need for surgical repair of intraoperative CSF leaks using autologous fat graft placement, other operative techniques, postoperative lumbar drainage, and/or reoperation in most patients and is associated with minimal risks.

Long-term outcome after resection of brainstem hemangioblastomas in von Hippel-Lindau disease

OBJECT Brainstem hemangioblastomas are frequently encountered in patients with von Hippel-Lindau (VHL) disease. These tumors can cause significant morbidity, and their optimal management has not been defined. To better define the outcome and management of these tumors, the authors analyzed the long-term results in patients who underwent resection of brainstem hemangioblastomas. METHODS Consecutive patients with VHL disease who underwent resection of brainstem hemangioblastomas with a follow-up of 12 months or more were included in this study. Serial functional assessments, radiographic examinations, and operative records were analyzed. RESULTS Forty-four patients (17 male and 27 female) underwent 51 operations for resection of 71 brainstem hemangioblastomas. The most common presenting symptoms were headache, swallowing difficulties, singultus, gait difficulties, and sensory abnormalities. The mean follow-up was 5.9 ± 5.0 years (range 1.0-20.8 years). Immediately after 34 operations (66.7%), the patients remained at their preoperative functional status; they improved after 8 operations (15.7%) and worsened after 9 operations (17.6%) as measured by the McCormick scale. Eight (88.9%) of the 9 patients who were worse immediately after resection returned to their preoperative status within 6 months. Two patients experienced functional decline during long-term follow-up (beginning at 2.5 and 5 years postoperatively) caused by extensive VHL disease-associated CNS disease. CONCLUSIONS Generally, resection of symptomatic brainstem hemangioblastomas is a safe and effective management strategy in patients with VHL disease. Most patients maintain their preoperative functional status, although long-term decline in functional status may occur due to VHL disease-associated progression.

The effect of a PP2A inhibitor on the nuclear receptor corepressor pathway in glioma

OBJECT Nuclear receptor corepressor (N-CoR) forms a complex that maintains neural stem cells in an undifferentiated state through transcriptional repression. Recently, it has been shown that N-CoR is overexpressed in glioblastoma multiforme (GBM) tumor stem cells and has a putative role in maintaining these cells in an undifferentiated immortal state. To determine the effects of disruption of N-CoR complex function by serine/threonine protein phosphatase 2A (PP2A) inhibition on GBM tumor cell differentiation and proliferation, the authors developed and investigated a competitive small molecule inhibitor (LB1) of PP2A in GBM. METHODS The authors investigated the effects of LB1 on GBM proliferation and molecular differentiation pathways using in vitro and in vivo studies. RESULTS The LB1 inhibited PP2A, leading to increased levels of phosphorylated Akt kinase and decreased NCoR expression, as well as dose-dependent antiproliferative activity in cultured U87 and U251 malignant glioma cells (dose range 1-10 microM). Systemic LB1 treatment (1.5 mg/kg/day for 21 days) had significant tumor antiproliferative effects in mice harboring U87 glioma xenografts (73% mean reduction in tumor volume compared with controls; p < 0.001). Moreover, a reduction in PP2A expression and activity after LB1 treatment in vivo correlated with increased Akt phosphorylation, reduced nuclear N-CoR expression and N-CoR cytoplasmic translocation, and increased accumulation of acetylated core histones, which coincided with the appearance of glial fibrillary acidic protein-expressing tumor cells. CONCLUSIONS These findings indicate that PP2A inhibition effectively disrupts N-CoR complex function/expression and leads to cytoplasmic translocation of N-CoR with subsequent tumor cell differentiation and/or death. Therapeutic paradigms that target N-CoR function in the cancer stem cell component of malignant gliomas may have treatment utility.

Acromegaly without Imaging Evidence of Pituitary Adenoma

CONTEXT GH-secreting pituitary adenomas are nearly always visible on conventional magnetic resonance (MR) imaging. However, management and outcome of acromegalic patients lacking imaging evidence of GH-secreting pituitary adenomas are undefined. OBJECTIVE The aim was to evaluate surgical exploration for MR-invisible GH-secreting pituitary adenomas. DESIGN AND SETTING We conducted a retrospective review at two tertiary care centers. PATIENTS OR OTHER PARTICIPANTS Consecutive acromegalic patients without imaging evidence of a pituitary adenoma on pre- and postcontrast, spin echo T1-weighted MR imaging and who lacked evidence of an ectopic (nonpituitary) source causing GH excess were included. INTERVENTIONS Surgical exploration with identification and resection of a pituitary adenoma was performed. MAIN OUTCOME MEASURES Laboratory values (GH, IGF-I), surgical findings, and clinical outcome were analyzed. RESULTS Six patients (three males, three females; 3% of all patients) with suspected GH-secreting adenomas did not demonstrate imaging evidence of pituitary adenoma on conventional MR imaging. Three patients underwent a postcontrast, volumetric interpolated breath-hold examination MR-imaging sequence (1.2-mm slice thickness), which revealed a 4-mm pituitary adenoma not seen on the spin echo T1-weighted MR imaging in one patient. A pituitary adenoma was identified and removed in all patients (mean diameter, 5.6 mm; range, 5 to 6.7 mm). Histological analysis confirmed that the lesions were GH-secreting adenomas. All patients achieved biochemical remission after surgical resection. CONCLUSION Acromegaly can be caused by GH-secreting pituitary adenomas that are not evident on conventional MR imaging. Adenomas in some of these patients become evident using volumetric interpolated breath-hold examination MR imaging. Surgical exploration of the pituitary gland in acromegalic patients with endocrine findings consistent with a GH-secreting adenoma but negative MR imaging can lead to identification and removal of an adenoma.

Type I cytokines synergize with oncogene inhibition to induce tumor growth arrest

Acquavella, Clever, and colleagues show that IFNγ and TNFα synergize with vemurafenib to induce tumor growth arrest, supporting further study of the intersection between immunologic and oncogenic signaling in cancer cells and of treatment strategies combining vemurafenib and T-cell–based immunotherapy. Both targeted inhibition of oncogenic driver mutations and immune-based therapies show efficacy in treatment of patients with metastatic cancer, but responses can be either short lived or incompletely effective. Oncogene inhibition can augment the efficacy of immune-based therapy, but mechanisms by which these two interventions might cooperate are incompletely resolved. Using a novel transplantable BRAFV600E-mutant murine melanoma model (SB-3123), we explored potential mechanisms of synergy between the selective BRAFV600E inhibitor vemurafenib and adoptive cell transfer (ACT)–based immunotherapy. We found that vemurafenib cooperated with ACT to delay melanoma progression without significantly affecting tumor infiltration or effector function of endogenous or adoptively transferred CD8+ T cells, as previously observed. Instead, we found that the T-cell cytokines IFNγ and TNFα synergized with vemurafenib to induce cell-cycle arrest of tumor cells in vitro. This combinatorial effect was recapitulated in human melanoma–derived cell lines and was restricted to cancers bearing a BRAFV600E mutation. Molecular profiling of treated SB-3123 indicated that the provision of vemurafenib promoted the sensitization of SB-3123 to the antiproliferative effects of T-cell effector cytokines. The unexpected finding that immune cytokines synergize with oncogene inhibitors to induce growth arrest has major implications for understanding cancer biology at the intersection of oncogenic and immune signaling and provides a basis for design of combinatorial therapeutic approaches for patients with metastatic cancer. Cancer Immunol Res; 3(1); 37–47. ©2014 AACR. See related commentary by Riddell, p. 23

The history of pituitary surgery for Cushing disease

Although he never performed a pituitary operation for the disease, Harvey Cushing was the first to describe and treat patients with Cushing disease (CD). Other surgeons at the time were reluctant to operate on the pituitary due to the normal sella on skull radiographs in CD and the unclear etiology of the disorder. To better define and understand factors influencing the history of pituitary surgery for CD, the authors analyzed historical texts related to CD biology, diagnosis, and treatment. Cushings monograph on basophilic pituitary adenomas and cortisol excess appeared in 1932. One year later in 1933, Alfred Pattison performed the first successful pituitary operation for CD by implanting radon seeds in the sella. Resection of a pituitary adenoma for CD was attempted 1 month later in 1933 by Howard Naffziger, resulting in only transient improvement that corresponded to the lack of tumor in the resected tissue. Soon thereafter, Susman in 1935 and Costello in 1936 described pituitary basophilic adenomas at autopsy in patients without premorbid endocrinopathy. They concluded that the adrenal gland was the cause of CD, which resulted in a 3-decade abandonment of pituitary surgery for CD. Jules Hardy in 1963 used the operating microscope to perform the first selective removal of an adrenocorticotropic hormone (ACTH)-secreting microadenoma, which established a pituitary cause and defined the modern treatment of CD. Subsequent reports by Hardy, Laws, and Wilson resulted in widespread acceptance of pituitary surgery for CD. Initial reluctance to operate on the pituitary for CD was multifaceted and included general uncertainty surrounding the etiology of Cushing syndrome as well as a lack of early surgical success, both due to the small size of ACTH-secreting adenomas. Selective removal of ACTH-secreting adenomas identified the source of CD and ended the delay in acceptance of pituitary surgery for CD.