Annette M. Molinaro

Glioma Groups Based on 1p/19q, IDH, and TERT Promoter Mutations in Tumors

BACKGROUND The prediction of clinical behavior, response to therapy, and outcome of infiltrative glioma is challenging. On the basis of previous studies of tumor biology, we defined five glioma molecular groups with the use of three alterations: mutations in the TERT promoter, mutations in IDH, and codeletion of chromosome arms 1p and 19q (1p/19q codeletion). We tested the hypothesis that within groups based on these features, tumors would have similar clinical variables, acquired somatic alterations, and germline variants. METHODS We scored tumors as negative or positive for each of these markers in 1087 gliomas and compared acquired alterations and patient characteristics among the five primary molecular groups. Using 11,590 controls, we assessed associations between these groups and known glioma germline variants. RESULTS Among 615 grade II or III gliomas, 29% had all three alterations (i.e., were triple-positive), 5% had TERT and IDH mutations, 45% had only IDH mutations, 7% were triple-negative, and 10% had only TERT mutations; 5% had other combinations. Among 472 grade IV gliomas, less than 1% were triple-positive, 2% had TERT and IDH mutations, 7% had only IDH mutations, 17% were triple-negative, and 74% had only TERT mutations. The mean age at diagnosis was lowest (37 years) among patients who had gliomas with only IDH mutations and was highest (59 years) among patients who had gliomas with only TERT mutations. The molecular groups were independently associated with overall survival among patients with grade II or III gliomas but not among patients with grade IV gliomas. The molecular groups were associated with specific germline variants. CONCLUSIONS Gliomas were classified into five principal groups on the basis of three tumor markers. The groups had different ages at onset, overall survival, and associations with germline variants, which implies that they are characterized by distinct mechanisms of pathogenesis. (Funded by the National Institutes of Health and others.).

Biomarker Expression and Risk of Subsequent Tumors After Initial Ductal Carcinoma In Situ Diagnosis

BACKGROUND Studies have failed to identify characteristics of women who have been diagnosed with ductal carcinoma in situ (DCIS) and have a high or low risk of subsequent invasive cancer. METHODS We conducted a nested case-control study in a population-based cohort of 1162 women who were diagnosed with DCIS and treated by lumpectomy alone from 1983 to 1994. We collected clinical characteristics and information on subsequent tumors, defined as invasive breast cancer or DCIS diagnosed in the ipsilateral breast containing the initial DCIS lesion or at a regional or distant site greater than 6 months after initial treatment of DCIS (N = 324). We also conducted standardized pathology reviews and immunohistochemical staining for the estrogen receptor (ER), progesterone receptor, Ki67 antigen, p53, p16, epidermal growth factor receptor-2 (ERBB2, HER2/neu oncoprotein), and cyclooxygenase-2 (COX-2) on the initial paraffin-embedded DCIS tissue. Competing risk models were used to determine factors associated with risk of subsequent invasive cancer vs DCIS, and cumulative incidence survival functions were used to estimate 8-year risk. RESULTS Factors associated with subsequent invasive cancer differed from those associated with subsequent DCIS. Eight-year risk of subsequent invasive cancer was statistically significantly (P = .018) higher for women with initial DCIS lesions that were detected by palpation or that were p16, COX-2, and Ki67 triple positive (p16(+)COX-2(+)Ki67(+)) (19.6%, 95% confidence interval [CI] = 18.0% to 21.3%) than for women with initial lesions that were detected by mammography and were p16, COX-2, and Ki67 triple negative (p16(-)COX-2(-)Ki67(-)) (4.1%, 95% CI = 3.4% to 5.0%). In a multivariable model, DCIS lesions that were p16(+)COX-2(+)Ki67(+) or those detected by palpation were statistically significantly associated with subsequent invasive cancer, but nuclear grade was not. Eight-year risk of subsequent DCIS was highest for women with DCIS lesions that had disease-free margins of 1 mm or greater combined with either ER(-)ERBB2(+)Ki67(+) or p16(+)COX-2(-)Ki67(+) status (23.6%, 95% CI = 18.1% to 34.0%). CONCLUSION Biomarkers can identify which women who were initially diagnosed with DCIS are at high or low risk of subsequent invasive cancer, whereas histopathology information cannot.

Hypoxia-Induced Autophagy Promotes Tumor Cell Survival and Adaptation to Antiangiogenic Treatment in Glioblastoma

Antiangiogenic therapy leads to devascularization that limits tumor growth. However, the benefits of angiogenesis inhibitors are typically transient and resistance often develops. In this study, we explored the hypothesis that hypoxia caused by antiangiogenic therapy induces tumor cell autophagy as a cytoprotective adaptive response, thereby promoting treatment resistance. Hypoxia-induced autophagy was dependent on signaling through the hypoxia-inducible factor-1α (HIF-1α)/AMPK pathway, and treatment of hypoxic cells with autophagy inhibitors caused a shift from autophagic to apoptotic cell death in vitro. In glioblastomas, clinically resistant to the VEGF-neutralizing antibody bevacizumab, increased regions of hypoxia and higher levels of autophagy-mediating BNIP3 were found when compared with pretreatment specimens from the same patients. When treated with bevacizumab alone, human glioblastoma xenografts showed increased BNIP3 expression and hypoxia-associated growth, which could be prevented by addition of the autophagy inhibitor chloroquine. In vivo targeting of the essential autophagy gene ATG7 also disrupted tumor growth when combined with bevacizumab treatment. Together, our findings elucidate a novel mechanism of resistance to antiangiogenic therapy in which hypoxia-mediated autophagy promotes tumor cell survival. One strong implication of our findings is that autophagy inhibitors may help prevent resistance to antiangiogenic therapy used in the clinic.

Genome-wide screen of promoter methylation identifies novel markers in melanoma

DNA methylation is an important component of epigenetic modifications, which influences the transcriptional machinery aberrant in many human diseases. In this study we present the first genome-wide integrative analysis of promoter methylation and gene expression for the identification of methylation markers in melanoma. Genome-wide promoter methylation and gene expression of eight early-passage human melanoma cell strains were compared with newborn and adult melanocytes. We used linear mixed effect models (LME) in combination with a series of filters based on the localization of promoter methylation relative to the transcription start site, overall promoter CpG content, and differential gene expression to discover DNA methylation markers. This approach identified 76 markers, of which 68 were hyper- and eight hypomethylated (LME, P < 0.05). Promoter methylation and differential gene expression of five markers (COL1A2, NPM2, HSPB6, DDIT4L, MT1G) were validated by sequencing of bisulfite-modified DNA and real-time reverse transcriptase PCR, respectively. Importantly, the incidence of promoter methylation of the validated markers increased moderately in early and significantly in advanced-stage melanomas, using early-passage cell strains and snap-frozen tissues (n = 18 and n = 24, respectively) compared with normal melanocytes and nevi (n = 11 and n = 9, respectively). Our approach allows robust identification of methylation markers that can be applied to other studies involving genome-wide promoter methylation. In conclusion, this study represents the first unbiased systematic effort to determine methylation markers in melanoma and revealed several novel genes regulated by promoter methylation that were not described in cancer cells before.

High levels of vascular endothelial growth factor and its receptors (VEGFR-1, VEGFR-2, neuropilin-1) are associated with worse outcome in breast cancer

Vascular endothelial growth factor has been shown to be up-regulated in breast cancers. Vascular endothelial growth factor receptors, VEGFR-1 and VEGFR-2, are the principal mediators of its effects. Together with VEGFR-1 and VEGFR-2, neuropilin-1 may act as a coreceptor for vascular endothelial growth factor. Although vascular endothelial growth factor exerts important effects on endothelial cells, VEGFRs are likely present on tumor cells as well. We used AQUA to analyze tumor-specific expression of vascular endothelial growth factor, VEGFR-1, VEGFR-2, and neuropilin-1 on a large cohort of breast cancer tissue microarray. Two-fold redundant arrays were constructed from 642 cases of primary breast adenocarcinomas. Automated image analysis with AQUA (Automated Quantitative Analysis) was then performed to determine a quantitative expression score. Scores from redundant arrays were normalized and averaged. Kaplan-Meier survival analysis showed that high levels of vascular endothelial growth factor, VEGFR-1, VEGFR-2, and neuropilin-1 were all significantly associated with survival (Miller Siegmeund corrected P = .0020, .0160, and .0320, respectively). In addition, vascular endothelial growth factor and neuropilin-1 retained a significant association with survival independent of other standard prognostic factors. Vascular endothelial growth factor, VEGFR-1 and -2, and neuropilin-1 are expressed to varying degrees in primary breast cancers and have prognostic significance. Further study of the functional significance of this finding is warranted as well as the prognostic value of these biomarkers in other tumor microenvironment-specific compartments (eg, vessels).

Awake craniotomy to maximize glioma resection: methods and technical nuances over a 27-year period

OBJECT Awake craniotomy is currently a useful surgical approach to help identify and preserve functional areas during cortical and subcortical tumor resections. Methodologies have evolved over time to maximize patient safety and minimize morbidity using this technique. The goal of this study is to analyze a single surgeons experience and the evolving methodology of awake language and sensorimotor mapping for glioma surgery. METHODS The authors retrospectively studied patients undergoing awake brain tumor surgery between 1986 and 2014. Operations for the initial 248 patients (1986-1997) were completed at the University of Washington, and the subsequent surgeries in 611 patients (1997-2014) were completed at the University of California, San Francisco. Perioperative risk factors and complications were assessed using the latter 611 cases. RESULTS The median patient age was 42 years (range 13-84 years). Sixty percent of patients had Karnofsky Performance Status (KPS) scores of 90-100, and 40% had KPS scores less than 80. Fifty-five percent of patients underwent surgery for high-grade gliomas, 42% for low-grade gliomas, 1% for metastatic lesions, and 2% for other lesions (cortical dysplasia, encephalitis, necrosis, abscess, and hemangioma). The majority of patients were in American Society of Anesthesiologists (ASA) Class 1 or 2 (mild systemic disease); however, patients with severe systemic disease were not excluded from awake brain tumor surgery and represented 15% of study participants. Laryngeal mask airway was used in 8 patients (1%) and was most commonly used for large vascular tumors with more than 2 cm of mass effect. The most common sedation regimen was propofol plus remifentanil (54%); however, 42% of patients required an adjustment to the initial sedation regimen before skin incision due to patient intolerance. Mannitol was used in 54% of cases. Twelve percent of patients were active smokers at the time of surgery, which did not impact completion of the intraoperative mapping procedure. Stimulation-induced seizures occurred in 3% of patients and were rapidly terminated with ice-cold Ringers solution. Preoperative seizure history and tumor location were associated with an increased incidence of stimulation-induced seizures. Mapping was aborted in 3 cases (0.5%) due to intraoperative seizures (2 cases) and patient emotional intolerance (1 case). The overall perioperative complication rate was 10%. CONCLUSIONS Based on the current best practice described here and developed from multiple regimens used over a 27-year period, it is concluded that awake brain tumor surgery can be safely performed with extremely low complication and failure rates regardless of ASA classification; body mass index; smoking status; psychiatric or emotional history; seizure frequency and duration; and tumor site, size, and pathology.

Regional variation in histopathologic features of tumor specimens from treatment-naive glioblastoma correlates with anatomic and physiologic MR Imaging

Histopathologic evaluation of glioblastoma multiforme (GBM) at initial diagnosis is typically performed on tissue obtained from regions of contrast enhancement (CE) as depicted on gadolinium-enhanced, T1-weighted images. The non-enhancing (NE) portion of the lesion, which contains both reactive edema and infiltrative tumor, is only partially removed due to concerns about damaging functioning brain. The purpose of this study was to evaluate histopathologic and physiologic MRI features of image-guided tissue specimens from CE and NE regions to investigate correlations between imaging and histopathologic parameters. One hundred nineteen tissue specimens (93 CE and 26 NE regions) were acquired from 51 patients with newly diagnosed GBM by utilizing stereotactic image-guided sampling. Variables of anatomic, diffusion-weighted imaging (DWI), and dynamic susceptibility-weighted, contrast-enhanced perfusion imaging (DSC) from each tissue sample location were obtained and compared with histopathologic features such as tumor score, cell density, proliferation, architectural disruption, hypoxia, and microvascular hyperplasia. Tissue samples from CE regions had increased tumor score, cellular density, proliferation, and architectural disruption compared with NE regions. DSC variables such as relative cerebral blood volume, peak height, and recovery factor were significantly higher, and the percentage of signal intensity recovery was significantly lower in the CE compared with the NE regions. DWI variables were correlated with histopathologic features of GBM within NE regions. Image-guided tissue acquisition and assessment of residual tumor from treatment-naive GBM should be guided by DSC in CE regions and by DWI in NE regions.

A phase 2 trial of dasatinib in advanced melanoma

Inhibiting src kinases (non‐receptor tyrosine kinase signaling intermediates) reduces melanoma cell proliferation and invasion. Dasatinib inhibits c‐kit, PDGFβR, and EPHA2 and src kinases c‐src, c‐Yes, Lck, and Fyn. A phase 2 trial of dasatinib in melanoma was conducted to assess response rate (RR), progression‐free survival (PFS), and toxicity.

DNA Methylation and Somatic Mutations Converge on the Cell Cycle and Define Similar Evolutionary Histories in Brain Tumors

The evolutionary history of tumor cell populations can be reconstructed from patterns of genetic alterations. In contrast to stable genetic events, epigenetic states are reversible and sensitive to the microenvironment, prompting the question whether epigenetic information can similarly be used to discover tumor phylogeny. We examined the spatial and temporal dynamics of DNA methylation in a cohort of low-grade gliomas and their patient-matched recurrences. Genes transcriptionally upregulated through promoter hypomethylation during malignant progression to high-grade glioblastoma were enriched in cell cycle function, evolving in parallel with genetic alterations that deregulate the G1/S cell cycle checkpoint. Moreover, phyloepigenetic relationships robustly recapitulated phylogenetic patterns inferred from somatic mutations. These findings highlight widespread co-dependency of genetic and epigenetic events throughout brain tumor evolution.

Melanoma Prognostic Model Using Tissue Microarrays and Genetic Algorithms

PURPOSE As a result of the questionable risk-to-benefit ratio of adjuvant therapies, stage II melanoma is currently managed by observation because available clinicopathologic parameters cannot identify the 20% to 60% of such patients likely to develop metastatic disease. Here, we propose a multimarker molecular prognostic assay that can help triage patients at increased risk of recurrence. METHODS Protein expression for 38 candidates relevant to melanoma oncogenesis was evaluated using the automated quantitative analysis (AQUA) method for immunofluorescence-based immunohistochemistry in formalin-fixed, paraffin-embedded specimens from a cohort of 192 primary melanomas collected during 1959 to 1994. The prognostic assay was built using a genetic algorithm and validated on an independent cohort of 246 serial primary melanomas collected from 1997 to 2004. RESULTS Multiple iterations of the genetic algorithm yielded a consistent five-marker solution. A favorable prognosis was predicted by ATF2 ln(non-nuclear/nuclear AQUA score ratio) of more than -0.052, p21(WAF1) nuclear compartment AQUA score of more than 12.98, p16(INK4A) ln(non-nuclear/nuclear AQUA score ratio) of < or = -0.083, beta-catenin total AQUA score of more than 38.68, and fibronectin total AQUA score of < or = 57.93. Primary tumors that met at least four of these five conditions were considered a low-risk group, and those that met three or fewer conditions formed a high-risk group (log-rank P < .0001). Multivariable proportional hazards analysis adjusting for clinicopathologic parameters shows that the high-risk group has significantly reduced survival on both the discovery (hazard ratio = 2.84; 95% CI, 1.46 to 5.49; P = .002) and validation (hazard ratio = 2.72; 95% CI, 1.12 to 6.58; P = .027) cohorts. CONCLUSION This multimarker prognostic assay, an independent determinant of melanoma survival, might be beneficial in improving the selection of stage II patients for adjuvant therapy.