John B. McIntyre

An analysis of image texture, tumor location, and MGMT promoter methylation in glioblastoma using magnetic resonance imaging

In glioblastoma (GBM), promoter methylation of the DNA repair gene O(6)-methylguanine-DNA methyltransferase (MGMT) is associated with benefit from chemotherapy. Correlations between MGMT promoter methylation and visually assessed imaging features on magnetic resonance (MR) have been reported suggesting that noninvasive detection of MGMT methylation status might be possible. Our study assessed whether MGMT methylation status in GBM could be predicted using MR imaging. We conducted a retrospective analysis of MR images in patients with newly diagnosed GBM. Tumor texture was assessed by two methods. First, we analyzed texture by expert consensus describing the tumor borders, presence or absence of cysts, pattern of enhancement, and appearance of tumor signal in T2-weighted images. Then, we applied space-frequency texture analysis based on the S-transform. Tumor location within the brain was determined using automatized image registration and segmentation techniques. Their association with MGMT methylation was analyzed. We confirmed that ring enhancement assessed visually is significantly associated with unmethylated MGMT promoter status (P=0.006). Texture features on T2-weighted images assessed by the space-frequency analysis were significantly different between methylated and unmethylated cases (P<0.05). However, blinded classification of MGMT promoter methylation status reached an accuracy of only 71%. There were no significant differences in the locations of methylated and unmethylated GBM tumors. Our results provide further evidence that individual MR features are associated with MGMT methylation but better algorithms for predicting methylation status are needed. The relevance of this study lies on the application of novel techniques for the analysis of anatomical MR images of patients with GBM allowing the evaluation of subtleties not seen by an observer and facilitating the standardization of the methods, decreasing the potential for interobserver bias.

POLE exonuclease domain mutation predicts long progression-free survival in grade 3 endometrioid carcinoma of the endometrium.

OBJECTIVE POLE exonuclease domain mutations were recently found to occur in a subset of endometrial carcinomas and result in defective proof-reading function during DNA replication. The aim of this study is to further characterize the clinical and pathologic significance of POLE exonuclease domain mutations in high-grade endometrial carcinomas. METHODS We assessed for mutations in the exonuclease domain of POLE by Sanger sequencing in 53 grade 3 endometrioid, 25 serous, 16 clear cell and 5 dedifferentiated carcinomas. We correlated POLE mutation status with clinicopathologic features and molecular parameters. Univariate and multivariate survival analyses were performed using Kaplan-Meier and cox regression analyses. RESULTS POLE exonuclease domain mutations were identified in 8 of 53 (15%) grade 3 endometrioid carcinomas and not in any other histotypes examined. Only 1 of the 8 grade 3 endometrioid carcinomas with POLE exonuclease domain mutation displayed deficient mismatch repair protein expression by immunohistochemistry (MSH6 loss), compared to 21 of 45 grade 3 endometrioid carcinomas with wild-type exonuclease domain. When analyzed together with published grade 3 endometrioid carcinomas by The Cancer Genome Atlas, the presence of POLE exonuclease domain mutation was associated with significantly better progression-free survival in univariate (p=0.025) and multivariate (p=0.010) analyses, such that none of the patients with POLE mutated tumors experienced disease progression CONCLUSIONS POLE exonuclease domain mutations occur in a subset of grade 3 endometrioid carcinomas and are associated with good clinical outcome. It can serve as an important prognostic molecular marker to guide the management of patients with grade 3 endometrioid carcinomas.

PIK3CA mutational status and overall survival in patients with cervical cancer treated with radical chemoradiotherapy

OBJECTIVE Mutational activation of PIK3CA is associated with poor prognosis in patients with solid tumors, and may predict favorable response to PI3K/AKT/mTOR pathway inhibitors. However, PIK3CA mutational status has not previously been evaluated in patients with cervical carcinoma treated with radical chemoradiotherapy (CRT). The aims of this study were (1) to evaluate the frequency of PIK3CA mutations in patients with cervical cancer treated with radical CRT and (2) to examine the effect of tumor PIK3CA mutational status in pre-treatment biopsies on overall survival (OS) and progression-free survival (PFS). METHODS Patients with cervical cancer, treated at a single institution with radical CRT, from 1999 to 2008, were eligible for this retrospective study. Pre-treatment tumor biopsies (n=157) were retrieved. Genomic DNA was extracted from tumor blocks, and exons 9 and 20 of the PIK3CA gene were sequenced for mutations. RESULTS Eighty-two tumors were sequenced for both exon 9 and exon 20. 19/82 (23%) tumors were PIK3CA mutation positive; of these 84% were squamous cell carcinomas. 79% of mutations were in exon 9. PIK3CA mutation status was strongly associated with overall survival (OS) in FIGO stage IB/II patients, unadjusted HR 6.0 (95% CI 2.1-17.5), p=0.0002, but not stage III/IVA patients, unadjusted HR 1.0 (95% CI 0.32-3.1), p=0.98. CONCLUSIONS In cervical cancer patients treated with CRT, tumor PIK3CA mutation status was associated with overall survival in FIGO stage IB/II cervix cancers. Further evaluation with a larger dataset will be required to validate these findings to inform potential clinical trials designs involving PI3K/AKT/mTOR pathway inhibitors.

Population-Based Study of Pseudoprogression after Chemoradiotherapy in GBM

INTRODUCTION Chemoradiotherapy followed by monthly temozolomide (TMZ) is the standard of care for patients with glioblastoma multiforme (GBM). Case reports have identified GBM patients who experienced transient radiological deterioration after concurrent chemoradiotherapy which stabilized or resolved after additional cycles of adjuvant TMZ, a phenomenon known as radiographic pseudoprogression. Little is known about the natural history of radiographic pseudoprogression. METHODS We retrospectively evaluated the incidence of radiographic pseudoprogression in a population-based cohort of GBM patients and determined its relationship with outcome and MGMT promoter methylation status. RESULTS Out of 43 evaluable patients, 25 (58%) exhibited radiographic progression on the first MRI after concurrent treatment. Twenty of these went on to receive adjuvant TMZ, and subsequent investigation demonstrated radiographic pseudoprogression in 10 cases (50%). Median survival (MS) was better in patients with pseudoprogression (MS 14.5 months) compared to those with true radiologic progression (MS 9.1 months, p=0.025). The MS of patients with pseudoprogression was similar to those who stabilized/responded during concurrent treatment (p=0.31). Neither the extent of the initial resection nor dexamethasone dosing was associated with pseudoprogression. CONCLUSIONS These data suggest that physicians should continue adjuvant TMZ in GBM patients when early MRI scans show evidence of progression following concurrent chemoradiotherapy, as up to 50% of these patients will experience radiologic stability or improvement in subsequent treatment cycles.

Targeted mutation analysis of endometrial clear cell carcinoma

Endometrial clear cell carcinomas (CCC) constitute fewer than 5% of all carcinomas of the endometrium. Currently, little is known regarding the genetic basis of endometrial CCC.

The diagnostic utility of TP53 and CDKN2A to distinguish ovarian high-grade serous carcinoma from low-grade serous ovarian tumors

Low-grade serous carcinomas and serous borderline tumors, combined herein and referred to as low-grade serous tumors, show distinct molecular alterations and clinical behaviors compared with high-grade serous carcinomas. The discrimination between low-grade serous tumors and high-grade serous carcinomas can be challenging on small tissue samples, such as cell blocks of paracentesis fluid or biopsies from omental disease. The purpose of this study was to test the ability of TP53 and CDKN2A immunohistochemistry to distinguish between high-grade serous carcinomas and low-grade serous tumors on small tissue samples. Tissue microarrays containing 582 high-grade serous carcinomas, 45 low-grade serous carcinomas, and 49 serous borderline tumors, confirmed by contemporary histopathological review, were stained for TP53 and CDKN2A (DO7 and E6H4 antibody clones, respectively). TP53 was scored as completely absent, wild-type pattern or overexpressed (>60%), and CDKN2A was scored as either negative/patchy (<90%) or block expression (>90%). The combination of the two markers, ie, the TP53 wild-type pattern and CDKN2A patchy expression, had sensitivity for low-grade serous tumors of 89%, a specificity of 93%, a positive predictive value of 68%, and a negative predictive value of 98%. These markers can, therefore, be used on small biopsies/cell blocks to refute a diagnosis of low-grade serous tumors. These findings may inform emerging neoadjuvant therapeutic strategies in advanced ovarian cancers and may be crucial for future clinical trials on molecular-based therapies.

Methylation status of MGMT gene promoter in meningiomas

Meningiomas are usually cured by surgical resection. However, approximately 10% are characterized by more aggressive clinical behavior and higher risk of recurrence. Typically, recurrent meningiomas require further surgical resection followed, in some cases, by radiotherapy. To date, no chemotherapeutic agent has proven to be effective in either preventing or treating recurrence. The alkylating chemotherapeutic agent, Temozolomide (TMZ) has shown to increase overall survival in patients with glioblastoma (GBM) but its effectiveness for other types of brain tumor is less known. The clinical benefit of TMZ seems to be limited to those GBM tumors with promoter methylation of the MGMT gene. In this study, we assessed if a biologic rationale exists to support the use of TMZ as a treatment for meningiomas by assessing the MGMT promoter methylation status in these tumors using methylation specific PCR. We investigated the MGMT promoter methylation status in 36 tumors (32 newly diagnosed; 4 recurrent). Histologically, the majority were grade I. Patients were primarily female (64%) with a mean age of 52. None of the meningiomas in our series showed MGMT gene promoter methylation. Based on these data, we conclude that there is no biological rational to suggest that TMZ might have significant anti-meningioma activity.

PIK3CA missense mutation is associated with unfavorable outcome in grade 3 endometrioid carcinoma but not in serous endometrial carcinoma.

OBJECTIVE To evaluate the outcome association of PIK3CA mutational status within histological types of rigorously classified high-grade endometrial carcinomas. METHODS We assessed PIK3CA mutational status in exon 9 and exon 20 hot spots by Sanger sequencing of DNA derived from formalin fixed paraffin embedded tissue of 57 grade 3 endometrioid, 26 serous, 11 clear cell and 5 dedifferentiated carcinomas. We correlated PIK3CA mutation status with clinicopathological and other molecular parameters. Univariate and multivariate disease specific survival analysis was performed using Kaplan-Meier and Cox regression analyses. RESULTS PIK3CA exon 9 or exon 20 missense mutations were identified in 20 of 99 (20%) high-grade endometrial carcinomas without significant difference across histological types (p=0.22). Presence of PIK3CA exon 9 or exon 20 missense mutations was associated with shorter disease specific survival within grade 3 endometrioid (p=0.0029) but not endometrial serous (p=0.57) carcinoma based on univariate analysis. Within grade 3 endometrioid carcinoma, PIK3CA exon 9 or exon 20 missense mutations were more commonly observed in cases that were deficient for mismatch repair protein expression (p=0.0058) and showed loss of ARID1A expression (p=0.037). CONCLUSIONS PIK3CA exon 9 or exon 20 missense mutations are present across all histological types of high-grade endometrial carcinomas but a significant outcome association is only seen in grade 3 endometrioid carcinoma, suggesting a greater biological importance in this tumor type.

Specific and Sensitive Hydrolysis Probe-Based Real-Time PCR Detection of Epidermal Growth Factor Receptor Variant III in Oral Squamous Cell Carcinoma

Background The tumor-specific EGFR deletion mutant, EGFRvIII, is characterised by ligand-independent constitutive signalling. Tumors expressing EGFRvIII are resistant to current EGFR-targeted therapy. The frequency of EGFRvIII in head and neck squamous cell carcinoma (HNSCC) is disputed and may vary by specific sub-site. The purpose of this study was to measure the occurrence of EGFRvIII mutations in a specific HNSCC subsite, oral squamous cell carcinoma (OSCC), using a novel real-time PCR assay. Methodology Pre-treatment Formalin Fixed Paraffin Embedded (FFPE) cancer specimens from 50 OSCC patients were evaluated for the presence of EGFRvIII using a novel hydrolysis probe-based real-time PCR assay. EGFR protein expression in tumor samples was quantified using fluorescent immunohistochemistry (IHC) and AQUA® technology. Principal findings We detected EGFRvIII in a single OSCC patient in our cohort (2%). We confirmed the validity of our detection technique in an independent cohort of glioblastoma patients. We also compared the sensitivity and specificity of our novel real-time EGFRvIII detection assay to conventional RT-PCR and direct sequencing. Our assay can specifically detect EGFRvIII and can discriminate against wild-type EGFR in FFPE tumor samples. AQUAnalysis® revealed that the presence of EGFRvIII transcript is associated with very high EGFR protein expression (98th percentile). Contrary to previous reports, only 44% of OSCC over-expressed EGFR in our study. Conclusion and Significance Our results suggest that the EGFRvIII mutation is rare in OSCC and corroborate previous reports of EGFRvIII expression only in tumors with extreme over-expression of EGFR. We conclude that EGFRvIII-specific therapies may not be ideally suited as first-line treatment in OSCC. Furthermore, highly specific and sensitive methods, such as the real-time RT-PCR assay and AQUAnalysis® described here, will provide accurate assessment of EGFR mutation frequency and EGFR expression, and will facilitate the selection of optimal tailored therapies for OSCC patients.

Molecular alterations in indolent, aggressive and recurrent ovarian low-grade serous carcinoma.

The clinical courses of patients with low‐grade serous carcinoma (LGSC) can be substantially different. The purpose of this study was to explore whether molecular or pathological features could identify patients who follow a more aggressive course.