Validation of Next-Generation Sequencing of Entire Mitochondrial Genomes and the Diversity of Mitochondrial DNA Mutations in Oral Squamous Cell Carcinoma

Background

Oral squamous cell carcinoma (OSCC) is mainly caused by smoking and alcohol abuse and shows a five-year survival rate of ~50%. We aimed to explore the variation of somatic mitochondrial DNA (mtDNA) mutations in primary oral tumors, recurrences and metastases.

Methods

We performed an in-depth validation of mtDNA next-generation sequencing (NGS) on an Illumina HiSeq 2500 platform for its application to cancer tissues, with the goal to detect low-level heteroplasmies and to avoid artifacts. Therefore we genotyped the mitochondrial genome (16.6 kb) from 85 tissue samples (tumors, recurrences, resection edges, metastases and blood) collected from 28 prospectively recruited OSCC patients applying both Sanger sequencing and high-coverage NGS (~35,000 reads per base).

Results

We observed a strong correlation between Sanger sequencing and NGS in estimating the mixture ratio of heteroplasmies (r = 0.99; p<0.001). Non-synonymous heteroplasmic variants were enriched among cancerous tissues. The proportions of somatic and inherited variants in a given gene region were strongly correlated (r = 0.85; p<0.001). Half of the patients shared mutations between benign and cancerous tissue samples. Low level heteroplasmies (<10%) were more frequent in benign samples compared to tumor samples, where heteroplasmies >10% were predominant. Four out of six patients who developed a local tumor recurrence showed mutations in the recurrence that had also been observed in the primary tumor. Three out of five patients, who had tumor metastases in the lymph nodes of their necks, shared mtDNA mutations between primary tumors and lymph node metastases. The percentage of mutation heteroplasmy increased from the primary tumor to lymph node metastases.

Conclusions

We conclude that Sanger sequencing is valid for heteroplasmy quantification for heteroplasmies ≥10% and that NGS is capable of reliably detecting and quantifying heteroplasmies down to the 1%-level. The finding of shared mutations between primary tumors, recurrences and metastasis indicates a clonal origin of malignant cells in oral cancer.     

Clonal Analysis in Recurrent Astrocytic, Oligoastrocytic and Oligodendroglial Tumors Implicates IDH1- Mutation as Common Tumor Initiating Event

Background

To investigate the dynamics of inter- and intratumoral molecular alterations during tumor progression in recurrent gliomas.

Methodology/Principal Findings

To address intertumoral heterogeneity we investigated non- microdissected tumor tissue of 106 gliomas representing 51 recurrent tumors. To address intratumoral heterogeneity a set of 16 gliomas representing 7 tumor pairs with at least one recurrence, and 4 single mixed gliomas were investigated by microdissection of distinct oligodendroglial and astrocytic tumor components. All tumors and tumor components were analyzed for allelic loss of 1p/19q (LOH 1p/19q), for TP53- mutations and for R132 mutations in the IDH1 gene. The investigation of non- microdissected tumor tissue revealed clonality in 75% (38/51). Aberrant molecular alterations upon recurrence were detected in 25% (13/51). 64% (9/14) of these were novel and associated with tumor progression. Loss of previously detected alterations was observed in 36% (5/14). One tumor pair (1/14; 7%) was significant for both. Intratumoral clonality was detected in 57% (4/7) of the microdissected tumor pairs and in 75% (3/4) of single microdissected tumors. 43% (3/7) of tumor pairs and one single tumor (25%) revealed intratumoral heterogeneity. While intratumoral heterogeneity affected both the TP53- mutational status and the LOH1p/19q status, all tumors with intratumoral heterogeneity shared the R132 IDH1- mutation as a common feature in both their microdissected components.

Conclusions/Significance

The majority of recurrent gliomas are of monoclonal origin. However, the detection of divertive tumor cell clones in morphological distinct tumor components sharing IDH1- mutations as early event may provide insight into the tumorigenesis of true mixed gliomas.     

Microenvironmental Heterogeneity Parallels Breast Cancer Progression: A Histology–Genomic Integration Analysis

Background

The intra-tumor diversity of cancer cells is under intense investigation; however, little is known about the heterogeneity of the tumor microenvironment that is key to cancer progression and evolution. We aimed to assess the degree of microenvironmental heterogeneity in breast cancer and correlate this with genomic and clinical parameters.

Methods and Findings

We developed a quantitative measure of microenvironmental heterogeneity along three spatial dimensions (3-D) in solid tumors, termed the tumor ecosystem diversity index (EDI), using fully automated histology image analysis coupled with statistical measures commonly used in ecology. This measure was compared with disease-specific survival, key mutations, genome-wide copy number, and expression profiling data in a retrospective study of 510 breast cancer patients as a test set and 516 breast cancer patients as an independent validation set. In high-grade (grade 3) breast cancers, we uncovered a striking link between high microenvironmental heterogeneity measured by EDI and a poor prognosis that cannot be explained by tumor size, genomics, or any other data types. However, this association was not observed in low-grade (grade 1 and 2) breast cancers. The prognostic value of EDI was superior to known prognostic factors and was enhanced with the addition of TP53 mutation status (multivariate analysis test set, p = 9 × 10⁻⁴, hazard ratio = 1.47, 95% CI 1.17–1.84; validation set, p = 0.0011, hazard ratio = 1.78, 95% CI 1.26–2.52). Integration with genome-wide profiling data identified losses of specific genes on 4p14 and 5q13 that were enriched in grade 3 tumors with high microenvironmental diversity that also substratified patients into poor prognostic groups. Limitations of this study include the number of cell types included in the model, that EDI has prognostic value only in grade 3 tumors, and that our spatial heterogeneity measure was dependent on spatial scale and tumor size.

Conclusions

To our knowledge, this is the first study to couple unbiased measures of microenvironmental heterogeneity with genomic alterations to predict breast cancer clinical outcome. We propose a clinically relevant role of microenvironmental heterogeneity for advanced breast tumors, and highlight that ecological statistics can be translated into medical advances for identifying a new type of biomarker and, furthermore, for understanding the synergistic interplay of microenvironmental heterogeneity with genomic alterations in cancer cells.     

Use of Ethanol in the Trans-Arterial Lipiodol Embolization (TAELE) of Intermediated-Stage HCC: Is This Safer than Conventional Trans-Arterial Chemo-Embolization (c-TACE)?

Purpose

To evaluate safety and efficacy of Trans-Arterial Ethanol-Lipiodol Embolization (TAELE) compared with conventional Trans-Arterial Chemo-Embolization (cTACE) in the treatment of small intermediate-HCC (BCLC-Stage B).

Materials and Methods

A random sample of 87 patients (37.93% male; 62.07% female; age range, 36–86 years) with documented small intermediate-HCC and treated with TAELE (mixture 1:1 of Ethanol and Lipiodol) or cTACE (mixture of 50mg-Epirubicin and 5cc-Lipiodol) were retrospectively studied in an institutional review board approved protocol. The two procedures were compared with χ²-test, χ²-test with Yates correction, McNemar’s exact test, ANOVA test and log-rank test.

Results

TAELE and cTACE therapies were performed in 45 and 42 patients, respectively. Thirty days after the procedure, a Multi-Detector Computed Tomography (MDCT) showed no significant difference in the number of patients with partial and complete response between the two groups (p-value = 0.958), according to mRECIST. Contrary, significant differences were found in tumor-devascularization, lesion-reduction and post-embolization syndrome occurrence (p-value = 0.0004, p-value = 0.0003 and p-value = 0.009, respectively). Similar survival was observed during 36-month follow-up (p-value = 0.884).

Conclusion

Compared to cTACE, TAELE showed a better toxicity profile with similar 36-month survival and similar one-month anti-tumor effects, which makes it better tolerated by patients, especially in case of more than one treatment.     

Individualized Mutation Detection in Circulating Tumor DNA for Monitoring Colorectal Tumor Burden Using a Cancer-Associated Gene Sequencing Panel

Background

Circulating tumor DNA (ctDNA) carries information on tumor burden. However, the mutation spectrum is different among tumors. This study was designed to examine the utility of ctDNA for monitoring tumor burden based on an individual mutation profile.

Methodology

DNA was extracted from a total of 176 samples, including pre- and post-operational plasma, primary tumors, and peripheral blood mononuclear cells (PBMC), from 44 individuals with colorectal tumor who underwent curative resection of colorectal tumors, as well as nine healthy individuals. Using a panel of 50 cancer-associated genes, tumor-unique mutations were identified by comparing the single nucleotide variants (SNVs) from tumors and PBMCs with an Ion PGM sequencer. A group of the tumor-unique mutations from individual tumors were designated as individual marker mutations (MMs) to trace tumor burden by ctDNA using droplet digital PCR (ddPCR). From these experiments, three major objectives were assessed: (a) Tumor-unique mutations; (b) mutation spectrum of a tumor; and (c) changes in allele frequency of the MMs in ctDNA after curative resection of the tumor.

Results

A total of 128 gene point mutations were identified in 27 colorectal tumors. Twenty-six genes were mutated in at least 1 sample, while 14 genes were found to be mutated in only 1 sample, respectively. An average of 2.7 genes were mutated per tumor. Subsequently, 24 MMs were selected from SNVs for tumor burden monitoring. Among the MMs found by ddPCR with > 0.1% variant allele frequency in plasma DNA, 100% (8 out of 8) exhibited a decrease in post-operation ctDNA, whereas none of the 16 MMs found by ddPCR with < 0.1% variant allele frequency in plasma DNA showed a decrease.

Conclusions

This panel of 50 cancer-associated genes appeared to be sufficient to identify individual, tumor-unique, mutated ctDNA markers in cancer patients. The MMs showed the clinical utility in monitoring curatively-treated colorectal tumor burden if the allele frequency of MMs in plasma DNA is above 0.1%.     

Estimation of 24-Hour Intraocular Pressure Peak Timing and Variation Using a Contact Lens Sensor

Purpose

To compare estimates of 24-hour intraocular pressure (IOP) peak timing and variation obtained using a contact lens sensor (CLS) and using a pneumatonometer.

Methods

Laboratory data collected from 30 healthy volunteers (ages, 20-66 years) in a randomized, controlled clinical trial were analyzed. Participants were housed for 24 hours in a sleep laboratory. One randomly selected right or left eye was fitted with a CLS that monitored circumferential curvature in the corneoscleral region related to the change of IOP. Electronic output signals of 30 seconds were averaged and recorded every 5 minutes. In the contralateral eye, habitual IOP measurements were taken using a pneumatonometer once every two hours. Simulated 24-hour rhythms in both eyes were determined by cosinor fitting. Simulated peak timings (acrophases) and simulated data variations (amplitudes) were compared between the paired eyes.

Results

Bilateral change patterns of average 24-hour data for the group were in parallel. The simulated peak timing in the CLS fitted eye occurred at 4:44 AM ± 210 min (mean ± SD) and the IOP peak timing in the contralateral eye at 4:11 AM ± 120 min (P=0.256, Wilcoxon signed-rank test). There was no significant correlation between the simulated data variations in the paired eyes (P=0.820, linear regression).

Conclusions

The 24-hour CLS data showed a simulated peak timing close to the 24-hour IOP peak timing obtained using the pneumatonometer. However, the simulated variations of 24-hour data in the paired eyes were not correlated. Estimated 24-hour IOP rhythms using the two devices should not be considered interchangeable.     

Classification of Epidermal Growth Factor Receptor Gene Mutation Status Using Serum Proteomic Profiling Predicts Tumor Response in Patients with Stage IIIB or IV Non-Small-Cell Lung Cancer

Objectives

Epidermal growth factor receptor (EGFR) gene mutations in tumors predict tumor response to EGFR tyrosine kinase inhibitors (EGFR-TKIs) in non-small-cell lung cancer (NSCLC). However, obtaining tumor tissue for mutation analysis is challenging. Here, we aimed to detect serum peptides/proteins associated with EGFR gene mutation status, and test whether a classification algorithm based on serum proteomic profiling could be developed to analyze EGFR gene mutation status to aid therapeutic decision-making.

Patients and Methods

Serum collected from 223 stage IIIB or IV NSCLC patients with known EGFR gene mutation status in their tumors prior to therapy was analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and ClinProTools software. Differences in serum peptides/proteins between patients with EGFR gene TKI-sensitive mutations and wild-type EGFR genes were detected in a training group of 100 patients; based on this analysis, a serum proteomic classification algorithm was developed to classify EGFR gene mutation status and tested in an independent validation group of 123 patients. The correlation between EGFR gene mutation status, as identified with the serum proteomic classifier and response to EGFR-TKIs was analyzed.

Results

Nine peptide/protein peaks were significantly different between NSCLC patients with EGFR gene TKI-sensitive mutations and wild-type EGFR genes in the training group. A genetic algorithm model consisting of five peptides/proteins (m/z 4092.4, 4585.05, 1365.1, 4643.49 and 4438.43) was developed from the training group to separate patients with EGFR gene TKI-sensitive mutations and wild-type EGFR genes. The classifier exhibited a sensitivity of 84.6% and a specificity of 77.5% in the validation group. In the 81 patients from the validation group treated with EGFR-TKIs, 28 (59.6%) of 47 patients whose matched samples were labeled as “mutant” by the classifier and 3 (8.8%) of 34 patients whose matched samples were labeled as “wild” achieved an objective response (p<0.0001). Patients whose matched samples were labeled as “mutant” by the classifier had a significantly longer progression-free survival (PFS) than patients whose matched samples were labeled as “wild” (p=0.001).

Conclusion

Peptides/proteins related to EGFR gene mutation status were found in the serum. Classification of EGFR gene mutation status using the serum proteomic classifier established in the present study in patients with stage IIIB or IV NSCLC is feasible and may predict tumor response to EGFR-TKIs.     

Efficacy of a Contact Lens Sensor for Monitoring 24-H Intraocular Pressure Related Patterns

Purpose

To study performance of a contact lens sensor (CLS) for 24-hour monitoring of IOP-related short-term patterns and compare with IOP obtained by pneumatonometry.

Methods

Prospective clinical trial. Thirty-one healthy volunteers and 2 glaucoma patients were housed for 24 hours in a sleep laboratory. One randomly selected eye was fitted with a CLS (Triggerfish, Sensimed, Switzerland), which measures changes in ocular circumference. In the contralateral eye, IOP measurements were taken using a pneumatonometer every two hours with subjects in the habitual body positions. Heart rate (HR) was measured 3 times during the night for periods of 6 minutes separated by 2 hours. Performance of CLS was defined in two ways: 1) recording the known pattern of IOP increase going from awake (sitting position) to sleep (recumbent), defined as the wake/sleep (W/S) slope and 2) accuracy of the ocular pulse frequency (OPF) concurrent to that of the HR interval. Strength of association between overall CLS and pneumatonometer curves was assessed using coefficients of determination (R²).

Results

The W/S slope was statistically significantly positive in both eyes of each subject (CLS, 57.0 ± 40.5 mVeq/h, p<0.001 and 1.6 ± 0.9 mmHg/h, p<0.05 in the contralateral eye). In all, 87 CLS plots concurrent to the HR interval were evaluated. Graders agreed on evaluability for OPF in 83.9% of CLS plots. Accuracy of the CLS to detect the OPF was 86.5%. Coefficient of correlation between CLS and pneumatonometer for the mean 24-h curve was R² = 0.914.

Conclusions

CLS measurements compare well to the pneumatonometer and may be of practical use for detection of sleep-induced IOP changes. The CLS also is able to detect ocular pulsations with good accuracy in a majority of eyes.

Trial Registration

ClinicalTrials.gov NCT01390779     

Deep sequencing reveals clonal evolution patterns and mutation events associated with relapse in B-cell lymphomas

Background

Molecular mechanisms associated with frequent relapse of diffuse large B-cell lymphoma (DLBCL) are poorly defined. It is especially unclear how primary tumor clonal heterogeneity contributes to relapse. Here, we explore unique features of B-cell lymphomas - VDJ recombination and somatic hypermutation - to address this question.

Results

We performed high-throughput sequencing of rearranged VDJ junctions in 14 pairs of matched diagnosis-relapse tumors, among which 7 pairs were further characterized by exome sequencing. We identify two distinctive modes of clonal evolution of DLBCL relapse: an early-divergent mode in which clonally related diagnosis and relapse tumors diverged early and developed in parallel; and a late-divergent mode in which relapse tumors developed directly from diagnosis tumors with minor divergence. By examining mutation patterns in the context of phylogenetic information provided by VDJ junctions, we identified mutations in epigenetic modifiers such as KMT2D as potential early driving events in lymphomagenesis and immune escape alterations as relapse-associated events.

Conclusions

Altogether, our study for the first time provides important evidence that DLBCL relapse may result from multiple, distinct tumor evolutionary mechanisms, providing rationale for therapies for each mechanism. Moreover, this study highlights the urgent need to understand the driving roles of epigenetic modifier mutations in lymphomagenesis, and immune surveillance factor genetic lesions in relapse.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0432-0) contains supplementary material, which is available to authorized users.     

Knowledge and Adherence to Medications among Palestinian Geriatrics Living with Chronic Diseases in the West Bank and East Jerusalem

Background

Adequate patient knowledge about medications is essential for appropriate drug taking behavior and patient adherence. This study aims to assess and quantify the level of knowledge and adherence to medications among Palestinian geriatrics living with chronic diseases and to investigate possible associated socio-demographic characteristics.

Methods and Findings

We conducted a cross-sectional study during June 2013 and January 2014 among Palestinian geriatrics ≥60 years old living with chronic disease in the West Bank and East Jerusalem. A stratified random sample was selected and a questionnaire-assisted interview was applied for data collection. T-test was applied for bivariate analyzing and one-way ANOVA test was applied for multivariate analyses.

Results

A total of 1192 Palestinian geriatrics were studied. The average age was 70.3 (SD=8.58) years and ranged from 60-110 years. The sample comprised 659 (55.3%) females and 533 (44.7%) males. The global knowledge and global adherence scores were (67.57%) and (89.29%), respectively. Adequate levels of knowledge were 71.4%, and of adherence 75%, which were recorded for 705 (59.1%) and 1088 (91.3%) participants, respectively. Significant higher levels of global knowledge and global adherence were recorded for males, and for participants who hold a Bachelor’s degree, those who live on their own, and did physical activity for more than 40 hours/week (p-value <0.05). Furthermore, workers, participants with a higher monthly income, and non-smokers have a higher knowledge level with (p-value <0.05). We found positive correlation between participants’ global adherence and global knowledge (r=0.487 and p-value <0.001). Negative correlation was found between participants’ global knowledge and adherence with age (r= -0.236, p-value <0.001 and r= -0.211 and p-value <0.001, respectively. Negative correlation between global knowledge and the number of drugs taken (r= -0.130, p-value <0.001) was predicted.

Conclusion

We concluded that patients with a higher level of knowledge are more adherent to their medications and that better understanding of socio-demographic factors has a clear influence on the level of knowledge and adherence to medications and thus contributes to the development of guidelines for treatment and may consequently lead to favourable clinical outcomes and savings of health care costs.     

Overexpression of Periostin in Stroma Positively Associated with Aggressive Prostate Cancer

Background

Periostin is an important extracellular matrix protein involved in cell development and adhesion. Previously, we identified periostin to be up-regulated in aggressive prostate cancer (CaP) using quantitative glycoproteomics and mass spectrometry. The expression of periostin was further evaluated in primary radical prostatectomy (RP) prostate tumors and adjacent non-tumorous prostate tissues using immunohistochemistry (IHC). Our IHC results revealed a low background periostin levels in the adjacent non-tumorous prostate tissues, but overexpressed periostin levels in the peritumoral stroma of primary CaP tumors.

Methods

In this study, periostin expression in CaP was further examined on multiple tissue microarrays (TMAs), which were conducted in four laboratories. To achieve consistent staining, all TMAs were stained with same protocol and scored by same image computation tool to determine the total periostin staining intensities. The TMAs were further scored by pathologists to characterize the stromal staining and epithelial staining.

Results

The periostin staining was observed mainly in peritumoral stromal cells and in some cases in tumor epithelial cells though the stronger staining was found in peritumoral stromal cells. Both periostin stromal staining and epithelial staining can differentiate BPH from CaP including low grade CaP (Gleason score ≤6), with significant p-value of 2.2e-16 and 0.001, respectively. Periostin epithelial staining differentiated PIN from low grade CaP (Gleason score ≤6) (p=0.001), while periostin stromal staining differentiated low grade Cap (Gleason score ≤6) from high grade Cap (Gleason score ≤6) (p=1.7e-05). In addition, a positive correlation between total periostin staining and Gleason score was observed (r=0.87, p=0.002).

Conclusions

The results showed that periostin staining was positively correlated with increasing Gleason score and the aggressiveness of prostate disease.     

Salivary Heparanase Level Is a Potential Biomarker to Diagnose and Prognose the Malignant Salivary Gland Tumor

Background

Upregulation of heparanase has been reported in an increasing number of human cancer tissues. However, the level of salivary heparanase and its clinical significance in patients with salivary gland tumors remain unclear.

Methods

Salivary heparanase levels in patients with salivary gland tumors were detected using enzyme-linked immunosorbent assays (ELISAs) and the clinical significance was evaluated by analyzing the correlations among salivary heparanase levels, clinicopathological parameters, and clinical outcomes.

Results

The levels of salivary heparanase were significantly higher in patients with malignant salivary gland tumors than in benign tumors and normal controls (P<0.0001). High salivary heparanase levels were positively correlated with increased lymph node metastasis (P = 0.0235) and poorer tumor node metastasis stage (TNM) (P = 0.0183). Survival analyses revealed that high salivary heparanase levels were associated with worse overall survival (P = 0.0023) and disease-free survival (DFS) (P = 0.0025).

Conclusions

The study shows that salivary heparanase levels, as detected by the ELISAs, can be used to diagnose and provide an accurate prognosis for malignant salivary gland tumors. Salivary heparanase level was an independent predictor in patients with malignant salivary gland tumors.     

MicroRNA Signature Characterizes Primary Tumors That Metastasize in an Esophageal Adenocarcinoma Rat Model

Objective

To establish a miRNA signature for metastasis in an animal model of esophageal adenocarcinoma (EAC).

Background

The incidence of esophageal adenocarcinoma (EAC) has dramatically increased and esophageal cancer is now the sixth leading cause of cancer deaths worldwide. Mortality rates remain high among patients with advanced stage disease and esophagectomy is associated with high complication rates. Hence, early identification of potentially metastatic disease would better guide treatment strategies.

Methods

The modified Levrat’s surgery was performed to induce EAC in Sprague-Dawley rats. Primary EAC and distant metastatic sites were confirmed via histology and immunofluorescence. miRNA profiling was performed on primary tumors with or without metastasis. A unique subset of miRNAs expressed in primary tumors and metastases was identified with Ingenuity Pathway Analysis (IPA) along with upstream and downstream targets. miRNA-linked gene expression analysis was performed on a secondary cohort of metastasis positive (n=5) and metastasis negative (n=28) primary tumors.

Results

The epithelial origin of distant metastasis was established by IF using villin (VIL1) and mucin 5AC (MUC5AC) antibodies. miRNome analysis identified four down-regulated miRNAs in metastasis positive primary tumors compared to metastasis negative tumors: miR-92a-3p (p=0.0001), miR-141-3p (p=0.0022), miR-451-1a (p=0.0181) and miR133a-3p (p=0.0304). Six target genes identified in the top scoring networks by IPA were validated as significantly, differentially expressed in metastasis positive primary tumors: Ago2, Akt1, Kras, Bcl2L11, CDKN1B and Zeb2.

Conclusion

In vivo metastasis was confirmed in the modified Levrat’s model. Analysis of the primary tumor identified a distinctive miRNA signature for primary tumors that metastasized.     

High-Throughput Mutation Profiling Changes before and 3 Weeks after Chemotherapy in Newly Diagnosed Breast Cancer Patients

Background

Changes in tumor DNA mutation status during chemotherapy can provide insights into tumor biology and drug resistance. The purpose of this study is to analyse the presence or absence of mutations in cancer-related genes using baseline breast tumor samples and those obtained after exposure to one cycle of chemotherapy to determine if any differences exist, and to correlate these differences with clinical and pathological features.

Methods

Paired breast tumor core biopsies obtained pre- and post-first cycle doxorubicin (n = 18) or docetaxel (n = 22) in treatment-naïve breast cancer patients were analysed for 238 mutations in 19 cancer-related genes by the Sequenom Oncocarta assay.

Results

Median age of patients was 48 years (range 32–64); 55% had estrogen receptor-positive tumors, and 60% had tumor reduction ≥25% after cycle 1. Mutations were detected in 10/40 (25%) pre-treatment and 11/40 (28%) post-treatment samples. Four mutation pattern categories were identified based on tumor mutation status pre- → post-treatment: wildtype (WT)→WT, n = 24; mutant (MT)→MT, n = 5; MT→WT, n = 5; WT→MT, n = 6. Overall, the majority of tumors were WT at baseline (30/40, 75%), of which 6/30 (20%) acquired new mutations after chemotherapy. Pre-treatment mutations were predominantly in PIK3CA (8/10, 80%), while post-treatment mutations were distributed in PIK3CA, EGFR, PDGFRA, ABL1 and MET. All 6 WT→MT cases were treated with docetaxel. Higher mutant allele frequency in baseline MT tumors (n = 10; PIK3CA mutations n = 8) correlated with less tumor reduction after cycle 1 chemotherapy (R = -0.667, p = 0.035). No other associations were observed between mutation pattern category with treatment, clinicopathological features, and tumor response or survival.

Conclusion

Tumor mutational profiles can change as quickly as after one cycle of chemotherapy in breast cancer. Understanding of these changes can provide insights on potential therapeutic options in residual resistant tumors.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00212082","term_id":"NCT00212082"}}NCT00212082     

Trends in Lung Cancer Incidence Rates,Oklahoma 2005–2010

Purpose

Lung cancer is the second most frequently diagnosed cancer among men and women in the United States. With cigarette smoking causing the majority of cases, patterns in lung cancer are often monitored to understand the impact of anti-tobacco efforts. The purpose of this research was to investigate trends in lung cancer incidence rates for the period 2005–2010 in Oklahoma.

Methods

Data on Oklahoma’s incident cases of lung cancer (2005–2010) were obtained from the Centers for Disease Control and Prevention WONDER system. Annual percent change (APC) was calculated by linear regression to characterize trends in lung cancer incidence rates over time for the overall population, by gender, by age group, and by age group within gender. Rates were considered to increase or decrease if the p-value for trend was <0.05.

Results

From 2005 through 2010, lung cancer incidence rates declined from 81.96 to 68.19 per 100,000 population, with an APC of -3.58% (p-value: 0.0220). When subgroups were examined, declines were observed among all males (APC: -4.25%; p-value: 0.0270), males <65 years (APC: -5.32%; p-value: 0.0008), females <65 years (APC: -4.85%; p-value: 0.0044), and persons aged 55–64 years (APC: -6.38%; p-value: 0.0017).

Conclusions

Declines in lung cancer incidence rates occurred during 2005–2010 among the overall population and within select demographic groups in Oklahoma. Although trends were stable for several demographic groups, rates of lung cancer incidence were lower in 2010 compared to 2005. Continued evidence-based tobacco control efforts are needed to ensure further reductions in lung cancer incidence rates in the state of Oklahoma.     

Polarisation of Tumor-Associated Macrophages toward M2 Phenotype Correlates with Poor Response to Chemoradiation and Reduced Survival in Patients with Locally Advanced Cervical Cancer

Objective

we investigate the prognostic role of pre-treatment ratio between Type 1 (M1) and Type 2 (M2) tumor-associated macrophages (TAMs) in locally advanced cervical cancer (LACC) patients treated with chemoradiation (CT/RT).

Methods

84 consecutive LACC patients treated with cisplatin-based CT/RT for a total dose of 50.0 Gy, followed by radical surgery were analysed. Double-staining immunohistochemistry of CD163/p-STAT, CD68/pSTAT1, CD163/c-MAF, and CD68/c-MAF was performed on tumor samples taken at the time of diagnosis. TAMs with CD163+pSTAT1+, or CD68+pSTAT1+ were defined M1; CD163+c-MAF+ or CD68+c-MAF+ defined the M2 phenotype. The number of M1 and M2 cells was counted at low magnification by evaluating for each case the same tumour area. The ratio between M1 and M2 (M1/M2) was finally calculated.

Results

At diagnosis, we observed a direct correlation between the number of circulating monocytes and of TAMs (p-value = 0.001). Patients with high M1/M2 experienced more frequently complete pathologic response (no residual tumor) to CT/RT, compared to cases with low M1/M2 (55.0% Vs 29.5%; p-value = 0.029). At multivariate analysis M1/M2 (OR = 2.067; p-value = 0.037) emerged as independent predictor of pathologic response to CT/RT. Women with high M1/M2 showed a longer 5-yrs Disease-free (67.2% Vs. 44.3%; p-value = 0.019), and 5-yrs Overall (69.3% Vs. 46.9%; p-value = 0.037) survival, compared to cases with low M1/M2. The presence of a high M1/M2 ratio was independently associated with an unfavourable survival outcome in multivariate analysis.

Conclusions

polarisation of TAMs toward a M2 phenotype, as reflected by a lower M1/M2 ratio, is an independent predictor of poor response to CT/RT, and shorter survival in LACC.     

Profiling Critical Cancer Gene Mutations in Clinical Tumor Samples

Background

Detection of critical cancer gene mutations in clinical tumor specimens may predict patient outcomes and inform treatment options; however, high-throughput mutation profiling remains underdeveloped as a diagnostic approach. We report the implementation of a genotyping and validation algorithm that enables robust tumor mutation profiling in the clinical setting.

Methodology

We developed and implemented an optimized mutation profiling platform (“OncoMap”) to interrogate ∼400 mutations in 33 known oncogenes and tumor suppressors, many of which are known to predict response or resistance to targeted therapies. The performance of OncoMap was analyzed using DNA derived from both frozen and FFPE clinical material in a diverse set of cancer types. A subsequent in-depth analysis was conducted on histologically and clinically annotated pediatric gliomas. The sensitivity and specificity of OncoMap were 93.8% and 100% in fresh frozen tissue; and 89.3% and 99.4% in FFPE-derived DNA. We detected known mutations at the expected frequencies in common cancers, as well as novel mutations in adult and pediatric cancers that are likely to predict heightened response or resistance to existing or developmental cancer therapies. OncoMap profiles also support a new molecular stratification of pediatric low-grade gliomas based on BRAF mutations that may have immediate clinical impact.

Conclusions

Our results demonstrate the clinical feasibility of high-throughput mutation profiling to query a large panel of “actionable” cancer gene mutations. In the future, this type of approach may be incorporated into both cancer epidemiologic studies and clinical decision making to specify the use of many targeted anticancer agents.     

Multi-Parametric MRI and Texture Analysis to Visualize Spatial Histologic Heterogeneity and Tumor Extent in Glioblastoma

Background

Genetic profiling represents the future of neuro-oncology but suffers from inadequate biopsies in heterogeneous tumors like Glioblastoma (GBM). Contrast-enhanced MRI (CE-MRI) targets enhancing core (ENH) but yields adequate tumor in only ~60% of cases. Further, CE-MRI poorly localizes infiltrative tumor within surrounding non-enhancing parenchyma, or brain-around-tumor (BAT), despite the importance of characterizing this tumor segment, which universally recurs. In this study, we use multiple texture analysis and machine learning (ML) algorithms to analyze multi-parametric MRI, and produce new images indicating tumor-rich targets in GBM.

Methods

We recruited primary GBM patients undergoing image-guided biopsies and acquired pre-operative MRI: CE-MRI, Dynamic-Susceptibility-weighted-Contrast-enhanced-MRI, and Diffusion Tensor Imaging. Following image coregistration and region of interest placement at biopsy locations, we compared MRI metrics and regional texture with histologic diagnoses of high- vs low-tumor content (≥80% vs <80% tumor nuclei) for corresponding samples. In a training set, we used three texture analysis algorithms and three ML methods to identify MRI-texture features that optimized model accuracy to distinguish tumor content. We confirmed model accuracy in a separate validation set.

Results

We collected 82 biopsies from 18 GBMs throughout ENH and BAT. The MRI-based model achieved 85% cross-validated accuracy to diagnose high- vs low-tumor in the training set (60 biopsies, 11 patients). The model achieved 81.8% accuracy in the validation set (22 biopsies, 7 patients).

Conclusion

Multi-parametric MRI and texture analysis can help characterize and visualize GBM’s spatial histologic heterogeneity to identify regional tumor-rich biopsy targets.     

Establishment and Biological Characterization of a Panel of Glioblastoma Multiforme (GBM) and GBM Variant Oncosphere Cell Lines

Objective

Human tumor cell lines form the basis of the majority of present day laboratory cancer research. These models are vital to studying the molecular biology of tumors and preclinical testing of new therapies. When compared to traditional adherent cell lines, suspension cell lines recapitulate the genetic profiles and histologic features of glioblastoma multiforme (GBM) with higher fidelity. Using a modified neural stem cell culture technique, here we report the characterization of GBM cell lines including GBM variants.

Methods

Tumor tissue samples were obtained intra-operatively and cultured in neural stem cell conditions containing growth factors. Tumor lines were characterized in vitro using differentiation assays followed by immunostaining for lineage-specific markers. In vivo tumor formation was assayed by orthotopic injection in nude mice. Genetic uniqueness was confirmed via short tandem repeat (STR) DNA profiling.

Results

Thirteen oncosphere lines derived from GBM and GBM variants, including a GBM with PNET features and a GBM with oligodendroglioma component, were established. All unique lines showed distinct genetic profiles by STR profiling. The lines assayed demonstrated a range of in vitro growth rates. Multipotency was confirmed using in vitro differentiation. Tumor formation demonstrated histologic features consistent with high grade gliomas, including invasion, necrosis, abnormal vascularization, and high mitotic rate. Xenografts derived from the GBM variants maintained histopathological features of the primary tumors.

Conclusions

We have generated and characterized GBM suspension lines derived from patients with GBMs and GBM variants. These oncosphere cell lines will expand the resources available for preclinical study.