An Immune Gene Expression Risk Score for Distant Metastases after Radiotherapy for Cervical Cancer.

PURPOSE: To develop an immune-based gene expression risk score to identify patients with cervical cancer at increased risk of distant metastases (DM). EXPERIMENTAL DESIGN: Tumor biopsies were obtained from 81 patients prior to chemoradiotherapy. Whole-transcriptome RNA sequencing was performed (Illumina NextSeq500). Beginning with 4,723 immune-related genes, a 55-gene risk score for DM was derived using Cox modeling and principal component analysis. It was validated in independent cohorts of 274 patients treated at the Norwegian Radium Hospital (NRH) and 206 patients from The Cancer Genome Atlas (TCGA). RESULTS: The risk score was predictive of DM (HR, 2.7; P < 0.0001) and lower cause-specific survival (CSS) by univariate analysis (HR, 2.0; P = 0.0003) and multivariate analysis adjusted for clinical factors (DM HR, 3.0; P < 0.0001; CSS HR, 2.2; P = 0.0004). The risk score predicted DM (HR, 1.4; P = 0.05) and CSS (HR, 1.48; P = 0.013) in the NRH cohort and CSS (HR, 1.4; P = 0.03) in TCGA cohort. Higher risk scores were associated with lower CIBERSORT estimates of tumor-infiltrating immune cells, including CD8 T cells and M1 and M2 macrophages (all P < 0.001). Higher risk scores were associated with lower expression (all P < 0.001) of important chemokines (CXCL12, CXCR4), IFN-regulated genes (IRF1, STAT1, IDO1), and immune checkpoint regulators (PD-1, PD-L1, CTLA-4). CONCLUSIONS: The immune metastatic risk score addresses important challenges in the treatment of cervical cancer-identifying patients at high risk of DM after radiotherapy. The findings of this study indicate that high tumor mutational burden and a "cold," immune-excluded tumor microenvironment influence distant metastatic recurrence. Further validation of the risk score is needed.

Mitochondrial Aconitase ACO2 Links Iron Homeostasis with Tumorigenicity in Non-Small Cell Lung Cancer.

The ability of a patient tumor to engraft an immunodeficient mouse is the strongest known independent indicator of poor prognosis in early-stage non-small cell lung cancer (NSCLC). Analysis of primary NSCLC proteomes revealed low-level expression of mitochondrial aconitase (ACO2) in the more aggressive, engrafting tumors. Knockdown of ACO2 protein expression transformed immortalized lung epithelial cells, whereas upregulation of ACO2 in transformed NSCLC cells inhibited cell proliferation in vitro and tumor growth in vivo. High level ACO2 increased iron response element binding protein 1 (IRP1) and the intracellular labile iron pool. Impaired cellular proliferation associated with high level ACO2 was reversed by treatment of cells with an iron chelator, whereas increased cell proliferation associated with low level ACO2 was suppressed by treatment of cells with iron. Expression of CDGSH iron-sulfur (FeS) domain-containing protein 1 [CISD1; also known as mitoNEET (mNT)] was modulated by ACO2 expression level and inhibition of mNT by RNA interference or by treatment of cells with pioglitazone also increased iron and cell death. Hence, ACO2 is identified as a regulator of iron homeostasis and mNT is implicated as a target in aggressive NSCLC. IMPLICATIONS: FeS cluster-associated proteins including ACO2, mNT (encoded by CISD1), and IRP1 (encoded by ACO1) are part of an "ACO2-Iron Axis" that regulates iron homeostasis and is a determinant of a particularly aggressive subset of NSCLC.

Optimizing Drug Response Study Design in Patient-Derived Tumor Xenografts.

Patient-derived tumor xenograft (PDX) models were used to evaluate the effectiveness of preclinical anticancer agents. A design using 1 mouse per patient per drug (1 × 1 × 1) was considered practical for large-scale drug efficacy studies. We evaluated modifiable parameters that could increase the statistical power of this design based on our consolidated PDX experiments. Real studies were used as a reference to investigate the relationship between statistical power with treatment effect size, inter-mouse variation, and tumor measurement frequencies. Our results showed that large effect sizes could be detected at a significance level of .2 or .05 under a 1 × 1 × 1 design. We found that the minimum number of mice required to achieve 80% power at an alpha level of .05 under all situations explored was 21 mice per group for a small effect size and 5 mice per group for a medium effect size.

Digital quantitative tissue image analysis of hypoxia in resected pancreatic ductal adenocarcinomas.

Background: Tumor hypoxia is theorized to contribute to the aggressive biology of pancreatic ductal adenocarcinoma (PDAC). We previously reported that hypoxia correlated with rapid tumor growth and metastasis in patient-derived xenografts. Anticipating a prognostic relevance of hypoxia in patient tumors, we developed protocols for automated semi-quantitative image analysis to provide an objective, observer-independent measure of hypoxia. We further validated this method which can reproducibly estimate pimonidazole-detectable hypoxia in a high-through put manner. Methods: We studied the performance of three automated image analysis platforms in scoring pimonidazole-detectable hypoxia in resected PDAC (n = 10) in a cohort of patients enrolled in PIMO-PANC. Multiple stained tumor sections were analyzed on three independent image-analysis platforms, Aperio Genie (AG), Definiens Tissue Studio (TS), and Definiens Developer (DD), which comprised of a customized rule set. Results: The output from Aperio Genie (AG) had good concordance with manual scoring, but the workflow was resource-intensive and not suited for high-throughput analysis. TS analysis had high levels of variability related to misclassification of cells class, while the customized rule set of DD had a high level of reliability with an intraclass coefficient of more than 85%. Discussion: This work demonstrates the feasibility of developing a robust, high-performance pipeline for an automated, quantitative scoring of pimonidazole-detectable hypoxia in patient tumors.

A Phase II Randomized Trial of Chemoradiation with or without Metformin in Locally Advanced Cervical Cancer.

PURPOSE: Tumor hypoxia is associated with poor response to radiation (RT). We previously discovered a novel mechanism of metformin: enhancing tumor RT response by decreasing tumor hypoxia. We hypothesized that metformin would decrease tumor hypoxia and improve cervical cancer response to RT. PATIENTS AND METHODS: A window-of-opportunity, phase II randomized trial was performed in stage IB-IVA cervical cancer. Patients underwent screening positron emission tomography (PET) imaging with hypoxia tracer fluoroazomycin arabinoside (FAZA). Only patients with FAZA uptake (hypoxic tumor) were included and randomized 2:1 to receive metformin in combination with chemoRT or chemoRT alone. A second FAZA-PET/CT scan was performed after 1 week of metformin or no intervention (control). The primary endpoint was a change in fractional hypoxic volume (FHV) between FAZA-PET scans, compared using the Wilcoxon signed-rank test. The study was closed early due to FAZA availability and the COVID-19 pandemic. RESULTS: Of the 20 consented patients, 6 were excluded due to no FAZA uptake and 1 withdrew. FHV of 10 patients in the metformin arm decreased by an average of 10.2% (44.4%-34.2%) ± SD 16.9% after 1 week of metformin, compared with an average increase of 4.7% (29.1%-33.8%) ± 11.5% for the 3 controls (P = 0.027). Those with FHV reduction after metformin had significantly lower MATE2 expression. With a median follow-up of 2.8 years, the 2-year disease-free survival was 67% for the metformin arm versus 33% for controls (P = 0.09). CONCLUSIONS: Metformin decreased cervical tumor hypoxia in this trial that selected for patients with hypoxic tumor. See related commentary by Lyng et al., p. 5233.

Repurposing Itraconazole and Hydroxychloroquine to Target Lysosomal Homeostasis in Epithelial Ovarian Cancer.

Drug repurposing is an attractive option for oncology drug development. Itraconazole is an antifungal ergosterol synthesis inhibitor that has pleiotropic actions including cholesterol antagonism, inhibition of Hedgehog and mTOR pathways. We tested a panel of 28 epithelial ovarian cancer (EOC) cell lines with itraconazole to define its spectrum of activity. To identify synthetic lethality in combination with itraconazole, a whole-genome drop-out genome-scale clustered regularly interspaced short palindromic repeats sensitivity screen in two cell lines (TOV1946 and OVCAR5) was performed. On this basis, we conducted a phase I dose-escalation study assessing the combination of itraconazole and hydroxychloroquine in patients with platinum refractory EOC (NCT03081702). We identified a wide spectrum of sensitivity to itraconazole across the EOC cell lines. Pathway analysis showed significant involvement of lysosomal compartments, the trans-golgi network and late endosomes/lysosomes; similar pathways are phenocopied by the autophagy inhibitor, chloroquine. We then demonstrated that the combination of itraconazole and chloroquine displayed Bliss defined synergy in EOC cancer cell lines. Furthermore, there was an association of cytotoxic synergy with the ability to induce functional lysosome dysfunction, by chloroquine. Within the clinical trial, 11 patients received at least one cycle of itraconazole and hydroxychloroquine. Treatment was safe and feasible with the recommended phase II dose of 300 and 600 mg twice daily, respectively. No objective responses were detected. Pharmacodynamic measurements on serial biopsies demonstrated limited pharmacodynamic impact. In vitro, itraconazole and chloroquine have synergistic activity and exert a potent antitumor effect by affecting lysosomal function. The drug combination had no clinical antitumor activity in dose escalation. Significance: The combination of the antifungal drug itraconazole with antimalarial drug hydroxychloroquine leads to a cytotoxic lysosomal dysfunction, supporting the rational for further research on lysosomal targeting in ovarian cancer.

Subpathologies and genomic classifier for treatment individualization of post-prostatectomy radiotherapy.

PURPOSE/OBJECTIVE: Risk-stratification for post-prostatectomy radiotherapy (PORT) using conventional clinicopathologic indexes leads to substantial over- and under-treatment. Better patient selection could spare unnecessary toxicities and improve outcomes. We investigated the prognostic utility of unfavorable subpathologies intraductal carcinoma and cribriform architecture (IDC/CA), and a 22-gene Decipher genomic classifier (GC) in prostate cancer (PCa) patients receiving PORT. MATERIAL/METHODS: A cohort of 302 men who received PORT at 2 academic institutions was pooled. PORT was predominately delivered as salvage (62% of cases); 20% received HT+PORT. Specimens were centrally reviewed for IDC/CA presence. In 104 cases, GC scores were determined. Endpoints were biochemical relapse-free (bRFR) and metastasis-free (mFR) rates. RESULTS: After a median follow-up of 6.49-years, 135 (45%) and 40 (13%) men experienced biochemical relapse and metastasis, respectively. IDC/CA were identified in 160 (53%) of cases. Men harboring IDC/CA experienced inferior bRFR (HR 2.6, 95%CI 1.8-3.2, P<0.001) and mFR (HR 3.1, 95%CI 1.5-6.4, P = 0.0014). Patients with GC scores, 22 (21%) were stratified low-, 30 (29%) intermediate-, and 52 (50%) high-risk. GC low-risk was associated with superior bRFR (HR 0.25, 95%CI 0.1-0.5, P<0.001) and mFR (HR 0.15, 95%CI 0.03-0.8, P = 0.025). On multivariable analyses, IDC/CA and GC independently predicted for bRFR, corresponding to improved discrimination (C-index = 0.737 (95%CI 0.662-0.813)). CONCLUSIONS: IDC/CA subpathologies and GC predict for biochemical relapse and metastasis beyond conventional clinicopathologic indexes in the PORT setting. Patients harboring IDC/CA are at higher risk of relapse after maximal local therapies, thus warranting consideration for treatment intensification strategies. Conversely, for men with absence of IDC/CA and low GC scores, de-intensification strategies could be explored.

Patient-derived tumor xenograft and organoid models established from resected pancreatic, duodenal and biliary cancers.

Patient-derived xenograft (PDX) and their xenograft-derived organoid (XDO) models that recapitulate the genotypic and phenotypic landscape of patient cancers could help to advance research and lead to improved clinical management. PDX models were established from 276 pancreato-duodenal and biliary cancer resections. Initial, passage 0 (P0) engraftment rates were 59% (118/199) for pancreatic, 86% (25/29) for duodenal, and 35% (17/48) for biliary ductal tumors. Pancreatic ductal adenocarcinoma (PDAC), had a P0 engraftment rate of 62% (105/169). KRAS mutant and wild-type PDAC models were molecularly profiled, and XDO models were generated to perform initial drug response evaluations. Subsets of PDAC PDX models showed global copy number variants and gene expression profiles that were retained with serial passaging, and they showed a spectrum of somatic mutations represented in patient tumors. PDAC XDO models were established, with a success rate of 71% (10/14). Pathway activation of KRAS-MAPK in PDXs was independent of KRAS mutational status. Four wild-type KRAS models were characterized by one with EGFR (L747-P753 del), two with BRAF alterations (N486_P490del or V600E), and one with triple negative KRAS/EGFR/BRAF. Model OCIP256, characterized by BRAF (N486-P490 del), had activated phospho-ERK. A combination treatment of a pan-RAF inhibitor (LY3009120) and a MEK inhibitor (trametinib) effectively suppressed phospho-ERK and inhibited growth of OCIP256 XDO and PDX models. PDAC/duodenal adenocarcinoma have high success rates forming PDX/organoid and retaining their phenotypic and genotypic features. These models may be effective tools to evaluate novel drug combination therapies.

Predicting Toxicity and Response to Pembrolizumab Through Germline Genomic HLA Class 1 Analysis.

Background: Human leukocyte antigen class 1 (HLA-1)-dependent immune activity is linked to autoimmune diseases. HLA-1-dependent CD8+ T cells are required for immune checkpoint blockade antitumor activity. It is unknown if HLA-1 genotype is predictive of toxicity to immune checkpoint blockade. Methods: Patients with advanced solid tumors stratified into 5 cohorts received single agent pembrolizumab (anti-programmed cell death-1) 200 mg intravenously every 3 weeks in an investigator-initiated phase II trial (Investigator-Initiated Phase II Study of Pembrolizumab Immunological Response Evaluation study, NCT02644369). Germline whole-exome sequencing of peripheral blood mononuclear cells was performed using the Illumina HiSeq2500 platform. HLA-1 haplotypes were predicted from whole-exome sequencing using HLAminer and HLAVBSeq. Heterozygosity of HLA-A, -B, and -C, individual HLA-1 alleles, and HLA haplotype dimorphism at positions -21 M and -21 T of the HLA-A and -B leader sequence were analyzed as predictors of toxicity defined as grade 2 or greater immune-related adverse events and clinical benefit defined as complete or partial response, or stable disease for 6 or more cycles of pembrolizumab. Statistical significance tests were 2-sided. Results: In the overall cohort of 101 patients, the frequency of toxicity and clinical benefit from pembrolizumab was 22.8% and 25.7%, respectively. There was no association between any of the HLA-1 loci or alleles with toxicity. HLA-C heterozygosity had an association with decreased clinical benefit relative to HLA-C homozygosity when controlling for cohort (odds ratio = 0.28, 95% confidence interval = 0.09 to 0.91, P = .04). HLA-A and -B haplotype -21 M/T dimorphism and heterozygosity of HLA-A, -B, and -C were not predictive of outcomes. Conclusions: HLA-C heterozygosity may predict decreased response to pembrolizumab. Prospective validation is required.

Predicting response and toxicity to PD-1 inhibition using serum autoantibodies identified from immuno-mass spectrometry.

Background: Validated biomarkers are needed to identify patients at increased risk of immune-related adverse events (irAEs) to immune checkpoint blockade (ICB). Antibodies directed against endogenous antigens can change after exposure to ICB. Methods: Patients with different solid tumors stratified into cohorts received pembrolizumab every 3 weeks in a Phase II trial (INSPIRE study). Blood samples were collected prior to first pembrolizumab exposure (baseline) and approximately 7 weeks (pre-cycle 3) into treatment. In a discovery analysis, autoantibody target immuno-mass spectrometry was performed in baseline and pre-cycle 3 pooled sera of 24 INSPIRE patients based on clinical benefit (CBR) and irAEs. Results: Thyroglobulin (Tg) and thyroid peroxidase (TPO) were identified as the candidate autoantibody targets. In the overall cohort of 78 patients, the frequency of CBR and irAEs from pembrolizumab was 31% and 24%, respectively. Patients with an anti-Tg titer increase ≥1.5x from baseline to pre-cycle 3 were more likely to have irAEs relative to patients without this increase in unadjusted, cohort adjusted, and multivariable models (OR=17.4, 95% CI 1.8-173.8, p=0.015). Similarly, patients with an anti-TPO titer ≥ 1.5x from baseline to pre-cycle 3 were more likely to have irAEs relative to patients without the increase in unadjusted and cohort adjusted (OR=6.1, 95% CI 1.1-32.7, p=0.035) models. Further, the cohort adjusted analysis showed patients with anti-Tg titer greater than median (10.0 IU/mL) at pre-cycle 3 were more likely to have irAEs (OR=4.7, 95% CI 1.2-17.8, p=0.024). Patients with pre-cycle 3 anti-TPO titers greater than median (10.0 IU/mL) had a significant difference in overall survival (23.8 vs 11.5 months; HR=1.8, 95% CI 1.0-3.2, p=0.05). Conclusions: Patient increase ≥1.5x of anti-Tg and anti-TPO titers from baseline to pre-cycle 3 were associated with irAEs from pembrolizumab, and patients with elevated pre-cycle 3 anti-TPO titers had an improvement in overall survival.

Circulating miR-16-5p, miR-92a-3p, and miR-451a in Plasma from Lung Cancer Patients: Potential Application in Early Detection and a Regulatory Role in Tumorigenesis Pathways.

BACKGROUND: Micro(mi)RNAs, potent gene expression regulators associated with tumorigenesis, are stable, abundant circulating molecules, and detectable in plasma. Thus, miRNAs could potentially be useful in early lung cancer detection. We aimed to identify circulating miRNA signatures in plasma from patients with lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC), and to verify whether miRNAs regulate lung oncogenesis pathways. METHODS: RNA isolated from 139 plasma samples (40 LUAD, 38 LUSC; 61 healthy/non-diseased individuals) were divided into discovery (38 patients; 21 controls for expression quantification using an 800-miRNA panel; Nanostring nCounter®) and validation (40 patients; 40 controls; TaqMan® RT-qPCR) cohorts. Elastic net, Maximizing-R-Square Analysis (MARSA), and C-Statistics were applied for miRNA signature identification. RESULTS: When compared to healthy individuals, 580 of 606 deregulated miRNAs in LUAD and 221 of 226 deregulated miRNAs in LUSC had significantly increased levels. Among the 10 most significantly overexpressed miRNAs, 6 were common to patients with LUAD and LUSC. Further analysis identified three signatures composed of 12 miRNAs. Signatures included miRNAs commonly overexpressed in patient plasma. Enriched pathways included target genes modulated by three miRNAs in the C-Statistics signature: miR-16-5p, miR-92a-3p, and miR-451a. CONCLUSIONS: The 3-miRNA signature (miR-16-5p, miR-92a-3p, miR-451a) had high specificity (100%) and sensitivity (84%) to predict cancer (LUAD and LUSC). These miRNAs are predicted to modulate genes and pathways with known roles in lung tumorigenesis, including EGFR, K-RAS, and PI3K/AKT signaling, suggesting that the 3-miRNA signature is biologically relevant in adenocarcinoma and squamous cell carcinoma of the lung.

Evaluation of Lymphadenopathy and Suspected Lymphoma in a Lymphoma Rapid Diagnosis Clinic.

PURPOSE: Lymphomas often present a diagnostic challenge, and for some a delay in diagnosis can negatively influence outcomes of therapy. We established a nurse practitioner-led lymphoma rapid diagnosis clinic (LRDC) with the goal of reducing wait times to definitive diagnosis. We examined the initial 30-month experience of the LRDC, and results were compared with time periods before implementation of the clinic to determine program impact. METHODS: All patients referred to LRDC with suspicion of lymphoma from June 1, 2015 to Nov 30, 2017 were evaluated. Time from initial consultation to diagnosis was compared with patients diagnosed at our center with lymphoma in 2008 and 2012. Patient symptoms and relevant laboratory/imaging findings were collected to identify patterns of presentation and predictive factors for benign diagnoses. RESULTS: Of the 126 patients evaluated, 66 (52%) had confirmation of lymphoma diagnosis. Median time to lymphoma diagnosis was 16 days for patients assessed in LRDC and 28 days for historical controls (P < .001). By univariable analysis, lymph node size greater than 3.4 cm and presence of mediastinal or abdominal adenopathy increased the likelihood of a diagnosis of malignancy, whereas younger age, being a nonsmoker, and prior rheumatologic condition were associated with a nonmalignant diagnosis. In multivariable analysis, lymph node size, age, and prior rheumatologic diagnosis remained significant. CONCLUSION: Establishing a nurse practitioner-led LRDC was effective in shortening time to diagnosis of lymphoma. Younger age, smaller lymph node size, and prior rheumatologic disorder reduced the likelihood of a cancer diagnosis in our patient population.

HPV-independent Vulvar Squamous Cell Carcinoma is Associated With Significantly Worse Prognosis Compared With HPV-associated Tumors.

Vulvar squamous cell carcinomas (VSCC) represent the most common carcinoma of the female external genitalia, with increasing incidence. Although high-risk human papillomavirus (HPV) infection has long been implicated in the majority of cervical and anal squamous cell carcinomas, there is uncertainty about its prevalence and prognostic impact in VSCC. In this study, we conducted a retrospective integrated morphologic and multimodal HPV analysis of a cohort of 114 VSCC cases treated at the Princess Margaret Cancer Centre/University Health Network, Toronto, Canada between 2000 and 2010. VSCC histology was reviewed. We analyzed the cohort for HPV using polymerase chain reaction based method, and tissue microarray DNA and RNA in situ hybridization (ISH), and p16 immunohistochemistry. Among the 114 cases (age 70±16 yr), 36.7% of cases were classified as having histomorphology of HPV infection. HPV was detected in 31.9% (polymerase chain reaction), 14.0% (DNA ISH), and 27.3% (RNA ISH) of cases. p16 immunohistochemistry was positive in 37.8% of cases. On univariate analysis, HPV morphology (P=0.009), p16+ (P=0.00013), DNA ISH+ (P=0.021), and RNA ISH+ (P=0.00061) were associated with better 5-yr progression-free survival. DNA ISH+ (P=0.049) was associated with better 5-yr overall survival. On multivariate analysis, HPV morphology (P=0.033), p16+ (P=0.01), and RNA ISH+ (P=0.035) were associated with better 5-yr progression-free survival. In conclusion, a subset of VSCC is associated with HPV, which correlates with better outcome. Relatively inexpensive tests such as histomorphologic evaluation, p16 immunohistochemistry, and HPV RNA ISH can be used to predict outcome in VSCC. Therefore, routine reporting of HPV status in VSCC is recommended.

Intratumoral heterogeneity and hypoxia gene expression signatures: Is a single biopsy adequate?

BACKGROUND AND PURPOSE: Gene expression signatures are often used to identify hypoxic tumors. However, intratumoral heterogeneity raises concern that multiple biopsies may be necessary to assess global hypoxia status. The objective of this study was to compare the impact of heterogeneity on the discriminative capacity of several previously described hypoxia gene signatures and determine if a single biopsy is sufficient to obtain a reliable estimate of hypoxia in cervical cancer. MATERIALS AND METHODS: Multiple biopsies (33) were obtained from 11 locally advanced (FIGO IB to IVB) cervical cancers prior to treatment. Ten hypoxia gene signatures were analyzed. Variance component analysis was used to determine the ratio of within-tumor variability to total-tumor variability when one to five biopsies are available for analysis (W/T1-5). The mean standardized error in the signature scores was estimated by comparing the score using one biopsy randomly selected from each tumor to the 'global' score using all available biopsies. RESULTS: The ten hypoxia signatures were comprised of 6-99 genes each. The W/T1 ratios for individual genes commonly found in the signatures ranged from 0.17 to 0.73. W/T1 ratios for the signatures were generally lower (0.21-0.45), implying greater capacity to discriminate among tumors. With additional biopsies, the signature W/T ratios (ie W/T2-5) decreased further. The mean error in the signature scores varied from 0.27 to 0.40 of one standard deviation, suggesting high capacity to discriminate among tumors with different global hypoxia scores. CONCLUSIONS: Compared with individual probes, hypoxia gene expression signatures are generally more consistent across multiple biopsies from different regions of a tumor and more tolerant of intratumoral heterogeneity.

Correction: Targeting CXCL12/CXCR4 and myeloid cells to improve the therapeutic ratio in patient-derived cervical cancer models treated with radio-chemotherapy.

Since the publication of this paper, the authors have reported that an incorrect version of Figure 1 was presented. The correct version of Figure 1 is provided.An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Anaplastic lymphoma kinase 5A4 immunohistochemistry as a diagnostic assay in lung cancer: A Canadian reference testing center's results in population-based reflex testing.

BACKGROUND: The presence of anaplastic lymphoma kinase (ALK) rearrangement predicts response to ALK tyrosine kinase inhibitor (TKI) therapy. Fluorescence in situ hybridization (FISH) was the initial reference standard to detect ALK rearrangement, but immunohistochemistry (IHC) using D5F3 has gained acceptance as an alternative diagnostic method. ALK IHC assays using other ALK antibodies have also been used as screening methods, but data supporting their utility as diagnostic tests have not been widely reported. METHODS: Data from reflexive clinical ALK IHC test using the 5A4 clone concurrent with epidermal growth factor receptor (EGFR) mutation testing were analyzed. ALK IHC results were reported as negative (-), equivocal, or positive (+), with equivocal or positive staining validated by FISH break-apart probe testing. Treatment outcomes were reviewed for ALK IHC+ patients. RESULTS: Between 2012 and 2015, 146 (2.5%) cases were reported as ALK IHC+, 188 (3.2%) were reported as equivocal, and 5624 (94.4%) were reported as ALK IHC-. Of the ALK IHC+ cases, 131/143(91.6%) were ALK FISH+. Excluding 6 cases in which FISH was inconclusive or not performed, the positive predictive value was 95.6%, and the negative predictive value was 100%. Most specimens (n = 5352 [89.6%]) were also successfully tested for EGFR. Clinical responses to ALK TKIs were noted in 49 ALK IHC+ patients, with a median progression-free survival of 9.9 months. CONCLUSIONS: ALK 5A4 IHC can serve as a robust diagnostic test for ALK-rearranged lung cancer and is associated with treatment response and survival. Optimized tissue allocation resulted in high success rates of combined reflex EGFR and ALK testing.

Somatic Alteration Burden Involving Non-Cancer Genes Predicts Prognosis in Early-Stage Non-Small Cell Lung Cancer.

The burden of somatic mutations and neoantigens has been associated with improved survival in cancer treated with immunotherapies, especially non-small cell lung cancer (NSCLC). However, there is uncertainty about their effect on outcome in early-stage untreated cases. We posited that the burden of mutations in a specific set of genes may also contribute to the prognosis of early NSCLC patients. From a small cohort of 36 NSCLC cases, we were able to identify somatic mutations and copy number alterations in 865 genes that contributed to patient overall survival. Simply, the number of altered genes (NAG) among these 865 genes was associated with longer disease-free survival (hazard ratio (HR) = 0.153, p = 1.48 × 10-4). The gene expression signature distinguishing patients with high/low NAG was also prognostic in three independent datasets. Patients with a high NAG could be further stratified based on the presence of immunogenic mutations, revealing a further subgroup of stage I NSCLC with even better prognosis (85% with >5 years survival), and associated with cytotoxic T-cell expression. Importantly, 95% of the highly-altered genes lacked direct relation to cancer, but were implicated in pathways regulating cell proliferation, motility and immune response.

Targeting CXCL12/CXCR4 and myeloid cells to improve the therapeutic ratio in patient-derived cervical cancer models treated with radio-chemotherapy.

BACKGROUND: The CXCL12/CXCR4 chemokine pathway is involved in cervical cancer pathogenesis and radiation treatment (RT) response. We previously reported that radiochemotherapy (RTCT) and concurrent administration of the CXCR4 inhibitor plerixafor improved primary tumour response. The aims of this study were to determine optimal sequencing of RTCT and plerixafor, the mechanisms responsible for improved response and the effect of plerixafor on late intestinal toxicity. METHODS: Orthotopic cervical cancer xenografts were treated with RTCT (30 Gy in 2 Gy fractions and cisplatin) with or without concurrent, adjuvant or continuous plerixafor. The endpoints were growth delay and molecular and immune cell changes at the end of treatment. Late intestinal toxicity was assessed by histologic examination of the rectum 90 days after a single 20 Gy fraction. RESULTS: RTCT increased CXCL12/CXCR4 signalling and the intratumoral accumulation of myeloid cells; the addition of plerixafor mitigated these effects. All of the RTCT and plerixafor arms showed prolonged tumour growth delay compared to RTCT alone, with the adjuvant arm showing the greatest improvement. Plerixafor also reduced late intestinal toxicity. CONCLUSION: Adding Plerixafor to RTCT blunts treatment-induced increases in CXCL12/CXCR4 signalling, improves primary tumour response and reduces intestinal side effects. This combination warrants testing in future clinical trials.

Repeatability and reproducibility of MRI-based radiomic features in cervical cancer.

PURPOSE: The aims of this study are to evaluate the stability of radiomic features from T2-weighted MRI of cervical cancer in three ways: (1) repeatability via test-retest; (2) reproducibility between diagnostic MRI and simulation MRI; (3) reproducibility in inter-observer setting. MATERIALS AND METHODS: This retrospective cohort study included FIGO stage IB-IVA cervical cancer patients treated with chemoradiation between 2005 and 2014. There were three cohorts of women corresponding to each aim of the study: (1) 8 women who underwent test-retest MRI; (2) 20 women who underwent MRI on different scanners (diagnostic and simulation MRI); (3) 34 women whose diagnostic MRIs were contoured by three observers. Radiomic features based on first-order statistics, shape features and texture features were extracted from the original, Laplacian of Gaussian (LoG)-filtered and wavelet-filtered images, for a total of 1761 features. Stability of radiomic features was assessed using intraclass correlation coefficient (ICC). RESULTS: The inter-observer cohort had the most reproducible features (95.2% with ICC ≥0.75) whereas the diagnostic-simulation cohort had the fewest (14.1% with ICC ≥0.75). Overall, 229 features had ICC ≥0.75 in all three tests. Shape features emerged as the most stable features in all cohorts. CONCLUSION: The diagnostic-simulation test resulted in the fewest reproducible features. Further research in MRI-based radiomics is required to validate the use of reproducible features in prognostic models.

Characterization of Distinct Populations of Carcinoma-Associated Fibroblasts from Non-Small Cell Lung Carcinoma Reveals a Role for ST8SIA2 in Cancer Cell Invasion.

Carcinoma-associated fibroblasts (CAFs) are abundant stromal cells in tumor microenvironment that are critically involved in cancer progression. Contrasting reports have shown that CAFs can have either pro- or antitumorigenic roles, indicating that CAFs are functionally heterogeneous. Therefore, to precisely target the cancer-promoting CAF subsets, it is necessary to identify specific markers to define these subpopulations and understand their functions. We characterized two CAFs subsets from 28 non-small cell lung cancer (NSCLC) patient tumors that were scored and classified based on desmoplasia [mainly characterized by proliferating CAFs; high desmoplastic CAFs (HD-CAF; n = 15) and low desmoplastic CAFs (LD-CAF; n = 13)], which is an independent prognostic factor. Here, for the first time, we demonstrate that HD-CAFs and LD-CAFs show different tumor-promoting abilities. HD-CAFs showed higher rate of collagen matrix remodeling, invasion, and tumor growth compared to LD-CAFs. Transcriptomic analysis identified 13 genes that were differentially significant (fold ≥1.5; adjusted P value < .1) between HD-CAFs and LD-CAFs. The top upregulated differentially expressed gene, ST8SIA2 (11.3 fold; adjusted P value = .02), enhanced NSCLC tumor cell invasion in 3D culture compared to control when it was overexpressed in CAFs, suggesting an important role of ST8SIA2 in cancer cell invasion. We confirmed the protumorigenic role of ST8SIA2, showing that ST8SIA2 was significantly associated with the risk of relapse in three independent NSCLC clinical datasets. In summary, our studies show that functional heterogeneity in CAF plays key role in promoting cancer cell invasion in NSCLC.