Role of Lactate in the Regulation of Transcriptional Activity of Breast Cancer-Related Genes and Epithelial-to-Mesenchymal Transition Proteins: A Compassion of MCF7 and MDA-MB-231 Cancer Cell Lines.

The Warburg Effect is characterized by accelerated glycolytic metabolism and lactate production and under fully aerobic conditions is a hallmark of cancer cells. Recently, we have demonstrated the role of endogenous, glucose-derived lactate as an oncometabolite which regulates gene expression in the estrogen receptor positive (ER+) MCF7 cell line cultivated in glucose media. Presently, with the addition of a triple negative breast cancer (TNBC) cell line, MDA-MB-231, we further confirm the effect of lactate on gene expression patterns and extend results to include lactate effects on protein expression. As well, we report effects of lactate on the expression of E-cadherin and vimentin, proteins associated with epithelial-to-mesenchymal transition (EMT). Endogenous lactate regulates the expression of multiple genes involved in carcinogenesis. In MCF7 cells, lactate increased the expression of EGFR, VEGF, HIF-1a, KRAS, MIF, mTOR, PIK3CA, TP53, and CDK4 as well as decreased the expression of ATM, BRCA1, BRCA2, E2F1, MET, MYC, and RAF mainly after 48h of exposure. On the other hand, in the MDA-MB-231 cell line, lactate increased the expressions of PIK3CA, VEGF, EGFR, mTOR, HIF-1α, ATM, E2F1, TP53 and decreased the expressions of BRCA1, BRCA2, CDK4, CDK6, MET, MIF, MYC, and RAF after 48h of exposure. In response to endogenous lactate, changes in protein expression of representative genes corroborated changes in mRNA expressions. Finally, lactate exposure decreased E-cadherin protein expression in MCF7 cells and increased vimentin expression in MDA-MB-231 cells. Furthermore, by genetically silencing LDHA in MCF7 cells, we show suppression of protein expression of EGFR and HIF-1α, while full protein expression occurred under glucose and glucose + exogenous lactate exposure. Hence, endogenous, glucose-derived lactate, and not glucose, elicited changes in gene and protein expression levels. In this study, we demonstrate that endogenous lactate produced under aerobic conditions (Warburg Effect) elicits important changes in gene and protein expression in both ER+ and TNBC cell lines. The widespread regulation of multiple genes by lactate and involves those involved in carcinogenesis including DNA repair, cell growth, proliferation, angiogenesis, and metastasis. Furthermore, lactate affected the expression of two relevant EMT biomarkers, E-cadherin and vimentin, which could contribute to the complex process of EMT and a shift towards a more mesenchymal phenotype in the two cancer cell lines studied.

Protein Expression of immune checkpoints STING and MHCII in small cell lung cancer.

BACKGROUND: SCLC is an aggressive malignancy where immunotherapies show limited efficacy. We aimed to characterize the SCLC microenvironment according to the expression patterns of SCLC subtype markers and novel immune checkpoints to identify therapeutic vulnerabilities. METHODS: We included SCLC tissue samples from 219 surgically resected, limited-stage patients in this cross-sectional study. We performed immunohistochemistry for STING and MHCII, as well as for the novel subtype markers (ASCL1, NEUROD1, POU2F3, YAP1). Moreover, we assessed CD45 + , CD8 + and CD68 + immune cell infiltration. RESULTS: 36% of SCLC tumors showed significant stromal or intraepithelial CD45 + immune cell infiltration. These patients exhibited significantly increased overall survival (OS) (vs. patients with immune-deserted tumors). High CD8 expression was associated with increased median OS. We found STING expression on cancer-associated fibroblasts in the stroma and on T-cells and macrophages in both tumorous and stromal compartments. STING expression positively correlated with immune cell infiltration. Increased STING-positivity in tumor nests was an independent favorable prognosticator for OS. ASCL1 was the most frequently expressed subtype-specific protein. Concomitant expression of three or four subtype-defining markers was seen in 13.8% of the included samples, whereas 24.1% of the cases were classified as quadruple negative tumors. YAP1 expression was associated with increased immune infiltrates. Tumor cell MHCII expression positively correlated with immune cell infiltration and with STING- and YAP1 expressions. CONCLUSIONS: STING and MHCII are expressed in SCLC. The majority of immune-infiltrated SCLCs exhibit increased STING expression. Immune infiltration and STING expression are prognostic in limited-stage SCLC, making STING a potential therapeutic target.

Expression patterns and prognostic relevance of subtype-specific transcription factors in surgically resected small-cell lung cancer: an international multicenter study.

The tissue distribution and prognostic relevance of subtype-specific proteins (ASCL1, NEUROD1, POU2F3, YAP1) present an evolving area of research in small-cell lung cancer (SCLC). The expression of subtype-specific transcription factors and P53 and RB1 proteins were measured by immunohistochemistry (IHC) in 386 surgically resected SCLC samples. Correlations between subtype-specific proteins and in vitro efficacy of various therapeutic agents were investigated by proteomics and cell viability assays in 26 human SCLC cell lines. Besides SCLC-A (ASCL1-dominant), SCLC-AN (combined ASCL1/NEUROD1), SCLC-N (NEUROD1-dominant), and SCLC-P (POU2F3-dominant), IHC and cluster analyses identified a quadruple-negative SCLC subtype (SCLC-QN). No unique YAP1-subtype was found. The highest overall survival rates were associated with non-neuroendocrine subtypes (SCLC-P and SCLC-QN) and the lowest with neuroendocrine subtypes (SCLC-A, SCLC-N, SCLC-AN). In univariate analyses, high ASCL1 expression was associated with poor prognosis and high POU2F3 expression with good prognosis. Notably, high ASCL1 expression influenced survival outcomes independently of other variables in a multivariate model. High POU2F3 and YAP1 protein abundances correlated with sensitivity and resistance to standard-of-care chemotherapeutics, respectively. Specific correlation patterns were also found between the efficacy of targeted agents and subtype-specific protein abundances. In conclusion, we investigated the clinicopathological relevance of SCLC molecular subtypes in a large cohort of surgically resected specimens. Differential IHC expression of ASCL1, NEUROD1, and POU2F3 defines SCLC subtypes. No YAP1-subtype can be distinguished by IHC. High POU2F3 expression is associated with improved survival in a univariate analysis, whereas elevated ASCL1 expression is an independent negative prognosticator. Proteomic and cell viability assays of human SCLC cell lines revealed distinct vulnerability profiles defined by transcription regulators. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Loss of STING expression is prognostic in non-small cell lung cancer.

BACKGROUND: Stimulator of interferon (IFN) genes (STING) is a protein that promotes type I IFN production essential for T-cell activation. In this study, we aim to characterize STING expression comprehensively using The Cancer Genome Atlas (TCGA) database, cell lines, and patient tumor samples stained with immunohistochemistry. METHODS: Two cohorts were evaluated comprising 721 non-small cell lung cancer (NSCLC) patients and 55 NSCLC cell lines for STING and cyclic GMP-AMP synthase (cGAS) expression using immunohistochemistry. Moreover, an independent cohort of n = 499 patients from the TCGA database was analyzed. Methylation was evaluated on STING and cGAS in five STING-negative NSCLC cell lines. RESULTS: STING RNA expression positively correlates with T cell function and development genes, negatively correlates with cell proliferation and associated with increased survival (5-year-overall survival [OS] 47.3% vs. 38.8%, p = 0.033). STING protein expression is significantly higher in adenocarcinoma (AC) and is lost with increasing stages of AC. STING-positivity is significantly higher in mutant EGFR and KRAS tumors. STING-positive NSCLC patients identified with immunohistochemistry (H-score > 50) have increased survival (median OS: 58 vs. 35 months, p = 0.02). Treatment of STING-negative cell lines with a demethylating agent restores STING expression. CONCLUSIONS: STING is ubiquitously expressed in NSCLC and associated with T cell function genes, AC histology, EGFR, and KRAS mutations and improved overall survival.

Characterization of Tumor-Associated Macrophages and the Immune Microenvironment in Limited-Stage Neuroendocrine-High and -Low Small Cell Lung Cancer.

This study aims to characterize tumor-infiltrating macrophages (TAMs), myeloid-derived suppressor cells (MDSC), and the related molecular milieu regulating anti-tumor immunity in limited-stage neuroendocrine (NE)-high and NE-low small cell lung cancer. Primary tumors and matched lymph node (LN) metastases of 32 resected, early-stage SCLC patients were analyzed by immunohistochemistry (IHC) with antibodies against pan-macrophage marker CD68, M2-macrophage marker CD163, and MDSC marker CD33. Area-adjusted cell counting on TMAs showed that TAMs are the most abundant cell type in the TME, and their number in tumor nests exceeds the number of CD3 + T-cells (64% vs. 38% in NE-low and 71% vs. 18% in NE-high). Furthermore, the ratio of CD163-expressing M2-polarized TAMs in tumor nests was significantly higher in NE-low vs. NE-high tumors (70% vs. 31%). TAM density shows a strong positive correlation with CD45 and CD3 in tumor nests, but not in the stroma. fGSEA analysis on a targeted RNAseq oncological panel of 2560 genes showed that NE-high tumors exhibited increased enrichment in pathways related to cell proliferation, whereas in NE-low tumors, immune response pathways were significantly upregulated. Interestingly, we identified a subset of NE-high tumors representing an immune-oasis phenotype, but with a different gene expression profile compared to NE-low tumors. In contrast, we found that a limited subgroup of NE-low tumors is immune-deserted and express distinct cellular pathways from NE-high tumors. Furthermore, we identified potential molecular targets based on our expression data in NE-low and immune-oasis tumor subsets, including CD70, ANXA1, ITGB6, TP63, IFI27, YBX3 and CXCR2.

Programmed Cell Death Ligand 1 Expression in Resected Non-Small Cell Lung Cancer.

BACKGROUND: Recently, anti-programmed cell death 1 (PD-1) and anti-programmed cell death ligand 1 (PD-L1) immunotherapies have yielded promising outcomes for patients with advanced non-small cell lung cancer (NSCLC) and led to great interest in applying these agents to treat resectable early-stage NSCLC. The objective of our study was to evaluate PD-L1 protein expression in resectable early-stage NSCLC specimens from a large Northern European cohort, examine the relationship to clinical characteristics, and demonstrate the prognostic role in resected NSCLC. MATERIALS AND METHODS: A large cohort of 875 NSCLC tumors consisted of 337 patients from Sweden and 538 patients from Norway was studied. All the patients had undergone pulmonary resection, and most patients had had early-stage NSCLC. PD-L1 protein expression was assessed by immunohistochemistry using the Dako PD-L1 22C3 pharmDx kit. The tumor proportion score for PD-L1 protein expression was compared with comprehensive demographic and clinicopathologic data. RESULTS: The overall prevalence of PD-L1 protein expression in the resectable NSCLC cohort was 9.5% at a tumor proportion score cutoff of ≥ 50%. Stage I NSCLC had lower PD-L1 expression compared with that of the other stages (P = .0012). PD-L1 expression correlated with wild-type EGFR gene expression (P = .0156) and mutated KRAS gene expression (P = .0004). No significant association was found between PD-L1 expression and mortality after multivariable adjustment for clinical characteristics, although the survival curves showed PD-L1 expression significantly correlated with a poor prognosis in the total NSCLC cohort and in the adenocarcinoma subgroup. CONCLUSION: PD-L1 expression in the present large cohort of resectable NSCLC was relatively low compared with data from clinical trials of advanced NSCLC. PD-L1 expression correlated positively with tumor stage, wild-type EGFR, and KRAS mutation. PD-L1 expression was not found as an independent prognostic factor in the present study. These findings could be important in the future when evaluating the role of anti-PD-1/PD-L1 immunotherapy in the setting of neoadjuvant or adjuvant trials for early-stage resectable NSCLC.

FGFR1 Is Critical for RBL2 Loss-Driven Tumor Development and Requires PLCG1 Activation for Continued Growth of Small Cell Lung Cancer.

Small cell lung cancer (SCLC) remains a recalcitrant disease where limited therapeutic options have not improved overall survival, and approved targeted therapies are lacking. Amplification of the tyrosine kinase receptor FGFR1 (fibroblast growth factor receptor 1) is one of the few actionable alterations found in the SCLC genome. However, efforts to develop targeted therapies for FGFR1-amplified SCLC are hindered by critical gaps in knowledge around the molecular origins and mediators of FGFR1-driven signaling as well as the physiologic impact of targeting FGFR1. Here we show that increased FGFR1 promotes tumorigenic progression in precancerous neuroendocrine cells and is required for SCLC development in vivo. Notably, Fgfr1 knockout suppressed tumor development in a mouse model lacking the retinoblastoma-like protein 2 (Rbl2) tumor suppressor gene but did not affect a model with wild-type Rbl2. In support of a functional interaction between these two genes, loss of RBL2 induced FGFR1 expression and restoration of RBL2 repressed it, suggesting a novel role for RBL2 as a regulator of FGFR1 in SCLC. Additionally, FGFR1 activated phospholipase C gamma 1 (PLCG1), whereas chemical inhibition of PLCG1 suppressed SCLC growth, implicating PLCG1 as an effector of FGFR1 signaling in SCLC. Collectively, this study uncovers mechanisms underlying FGFR1-driven SCLC that involve RBL2 upstream and PLCG1 downstream, thus providing potential biomarkers for anti-FGFR1 therapy. SIGNIFICANCE: This study identifies RBL2 and PLCG1 as critical components of amplified FGFR1 signaling in SCLC, thus representing potential targets for biomarker analysis and therapeutic development in this disease.

Fructose contributes to the Warburg effect for cancer growth.

Obesity and metabolic syndrome are strongly associated with cancer, and these disorders may share a common mechanism. Recently, fructose has emerged as a driving force to develop obesity and metabolic syndrome. Thus, we assume that fructose may be the mechanism to explain why obesity and metabolic syndrome are linked with cancer. Clinical and experimental evidence showed that fructose intake was associated with cancer growth and that fructose transporters are upregulated in various malignant tumors. Interestingly, fructose metabolism can be driven under low oxygen conditions, accelerates glucose utilization, and exhibits distinct effects as compared to glucose, including production of uric acid and lactate as major byproducts. Fructose promotes the Warburg effect to preferentially downregulate mitochondrial respiration and increases aerobic glycolysis that may aid metastases that initially have low oxygen supply. In the process, uric acid may facilitate carcinogenesis by inhibiting the TCA cycle, stimulating cell proliferation by mitochondrial ROS, and blocking fatty acid oxidation. Lactate may also contribute to cancer growth by suppressing fat oxidation and inducing oncogene expression. The ability of fructose metabolism to directly stimulate the glycolytic pathway may have been protective for animals living with limited access to oxygen, but may be deleterious toward stimulating cancer growth and metastasis for humans in modern society. Blocking fructose metabolism may be a novel approach for the prevention and treatment of cancer.

Multi-Institutional Prospective Validation of Prognostic mRNA Signatures in Early Stage Squamous Lung Cancer (Alliance).

INTRODUCTION: Surgical resection is curative for some patients with early lung squamous cell carcinoma. Staging and clinical factors do not adequately predict recurrence risk. We sought to validate the discriminative performance of proposed prognostic gene expression signatures at a level of rigor sufficient to support clinical use. METHODS: The two-stage validation used independent core laboratories, objective quality control standards, locked test parameters, and large multi-institutional specimen and data sets. The first stage validation confirmed a signature's ability to stratify patient survival. The second-stage validation determined which signature(s) optimally improved risk discrimination when added to baseline clinical predictors. Participants were prospectively enrolled in institutional (cohort I) or cooperative group (cohort II) biospecimen and data collection protocols. All cases underwent a central review of clinical, pathologic, and biospecimen parameters using objective criteria to determine final inclusion (cohort I: n = 249; cohort II: n = 234). Primary selection required that a signature significantly predict a 3-year survival after surgical resection in cohort I. Signatures meeting this criterion were further tested in cohort II, comparing risk prediction using baseline risk factors alone versus in combination with the signature. RESULTS: Male sex, advanced age, and higher stage were associated with shorter survival in cohort I and established a baseline clinical model. Of the three signatures validated in cohort I, one signature was validated in cohort II and statistically significantly enhanced the prognosis relative to the baseline model (C-index difference 0.122; p < 0.05). CONCLUSIONS: These results represent the first rigorous validation of a test appropriate to direct adjuvant treatment or clinical trials for patients with lung squamous cell carcinoma.

Role of mTOR As an Essential Kinase in SCLC.

OBJECTIVES: SCLC represents 15% of all lung cancer diagnoses in the United States and has a particularly poor prognosis. We hypothesized that kinases regulating SCLC survival pathways represent therapeutically targetable vulnerabilities whose inhibition may improve SCLC outcome. METHODS: A short-hairpin RNA (shRNA) library targeting all human kinases was introduced in seven chemonaive patient-derived xenografts (PDX) and the cells were cultured in vitro and in vivo. On harvest, lost or depleted shRNAs were considered as regulating-cell survival pathways and deemed essential kinases. RESULTS: Unsupervised hierarchical cluster analysis of recovered shRNAs separated the PDXs into two clusters, suggesting kinase-based heterogeneity among the SCLC PDXs. A total of 23 kinases were identified as essential in two or more PDXs, with mechanistic Target of Rapamycin (mTOR) a candidate essential kinase in four. mTOR phosphorylation status correlated with PDX sensitivity to mTOR kinase inhibition, and mTOR inhibition sensitized the PDX to cisplatin and etoposide. In the PDX in which mTOR was defined as essential, mTOR inhibition caused a 43% decrease in tumor volume at 21 days (p < 0.01). Combining mTOR inhibition with cisplatin and etoposide decreased PDX tumor volume 96% compared with cisplatin and etoposide alone at 70 days (p < 0.002). Chemoresistance did not develop with the combination of mTOR inhibition and cisplatin and etoposide in mTOR-essential PDX over 105 days. The prevalence of phospho-mTOR-Ser-2448 in a tissue microarray of chemonaive SCLC was 27%, thus, identifying an important SCLC subtype that might benefit from the addition of mTOR inhibition to standard chemotherapy. CONCLUSIONS: These studies reveal that kinases can define SCLC subgroups, can identify therapeutic vulnerabilities, and can potentially be used to optimize therapeutic approaches. Significance We used functional genomics to identify kinases regulating SCLC survival. mTOR was identified as essential in a subset of PDXs. mTOR inhibition decreased PDX growth, sensitized PDX to cisplatin and etoposide, and prevented chemoresistance.

Neuroendocrine subtypes of small cell lung cancer differ in terms of immune microenvironment and checkpoint molecule distribution.

Small cell lung cancer (SCLC) has recently been subcategorized into neuroendocrine (NE)-high and NE-low subtypes showing 'immune desert' and 'immune oasis' phenotypes, respectively. Here, we aimed to characterize the tumor microenvironment according to immune checkpoints and NE subtypes in human SCLC tissue samples at the protein level. In this cross-sectional study, we included 32 primary tumors and matched lymph node (LN) metastases of resected early-stage, histologically confirmed SCLC patients, which were previously clustered into NE subtypes using NE-associated key RNA genes. Immunohistochemistry (IHC) was performed on formalin-fixed paraffin-embedded TMAs with antibodies against CD45, CD3, CD8, MHCII, TIM3, immune checkpoint poliovirus receptor (PVR), and indoleamine 2,3-dioxygenase (IDO). The stroma was significantly more infiltrated by immune cells both in primary tumors and in LN metastases compared to tumor nests. Immune cell (CD45+ cell) density was significantly higher in tumor nests (P = 0.019), with increased CD8+ effector T-cell infiltration (P = 0.003) in NE-low vs NE-high tumors. The expression of IDO was confirmed on stromal and endothelial cells and was positively correlated with higher immune cell density both in primary tumors and in LN metastases, regardless of the NE pattern. Expression of IDO and PVR in tumor nests was significantly higher in NE-low primary tumors (vs NE-high, P < 0.05). We also found significantly higher MHC II expression by malignant cells in NE-low (vs NE-high, P = 0.004) tumors. TIM3 expression was significantly increased in NE-low (vs NE-high, P < 0.05) tumors and in LN metastases (vs primary tumors, P < 0.05). To our knowledge, this is the first human study that demonstrates in situ that NE-low SCLCs are associated with increased immune cell infiltration compared to NE-high tumors. PVR, IDO, MHCII, and TIM3 are emerging checkpoints in SCLC, with increased expression in the NE-low subtype, providing key insight for further prospective studies on potential biomarkers and targets for SCLC immunotherapies.

Comprehensive Immune Profiling of Medullary Thyroid Cancer.

Background: Despite advances in targeted kinase inhibitor development for patients with medullary thyroid cancer (MTC), most patients develop resistance and would benefit from alternative approaches. Immune-based therapies are now considered for patients with progressive MTC. This study is the first comprehensive assessment of the immune milieu, immune-suppressive molecules, and potential tumor antigens in patients with MTC. Methods: Primary and/or regionally metastatic tumor tissues from 46 patients with MTC were screened for immune infiltrates by using standard immunohistochemistry (IHC) and further analyzed by multispectral imaging for T cell and myeloid markers. RNASeq expression profiling was performed in parallel. RNASeq, targeted sequencing, and IHC techniques identified cancer-associated mutations and MTC-enriched proteins. Results: Organized immune infiltration was observed in 49% and 90% of primary and metastatic tumors, respectively. CD8+ cells were the dominant T cell subtype in most samples, while CD163+ macrophages were most frequent among myeloid infiltrates. PD-1+ T cells were evident in 24% of patients. Myeloid subsets were largely major histocompatibility complex II (MHCII-), suggesting a dysfunctional phenotype. Expression profiling confirmed enrichment in T cell, macrophage, and inflammatory profiles in a subset of samples. PD-L1 was expressed at low levels in a small subset of patients, while the immune regulatory molecules CD155 and CD47 were broadly expressed. Calcitonin, GRP, HIST1H4E, NOMO3, and NPIPA2 were highly and specifically expressed in MTC. Mutations in tumor suppressors, PTEN and p53, and mismatch repair genes, MSH2 and MSH6, may be relevant to disease progression and antigenicity. Conclusions: This study suggests that MTC is a more immunologically active tumor that has been previously reported. Patients with advanced MTC should be screened for targetable antigens and immune checkpoints to determine their eligibility for current clinical trials. Additional studies are necessary to fully characterize the antigenic potential of MTC and may encourage the development of adoptive T cells therapies for this rare tumor.

Fructose tolerance test in obese people with and without type 2 diabetes.

BACKGROUND: Fructose is distinct among common sugars in its ability to raise serum uric acid, and some studies suggest fructose-induced uric acid production may have a role in the ability of this sugar to induce metabolic syndrome. A fructose tolerance test has been previously developed to evaluate the relative ability of fructose to raise uric acid in individuals. However, the effect of fructose to raise uric acid in people with diabetes has not been studied. METHODS: People with type 2 diabetes (n = 143) and without diabetes controls (n = 132) with similar body mass index (BMI) underwent an oral fructose tolerance test. As a comparison, participants also had their uric acid levels measured after an oral glucose tolerance test on a different day. RESULTS: Serum uric acid was lower in people with type 2 diabetes compared to controls with a similar BMI, especially those with poor glucose control (glycosylated hemoglobin [HbA1c] ≥ 8%). Fructose administration raised serum uric acid in both groups, with a lower absolute rise in people with diabetes. People with diabetes with a blunted rise in serum uric acid had higher baseline serum uric acid concentrations and a higher BMI. People without diabetes with a higher BMI also showed a blunted serum uric acid response. Oral glucose administration lowered serum uric acid in both participants, with a greater fall in those with diabetes. CONCLUSION: Both the presence of diabetes and obesity blunt the serum uric acid response to fructose ingestion. These data demonstrate altered fructose-dependent urate metabolism in type 2 diabetes.

Round Robin Evaluation of MET Protein Expression in Lung Adenocarcinomas Improves Interobserver Concordance.

INTRODUCTION: Overexpression of the mesenchymal-epithelial transition (MET) receptor, a receptor tyrosine kinase, can propel the growth of cancer cells and portends poor prognoses for patients with lung cancer. Evaluation of MET by immunohistochemistry is challenging, with MET protein overexpression varying from 20% to 80% between lung cancer cohorts. Clinical trials using MET protein expression to select patients have also reported a wide range of positivity rates and outcomes. MATERIALS AND METHODS: To overcome this variability, the Lung Cancer Mutation Consortium Pathologist Panel endeavored to standardize the evaluation of MET protein expression with "Round Robin" conferences. This panel used randomly selected Aperio-scanned formalin-fixed paraffin-embedded lung cancer specimens stained by MET immunohistochemistry for the Lung Cancer Mutation Consortium 2.0 study (N=838). Seven pathologists in separate laboratories scored images of 5 initial cases and 2 subsequent rounds of 39 cases. The pathologists' scores were compared for consistency using the intraclass correlation coefficient. Issues affecting reproducibility were discussed in Round Robin conferences between rounds, and steps were taken to improve scoring consistency, such as sharing reference materials and example images. RESULTS: The overall group intraclass correlation coefficient comparing the consistency of scoring improved from 0.50 (95% confidence interval, 0.37-0.64) for the first scoring round to 0.74 (95% confidence interval, 0.64-0.83) for the second round. DISCUSSION: We found that the consistency of MET immunohistochemistry scoring is improved by continuous training and communication between pathologists.

EGFR-directed monoclonal antibodies in combination with chemotherapy for treatment of non-small-cell lung cancer: an updated review of clinical trials and new perspectives in biomarkers analysis.

Lung cancer still represents one of the most common and fatal neoplasm, accounting for nearly 30% of all cancer-related deaths. Targeted therapies based on molecular tumor features and programmed death-1 (PD-1)/programmed death ligand-1 (PDL-1) blockade immunotherapy have offered new therapeutic options for patients with advanced non-small-cell lung cancer (NSCLC). Activation of the epidermal growth factor receptor (EGFR)-pathway promotes tumor growth and progression, including angiogenesis, invasion, metastasis and inhibition of apoptosis, providing a strong rationale for targeting this pathway. EGFR expression is detected in up to 85% of NSCLC and has been demonstrated to be associated with poor prognosis. Two approaches for blocking EGFR signaling are available: prevention of ligand binding to the extracellular domain with monoclonal antibodies (mAbs) and inhibition of the intracellular tyrosine kinase activity with small molecules. There is a strong rationale to consider the tumor's level of EGFR expression as one of the most significant predictive biomarkers in this setting. In this paper we provide an update focusing on the current status of EGFR-directed mAbs use for the treatment of patients with advanced NSCLC, through a review of all clinical trials involving anti-EGFR mAbs in combination with chemotherapy (CT) for advanced disease and with chemo-radiotherapy for stage III disease. Here we also discuss the current status of predictive biomarkers for anti-EGFR mAbs when added to first-line CT in patients with advanced NSCLC. Finally, we focused on the relevance of EGFR fluorescence in situ hybridization (FISH)+ and immunohistochemistry (IHC)-Score ≥ 200 as predictive biomarkers for the selection of patients who would be most likely to derive a clinical benefit from treatment with CT in combination with anti-EGFR mAbs, with particular reference also to histology.

Comparative expression analysis in small cell lung carcinoma reveals neuroendocrine pattern change in primary tumor versus lymph node metastases.

BACKGROUND: Recent preclinical data suggest that neuroendocrine (NE) subtype of small cell lung cancer (SCLC) has strong therapeutic relevance. NE high tumors are associated with immune desert and NE low tumors are considered to have an immune oasis phenotype. Our aim was to investigate the NE phenotypes of surgically resected SCLC tumors according to inter-tumor heterogeneity. METHODS: Expression analysis for 2,560 genes was performed in 32 surgically resected SCLC patients' primary tumors and corresponding lymph node (LN) metastases. To analyze tumor heterogeneity, we examined the differences in the gene expression of primary tumors versus LN metastases. We performed cluster analysis and heat map to divide patients into NE high and low subtypes by using the top NE-associated genes described in preclinical studies. RESULTS: We found 6% (n=154) genes with significant differences and only 13.1% (n=336) of all genes in the panel had a strong correlation between the primary tumor and LN metastases. Cluster analysis clearly distinguished SCLC NE high versus low subtypes both in primary tumor (20 vs. 12, respectively) and LNs (23 vs. 9, respectively). As for inter-tumor heterogeneity, in case of five patients, a change in the NE pattern was observed. Specifically, we found significant downregulation of the NE-associated genes CAV1 (P=0.004), CAV2 (P=0.029) and ANXA3 (P=0.035) in their LN metastases compared to their primary tumor. CONCLUSIONS: Our data confirm the results of preclinical studies and clearly distinguish NE low and high differentiation clusters in SCLC. Moreover, they highlight the gene expression discordance between primary tumors and corresponding LN metastases suggesting that the NE pattern of metastatic LNs might not reflect that of the primary tumor. Altogether, by shedding light on the diversity of SCLC, the current study might help to improve patient selection and treatment in this devastating disease. KEYWORDS: Small cell lung cancer (SCLC); neuroendocrine tumor; lymph node metastasis; tumor heterogeneity; RNA sequencing.

Correlation of PD-L1 Expression with Tumor Mutation Burden and Gene Signatures for Prognosis in Early-Stage Squamous Cell Lung Carcinoma.

OBJECTIVES: Anti-programmed cell death 1 (PD-1)/programmed death ligand 1 (PD-L1) immunotherapy has demonstrated success in the treatment of advanced NSCLC. Recently, PD-1/PD-L1 blockade also has demonstrated interesting results in small trials of neoadjuvant treatment in stage IB to IIIA NSCLC. In addition, several clinical trials using anti-PD-1/PD-L1 immunotherapy as an adjuvant or neoadjuvant treatment in patients with resectable stage NSCLC are ongoing. However, few analyses of anti-PD-1/PD-L1 immunotherapy-related biomarkers in early-stage squamous cell lung carcinoma (SqCLC) have been reported. In this study, we evaluated PD-L1 protein expression, tumor mutation burden, and expression of an immune gene signature in early-stage SqCLC, providing data for identifying the potential role for patients with anti-PD-1/PD-L1 treatment in early-stage SqCLC. METHODS: A total of 255 specimens from patients with early-stage SqCLC were identified within participating centers of the Strategic Partnering to Evaluate Cancer Signatures program. PD-L1 protein expression by immunohistochemistry was evaluated by using the Dako PD-L1 22C3 pharmDx kit on the Dako Link 48 auto-stainer (Dako, Carpinteria, CA). Tumor mutation burden (TMB) was calculated on the basis of data from targeted genome sequencing. The T-effector and interferon gamma (IFN-γ) gene signature was determined from Affymetrix gene chip data (Affymetrix, Santa Clara, CA) from frozen specimens. RESULTS: The prevalence of PD-L1 expression was 9.8% at a tumor proportion score cutoff of at least 50%. PD-L1 mRNA and programmed cell death 1 ligand 2 mRNA positively correlated with PD-L1 protein expression on tumor cells (TCs) and tumor-infiltrating immune cells. PD-L1 protein expression on tumor-infiltrating immune cells was correlated with the T-effector and IFN-γ gene signature (p < 0.001), but not with TMB. For TCs, all of these biomarkers were independent of each other and neither PD-L1 protein expression, TMB, or T-effector and IFN-γ gene signatures were independently prognostic for patient outcomes. CONCLUSIONS: Evaluation of PD-L1 expression, TMB, and T-effector and IFN-γ gene signatures in the cohort with early-stage SqCLC found them to be independent of each other, and none was associated with overall survival. Our results also support the hypothesis that PD-L1 expression is regulated by an intrinsic mechanism on TCs and an adaptive mechanism on immune cells.

The immune checkpoint, HVEM may contribute to immune escape in non-small cell lung cancer lacking PD-L1 expression.

BACKGROUND: Herpes Virus Entry Mediator (HVEM) is an important immune checkpoint in cancer recognition. HVEM expressed on tumor cell membranes activates immune cell signaling pathways leading to either inhibition of activity (B- and T-lymphocyte attenuator, BTLA) or activation of immune activity (LIGHT). The aim of this study is to investigate the prevalence of HVEM expression and its association with PDL1 expression in NSCLC. METHODS: A TMA of 527 resected NSCLC samples and 56 NSCLC cell lines were evaluated for HVEM and PD-L1 expression. The IHC assay for HVEM was optimized on the Dako Link48 autostainer using a polyclonal antibody from R&D Systems(AF356). PD-L1 IHC was performed on the Dako Link48 autostainer using the PD-L1 28-8 pharmDx kit. Scoring HVEM employed the H-score system while for PD-L1 the tumor proportion score (TPS) was used. RESULTS: HVEM expression in the NSCLC resected samples and cell lines revealed a positive H-score more than 1 was 18.6% (77/415) and 48.2% (27/56) respectively. HVEM expression was significantly higher in patients with lymph node N2 metastasis (25.5% vs 7.9% vs 17.5%, p = 0.046) when comparing with N1 or no lymph node metastasis, and was marginally significantly higher in patients with stage III/IV disease (24.5% vs 16.4%, p = 0.059). Subgroup analysis showed that HVEM (median 45 vs 36 months, p = 0.706) and PD-L1 expression (median 45 vs 48 months, p = 0.178) status was not predictive of overall survival. HVEM was found to have a significant negative correlation with PD-L1 expression (r=-0.274, p < 0.001) in patients with NSCLC and also a weak negative correlation in NSCLC cell lines (r=-0.162, p = 0.352). CONCLUSION: HVEM was found to be overexpressed in NSCLC patients of N2 lymph node metastasis or later stage and has a negative co-relationship with PD-L1 expression. HVEM was not prognostic for NSCLC patients.

Fructose increases risk for kidney stones: potential role in metabolic syndrome and heat stress.

BACKGROUND: Fructose intake, mainly as table sugar or high fructose corn syrup, has increased in recent decades and is associated with increased risk for kidney stones. We hypothesized that fructose intake alters serum and urinary components involved in stone formation. METHODS: We analyzed a previously published randomized controlled study that included 33 healthy male adults (40-65 years of age) who ingested 200 g of fructose (supplied in a 2-L volume of 10% fructose in water) daily for 2 weeks. Participants were evaluated at the Unit of Nephrology of the Mateo Orfila Hospital in Menorca. Changes in serum levels of magnesium, calcium, uric acid, phosphorus, vitamin D, and intact PTH levels were evaluated. Urine magnesium, calcium, uric acid, phosphorus, citrate, oxalate, sodium, potassium, as well as urinary pH, were measured. RESULTS: Ingestion of fructose was associated with an increased serum level of uric acid (p < 0.001), a decrease in serum ionized calcium (p = 0.003) with a mild increase in PTH (p < 0.05) and a drop in urinary pH (p = 0.02), an increase in urine oxalate (p = 0.016) and decrease in urinary magnesium (p = 0.003). CONCLUSIONS: Fructose appears to increase urinary stone formation in part via effects on urate metabolism and urinary pH, and also via effects on oxalate. Fructose may be a contributing factor for the development of kidney stones in subjects with metabolic syndrome and those suffering from heat stress. TRIAL REGISTRATION: ClinicalTrials.gov NCT00639756 March 20, 2008.

Effects of fructose-containing sweeteners on fructose intestinal, hepatic, and oral bioavailability in dual-catheterized rats.

OBJECTIVE: Fructose is commonplace in Western diets and is consumed primarily through added sugars as sucrose or high fructose corn syrup. High consumption of fructose has been linked to the development of metabolic disorders, such as cardiovascular diseases. The majority of the harmful effects of fructose can be traced to its uncontrolled and rapid metabolism, primarily within the liver. It has been speculated that the formulation of fructose-containing sweeteners can have varying impacts on its adverse effects. Unfortunately, there is limited data supporting this hypothesis. The objective of this study was to examine the impact of different fructose-containing sweeteners on the intestinal, hepatic, and oral bioavailability of fructose. METHODS: Portal and femoral vein catheters were surgically implanted in male Wistar rats. Animals were gavaged with a 1 g/kg carbohydrate solution consisting of fructose, 45% glucose/55% fructose, sucrose, glucose, or water. Blood samples were then collected from the portal and systemic circulation. Fructose levels were measured and pharmacokinetic parameters were calculated. RESULTS: Compared to animals that were gavaged with 45% glucose/55% fructose or sucrose, fructose-gavaged animals had a 40% greater fructose area under the curve and a 15% greater change in maximum fructose concentration in the portal circulation. In the systemic circulation of fructose-gavaged animals, the fructose area under the curve was 17% and 24% higher and the change in the maximum fructose concentration was 15% and 30% higher than the animals that received 45% glucose/55% fructose or sucrose, respectively. After the oral administration of fructose, 45% glucose/55% fructose, and sucrose, the bioavailability of fructose was as follows: intestinal availability was 0.62, 0.53 and 0.57; hepatic availability was 0.33, 0.45 and 0.45; and oral bioavailability was 0.19, 0.23 and 0.24, respectively. CONCLUSIONS: Our studies show that the co-ingestion of glucose did not enhance fructose absorption, rather, it decreased fructose metabolism in the liver. The intestinal, hepatic, and oral bioavailability of fructose was similar between 45% glucose/55% fructose and sucrose.