Genomic Profiling Reveals Novel Predictive Biomarkers for Chemo-Radiotherapy Efficacy and Thoracic Toxicity in Non-Small-Cell Lung Cancer.

Chemo-radiotherapy (CRT) remains the main treatment modality for non-small-cell lung cancer (NSCLC). However, its clinical efficacy is largely limited by individual variations in radio-sensitivity and radiotherapy-associated toxicity. There is an urgent need to identify genetic determinants that can explain patients' likelihood to develop recurrence and radiotherapy-associated toxicity following CRT. In this study, we performed comprehensive genomic profiling, using a 474-cancer- and radiotherapy-related gene panel, on pretreatment biopsy samples from patients with unresectable stage III NSCLCs who underwent definitive CRT. Patients' baseline clinical characteristics and genomic features, including tumor genetic, genomic and molecular pathway alterations, as well as single nucleotide polymorphisms (SNPs), were correlated with progression-free survival (PFS), overall survival (OS), and radiotherapy-associated pneumonitis and/or esophagitis development after CRT. A total of 122 patients were enrolled between 2014 and 2019, with 84 (69%) squamous cell carcinomas and 38 (31%) adenocarcinomas. Genetic analysis confirmed the association between the KEAP1-NRF2 pathway gene alterations and unfavorable survival outcome, and revealed alterations in FGFR family genes, MET, PTEN, and NOTCH2 as potential novel and independent risk factors of poor post-CRT survival. Combined analysis of such alterations led to improved stratification of the risk populations. In addition, patients with EGFR activating mutations or any oncogenic driver mutations exhibited improved OS. On the other hand, we also identified genetic markers in relation to radiotherapy-associated thoracic toxicity. SNPs in the DNA repair-associated XRCC5 (rs3835) and XRCC1 (rs25487) were associated with an increased risk of high-grade esophagitis and pneumonitis respectively. MTHFR (rs1801133) and NQO1 (rs1800566) were additional risk alleles related to higher susceptibility to pneumonitis and esophagitis overall. Moreover, through their roles in genome integrity and replicative fidelity, somatic alterations in ZNF217 and POLD1 might also serve as risk predictors of high-grade pneumonitis and esophagitis. Taken together, leveraging targeted next-generating sequencing, we identified a set of novel clinically applicable biomarkers that might enable prediction of survival outcomes and risk of radiotherapy-associated thoracic toxicities. Our findings highlight the value of pre-treatment genetic testing to better inform CRT outcomes and clinical actions in stage III unresectable NSCLCs.

Genomic Correlates of Unfavorable Outcome in Locally Advanced Cervical Cancer Treated with Neoadjuvant Chemoradiation.

PURPOSE: Neoadjuvant therapy modality can increase the operability rate and mitigate pathological risks in locally advanced cervical cancer, but treatment response varies widely. It remains unclear whether genetic alterations correlate with the response to neoadjuvant therapy and disease-free survival (DFS) in locally advanced cervical cancer. MATERIALS AND METHODS: A total of 62 locally advanced cervical cancer (stage IB-IIA) patients who received neoadjuvant chemoradiation plus radical hysterectomy were retrospectively analyzed. Patients' tumor biopsy samples were comprehensively profiled using targeted next generation sequencing. Pathologic response to neoadjuvant treatment and DFS were evaluated against the association with genomic traits. RESULTS: Genetic alterations of PIK3CA were most frequent (37%), comparable to that of Caucasian populations from The Cancer Genome Atlas. The mutation frequency of genes including TERT, POLD1, NOS2, and FGFR3 was significantly higher in Chinese patients whereas RPTOR, EGFR, and TP53 were underrepresented in comparison to Caucasians. Germline mutations were identified in 21% (13/62) of the cohort and more than half (57%) had mutations in DNA damage repair genes, including BRCA1/2, TP53 and PALB2. Importantly, high tumor mutation burden, TP53 polymorphism (rs1042522), and KEAP1 mutations were found to be associated with poor pathologic response to neoadjuvant chemoradiation treatment. KEAP1 mutations, PIK3CA-SOX2 co-amplification, TERC copy number gain, and TYMS polymorphism correlated with an increased risk of disease relapse. CONCLUSION: We report the genomic profile of locally advanced cervical cancer patients and the distinction between Asian and Caucasian cohorts. Our findings highlight genomic traits associated with unfavorable neoadjuvant chemoradiation response and a higher risk of early disease recurrence.

Comprehensive Next-Generation Sequencing Reveals Novel Predictive Biomarkers of Recurrence and Thoracic Toxicity Risks After Chemoradiation Therapy in Limited Stage Small Cell Lung Cancer.

PURPOSE: Although definitive chemoradiation therapy (dCRT) remains the most effective treatment modality in limited stage small cell lung cancer (SCLC), some patients progress quickly or develop serious radiation-induced thoracic toxicity (RITT). Molecular correlates of response to dCRT remain to be explored. METHODS AND MATERIALS: Genomic profiling was performed retrospectively on 231 patients with limited-stage SCLC treated with dCRT between 2015 and 2019 using a customized panel covering cancer and radiation therapy response-related genes. Exploratory associations of progression-free survival, overall survival, and RITT with clinical features, tumor genetics, genomic and molecular pathway alterations, and single nucleotide polymorphisms were conducted. RESULTS: In addition to the common SCLC genes, such as TP53, RB1, and NOTCH1/2, potentially actionable mutations in EGFR, KRAS, and BRCA1/2 were among the top alterations in the cohort. At the single-gene level, CDK4 and GATA6 alterations were independent predictors of poor survival by multivariate analysis. At the genomic level, high tumor mutational burden was strongly associated with favorable survival outcome. Pathway-level analysis showed that activating mutations in the MAPK/ERK pathway genes, particularly those in EGFR/ERBB2, correlated with poor survival. Combined analysis enabled optimized risk stratification of post-dCRT survival. On the other hand, our study also confirmed that single nucleotide polymorphisms in MTHFR, CYP2B6, NQO1, and LIG4 were risk alleles of high-grade RITT. Remarkably, somatic loss-of-function mutations in the DNA damage repair pathway genes were associated with increased risk of high-grade RITT, particularly pneumonitis, which likely reflect a complex interplay between the tumor and its immune microenvironment. CONCLUSIONS: Taken together, by examining the mutational landscape of a large cohort of limited-stage SCLC, we identified novel molecular predictors of survival and RITT. Our findings also implicate several key molecular pathways, including the MAPK/ERK and DNA damage repair pathways, in the regulation of dCRT response.

Clinicopathologic analysis of microscopic tumor extension in glioma for external beam radiotherapy planning.

BACKGROUND: There is no consensus regarding the clinical target volume (CTV) margins in radiotherapy for glioma. In this study, we aimed to perform a complete macropathologic analysis examining microscopic tumor extension (ME) to more accurately define the CTV in glioma. METHODS: Thirty-eight supra-total resection specimens of glioma patients were examined on histologic sections. The ME distance, defined as the maximum linear distance from the tumor border to the invasive tumor cells, was measured at each section. We defined the CTV based on the relationships between ME distance and clinicopathologic features. RESULTS: Between February 2016 and July 2020, a total of 814 slides were examined, corresponding to 162 slides for low-grade glioma (LGG) and 652 slides for high-grade glioma (HGG). The ME value was 0.69 ± 0.43 cm for LGG and 1.29 ± 0.54 cm for HGG (P < 0.001). After multivariate analysis, tumor grade, O6-methylguanine-DNA-methyltransferase promoter methylated status (MGMTm), isocitrate dehydrogenase wild-type status (IDHwt), and 1p/19q non-co-deleted status (non-codel) were positively correlated with ME distance (all P < 0.05). We defined the CTV of glioma based on tumor grade. To take into account approximately 95% of the ME, a margin of 1.00 cm, 1.50 cm, and 2.00 cm were chosen for grade II, grade III, and grade IV glioma, respectively. Paired analysis of molecularly defined patients confirmed that tumors that had all three molecular alterations (i.e., MGMTm/IDHwt/non-codel) were the most aggressive subgroups (all P < 0.05). For these patients, the margin could be up to 1.50 cm, 2.00 cm, and 2.50 cm for grade II, grade III, and grade IV glioma, respectively, to cover the subclinical lesions in 95% of cases. CONCLUSIONS: The ME was different between the grades of gliomas. It may be reasonable to recommend 1.00 cm, 1.50 cm, and 2.00 cm CTV margins for grade II, grade III, and grade IV glioma, respectively. Considering the highly aggressive nature of MGMTm/IDHwt/non-codel tumors, for these patients, the margin could be further expanded by 0.5 cm. These recommendations would encompass microscopic disease extension in 95% of cases. TRIAL REGISTRATION: The trial was registered with Chinese Clinical Trial Registry ( ChiCTR2100049376 ).

Determining optimal clinical target volume margins in high-grade glioma based on microscopic tumor extension and magnetic resonance imaging.

INTRODUCTION: In this study, we performed a consecutive macropathologic analysis to assess microscopic extension (ME) in high-grade glioma (HGG) to determine appropriate clinical target volume (CTV) margins for radiotherapy. MATERIALS AND METHODS: The study included HGG patients with tumors located in non-functional areas, and supratotal resection was performed. The ME distance from the edge of the tumor to the microscopic tumor cells surrounding brain tissue was measured. Associations between the extent of ME and clinicopathological characteristics were evaluated by multivariate linear regression (MVLR) analysis. An ME predictive model was developed based on the MVLR model. RESULTS: Between June 2017 and July 2019, 652 pathologic slides obtained from 30 HGG patients were analyzed. The mean ME distance was 1.70 cm (range, 0.63 to 2.87 cm). The MVLR analysis identified that pathologic grade, subventricular zone (SVZ) contact and O6-methylguanine-DNA methyltransferase (MGMT) methylation, isocitrate dehydrogenase (IDH) mutation and 1p/19q co-deletion status were independent variables predicting ME (all P < 0.05). A multivariable prediction model was developed as follows: YME = 0.672 + 0.513XGrade + 0.380XSVZ + 0.439XMGMT + 0.320XIDH + 0.333X1p/19q. The R-square value of goodness of fit was 0.780. The receiver operating characteristic curve proved that the area under the curve was 0.964 (P < 0.001). CONCLUSION: ME was heterogeneously distributed across different grades of gliomas according to the tumor location and molecular marker status, which indicated that CTV delineation should be individualized. The model could predict the ME of HGG, which may help clinicians determine the CTV for individual patients. Trial registration The trial was registered with Chinese Clinical Trial Registry (ChiCTR2100046106). Registered 4 May 2021-Retrospectively registered.

PD-L1 in Combination with CD8+TIL and HIF-1α are Promising Prognosis Predictors of Head and Neck Squamous Cell Carcinoma.

OBJECTIVE: The aim of this study was to evaluate the prognosis effect of PD-L1 in combination with CD8+ tumor-infiltrating lymphocyte (TIL) or HIF-1α in head and neck squamous cell carcinoma (HNSCC). METHODS: A total of 63 patients who underwent surgical resection were included in this study. The level of PD-L1, CD8+ TIL, and HIF-1α was determined by immunohistochemical analysis. The survival of patients was evaluated by Kaplan-Meier analysis. The prognostic power of these parameters was evaluated by C-index. RESULTS: We observed that the survival of patients, who had a high level of PD-L1 in tumor cells, was significantly shorter than those who had a low level of PD-L1. However, the survival of patients who had a high level of PD-L1 in tumor microenvironment was significantly longer than patients with a low level of PD-L1 in tumor microenvironment. In addition, high level of CD8+ tumor-infiltrating lymphocyte or low level of HIF-1α level suggests a better prognosis. Moreover, we observed that PD-L1 in combination with CD8+ tumor-infiltrating lymphocyte and HIF-1α could significantly improve the prognostic effect of current TNM stage. CONCLUSION: The results of this study suggest that the level of PD-L1, CD8+TIL, and HIF-1α are useful prognostic biomarkers for patients with HNSCC. Incorporating these biomarkers into current TNM stage of HNSCC improve the discriminatory capability of TNM stage.

Genomic alterations of whole exome sequencing in esophageal squamous cell carcinoma before and after radiotherapy.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is among the leading causes of cancer mortality, especially in China. Advances in technology have resulted in significant clinical gains in the treatment of ESCC, with more precise radiotherapy now considered an integral part of standard patient care, either alone or in combination with chemotherapy. Though, a better understanding of tumoral radiosensitivity is still needed in order to develop strategies and further personalize radiation treatments. METHODS: We carried out whole-exome sequencing (WES) on paired tumors collected before and after radiotherapy from 11 patients with ESCC. A comprehensive analysis was performed to compare the somatic mutations, the driver genes mutations, the copy number variations (CNVs), the mutational signatures, the tumor's clonal composition, and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway between pre- and post-radiotherapy samples in this cohort. RESULTS: According to the analysis of WES results, more insertion/deletion mutations (indels) were discovered in the post-radiotherapy samples than in the pre-radiotherapy samples (Wilcoxon rank-sum test, P=0.014). The mutation rate of driver gene Ephrin-A2 (EPHA2) was significantly reduced after radiotherapy (Fisher's exact test, P=0.035). However, comparison between the pre- and post-radiotherapy groups reveals no significant differences in other content. CONCLUSIONS: Our study revealed the overall genomic profile of ESCC before and after radiotherapy and determined that a loss of EPHA2 mutations might make cancer cells resistance to radiotherapy.

Robust performance of a novel stool DNA test of methylated SDC2 for colorectal cancer detection: a multicenter clinical study.

BACKGROUND AND AIMS: Stool DNA testing is an emerging and attractive option for colorectal cancer (CRC) screening. We previously evaluated the feasibility of a stool DNA (sDNA) test of methylated SDC2 for CRC detection. The aim of this study was to assess its performance in a multicenter clinical trial setting. METHODS: Each participant was required to undergo a sDNA test and a reference colonoscopy. The sDNA test consists of quantitative assessment of methylation status of SDC2 promoter. Results of real-time quantitative methylation-specific PCR were dichotomized as positive and negative, and the main evaluation indexes were sensitivity, specificity, and kappa value. All sDNA tests were performed and analyzed independently of colonoscopy. RESULTS: Among the 1110 participants from three clinical sites analyzed, 359 and 38 were diagnosed, respectively, with CRC and advanced adenomas by colonoscopy. The sensitivity of the sDNA test was 301/359 (83.8%) for CRC, 16/38 (42.1%) for advanced adenomas, and 134/154 (87.0%) for early stage CRC (stage I-II). Detection rate did not vary significantly according to age, tumor location, differentiation, and TNM stage, except for gender. The follow-up testing of 40 postoperative patients with CRC returned negative results as their tumors had been surgically removed. The specificity of the sDNA test was 699/713 (98.0%), and unrelated cancers and diseases did not seem to interfere with the testing. The kappa value was 0.84, implying an excellent diagnostic consistency between the sDNA test and colonoscopy. CONCLUSION: Noninvasive sDNA test using methylated SDC2 as the exclusive biomarker is a clinically viable and accurate CRC detection method. CHINESE CLINICAL TRIAL REGISTRY: Chi-CTR-TRC-1900026409, retrospectively registered on October 8, 2019; http://www.chictr.org.cn/edit.aspx?pid=43888&htm=4 .

Correlation of hypoxia as measured by fluorine-18 fluoroerythronitroimidazole (18F-FETNIM) PET/CT and overall survival in glioma patients.

PURPOSE: Hypoxia is important in the biology of glioma in humans. Positron emission tomography/computed tomography (PET/CT) with a hypoxia tracer offers a noninvasive method to differentiate individual tumor biology and potentially modify treatment for patients with malignancies. The purpose of this study was to determine whether hypoxia, as measured by fluorine-18 fluoroerythronitroimidazole (18F-FETNIM) PET/CT, was associated with tumor grade, overall survival (OS), and immunohistochemical features related to hypoxia, proliferation, angiogenesis, and the invasion of gliomas. PROCEDURES: Twenty-five patients with gliomas in whom gross maximal resection could be safely attempted were analyzed. All patients underwent 18F-FETNIM PET/CT studies before surgery. The maximum standardized uptake value (SUVmax) was obtained from the PET images of tumor tissues. Tumor specimens were stereotactically obtained for the immunohistochemical staining of hypoxia-inducible factor-1 alpha (HIF-1α), Ki-67, vascular endothelial growth factor (VEGF), and matrix metalloproteinase 9 (MMP-9). RESULTS: A correlation between the SUVmax and glioma grade was found (r = 0.881, P < 0.001). The SUVmax was significantly correlated with the expression of HIF-1α, Ki-67, VEGF, and MMP-9 (r = 0.820, 0.747, 0.606, and 0.727; all P < 0.001). Patients with a high SUVmax had significantly worse 3-year OS than those with a low SUVmax (24.4% vs. 82.1%, P = 0.003). CONCLUSIONS: 18F-FETNIM PET/CT provides an excellent noninvasive assessment of hypoxia in glioma. It can be used to understand the mechanisms by which hypoxia affects the OS of glioma patients.

Expression and prognostic role of IKBKE and TBK1 in stage I non-small cell lung cancer.

BACKGROUND: The inhibitors of nuclear factor kappa-B kinase subunit epsilon (IKBKE) and TANK-binding kinase 1 (TBK1) are important members of the nonclassical IKK family that share the kinase domain. They are important oncogenes for activation of several signaling pathways in several tumors. This study aims to explore the expression of IKBKE and TBK1 and their prognostic role in stage I non-small cell lung cancer (NSCLC). PATIENTS AND METHODS: A total of 142 surgically resected stage I NSCLC patients were enrolled and immunohistochemistry of IKBKE and TBK1 was performed. RESULTS: IKBKE and TBK1 were expressed in 121 (85.2%) and 114 (80.3%) of stage I NSCLC patients respectively. IKBKE expression was significantly associated with TBK1 expression (P=0.004). Furthermore, multivariate regression analyses showed there was a significant relationship between patients with risk factors, the recurrence pattern of metastasis and IKBKE+/TBK1+ co-expression (P=0.032 and P=0.022, respectively).  In Kaplan-Meier survival curve analyses, the IKBKE+/TBK1+ co-expression subgroup was significantly associated with poor overall survival (P=0.014). CONCLUSIONS: This is the first study to investigate the relationship between IKBKE and TBK1 expression and clinicopathologic characteristics in stage I NSCLC patients. IKBKE+/TBK1+ co-expression was significantly obvious in patients with risk factors and with recurrence pattern of distant metastasis. Furthermore, IKBKE+/TBK1+ is also an effective prognostic predictor for poor overall survival.

Prevalence of ROS1 fusion in Chinese patients with non-small cell lung cancer.

BACKGROUND: The study was conducted to investigate the clinicopathological features and prevalence of ROS1 gene fusion in Chinese patients with non-small cell lung cancer (NSCLC). METHODS: The presence of ROS1 fusion was assessed by quantitative real-time PCR. Associations between ROS1 fusion and clinical characteristics were analyzed. RESULTS: In total, 6066 patients with pathologically confirmed NSCLC and ROS1 fusion test results were enrolled. The average age was 60.89 ± 10.60 years and fusion was detected in 157 (2.59%) patients. Fusion frequency was significantly correlated with age, gender, smoking status (all P < 0.001), pathology type (P = 0.017), and lymph node metastasis stage (P = 0.027). ROS1 fusion-positive patients were significantly younger (55.68 ± 11.34 vs. negative 61.02 ± 10.44 years; P < 0.01). Fusion frequency was higher in women (3.71% vs. men 1.81%), never-smokers (3.33% vs. smokers 1.21%), and patients with adenocarcinoma (2.77% vs. squamous lung cancer 0.93%) and at advanced node stages (1.31%, 1.40%, 2.07%, and 3.23% for N0, N1, N2, and N3, respectively). No significant correlation between ROS1 fusion status and pathological stage was found in subgroups classified by pathological, tumor, or metastasis stage (P > 0.05). Age, smoking status, and lymph node stage were statistically significantly correlated with ROS1 fusion frequency (all P < 0.05); gender and pathology type were not significantly correlated with ROS1 fusion status after adjusting for smoking status. CONCLUSION: An overall ROS1 fusion frequency of 2.59% was confirmed in this study. ROS1 fusion was more prevalent among younger patients, never-smokers, and those at advanced node stages.

Melanotic Xp11 translocation renal cancer: a report of a distinctive case and a review of the literature.

BACKGROUND: Melanotic Xp11 translocation renal cancer (TRC) is a newly described exceedingly rare tumor, and its characterization remains controversial. This study aimed to describe a case of distinctive melanotic Xp11 TRC and to elucidate its clinicopathological and molecular genetic features. CASE PRESENTATION: A 44-year-old Chinese female presented with a left renal mass. Abdominal ultrasonography and computed tomography (CT) scans revealed a 4.5 cm × 4.0 cm mass in the left kidney. Grossly, the well-demarcated mass was black with moderately firm consistency. Microscopic examination indicated that the tumor was characterized by the presence of nests and cords of polygonal cells with clear and granular eosinophilic cytoplasm, central round to oval nuclei and occasional nucleoli. Intracytoplasmic melanin was observed in approximately 45% of tumor cells. Uniquely, the tumor presented with intranuclear eosinophilic pseudoinclusions and thick-walled stromal blood vessels. IHC showed that tumor cells were diffusely positive for TFE3 and exhibited patchy and weak HMB45 staining. FISH confirmed the presence of TFE3 rearrangement. CONCLUSION: This case is the twentieth published case of melanotic Xp11 TRC. Moreover, the present patient had a favorable prognosis given that she was disease free at her 113-month postoperative follow-up. Our case adds to the small body of literature on these exceptionally rare tumors and widens their clinicopathological spectrum.

Relationship between expression of PD-L1 and tumor angiogenesis, proliferation, and invasion in glioma.

Programmed death ligand 1 (PD-L1) is highly expressed in many cancers. We investigated the expression of PD-L1 and its relationship with vascular endothelial growth factor (VEGF), matrix metalloproteinase-9 and KI-67 expression in 64 patients with primary glioma. The expression rate of PD-L1 in glioma patients was 78.12%. PD-L1 levels correlated with the tumor grade (p = 0.013), VEGF status (p = 0.002) and KI-67 status (p = 0.002). In addition, PD-L1 levels correlated positively with VEGF (r = 0.314, p = 0.011) and KI-67 (r = 0.391, p = 0.001) levels when the data were treated as continuous variables. This is the first report suggesting that PD-L1 is important for glioma angiogenesis and proliferation. Thus, further research should be conducted to assess the combination of targeted VEGF therapy and anti-PD-L1 immunotherapy for the treatment of glioma.

Nestin servers as a promising prognostic biomarker in non-small cell lung cancer.

Lung cancer is currently the leading cause of cancer-related death worldwide and it is important to identify the predictive and/or prognostic markers for the cancer. Nestin, a proliferative and multipotent biomarker has been reported to be associated with prognosis in non-small cell lung cancer (NSCLC) in a few studies. In the present study, we retrospectively recruited 153 patients with NSCLC. Nestin protein expression in tumor samples was determined by immunohistochemistry staining. Nestin expression was related with tumor differentiation (P=0.036), lymphatic metastasis (N stage, P=0.011), and p-TNM stage (P=0.013), while there was no significant association between Nestin expression level and age, smoking habits, gender, histologic type, and T stage. Nestin was an independent prognostic factor for overall survival in NSCLC with an adjusted hazard ratio of 2.701 (95% CI, 1.616-4.513, P<0.001) after controlling the confounding factors. Then we determined the effects of Nestin on cell proliferation, colony formation, invasion, and apoptosis by knockout of Nestin with a new developed method, CRISPR/Cas9 mediated genome editing. It was observed that knockout of Nestin caused enhancement of cancer cell apoptosis and inhibition of cell proliferation, colony formation, and invasion in A549 and H1299 cell lines. Furthermore, we examined the expression of epithelial-mesenchymal transition (EMT) related biomarkers such as E-cadherin and Vimentin in Nestin-depleted lung cancer cells and knockout of Nestin was found to inhibit EMT, suggesting the involvement of Nestin mediated EMT signaling in lung cancer. The finding above demonstrated that Nestin might serve as a prognostic factor and therapeutic target in NSCLCs.

A comparison study between gross tumor volumes defined by preoperative magnetic resonance imaging, postoperative specimens, and tumor bed for radiotherapy after breast-conserving surgery.

BACKGROUND: The identification and contouring of target volume is important for breast-conserving therapy. The aim of the study was to compare preoperative magnetic resonance imaging (MRI), postoperative pathology, excised specimens' (ES) size, and tumor bed (TB) delineation as methods for determining the gross tumor volume (GTV) for radiotherapy after breast-conserving surgery (BCS). METHODS: Thirty-three patients with breast cancer who underwent preoperative MRI and radiotherapy after BCS were enrolled. The GTVs determined by MRI, pathology, and the ES were defined as GTVMRI, GTVPAT, and GTVES, respectively. GTVMRI+1 was defined as a 1.0-cm margin around the GTVMRI. The radiation oncologist delineated GTV of the TB (GTVTB) using planning computed tomography according to ≥5 surgical clips placed in the lumpectomy cavity (LC). RESULTS: The median GTVMRI, GTVMRI+1, GTVPAT, GTVES, and GTVTB were 0.97 cm (range, 0.01-6.88), 12.58 cm (range, 3.90-34.13), 0.97 cm (range, 0.01-6.36), 15.46 cm (range, 1.15-70.69), and 19.24 cm (range, 4.72-54.33), respectively. There were no significant differences between GTVMRI and GTVPAT, GTVMRI+1 and GTVES, GTVES and GTVTB (P = 0.188, 0.070, and 0.264, respectively). GTVMRI is positively related with GTVPAT. However, neither GTVES nor GTVTB correlated with GTVMRI (P = 0.071 and 0.378, respectively). Furthermore, neither GTVES nor GTVTB correlated with GTVMRI+1 (P = 0.068 and 0.375, respectively). CONCLUSION: When ≥5 surgical clips were placed in the LC for BCS, the volume of TB was consistent with the volume of ES. Neither the volume of TB nor the volume of ES correlated significantly with the volume of tumor defined by preoperative MRI.

[Gene Expression and Clinical Characteristics of Molecular Targeted Therapy 
in Non-small Cell Lung Cancer Patients in Shandong].

BACKGROUND: Molecular targeted therapy has gradually become an important treatment for lung cancer, the aim of this research is to analyze the clinicopathologic features associated with the gene mutation status of epidermal growth factor receptor (EGFR), echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase (EML4-ALK), ROS proto-oncogene 1, receptor tyrosine kinase (ROS1) and Kirsten rat sarcoma viral oncogene (KRAS) in non-small cell lung cancer (NSCLC) patients and determine the most likely populations to benefit from molecular target therapy treatment. METHODS: The mutation status of EGFR, EML4-ALK fusion gene, ROS1 and KARS gene were determined by Real-time PCR, the relationship between clinical pathologic features and concomitant gene were analyzed with χ2 test by SPSS software 19.0. RESULTS: A total of 514 specimens from Shandong tumor hospital were collected from NSCLC patients between January 2014 and May 2016. The total mutation rate of EGFR gene was 36.70%, major occurred in exon 19 (36.61%) and exon 21 (51.36%), respectively, and EGFR mutations usually occurred in female, non-smoking and adenocarcinoma patients (P<0.05). The total rearrangements rate of EML4-ALK fusion gene was 9.37%, EML4-ALK fusion gene usually occurred in younger age (≤60 yr) and non-smoking patients (P<0.05). Mutations were not related to gender and pathological type (P>0.05). ROS1 fusion gene was detected in 136 cases, the positive rate was 3.67%, all patients were 60 years old, and the difference was statistically significant (P<0.05). Only 23 samples were tested KARS gene mutations, two of them were positive and the positive rate was 8.70%. They all occurred in non-smoker and adenocarcinoma patients. No mutation was detected to coexist in EGFR, EML4-ALK and KARS gene mutation. CONCLUSIONS: EGFR, EML4-ALK, ROS1 and KRAS defines different molecular subset of NSCLC with distinct characteristic, which provides a new option for the clinical treatment of patients with NSCLC.

A clear cell adenocarcinoma of the gallbladder with hepatoid differentiation: case report and review of literature.

An 80-year-old male was referred to our department for a gallbladder mass. He denied any history of alcohol consumption or cholecystitis and smoking. Hepatitis B surface antigen test and antihepatitis C antibody test were found to be negative. Serum carbohydrate antigen 19-9 (CA19-9) and carcinoembryonic antigen were elevated (CA19-9 was 59.92 U/mL and carcinoembryonic antigen was 12.64 ng/mL), whereas alpha-fetoprotein was below the normal limit (2.46 ng/mL). Computed tomography scan revealed a solid mass with measurements of 4.6×5.6×7.1 cm, which nearly filled the whole gallbladder space. Radical cholecystectomy, including segments IV B and V of the liver and lymphadenectomy, was performed. The neoplasm in gallbladder was completely resected, and the patient obtained a negative margin. Histological and immunohistochemical profile suggested a clear cell adenocarcinoma of the gallbladder with hepatoid differentiation. After reviewing the literature, we reported that this case is the first identified case of cell adenocarcinoma of the gallbladder with extensive hepatoid differentiation. However, clinical features of clear cell adenocarcinoma with hepatoid differentiation remain unclear due to the extremely rare incidence. There was no indication of adjuvant chemotherapy and no literature has been reported on the application of chemotherapy. This case showed a promising clinical outcome after curative resection, which indicated that surgical treatment could be potentially considered for suitable patients.

Expressions of CD8+TILs, PD-L1 and Foxp3+TILs in stage I NSCLC guiding adjuvant chemotherapy decisions.

PURPOSE: Currently, adjuvant chemotherapy is recommended for patients with high risk stage I non-small cell lung cancer (NSCLC). However, identifying high risk patients remains a challenge. This study aims to identify the patient cohorts more likely to benefit from adjuvant chemotherapy based on the tumor micro-immune environment. RESULTS: CD8+TILs significantly associated with disease-free survival (DFS) and overall survial (OS) (p=0.002; 0.040). Patients with high risk factors may also predict shorter DFS (P=0.056). When compared together, patients with high-CD8+TILs showed better DFS than patients with low-CD8+TILs, no matter their risk factors status. There's no correlation between PD-L1 expressions and survival. PD-L1 was highly expressed in men, squamous and well differentiated carcinoma. In addition, Foxp3+TILs alone didn't show any prognostic effects, but low-Foxp3/high-CD8+TILs were associated with prolonged DFS (p=0.031). METHODS: A total of 126 patients with surgically resected stage I NSCLC were included to perform immunohistochemistry of CD8+ tumor infiltrating lymphocytes (TILs), programmed death ligand-1(PD-L1) and forkhead box P3 (Foxp3)+TILs. CONCLUSION: CD8+TILs are effective prognostic predictors. Patients with surgically resected stage I NSCLC showing low CD8+TILs could be considered for adjuvant chemotherapy, even if they have no high risk features.

CD8+/FOXP3+ ratio and PD-L1 expression associated with survival in pT3N0M0 stage esophageal squamous cell cancer.

Data describing relationships between the tumor immune microenvironment and patient outcome are limited for esophageal squamous cell cancer (ESCC). The present study investigated the prognostic values of programmed death-ligand 1 (PD-L1) expression and CD8+ or forkhead box protein 3+ (FOXP3+) tumor-infiltrating lymphocytes (TILs) in 133 pathological T3N0M0 stage ESCC patients who underwent radical resection without neoadjuvant or adjuvant therapy. CD8+ and FOXP3+ TIL densities as well as PD-L1 levels in tumor cells and lymphocytes, were assessed through immunohistochemical staining. Patient survival was not associated with CD8+ or FOXP3+ TILs alone, but PD-L1 expression and the CD8+/FOXP3+ ratio were independent predictors of both disease-free and overall survival. PD-L1 expression correlated with age (p = 0.029), tumor length (p < 0.001), tumor differentiation status (p = 0.002) and reduced intratumoral CD8+ TIL density (p < 0.001). Our results suggest pT3N0M0 ESCC clinical outcomes correlate with CD8+ and FOXP3+ TIL densities and PD-L1 levels. Moreover, an intrinsic mechanism for induction of PD-L1 overexpression may be occurring during early tumor oncogenesis. This information may be useful for stratifying patients and guide the application of checkpoint blockade therapy in ESCC.