Xiaohui Chen

Comparison of 22C3 PD-L1 Expression between Surgically Resected Specimens and Paired Tissue Microarrays in Non–Small Cell Lung Cancer

Introduction: The extent to which intratumoral heterogeneity of programmed death ligand 1 (PD‐L1) expression causes discordance of PD‐L1 expression between paired samples remains unclear. Here, PD‐L1 status was compared between whole sections from NSCLCs and the corresponding tissue microarrays (TMAs) serving as surrogate biopsy specimens. Methods: PD‐L1 expression was evaluated by 22C3 immunohistochemistry assay on 190 archival surgical specimens and matched to the TMA results. PD‐L1 expression was determined by the tumor proportion score (TPS) and classified as TPS lower than 1%, TPS of 1% to 49%, and TPS of 50% or higher. Agreement statistics were used. Results: The percentage of PD‐L1 expression on tumor cells differed greatly between individual TMAs and matched surgical specimens. When PD‐L1 TPS was adopted, a total of 36 of 190 discordance cases (18.9%) were observed, with a &kgr;‐value of 0.630 between paired samples. The TMAs underestimated or overestimated PD‐L1 status in 19 of 36 (52.8%) and 17 of 36 (47.2%) of the matched surgical specimens, respectively (p = 0.118). The discordance rate was much lower in cases with a PD‐L1 TPS lower than 1% compared with in cases with a TPS of 1% to 49% and TPS of 50% or higher (18.4% versus 56.7% and 43.3%, p < 0.001). When a TPS of 50% or higher was used as the cutoff, the discordance rate of PD‐L1 TPS less than 50% was further reduced to 7.5%. Such discrepancies were due mainly to intratumoral heterogeneity of PD‐L1 expression and nonsignificant association with clinicopathological features. Conclusions: PD‐L1 expression in TMAs correlates moderately well with that in the corresponding surgical specimens, indicating that evaluating PD‐L1 expression in diagnostic biopsy specimens could be misleading in defining sensitivity to pembrolizumab treatment yet may be reliable as a way to exclude patients with a PD‐L1 TPS less than 50% from first‐line pembrolizumab treatment.

The pattern of cervical lymph node metastasis in thoracic esophageal squamous cell carcinoma may affect the target decision for definitive radiotherapy

BACKGROUNDnMetastasis to lymph nodes is a key determinant of thoracic esophageal squamous cell carcinoma (TE-SCC) prognosis. We sought to identify factors linked with cervical lymph node metastasis, which could be used to inform the decision of surgical and definitive radiotherapy.nnnMETHODSnWe retrospectively reviewed records from 1715 patients who had had radical esophagectomy with three-field lymphadenectomy between January 1993 and March 2007 in our hospital. All patients included in the study had pathologically confirmed TE-SCC and no clinical evidence of cervical metastasis.nnnRESULTSnCervical node metastases were found in 547 patients (31.9%); rates of cervical-node positivity were 44.2% for those with upper-thoracic tumors, 31.5% for mid-thoracic tumors, and 14.4% for lower-thoracic tumors. Univariate analysis showed that cervical node metastasis was associated with tumor site, differentiation, and length, pathologic T status, and pN status (P<0.05); however, only tumor site and pN status retained significance in multivariate analysis (P<0.05). Positive cervical nodes were most often found in the paraesophageal region (72.3%), followed by supraclavicular (24.4%); involvement of deep cervical (2.4%) or retropharyngeal nodes (0.9%) was rare (P<0.0001). Positive cervical nodes were most often associated with upper TE-SCCs (60.1%), followed by middle TE-SCCs (31.2%) and lower TE-SCCs (10.6%).nnnCONCLUSIONSnUpper TE-SCC with multiple involved nodes at any site was associated with a high rate of cervical node metastasis. These findings provide critical information for clinical decision-making regarding the extent of nodal dissection or the size of radiation fields in definitive radiotherapy.

Prognostic factors in patients with thoracic esophageal carcinoma staged pT 1-4a N 0 M 0 undergone esophagectomy with three-field lymphadenectomy

BACKGROUNDnTo analyze prognostic factors in patients with thoracic esophageal carcinoma staged pT1-4aN0M0 and undergone esophagectomy with 3-field lymphadenectomy and to evaluate the effect of postoperative radiotherapy.nnnMETHODSnFrom January 1993 to March 2007, 770 patients with stage pT1-4aN0M0 underwent 3-field lymphadenectomy at Fujian Province Cancer Hospital, China were enrolled for analysis. The study consisted of 770 patients with stage pT1-4aN0M0 who underwent 3-field lymphadenectomy at Fujian Province Cancer Hospital, China. A total of 687 had received surgery only, and 83 patients had undergone surgery followed by postoperative radiotherapy. Radiation dose was 50 Gy in 25 fractions.nnnRESULTSnThe overall survival rates at 1, 3, 5, and 10 years were 92.9%, 80.8%, 71.7% and 57.4%, respectively. Univariate analysis showed that age and T staging were two independent factors on prognoses. Five-year survival in cases younger and older than 60 were 76.5% vs. 63.3% (P=0.001), while those of pT1, pT2, pT3 and pT4a were 83.8%, 78.8%, 67.8% and 54.1%, respectively (P=0.000). Five-year survival in group of simple surgery was 71.3%, compared with 74.5% in group of surgery plus postoperative radiotherapy (P=0.763), while stratified analysis indicated that postoperative radiotherapy was able to boost the survival of patients in pT4a which were 72.4% vs. 33.8% (P=0.036) and to lower relapse rate of tumor bed in patients with pT4a (P=0.005). Multivariate analysis showed that age and T staging were two independent factors on prognoses.nnnCONCLUSIONSnPatients with high T staging and at an age more than 60 turned out bad prognoses, neither could postoperative radiotherapy improve their survival.

TNF-related apoptosis-inducing ligand enhances vinorelbine-induced apoptosis and antitumor activity in a preclinical model of non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is relatively insensitive to chemotherapy. NP [vinorelbine (NVB)xa0+xa0cisplatin] is the standard chemotherapy regimen in clinical treatment; however, its side-effects are intolerable for most patients. In some reports, the TNF-related apoptosis-inducing ligand (TRAIL) can enhance the sensitivity of chemotherapy drugs by inducing apoptosis without toxicity to normal cells. In the present study, we evaluated the antitumor effects of the two drugs (TRAIL and NVB alone or in combination) by inducing apoptosis inxa0vitro and inxa0vivo. Using the human NSCLC cell line (A549) and a BALB/c nude mice model, we observed the cell viability (MTT assay), cell apoptosis [Hoechst staining, Annexinxa0V/propidium iodide (PI) staining assay, immunohistochemical staining, RT-PCR and western blotting] and cell proliferation (soft agar colony formation and cell cycle assay). The results showed that TRAIL and NVB alone inhibited tumor growth both inxa0vivo and inxa0vitro. However, the combination of the two drugs produced a more potent antitumor effect (P<0.05) and caused more severe apoptosis in tumor tissue (P<0.05). The key molecular protein level of the mitochondrial apoptotic pathway (Bax and caspase-3) was further upregulated by the combination of the two drugs (P<0.01) and either drug alone (P<0.05). The mRNA level of Bcl-2 and Bax in the combination group was downregulated and upregulated respectively, when compared with the control group (P<0.01). The combination group of A549 cells also showed lower viability compared with the one drug alone group (P<0.05). Moreover, the Hoechst staining assay and Annexinxa0V/PI staining assay showed that the combination of the two drugs induced a more potent apoptosis than either drug alone (P<0.05, early apoptosis P<0.01, respectively). In addition, the cell colony numbers were deduced after treatment with TRAIL or NVB alone (P<0.05) and the two drug combination (P<0.01). Cell cycle analysis showed that TRAIL and NVB alone markedly increased the percentage of cells in G1 phase (P<0.05) and the combination of the two drugs decreased the percentage of cells in G2 and S phase (P<0.05). Thus, the combination of TRAIL and NVB can inhibit lung cancer cell growth by affecting the level of key signaling protein expression of the apoptosis pathway.

Analysis of H3K27me3 expression and DNA methylation at CCGG sites in smoking and non-smoking patients with non-small cell lung cancer and their clinical significance

Smoking frequently leads to epigenetic alterations, including DNA methylation and histone modifications. The effect that smoking has on the DNA methylation levels at CCGG sites, the expression of trimethylation of histone H3 at lysine 27 (H3K27me3) and enhancer of zeste homolog 2 (EZH2), and their interactions in patients with non-small cell lung cancer (NSCLC) were analyzed. There were a total of 42 patients with NSCLC, 22 with adenocarcinomas and 20 with squamous cell carcinomas enrolled in the present study. Expression of H3K27me3, EZH2 and proliferating cellular nuclear antigen (PCNA) were immunohistochemically detected. DNA methylation at CCGG sites was evaluated via histoendonuclease-linked detection of DNA methylation sites. The apoptotic index of cancerous tissues obtained from patients of different smoking statuses was evaluated via the terminal deoxynucleotidyl-transferase-mediated dUTP-biotin nick end labeling method. The association with clinicopathological data was calculated relative to different smoking statuses. Compared with the non-smokers, smokers with NSCLC exhibited a significantly lower apoptotic index (P<0.05), and frequently had a lower level of DNA methylation at CCGG sites, lower H3K27me3 expression and a higher EZH2 expression (P<0.05). DNA methylation levels at CCGG sites were negatively correlated to the Brinkman index (P=0.017). Furthermore, there was a parallel association between the H3K27me3 and EZH2 expression levels in the majority of smokers, whereas in the majority of non-smokers, there was a diverging association (P=0.015). There was a diverging association between the PCNA and EZH2 expression levels in the majority of smokers; however, in the majority of non-smokers, there was a parallel association (P=0.048). In addition, the association between the CCGG methylation ratio and immunohistochemical expression of H3K27me3 was a parallel association in the majority of smokers, while in the majority of non-smokers there was a diverging association (P=0.049). Conclusively, patients with NSCLC and different smoking statuses exhibit different epigenetic characteristics. Additionally, DNA methylation levels at the CCGG sites may have the ability to determine associations between the expression levels of H3K27me3, EZH2 and PCNA.

Loss of expression rather than cytoplasmic mislocalization of RUNX3 predicts worse outcome in non-small cell lung cancer

Functional inactivation of human runt-related transcription factor 3 (RUNX3) through mutation or epigenetic silencing has been well-documented in many cancerous entities. In addition to gene mutation and promoter hypermethylation, cytoplasmic mislocalization has emerged as another major manifestation of RUNX3 dysfunction in malignancies including breast, colorectal and gastric cancers. The aim of the present study was to investigate whether patients with non-small cell lung cancer (NSCLC) and different RUNX3 expression patterns would have different overall survival (OS), and the associations between different patterns of clinicopathological parameters and clinical outcome. Expressions of RUNX3 and Ki-67 were immunohistochemically detected in normal lung tissue (n=5) and surgically resected tissues from NSCLC patients (n=188). The optimal cutoff of RUNX3 was determined by X-tile software associated with their survival. Apoptotic index in cancerous tissue was evaluated using the terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labelling method. The prognostic significance of different expression patterns of RUNX3 was determined by means of Kaplan-Meier survival estimates and log-rank tests. It was revealed that loss of RUNX3 expression in NSCLC was correlated with a low cancerous apoptotic index (P<0.001), shorter OS and worse prognosis (P=0.0142), while no statistical difference of apoptotic index (P=0.73) or survival (P=0.3781) was determined between patient subgroups with different localization of RUNX3 expression, which was quite different from the situation demonstrated in other malignancies. In conclusion, loss of expression rather than cytoplasmic mislocalization of RUNX3 predicted worse outcome in NSCLC, which was quite different from what manifested in other cancer types, and thus, the underlying mechanism may deserve further investigation.

Simultaneous VENTANA IHC and RT-PCR testing of ALK status in Chinese non-small cell lung cancer patients and response to crizotinib

BackgroundALK rearrangement-advanced NSCLC patients respond to crizotinib. ALK rearrangement is currently determined with RT-PCR. VENTANA IHC is a standard method to identify ALK protein overexpression in NSCLC; however, VENTANA IHC has rarely been used to determine the response to crizotinib in Chinese patients with NSCLC and ALK overexpression. To better clarify the clinical implication of VENTANA IHC to detect ALK rearrangements, we conducted this study to analyze VENTANA IHC and RT-PCR in a large cohort of Chinese patients with NSCLC undergoing screening for ALK rearrangements.MethodsA total of 1720 patients with NSCLC who had ALK rearrangements detected by VENTANA IHC and/or RT-PCR were included in this analysis. We compared the efficacy and survival of ALK-positive patients detected by VENTANA IHC and RT-PCR. We used NGS to identify patients in whom the two methods were inconsistent.ResultsAmong 1720 patients, 187 (10.87%) were shown to be ALK-positive by VENTANA IHC and/or RT-PCR, and 66 received crizotinib treatment. We identified 10.27% (172/1674) of patients as ALK-positive by the VENTANA IHC method, and 12.73% (41/322) of patients had ALK rearrangements by the RT-PCR method. Twenty-nine of 276 (10.51%) ALK-positive patients were simultaneously analyzed using VENTANA IHC and RT-PCR. The overall response rates were 65.90% (29/44) by VENTANA IHC and 55.88% (19/34) by RT-PCR. The disease control rates were 86.36% (38/44) by VENTANA IHC and 76.47% (26/34) by RT-PCR. In contrast, the median progression-free survival for VENTANA IHC and RT-PCR was 8.5 and 9.2xa0months, respectively. The VENTANA IHC and RT-PCR results obtained for 6 of 17 ALK-positive patients were inconsistent based on NGS; specifically, 4 patients had EML4-ALK fusions, 2 patients had non EML4-ALK fusions, 1 patient had a KCL1-ALK fusion, and one patient had a FBXO36-ALK fusion.ConclusionsVENTANA IHC is a reliable and rapid screening tool used in routine pathologic laboratories for the identification of suitable candidates for ALK-targeted therapy. VENTANA IHC has moderate sensitivity and a slightly higher association with response to therapy with ALK inhibitors, and some VENTANA IHC-positive, but RT-PCR-negative cases may benefit from crizotinib.

Histological characterisation and prognostic evaluation of 62 gastric neuroendocrine carcinomas

Aim of the study To determine the significance of expression of synaptophysin, chromogranin A, and Ki-67 and their association with clinicopathological parameters, and to find out the possible prognostic factors in gastric neuroendocrine carcinoma (G-NEC). Material and methods We investigated the immunohistochemical features and prognosis of 62 G-NECs, and evaluated the association among expressions of synaptophysin, chromogranin A, and Ki-67, clinicopathological variables, and outcome. Results Chromogranin A expression was found more commonly in small-cell NECs (9/9, 100%) than in large-cell NECs (27/53, 51%) (p = 0.008). No statistical significance was found in Ki-67 (p = 0.494) or synaptophysin (p > 0.1) expression between NEC cell types. Correlation analyses revealed that Ki-67 expression was significantly associated with mid-third disease of stomach (p = 0.005) and vascular involvement (p = 0.006), and had a trend of significant correlation with tumour relapse (p = 0.078). High expression of chromogranin A was significantly associated with histology of small-cell NECs (p = 0.008) and lesser tumour greatest dimension (p = 0.038). The prognostic significance was determined by means of Kaplan-Meier survival estimates and log-rank tests, and as a result, early TNM staging and postoperative chemotherapy were found to be correlated with longer overall survival (p < 0.05). Univariate analysis revealed associations between poor prognosis in NECs and several factors, including high TNM staging (p = 0.048), vascular involvement (p = 0.023), relapse (p = 0.004), and microscopic/macroscopic residual tumour (R1/2, p < 0.001). In a multivariate analysis, relapse was identified as the sole independent prognostic factor. Conclusions No significant correlation between survival and expression of synaptophysin, chromogranin A, or Ki-67 has been determined in G-NECs. Our study indicated that early diagnosis, no-residual-tumour resection, and postoperative chemotherapy were possible prognostic factors.

Comprehensive Predictors of Portal Pressure from Functional Liver Reserve in Patients Who Underwent Hepatectomy.

Portal hypertension is a major risk factor for hepatic failure or intestinal bleeding in patients with liver disease but cannot be measured indirectly. We attempted to comprehensively evaluate preoperative parameters of functional liver reserve that correlated with portal pressure (PP) in patients with various liver diseases. We examined 93 patients in whom portal pressure was directly measured during preoperative portal vein embolization (PVE) or operation. Background liver included chronic viral liver disease in 43 patients, obstructive jaundice in 29 patients, and normal liver in 21. Multivariate logistic analysis and linear regression analysis were applied to create a predictive formula for PP. Mean PP was 13.4u2009±u20094.9xa0cm H2O, and PP was significantly associated with severity of liver injury, hepatic fibrosis, intraoperative blood loss, and post-hepatectomy morbidity (pu2009<u20090.05 each). Mean PP after PVE (22.5u2009±u20097.8xa0cm H2O) was significantly increased compared to that before embolization (13.1u2009±u20094.7xa0cm H2O; pu2009<u20090.01). Univariate analysis identified seven significant parameters of preoperative liver function associated with PP: indocyanine green (ICG) test result, liver uptake and clearance index (HH15) on 99mTc-galactosyl serum albumin liver scintigraphy, total bilirubin level, prothrombin activity, and hyaluronate level. Using multiple linear regression analysis, the predictive formula using ICG and HH15 was as follows: Y (estimated PP)u2009=u20090.273u2009+u20090.086u2009×u2009ICGR15u2009+u20090.193u2009×u2009HH15. The calculated PP (11.5u2009±u20094.6xa0cm H2O (−1.9xa0cm H2O)) was lower than true PP, which was significantly associated with post-hepatectomy morbidity (pu2009<u20090.05). The correlation between true and calculated PP was weak, and prediction using the conventional liver functional parameters was limited at present and, however, estimating PP appears to be useful in evaluating portal hypertension and post-hepatectomy morbidity.