Wentao Yang

An Interobserver Reproducibility Analysis of Ki67 Visual Assessment in Breast Cancer

Background Ki67 labeling index (LI) is used as a predictive marker and is associated with prognosis in breast cancer. However, standardised methodologies for measurement are lacking which has limited its application in clinical practice. In this study, we evaluated the interobserver concordance of visual assessment of Ki67 LI in breast cancer. Methods Ki67- immunostained slides of 160 cases of primary invasive breast cancer were visual assessed by five breast pathologists with two different methods to choose the scoring fields: (1) hot-spot score, (2) average score. Proportions of positive invasive tumor cells at 10 % intervals were scored. The intra-class correlation coefficient (ICC) was used to assess the interobserver reproducibility. Results (1) A perfect concordance of Ki67 LI was demonstrated according to both score methods (P<0.0001). Average score method (ICC, 0.904) demonstrated a better correlation than hot-spot score method (ICC, 0.894). (2) By respective means according to two score methods, all cases were classified into three groups (≤10%, 11%-30% and >30% Ki-67 LI). The concordance was relatively low in intermediate Ki67 LI group compared with low and high Ki67 LI groups. (3) All cases were classified into three groups by paired-difference (d) between means of hot-spot score and average score (d<5, 5≤d<10, d≥10). The consistency was observed to decrease with increasing paired-difference according to both methods. Conclusions Visual assessment of Ki67 LI at 10 % intervals is a candidate for a standard method in breast cancer clinical practice. Average score and hot-spot score of visual assessment both demonstrated a perfect concordance, and an overall average assessment across the whole section including hot spots may be a better method. Interobserver concordance of intermediate Ki67 LI in which most cutoffs are located for making clinical decisions was relatively low.

Preoperative Neutrophil-to-Lymphocyte Ratio as a Predictive and Prognostic Factor for High-Grade Serous Ovarian Cancer

Objective We aimed to demonstrate the clinical and prognostic significance of the preoperative neutrophil-to-lymphocyte ratio (NLR) in high-grade serous ovarian cancer (HGSC). Methods We retrospectively investigated 875 patients who underwent primary staging or debulking surgery for HGSC between April 2005 and June 2013 at our institution. None of these patients received neoadjuvant chemotherapy. NLR was defined as the absolute neutrophil count divided by the absolute lymphocyte count. Progression-free survival (PFS) and overall survival (OS) were analyzed with the Kaplan-Meier method and log-rank tests for univariate analyses. For multivariate analyses, Cox regression analysis was used to evaluate the effects of the prognostic factors, which were expressed as hazard ratios (HRs). Results The NLRs ranged from 0.30 to 24.0. The median value was 3.24 and used as the cutoff value to discriminate between the high-NLR (≥3.24) and low-NLR (<3.24) groups. A high preoperative NLR level was associated with an advanced FIGO stage, increased CA125 level, more extensive ascites, worse cytoreduction outcome and chemoresistance. For univariate analyses, a high NLR was associated with reduced PFS (p<0.001) and OS (p<0.001). In multivariate analyses, a high NLR was still an independent predictor of PFS (p = 0.011), but not OS (p = 0.148). Conclusion Our study demonstrated that NLR could reflect tumor burden and clinical outcomes to a certain extent and should be regarded as a predictive and prognostic parameter for HGSC.

Prognostic impact of the time interval from primary surgery to intravenous chemotherapy in high grade serous ovarian cancer

OBJECTIVEnThe aim of our study was to investigate the prognostic influence of time to chemotherapy (TTC) in patients with high grade serous ovarian cancer (HGSC).nnnMETHODSnWe retrospectively investigated 625 consecutive patients with HGSC who underwent primary staging or debulking surgery followed by platinum-based intravenous chemotherapy between April 2005 and June 2013 in our center. TTC was defined as the time interval between primary surgery and initiation of chemotherapy.nnnRESULTSnThe median (range) TTC was 15 (4-62) days. TTC was longer for patients who underwent bowel resection (p<0.001). There were no differences in PFS and OS between patients initiating chemotherapy before and after 15days (p=0.604 and 0.826, respectively) or among 4 groups categorized by quartile values (<10days, 10-14days, 15-20days, or ≥21days after surgery) (p=0.471 and 0.516, respectively). When stratified by with and without residual disease, there were still no differences in PFS (p=0.592 and 0.755, respectively) and OS (p=0.962 and 0.640, respectively) between patients initiating chemotherapy before and after 15days. In multivariate analyses, TTC was also not associated with PFS and OS categorized by median (p=0.570 and 0.701, respectively), quartile values (p=0.432, 0.194, 0.737 and 0.799, 0.290, 0.743, respectively) or integrated as a continuous variable (p=0.550 and 0.430, respectively).nnnCONCLUSIONnThe time interval between surgery and chemotherapy seemed to have no prognostic impact on patients with HGSC within 6weeks.

A Comparison of Visual Assessment and Automated Digital Image Analysis of Ki67 Labeling Index in Breast Cancer

Background Ki67 labeling index (LI) is critical for treatment options and prognosis evaluation in breast cancer. Visual assessment (VA) is widely used to assess Ki67 LI, but has some limitations. In this study, we compared the consistency between VA and automated digital image analysis (DIA) of Ki67 LI in breast cancer, and to evaluate the application value of DIA in Ki67 LI assessment. Methods Ki67 immunostained slides of 155 cases of primary invasive breast cancer were eyeballing assessed by five breast pathologists and automated digital image analyzed by one breast pathologist respectively. Two score methods, hot-spot score and average score, were used to choose score areas. The intra-class correlation coefficient (ICC) was used to analyze the consistency between VA and DIA, and Wilcoxon signed-rank test was used to compare the median of paired-difference between VA and DIA values. Results (1) A perfect agreement was demonstrated between VA and DIA of Ki67 LI by ICC analysis (P<0.0001) in the whole cohort. A perfect agreement between VA and DIA of Ki67 LI was also showed in G2-G3, ER positive/HER2 negative cases. Average score and hot-spot score methods both demonstrated a perfect concordance between VA and DIA of Ki67 LI. (2) All cases were classified into three groups by VA values (≤10%, 11%-30% and >30% Ki67 LI). The concordance was relatively lower in intermediate Ki67 LI group (11%-30%) compared with high (>30%) Ki67 LI groups according to both methods. (3) All cases were classified into three groups by paired-difference (d) between VA values of hot-spot score and average score (d<5, 5≤d<10, d≥10) to evaluate the correlation between Ki67 staining distribution (heterogeneous or homogenous) and reproducibility of assessment. A perfect agreement was all demonstrated in three groups, and a slightly better Ki67 LI agreement between VA and DIA was indicated in homogenous staining slides than in heterogeneous staining ones. (4) VA values were relatively smaller than DIA values (average score: median of paired-difference -3.72; hot-spot score: median of paired-difference -9.12). Conclusions An excellent agreement between VA and DIA of Ki67 LI in breast cancer was demonstrated in the whole mixed cohort, suggesting that VA and DIA both could be used to assess Ki67 LI in clinical practice. Average score and hot-spot score methods both demonstrated a perfect concordance between VA and DIA of Ki67 LI. The almost perfect agreement between VA and DIA was observed in high Ki67 LI cases, displaying a homogenous staining pattern. The consistency between VA and DIA was relatively low in intermediate Ki67 LI group. The heterogeneity of tumors may slightly affect the concordance between VA and DIA of Ki67 LI. Assessment of VA provides lower Ki67 values than DIA, the biological importance of these values are not known at the moment.

Thrombocytosis and hyperfibrinogenemia are predictive factors of clinical outcomes in high-grade serous ovarian cancer patients

BackgroundOver 20xa0% of ovarian cancer patients have preoperative thrombocytosis or hyperfibrinogenemia. We aimed to demonstrate the clinical and prognostic significance of thrombocytosis and hyperfibrinogenemia in high-grade serous ovarian cancer (HGSC).MethodsWe retrospectively investigated HGSC patients who underwent primary staging or debulking surgery between April 2005 and June 2013 in our institution. None of these patients had received neoadjuvant chemotherapy. Data, including age, performance status, FIGO stage, serum CA125, platelet count, fibrinogen level, and surgical residual disease, were collected. Thrombocytosis was defined as a platelet count greater than 450u2009×u2009109/L, and hyperfibrinogenemia was defined as a fibrinogen level higher than 4.00xa0g/L. Progression-free survival (PFS) and overall survival (OS) were analyzed with the Kaplan-Meier method and log-rank tests for univariate analyses. For the multivariate analyses, Cox regression analysis was used to evaluate the effects of the prognostic factors, which are expressed as hazard ratios (HRs).ResultsA total of 875 consecutive HGSC patients were identified. The median follow-up time was 29 (1–115) months. The median (interquartile range, IQR) preoperative platelet count was 301 (235–383)u2009×u2009109/L, and 121 (13.8xa0%) women had thrombocytosis. The median (IQR) preoperative fibrinogen level was 3.85 (3.19–4.45) g/L, and 332 (45.9xa0%) of the patients had hyperfibrinogenemia. Both preoperative thrombocytosis and hyperfibrinogenemia were associated with an advanced FIGO stage (pu2009=u20090.008 and <0.001, respectively), an increased CA125 level (pu2009=u20090.004 and 0.001, respectively), more extensive ascites (pu2009<u20090.001 and <0.001, respectively), more extensive residual disease (pu2009<u20090.001 and <0.001, respectively) and chemosensitivity (pu2009=u20090.043 and <0.001, respectively). In the univariate analyses, hyperfibrinogenemia was associated with reduced PFS (pu2009<u20090.001) and OS (pu2009<u20090.001). However, thrombocytosis was not found to be a potential predictor of PFS (Pu2009=u20090.098) or OS (pu2009=u20090.894). In the multivariate analyses, hyperfibrinogenemia was an independent predictor of OS (pu2009=u20090.014) but not PFS (pu2009=u20090.062).ConclusionPreoperative thrombocytosis and hyperfibrinogenemia reflected tumor burden to some extent and thus influenced treatment outcomes, and the fibrinogen level was found to be useful as a prognostic predictor in the HGSC patients.

Evaluation of the prognostic value of tumor-infiltrating lymphocytes in triple-negative breast cancers.

Tumor-infiltrating lymphocytes (TILs) may be associated with clinical outcome in triple-negative breast cancers (TNBCs). However, lacking of standardized methodologies in TILs evaluation has hindered its application in clinical practice. To evaluate the prognostic role of TILs scored by methods recommended by International TILs Working Group 2014, we performed a retrospective study of TILs in 425 primary invasive TNBCs in a Chinese population with a median follow-up of 4 years. Intratumoral TILs (iTILs) and stromal TILs (sTILs) were scored respectively. The associations between TILs and disease-free survival (DFS), distant disease-free survival (DDFS) and overall survival (OS) were evaluated with COX models. ITILs were not associated with prognosis. Higher sTILs were associated with better prognosis; for every 10% increase in sTILs, a 5% reduction of risk of recurrence or death (P < 0.001), 5% reduction of risk of distant recurrence (P < 0.001), and 4% reduction of risk of death (P = 0.002) were observed. Multivariate analysis confirmed sTILs to be an independent prognostic marker. 3.5% of TNBCs had more than 50% lymphocytes (lymphocyte-predominant breast cancer, LPBC), and associations between LPBC status and prognosis were observed but did not reach statistical significance. TNBCs with more than 20% sTILs had a significantly better prognosis than the patients with no more than 20% sTILs. In conclusion, our study indicated that sTILs scored by methods recommended by International TILs Working Group 2014 were associated with the prognosis of TNBCs. STILs could be an independent prognostic biomarker in TNBCs, increasing sTILs predicting better prognosis.

HDAC5, a potential therapeutic target and prognostic biomarker, promotes proliferation, invasion and migration in human breast cancer

Purpose Histone deacetylase 5 (HDAC5) is an important protein in neural and cardiac diseases and a potential drug target. However, little is known regarding the specific role of HDAC5 in breast cancer (BC). We aimed to evaluate HDAC5 expression in human breast tumors and to determine the effects of the inhibition of HDAC5 expression in BC cells. Experimental design HDAC5 expression was evaluated in BC patients and was correlated with clinical features and with patient prognosis. Functional experiments were performed using shRNA and the selective HDAC inhibitor LMK-235 for HDAC5 knockdown and inhibition in BC cells. The synergistic effects of LMK-235 with the proteasome inhibitor bortezomib were also examined. Results HDAC5 was extensively expressed in human BC tissues, and high HDAC5 expression was associated with an inferior prognosis. Knockdown of HDAC5 inhibited cell proliferation, migration, invasion, and enhanced apoptosis. The HDAC5 inhibitor LMK-235 inhibited cell growth and induced apoptosis, while the inclusion of bortezomib synergistically enhanced the efficacy of LMK-235. Conclusions Our findings indicate that HDAC5 is a promising prognostic marker and drug target for BC and that the combination of LMK-235 and bortezomib presents a novel therapeutic strategy for BC.

A clinically applicable molecular classification for high-grade serous ovarian cancer based on hormone receptor expression

To establish an effective hormone receptor-based molecular classification of high-grade serous ovarian cancer (HGSC), we retrospectively examined 875 consecutive HGSC patients who underwent primary surgery at our hospital and constructed tissue microarrays from these specimens. The expression levels of the hormone receptors were as follows: ER 64.4%, PR 12.6%, AR 35.6%, FSHR 54.5%, LHR 34.8%, and GnRHR 88.3%. Based on clustering of their expression patterns, we classified patients into five subgroups with distinctive clinical features (PR+, PRu2009−u2009ERu2009+u2009AR+, PRu2009−u2009ERu2009+u2009AR−, PRu2009−u2009ERu2009−u2009AR+, and PRu2009−u2009ERu2009−u2009AR−). Patients in the PRu2009+u2009group were younger compared to those in the other groups (pu2009<u20090.001). More patients were of advanced stage in the PRu2009−u2009ERu2009+u2009AR− group than the other groups (pu2009=u20090.020). A greater proportion of patients were sensitive to platinum-based chemotherapy in the PRu2009−u2009ERu2009−u2009ARu2009+u2009group compared with the other groups (pu2009=u20090.034). A trend of increasing risk of death was observed among these subgroups (pu2009<u20090.001). In the multivariate analysis, patients also had orderly increased hazard ratios for death in the PRu2009+u2009(HRu2009=u20092.256, 95% CI, 0.983–5.175), PRu2009−u2009ERu2009+u2009ARu2009+u2009(HRu2009=u20092.188, 95% CI, 1.004–4.796), PRu2009−u2009ERu2009−u2009AR− (HRu2009=u20092.316, 95% CI, 1.097–5.082) and PRu2009−u2009ERu2009+u2009AR− (HRu2009=u20092.928, 95% CI, 1.366–6.276) subgroups compared to the PRu2009−u2009ERu2009−u2009AR+ subgroup. Our classification could help predict patient clinical outcomes, guide individual treatments and stratify patients in future clinical trials.

PIK3CA mutation analysis in Chinese patients with surgically resected cervical cancer.

The aim of this study was to evaluate the clinicopathological and prognostic relevance of PIK3CA mutations in Chinese patients with surgically resected cervical cancer. PIK3CA mutations were screened in 771 cervical cancer specimens using reverse transcription polymerase chain reaction and Sanger sequencing. In total, 13.6% (105 of 771) of patients harbored non-synonymous PIK3CA mutations. Patients harboring PIK3CA mutations were older than patients with wild-type PIK3CA (mean age: 50.7 years vs. 47.0 years, Pu2009<u20090.01). PIK3CA mutations were more commonly observed in postmenopausal patients than in premenopausal patients (19.6% vs. 10.2%, Pu2009<u20090.01). PIK3CA mutations were more common in squamous cell carcinomas than in non-squamous cell tumors (15.3% vs 7.3%, of Pu2009<u20090.01). The 3-year relapse-free survival was 90.2% for PIK3CA mutant patients and 80.9% for PIK3CA wild-type patients (Pu2009=u20090.03). PIK3CA mutation was confirmed as an independent predictor for better treatment outcome in the multivariate analyses (HRu2009=u20090.54, 95% CI: 0.29–0.99, Pu2009=u20090.048). PIK3CA mutations were significantly associated with less distant metastases (mutant-type: 8/105, wild-type: 98/666, pu2009=u20090.048). Thus, patients with mutant PIK3CA had distinct characteristics in age, menopausal status, and histological subtype and have better treatment outcome and less distant metastasis after surgery-based multimodal therapy.

Heterogeneity of PD-L1 expression in primary tumors and paired lymph node metastases of triple negative breast cancer

BackgroundProgrammed cell death ligand 1 (PD-L1) is a potential predictive biomarker of the response to anti-PD-L1/anti- programmed cell death 1 (PD-1) therapy in multiple cancers, including triple negative breast cancer(TNBC). The purpose of this study was to investigate whether PD-L1 expression is homogenous in primary tumors(PTs) and synchronous axillary lymph node metastases(LNMs) of TNBC.MethodsPD-L1 expression was immunohistochemically evaluated in 101 TNBC patients’ PTs and paired LNMs. PD-L1 expression in tumor cells and infiltrating immune cells or node lymphocytes in the PTs and associated LNMs was scored separately and was correlated with patients’ clinical parameters and prognoses.ResultsPD-L1 expression exhibited spatial heterogeneity in both the tumor cells and the infiltrating immune cells or node lymphocytes of PTs and LNMs. The PD-L1 expression levels were significantly higher in the lymphocytes and tumor cells of the LNMs than in the PTs. PD-L1 expression was also more frequent among the LNMs. PD-L1 expression was associated with high grade and more stromal tumor-infiltrating lymphocytes(TILs). Furthermore, the disease-free survival and overall survival were similar between the PT- negative/LNM- positive and PT- positive/LNM- positive patients, both of which exhibited worse disease-free survival(DFS) thanPT -negative/LNM -negative patients.ConclusionsThe differential expression of PD-L1 between the PTs and LNMs suggests that LNMs PD-L1 status may be used to indicate whether PD-1/PD-L1-targeted therapy would be suitable for a node-positive TNBC patient in the future.