AGR2 and FOXA1 as prognostic markers in ER-positive breast cancer.

BACKGROUND: The prognostic role of either forkhead box A1 (FOXA1) or anterior gradient 2 (AGR2) in breast cancer has been found separately. Considering that there were interplays between them depending on ER status, we aimed to assess the statistical interaction between AGR2 and FOXA1 on breast cancer prognosis and examine the prognostic role of the combination of them by ER status. METHODS: AGR2 and FOXA1 expression in tumor tissues were evaluated with tissue microarrays by immunohistochemistry in 915 breast cancer patients with follow up data. The expression levels of these two markers were treated as binary variables, and many different cutoff values were tried for each marker. Survival and Cox proportional hazard analyses were used to evaluate the relationship between AGR2, FOXA1 and prognosis, and the statistical interaction between them on the prognosis was assessed on multiplicative scale. RESULTS: Statistical interaction between AGR2 and FOXA1 on the PFS was significant with all the cutoff points in ER-positive breast cancer patients but not ER-negative ones. Among ER-positive patients, the poor prognostic role of the high level of FOXA1 was significant only in patients with the low level of AGR2, and vice versa. When AGR2 and FOXA1 were considered together, patients with low levels of both markers had significantly longer PFS compared with all other groups. CONCLUSIONS: There was a statistical interaction between AGR2 and FOXA1 on the prognosis of ER-positive breast cancer. The combination of AGR2 and FOXA1 was a more useful marker for the prognosis of ER-positive breast cancer patients.

Combined low levels of H4K16ac and H4K20me3 predicts poor prognosis in breast cancer.

BACKGROUND: Results of previous studies about the prognostic roles of histone H4 lysine 16 acetylation (H4K16ac) and histone H4 lysine 20 trimethylation (H4K20me3) in breast cancer were inconsistent. Cellular experiments revealed the interplays between H4K16ac and H4K20me3, but no population study explored the interaction between them on the prognosis. METHODS: H4K16ac and H4K20me3 levels in tumors were evaluated by immunohistochemistry for 958 breast cancer patients. Hazard ratios for overall survival (OS) and progression-free survival (PFS) were estimated using Cox regression models. Interaction was assessed on multiplicative scale. Concordance index (C-index) was calculated to verify the predictive performance. RESULTS: The prognostic roles of the low level of H4K16ac or H4K20me3 were significant only in patients with the low level of another marker and their interactions were significant. Moreover, compared with joint high levels of both them, only the combined low levels of both them was associated with a poor prognosis but not the low level of single one. The C-index of the clinicopathological model combined the joint expression of H4K16ac and H4K20me3 [0.739 for OS; 0.672 for PFS] was significantly larger than that of the single clinicopathological model [0.699 for OS, P < 0.001; 0.642 for PFS, P = 0.003] or the model combined with the single H4K16ac [0.712 for OS, P < 0.001; 0.646 for PFS, P < 0.001] or H4K20me3 [0.724 for OS, P = 0.031; 0.662 for PFS, P = 0.006]. CONCLUSIONS: There was an interaction between H4K16ac and H4K20me3 on the prognosis of breast cancer and the combination of them was a superior prognostic marker compared to the single one.

Matrix Gla protein (MGP), GATA3, and TRPS1: a novel diagnostic panel to determine breast origin.

BACKGROUND: Metastatic breast carcinoma is commonly considered during differential diagnosis when metastatic disease is detected in females. In addition to the tumor morphology and documented clinical history, sensitive and specific immunohistochemical (IHC) markers such as GCDFP-15, mammaglobin, and GATA3 are helpful for determining breast origin. However, these markers are reported to show lower sensitivity in certain subtypes, such as triple-negative breast cancer (TNBC). MATERIALS AND METHODS: Using bioinformatics analyses, we identified a potential diagnostic panel to determine breast origin: matrix Gla protein (MGP), transcriptional repressor GATA binding 1 (TRPS1), and GATA-binding protein 3 (GATA3). We compared MGP, TRPS1, and GATA3 expression in different subtypes of breast carcinoma of (n = 1201) using IHC. As a newly identified marker, MGP expression was also evaluated in solid tumors (n = 2384) and normal tissues (n = 1351) from different organs. RESULTS: MGP and TRPS1 had comparable positive expression in HER2-positive (91.2% vs. 92.0%, p = 0.79) and TNBC subtypes (87.3% vs. 91.2%, p = 0.18). GATA3 expression was lower than MGP (p < 0.001) or TRPS1 (p < 0.001), especially in HER2-positive (77.0%, p < 0.001) and TNBC (43.3%, p < 0.001) subtypes. TRPS1 had the highest positivity rate (97.9%) in metaplastic TNBCs, followed by MGP (88.6%), while only 47.1% of metaplastic TNBCs were positive for GATA3. When using MGP, GATA3, and TRPS1 as a novel IHC panel, 93.0% of breast carcinomas were positive for at least two markers, and only 9 cases were negative for all three markers. MGP was detected in 36 cases (3.0%) that were negative for both GATA3 and TRPS1. MGP showed mild-to-moderate positive expression in normal hepatocytes, renal tubules, as well as 31.1% (99/318) of hepatocellular carcinomas. Rare cases (0.6-5%) had focal MGP expression in renal, ovarian, lung, urothelial, and cholangiocarcinomas. CONCLUSIONS: Our findings suggest that MGP is a newly identified sensitive IHC marker to support breast origin. MGP, TRPS1, and GATA3 could be applied as a reliable diagnostic panel to determine breast origin in clinical practice.

α-Fetoprotein mRNA in situ hybridisation is a highly specific marker of hepatocellular carcinoma: a multi-centre study.

BACKGROUND: Pathologic diagnosis of hepatocellular carcinoma (HCC) can be challenging in differentiating from benign and non-hepatocytic malignancy lesions. The aim of this study was to investigate the potential utility of α-fetoprotein (AFP) mRNA RNAscope, a sensitive and specific method, in the diagnosis of HCC. METHODS: Three independent retrospective cohorts containing 2216 patients with HCC, benign liver lesions, and non-hepatocytic tumours were examined. AFP was detected using ELISA, IHC (Immunohistochemistry), and RNAscope. Glypican3 (GPC3), hepatocyte paraffin-1 (HepPar-1), and arginase-1 (Arg-1) proteins were detected using IHC. RESULTS: AFP RNAscope improved the HCC detection sensitivity by 24.7-32.7% compared with IHC. In two surgical cohorts, a panel of AFP RNAscope and GPC3 provided the best diagnostic value in differentiating HCC from benign hepatocytic lesions (AUC = 0.905 and 0.811), and a panel including AFP RNAscope, GPC3, HepPar-1, and Arg-1 yielded the best AUC (0.971 and 0.977) when distinguishing HCC from non-hepatocytic malignancies. The results from the liver biopsy cohort were similar, and additional application of AFP RNAscope improved the sensitivity by 18% when distinguishing HCC from benign hepatocytic lesions. CONCLUSIONS: AFP mRNA detected by RNAscope is highly specific for hepatocytic malignancy and may serve as a novel diagnostic biomarker for HCC.

Time-varying effects of FOXA1 on breast cancer prognosis.

PURPOSE: Results of previous studies on the associations between Forkhead box A1 (FOXA1) expression in breast cancer tissues and the prognosis varied depending on the follow-up durations. The present study would investigate whether there is a time-varying effect of FOXA1 in breast cancer tissues on the prognosis. METHODS: FOXA1 expressions were evaluated in 1041 primary invasive breast tumors with tissue microarrays by immunohistochemistry. Cox models with restricted cubic splines and Kaplan-Meier survival analysis were used to examine the associations between FOXA1 and the prognosis. Flexible parametric models were applied to explore the time-varying effect of FOXA1. RESULTS: Overall, the association between FOXA1 expression and the prognosis was not significant but varied on the time of follow-up. Compared to FOXA1 ≤ 270 of H-score, the hazard ratios (HRs) of death for those with 271-285 of FOXA1 expression increased from 0.35 (95% CI 0.14-0.86) at 6 months after diagnosis to 2.88 (95% CI 1.35-6.15) at 120 months with a crossover at around 36 months. Similar patterns were also observed for FOXA1 > 285 of H-score and for progression free survival (PFS). Moreover, when allowed both FOXA1 and estrogen receptor (ER) to change over time in the model (considering that ER had a similar time-varying effect), these time-varying effects remained for FOXA1 on both overall survival (OS) (P < 0.01) and PFS (P = 0.01) but were attenuated for ER (P = 0.13 for OS). CONCLUSIONS: This study revealed an independent time-varying effect of FOXA1 on breast cancer prognosis, which would provide an insight into the roles of FOXA1 as a marker of breast cancer prognosis and may help optimize the medication strategies.

A novel model of controlling PD-L1 expression in ALK+ anaplastic large cell lymphoma revealed by CRISPR screening.

The success of programmed cell death protein 1 (PD-1)/PD-L1-based immunotherapy highlights the critical role played by PD-L1 in cancer progression and reveals an urgent need to develop new approaches to attenuate PD-L1 function by gaining insight into how its expression is controlled. Anaplastic lymphoma kinase (ALK)-positive anaplastic large-cell lymphoma (ALK+ ALCL) expresses a high level of PD-L1 as a result of the constitutive activation of multiple oncogenic signaling pathways downstream of ALK activity, making it an excellent model in which to define the signaling processes responsible for PD-L1 upregulation in tumor cells. Here, using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 library screening, we sought a comprehensive understanding of the molecular effectors required for PD-L1 regulation in ALK+ ALCL. Indeed, we determined that PD-L1 induction is dependent on the nucleophosmin-ALK oncoprotein activation of STAT3, as well as a signalosome containing GRB2/SOS1, which activates the MEK-ERK and PI3K-AKT signaling pathways. These signaling networks, through STAT3 and the GRB2/SOS1, ultimately induce PD-L1 expression through the action of transcription factors IRF4 and BATF3 on the enhancer region of the PD-L1 gene. IRF4 and BATF3 are essential for PD-L1 upregulation, and IRF4 expression is correlated with PD-L1 levels in primary ALK+ ALCL tissues. Targeting this oncogenic signaling pathway in ALK+ ALCL largely inhibited the ability of PD-L1-mediated tumor immune escape when cocultured with PD-1-positive T cells and natural killer cells. Thus, our identification of this previously unrecognized regulatory hub not only accelerates our understanding of the molecular circuitry that drives tumor immune escape but also provides novel opportunities to improve immunotherapeutic intervention strategies.

Promiscuity of host cell proteins in the purification of histidine tagged recombinant xylanase A by IMAC procedures: A case study with a Ni2+-tacn-based IMAC system.

Determination of the extent of host cell protein (HCP) contamination is an essential pre-requisite to validate the chromatographic purification of recombinant proteins. This study explores how different experimental conditions affect the HCP profiles generated during the immobilised metal ion affinity chromatographic (IMAC) purification with a Ni2+-1,4,7-triaza-cyclononane (tacn) Sepharose FF™ sorbent of the Bacillus halodurans N- and C-terminal His6-tagged xylanase A, expressed by Escherichia coli BL21(DE3) cells, and captured directly from cell lysates. Comparative studies were also carried out under identical loading, wash and elution conditions using nitrilotriacetic acid (NTA), also immobilised onto an agarose support and complexed with Ni2+ ions. High-resolution tandem mass spectrometry of the tryptic peptides derived from the proteins present in the IMAC flow-through, wash and elution fractions confirmed that the E. coli BL21(DE3) HCP profiles were dependent on the choice of adsorbent. With feedstocks containing the N- or C-terminal His6-tagged xylanase A, in several instances the same E. coli BL21(DE3) HCPs were found to co-elute with the tagged protein from either adsorbent, indicating a preferential ability of some HCPs to bind to both the IMAC resin and to the recombinant protein. This promiscuous behaviour has been found to be due to factors other than just the presence of histidine-rich motifs within the amino acid sequences of these HCPs. This case study demonstrates that the choice of protein expression and separation conditions impact on the levels of HCP contamination when different IMAC systems are employed.

Comparison of Mismatch Repair Status Between Primary and Matched Metastatic Sites in Patients With Colorectal Cancer.

BACKGROUND: Differences between the features of primary cancer and matched metastatic cancer have recently drawn attention in research. This study investigated the concordance in microsatellite instability (MSI) and mismatch repair (MMR) status between primary and corresponding metastatic colorectal cancer (CRC). METHODS: Consecutive patients with metastatic CRC who had both primary and metastatic tumors diagnosed at our institution in January 2008 through December 2016 were identified. Immunohistochemistry was used to test the MMR status of both primary and matched metastatic tumors, and PCR analysis was performed to test MSI in patients with deficient MMR (dMMR) status. RESULTS: A total of 369 patients were included. Of the 46 patients with MSI-high primary tumors, 37 (80.4%) also had MSI-high metastatic tumors, whereas 9 (19.6%) had microsatellite stable (MSS) metastatic tumors. A high concordance was found in patients with liver, lung, or distant lymph node metastases. Interestingly, the discrepancy was more likely to be limited to peritoneal (5/20) or ovarian (4/4) metastasis (chi-square test, P<.001). These organ-specific features were also found in the pooled analysis. Along with the change of MSI-high in primary cancer to MSS in metastatic cancer, lymphocyte infiltration decreased significantly (P=.008). However, the change did not influence survival; the median overall survival of MSI-high and MSS metastatic tumors was 21.3 and 21.6 months, respectively (P=.774). The discrepancy rate was 1.6% for patients with proficient MMR primary tumors. CONCLUSIONS: For patients with dMMR primary tumors, the concordance of MSI and MMR status in primary CRC and corresponding metastatic cancer is potentially organ-specific. High concordance is found in liver, lung, and distant lymph node metastases, whereas discrepancy is more likely to occur in peritoneal or ovarian metastasis. Rebiopsy to evaluate MSI-high/dMMR status might be needed during the course of anti-PD-1 therapy in cases of peritoneal or ovarian metastasis.

The systemic immune-inflammation index is an independent predictor of survival for metastatic colorectal cancer and its association with the lymphocytic response to the tumor.

BACKGROUND: Systemic inflammation and immune dysfunction has been proved to be significantly associated with cancer progression and metastasis in many cancer types, including colorectal cancer. We examined the prognostic significance of the systemic immune-inflammation index (SII) in patients with metastatic colorectal cancer (mCRC) and the relationship between the lymphocytic response to the tumor and this index. METHODS: This retrospective study evaluated 240 consecutive patients with newly diagnosed stage IV mCRC who underwent surgical resection. The SII values were calculated based on preoperative laboratory data regarding platelet, neutrophil, and lymphocyte counts. Tumor-infiltrating lymphocytes were evaluated using the surgical specimens. The overall survival and their 95% confidence interval (95% CI) were estimated by regression analyses and the Kaplan-Meier method. RESULTS: After a mean follow-up of 26.7 (1.1-92.4) months, 146 patients (60.8%) died. In the univariate analysis, a high SII was significantly associated with poor overall survival (P = 0.009). The multivariable analysis also confirmed that a high SII was independently associated with poor overall survival (hazard ratio: 1.462, 95% confidence interval 1.049-2.038, P = 0.025). The SII value was significantly correlated with the TILs value at the tumor's center (P = 0.04), but not at the invasive margin (P = 0.39). When we evaluated overall survival for groupings of the tumor-infiltrating lymphocytes and SII values, we identified three distinct prognostic groups. The group with low tumor-infiltrating lymphocyte values and high SII values had the worst prognosis. CONCLUSIONS: A high SII value independently predicts poor clinical outcomes among patients with mCRC. In addition, combining the lymphocytic response to the tumor and SII could further enhance prognostication for mCRC.

Comparison of immunological characteristics between paired mismatch repair-proficient and -deficient colorectal cancer patients.

BACKGROUND: Currently, mismatch repair-deficient (dMMR) status is a promising candidate for targeted immune checkpoint inhibition therapy in colorectal cancer (CRC) patients, however, the potential immunological mechanism has not yet been well clarified and some other predictors need to be excavated as well. METHODS: We collected 330 CRC patients by the match of mismatch repair-proficient (167) and dMMR (163), explored the relationship between MMR status and some important immune molecules including MHC class I, CD3, CD4, CD8, CD56, programmed death-1 and programmed death ligand-1, and investigated the risk factors for dMMR status as well as low MHC class I expression. The Pearson Chi square test was used for analyzing the associations between clinicopathological and immune characteristics and MMR status, and two categories logistic regression model was used for univariate and multivariate analysis to predict the odds ratio of risk factors for dMMR status and low MHC class I expression. RESULTS: Multivariate logistic regression analysis showed that low MHC class I and CD4 expression and high CD8 expression were significant risk factors for dMMR status [odds ratio (OR) = 24.66, 2.94 and 2.97, respectively; all p < 0.05] and dMMR status was the only risk factor for low MHC class I expression (OR = 15.34; p < 0.001). CONCLUSIONS: High CD8 and low MHC class I expression suggests the contradiction and complexity of immune microenvironment in dMMR CRC patients. Some other immunocytes such as CD56+ cells might also participate in the process of immune checkpoint inhibition, whereas needs further investigations.

Use of peak sharpening effects to improve the separation of chiral compounds with molecularly imprinted porous polymer layer open-tubular capillaries.

This investigation demonstrates the application of a new peak sharpening technique to improve the separation of difficult-to-resolve racemic mixtures in capillary electro-chromatography. Molecularly imprinted porous layer open tubular (MIP-PLOT) capillaries, prepared by a layer-on-layer polymerization approach with Z-l-Asp-OH as the template, were selected to validate the approach. SEM revealed that the polymer film thickness can be varied by changes in both the polymer composition and the layer-on-layer regime. Capillaries made with methacrylic acid as the functional monomer could not separate the Z-Asp-OH racemate, due to weak interactions between the MIP-PLOT material and the target analytes. In contrast, MIP-PLOT capillaries prepared with 4-vinylpyridine as the functional monomer resulted in increased ionic interactions with the target analytes. Separation of the enantiomers could be enhanced when a peak zone sharpening effect was exploited through the use of specific BGE compositions and by taking advantage of eigenpeak phenomena. In this manner, the position of a sharpening zone and the peak shape of the sample analytes could be fine-tuned, so that when the sharpening zone and the target analyte co-migrated the separation of the Z-l-Asp-OH enantiomer from its d-enantiomer in a racemic mixture could be achieved under overloading conditions.

En bloc transurethral resection with 2-micron continuous-wave laser for primary non-muscle-invasive bladder cancer: a randomized controlled trial.

PURPOSE: To evaluate the practicability of en bloc transurethral resection with 2-micron continuous-wave laser as treatment for primary non-muscle-invasive bladder cancer (NMIBC). METHODS: This was a single-center, randomized, controlled trial involving 142 patients with newly diagnosed NMIBC. All patients were randomly assigned in a 1:1 ratio to receive either laser treatment or conventional transurethral resection of bladder tumor (TURBT). All patients received intravesical chemotherapy. Follow-up was performed in 18 months. Primary outcome measure was difference of tumor recurrence rate at the end of study. RESULTS: Baseline characteristics did not differ between patients in two groups. Operation time was longer in laser group than in TURBT group (56.5 ± 37.4 vs. 41.0 ± 29.4 min, P = 0.017). Obturator nerve reflection was noted during TURBT in 18 patients, whereas none was noted during laser resection. Number of T1 tumors was higher in the laser group (25 vs. 15, P = 0.047). According to Kaplan-Meier survival curves, there was no statistical difference in the rate of recurrence in 18 months (P = 0.383). All recurrences were out of the site of first resection, and there was no progression in tumor grade. CONCLUSION: Two-micron continuous-wave laser did not diminish tumor recurrence rate in primary NMIBC for 18-months observation. However, T1 tumors were significantly higher among laser group. Clear and complete tumor bases were easily conserved by laser resection, which may enable pathologists to distinguish the T stages of bladder cancer more easily. Further studies need to be done in future.

miR-720 inhibits tumor invasion and migration in breast cancer by targeting TWIST1.

Breast cancer is the leading cause of cancer death among females, with tumor metastasis being primarily responsible for breast cancer-associated mortality. Current literatures have shown that microRNAs (miRNAs) are implicated in tumor metastasis. In this study, we found that the expression of miR-720 was significantly downregulated in primary breast cancer, with greater downregulation in metastatic tumors. Statistical analysis of 105 cases of primary human breast cancer demonstrated that decreased expression of miR-720 was correlated with lymph node metastasis. Furthermore, reexpression of miR-720 in breast cancer cells remarkably inhibited cell invasiveness and migration both in vitro and in vivo. Mechanistically, downregulation of TWIST1, a promoter of metastasis that was identified as a direct functional target of miR-720, was attributed to the inhibition of metastasis. Consistent with the reduced TWIST1 levels in breast cancer, reexpression of miR-720 upregulated epithelial markers (E-cadherin and ß-catenin) and downregulated mesenchymal markers (N-cadherin, fibronectin, vimentin and matrix metalloproteinase-2). Expression of miR-720 was inversely associated with TWIST1 in human breast cancer tissues. Knockdown of TWIST1 expression by small interfering RNA exhibited similar effects to reintroduction of miR-720, whereas overexpression of TWIST1 (without the 3'-untranslated region) abrogated miR-720-mediated metastasis inhibition. Collectively, our data indicate that miR-720 is frequently decreased in breast cancer and manifests antimetastatic activity by downregulating TWIST1, presenting a novel mechanism of miRNA-mediated regulation of tumor metastasis.

H4K20me3, H3K4me2 and H3K9me2 mediate the effect of ER on prognosis in breast cancer.

Previous studies have indicated that histone methylations act as mediators in the relationship between oestrogen receptor (ER) and breast cancer prognosis, yet the mediating role has never been assessed. Therefore, we investigated seven histone methylations (H3K4me2, H3K4me3, H3K9me1, H3K9me2, H3K9me3, H3K27me3 and H4K20me3) to determine whether they mediate the prognostic impact of ER on breast cancer. Tissue microarrays were constructed from 1045 primary invasive breast tumours, and the expressions of histone methylations were examined by immunohistochemistry. Multifactorial logistic regression was used to analyse the associations between ER and histone methylations. Cox proportional hazard model was performed to assess the relationship between histone methylations and breast cancer prognosis. The mediation effects of histone methylations were evaluated by model-based causal mediation analysis. High expressions of H3K9me1, H3K9me2, H3K4me2, H3K27me3, H4K20me3 were associated with ER positivity, while high expression of H3K9me3 was associated ER negativity. Higher H3K9me2, H3K4me2 and H4K20me3 levels were associated with better prognosis. The association between ER and breast cancer prognosis was most strongly mediated by H4K20me3 (29.07% for OS; 22.42% for PFS), followed by H3K4me2 (11.5% for OS; 10.82% for PFS) and least by H3K9me2 (9.35% for OS; 7.34% for PFS). H4K20me3, H3K4me2 and H3K9me2 mediated the relationship between ER and breast cancer prognosis, which would help to further elucidate the impact of ER on breast cancer prognosis from an epigenetic perspective and provide new ideas for breast cancer treatment.

Selectivity differences of water-soluble vitamins separated on hydrophilic interaction stationary phases.

In this study, the retention behavior and selectivity differences of water-soluble vitamins were evaluated with three types of polar stationary phases (i.e. an underivatized silica phase, an amide phase, and an amino phase) operated in the hydrophilic interaction chromatographic mode with ESI mass spectrometric detection. The effects of mobile phase composition, including buffer pH and concentration, on the retention and selectivity of the vitamins were investigated. In all stationary phases, the neutral or weakly charged vitamins exhibited very weak retention under each of the pH conditions, while the acidic and more basic vitamins showed diverse retention behaviors. With the underivatized silica phase, increasing the salt concentration of the mobile phase resulted in enhanced retention of the acidic vitamins, but decreased retention of the basic vitamins. These observations thus signify the involvement of secondary mechanisms, such as electrostatic interaction in the retention of these analytes. Under optimized conditions, a baseline separation of all vitamins was achieved with excellent peak efficiency. In addition, the effects of water content in the sample on retention and peak efficiency were examined, with sample stacking effects observed when the injected sample contained a high amount of water.

Simultaneous separation of hydrophobic and polar bases using a silica hydride stationary phase.

In this study, the retention behavior of selected hydrophobic and polar bases on a minimally modified silica hydride phase was investigated. From these results and the associated retention plots, significant differences in the chromatographic dependencies of these two classes of basic compounds were evident. The polar bases exhibited strong retention with mobile phases of high organic solvent content, but displayed weak retention with mobile phases of high water content. In contrast, the hydrophobic bases showed "U-shape" retention dependencies, indicative of the interplay of both RP and normal-phase retention characteristics. These studies have demonstrated that hydrophobic and polar bases can be simultaneously separated on the same column either under typical RP-like or aqueous normal-phase-like conditions, respectively, with distinctive selectivity. Finally, the effects of temperature on the RP and aqueous normal phase modality of separations with these analytes were investigated, where discrete changes in retention behavior were also observed.

Analysis of polar peptides using a silica hydride column and high aqueous content mobile phases.

The retention behavior of a set of polar peptides separated on a silica hydride stationary phase was examined with a capillary HPLC system coupled to ESI-MS detection. The mobile phases consisted of formic acid or acetic acid/acetonitrile/water mixtures with the acetonitrile content ranging from 5 to 80% v/v. The effects on peptide retention of these two acidic buffer additives and their concentrations in the mobile phase were systematically investigated. Strong retention of the peptides on the silica hydride phase was observed with relatively high-organic low-aqueous mobile phases (i.e. under aqueous normal-phase conditions). However, when low concentrations of acetic acid were employed as the buffer additive, strong retention of the peptides was also observed even when high aqueous content mobile phases were employed. This unique feature of the stationary phase therefore provides an opportunity for chromatographic analysis of polar peptides with water-rich eluents, a feature usually not feasible with traditional RP sorbents, and thus under conditions more compatible with analytical green chemistry criteria. In addition, both isocratic and gradient elution procedures can be employed to optimize peptide separations with excellent reproducibility and resolution under these high aqueous mobile phase conditions with this silica hydride stationary phase.

Nomogram for predicting the biochemical recurrence of prostate cancer after neoadjuvant androgen deprivation therapy.

BACKGROUND: A predictive model for biochemical recurrence (BCR) of prostate cancer (PCa) after neoadjuvant androgen deprivation therapy (nADT) has not been established. This study was aimed at determining multiparameter variables that could be used to construct a nomogram to predict the post-nADT BCR of PCa. METHODS: Overall, 43 radical prostatectomy specimens from PCa patients who had undergone nADT were collected. Multiparameter variables were analyzed by univariate and then multivariate logistic analyses to identify the independent prognostic factors for predicting BCR. The predictive model was established using Lasso regression analysis. RESULTS: Univariate logistic analysis revealed six variables, pathology stage; margins; categorization as group A, B, or C; nucleolus grading; percentage of tumor involvement (PTI); and PTEN status were significantly associated with the BCR of PCa (all p < 0.05). Multivariate logistic regression analysis suggested that categorization as group C, severe nucleolus grading, PTI less than or equal to 5%, and PTEN loss were positively correlated with BCR (all p < 0.05). A nomogram comprising the four variables predicting BCR was constructed, and it exhibited good discrimination (AUC: 0.985; specificity: 86.2%; sensitivity: 100%). Calibration plots for the probability of freedom from BCR at 1 and 2 years showed a good match between the prediction by the nomogram. CONCLUSIONS: We constructed and validated a nomogram to predict the risk of BCR in PCa patients after nADT. This nomogram is a complement to the existing risk stratification systems for PCa, which could have marked implications for clinical decision-making for PCa patients after nADT.

Association of Enolase-1 with Prognosis and Immune Infiltration in Breast Cancer by Clinical Stage.

Purpose: Enolase-1 (ENO1) plays a key role in malignancies. Previous studies on the association between ENO1 expression and breast cancer prognosis had yielded inconsistent results. In the present study, we assessed the prognostic effect of ENO1 in breast cancer using Guangzhou Breast Cancer Study (GZBCS) cohort with full consideration of the potential confounders and the modification effects. The results were further validated in the TCGA-BRCA cohort and explained by tumor immunity. Methods: ENO1 protein expressions were evaluated by immunohistochemistry in tissue microarrays from 961 patients with primary invasive breast cancer. Chi-square tests were used to assess the association of ENO1 levels with the patient's characteristics. Cox regression models were applied to assess the prognostic effects. The TCGA-BRCA cohort was utilized to validate the results and explore the potential mechanisms. The immune infiltration was determined using the CIBERSORT and ssGSEA algorithms; the correlation between ENO1 expression and the abundance of tumor-infiltrating immune cells (TIICs) and scores of immune-related functions was evaluated by Wilcoxon signed-rank tests and Spearman's rank test. Results: ENO1 protein expression exerted a protective effect on OS in stage I/II patients (HR=0.58, 95% CI: 0.35-0.96) but not in stage III patients (HR=1.42, 95% CI: 0.81-2.49, P interaction=0.04) in GZBCS; consistent results were obtained at mRNA levels in TCGA cohort. Immune infiltration analyses revealed that ENO1 was positively correlated with multiple antitumor TIICs (including M1 macrophages, B cells, CD8 T cells, T helper 2 cells, and NK cells) only in stage I/II but not stage III patients. Conclusion: A higher expression of ENO1 was associated with a better prognosis only in early-stage breast cancer, which may be related to the different effects of ENO1 on immune infiltration, suggesting that ENO1 may be a promising target for precision immunotherapy in breast cancer.

Cutoff values of PD-L1 expression in urinary cytology samples for predicting response to immune checkpoint inhibitor therapy in upper urinary tract urothelial carcinoma.

BACKGROUND: The objective of this study was to determine the cutoff value of PD-L1 expression that can predict response to immune checkpoint inhibitor (ICI) immunotherapy for upper tract urothelial carcinoma (UTUC). METHODS: The concordance of PD-L1 expression between paired surgical resection specimens (SRSs) and urine cell blocks (UCBs) (cohort 1) was studied in a retrospective set of 58 UTUC patients to determine its suitability as a predictor of ICI immunotherapy efficacy. PD-L1 expression in UCBs obtained before neoadjuvant ICI immunotherapy was verified in a prospective set of 12 UTUC patients (cohort 2). PD-L1 (SP263 clone) expression was assessed for percentage (tumor proportional score) of tumor cell (TC) showing PD-L1 staining. RESULTS: The authors found an overall agreement of 94.4% (51 of 54) between UCBs and SRSs in cohort 1 (positive percent agreement = 100%, negative percent agreement = 93.8%, r value = 0.63). PD-L1 expression in <10% and ≥10% of tumor cells (TCs) of UCBs were the best predictors of negative (<25%) and positive (≥25%) expression in TCs of SRSs, respectively (concordance = 98.1%, r value = 0.93). These findings were verified in cohort 2: at the 10% cutoff for PD-L1 expression, the best response predictive value was 83.3% (5 of 6) in PD-L1-positive patients, and the nonresponse predictive value was 50% (3 of 6) in PD-L1-negative patients. The sensitivity, specificity, and area under the receiver operating characteristic curve values for predicting ICI immunotherapy efficacy based on PD-L1-expressing TCs in UCBs were 62.5%, 75%, and 0.688, respectively. CONCLUSIONS: Immunocytochemistry of UCBs is reliable for determining PD-L1 expression, which can predict the efficacy of ICI immunotherapy at a cutoff of 10%.