Expression In Biopsy Specimens Research Articles

Abstract 7607: Single-cell genomics study before and after neoadjuvant therapy for triple-negative breast cancer

Abstract Breast cancer has become the most common malignant tumor globally. Neoadjuvant chemotherapy (NAC) has become an important treatment option for locally advanced breast cancer, capable of eliminating circulating tumor cells (micrometastases) and achieving downstaging objectives. However, even after completing neoadjuvant chemotherapy or receiving local treatment, some patients may still have residual lesions, which are a key factor in distant recurrence and metastasis. The existing clinical biomarker tests such as CEA, CA-153, and imaging methods are unable to accurately assess the condition early on. Although tissue pathology biopsy of tumor specimens is the gold standard for clinical pathological diagnosis, it also has certain limitations. Traditional high-throughput sequencing technology for breast cancer primarily identifies "driver genes" associated with the occurrence and development of breast cancer. However, it has limitations in identifying genomic structural changes and subclones of breast cancer, and it overlooks the heterogeneity among tumor cells. The emergence of single-cell RNA sequencing (scRNA-seq) technology can detect these heterogeneous individuals, decode the heterogeneity of breast cancer cells, refine molecular subtypes, and open up new pathways to overcome drug resistance. We collected samples from 6 patients with locally advanced triple-negative breast cancer (ER-, PR-, HER2-negative, defined as HER2 immunohistochemistry 0, 1+ or 2+ but FISH-negative). We compared the cell subgroups and their gene expression in biopsy specimens before neoadjuvant chemotherapy with those in post-treatment specimens obtained during surgery, analyzing changes in cell composition in the tumor microenvironment before and after immunotherapy to identify specific cell types potentially contributing to the outcome of immunotherapy, particularly cell subtypes associated with T-cell clonal proliferation. We found that in the group with good neoadjuvant treatment response (Miller-Payne grade 4-5), B cells, CD4+ T cells, CD8+ T cells, and Tregs cells were all significantly downregulated, while endothelial cells and aSMA-positive fibroblast cells were significantly upregulated. By comparing changes in the immune microenvironment between patients with and without T-cell clonal proliferation before and after treatment, we revealed the differentiation patterns of various immune cells in immunotherapy and potential mechanisms of action. Citation Format: Yingkuan Shao, Kailun Xu. Single-cell genomics study before and after neoadjuvant therapy for triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7607.

Usefulness of Biopsy Specimens for Evaluating CD103+ Tumor-resident Memory T Cells in Esophageal Cancer.

CD103+ tissue-resident memory T cells (TRM) in tumor sites are associated with a favorable prognosis and predict the effectiveness of immune checkpoint inhibitors. The detection of CD103+ TRM infiltration in biopsy samples could be beneficial for patients without surgical indications. However, the usefulness of TRM detection in biopsy tissue and the difference in TRM status between biopsy and surgical specimens' post-neoadjuvant chemotherapy have not been elucidated. In the present study, we aimed to elucidate whether we can detect TRM in biopsy specimens and the impact of chemotherapy on TRM infiltration. Tissue sections were obtained from 46 patients with esophageal cancer who received neoadjuvant chemotherapy and underwent radical esophagectomy in 2017. Immunohistochemistry was performed using an anti-CD103 antibody for biopsy and surgical specimens. We examined the relationship between CD103 expression, clinicopathological features, and prognosis for each patient. TRM infiltration was detected in the biopsy specimens. CD103 expression in biopsy specimens correlated with that in surgical specimens. Although there was no statistical significance in clinicopathological findings between CD103high and CD103low, patients with CD103high biopsy specimens exhibited favorable prognosis. The number of CD103+ cells was increased by chemotherapy: though with no survival benefit. Regardless of surgical indication, we were able to determine the TRM status even in biopsy specimens. CD103 evaluation at biopsy may be more useful and practical than evaluation in surgical specimens, enabling prediction of prognosis and response to immune therapy.

Predictive factors associated with differential pathologic response to neoadjuvant chemohormonal therapy in high-risk localized prostate cancer

To explore the clinical parameters and molecular biomarkers that can predict differential pathologic response to neoadjuvant chemohormonal therapy (NCHT) in prostate cancer (CaP).A total of 128 patients with primary high-risk localized CaP who had received NCHT followed by radical prostatectomy (RP) were included. Androgen receptor (AR), AR splice variant-7 (AR-V7) and Ki-67 staining were evaluated in prostate biopsy specimens by immunohistochemistry. The pathologic response to NCHT in whole mount RP specimens was measured based on the reduction degree of tumor volume and cellularity compared to the paired pretreatment needle biopsy, and divided into 5 tier grades (Grades 0-4). Patients with Grades 2 to 4 (the reduction degree more than 30%) were defined as having a favorable response. Logistic regression was performed to explore the predictive factors associated with a favorable pathologic response. The predictive accuracy was evaluated by receiver operating characteristic (ROC) curve and area under the ROC curve (AUC).Ninety-seven patients (75.78%) had a favorable response to NCHT. Logistic regression showed that the preoperative PSA level, low AR expression and high Ki-67 expression in biopsy specimens were associated with a favorable pathologic response (P < 0.05). Furthermore, the AUC of the preoperative PSA level, AR and Ki-67 were 0.625, 0.624 and 0.723, respectively. Subgroup analysis revealed that the rate of favorable pathologic response to NCHT was 88.5% in patients with ARlowKi-67high, which was higher than patients with ARlowKi-67low, ARhighKi-67low, and ARhighKi-67high (88.5% vs. 73.9%, 72.9%, and 70.9%, all P < 0.05).A lower preoperative PSA level was an independent predictive factor for a favorable pathologic response. Moreover, the expression status of AR and Ki-67 in biopsy specimens were associated with differential pathologic response to NCHT, and AR low/Ki-67 high was also associated with favorable response but warrants further evaluation in this patient subgroup and future trial clinical trial design.

Expression of interferon-stimulated gene 20 (ISG20), an antiviral effector protein, in glomerular endothelial cells: possible involvement of ISG20 in lupus nephritis

Background In addition to regulating the antiviral response, increased expression of Toll-like receptor 3 (TLR3) in resident renal cells plays a role in developing some forms of glomerulonephritis. TLR3 activation leads to type I interferon (IFN) production, which induces the expression of IFN-stimulated genes (ISGs). However, the role of ISG20 expression in resident renal cells remains unclear. Methods Cultured normal human glomerular endothelial cells (GECs) were treated with polyinosinic-polycytidylic acid (poly IC), Escherichia coli lipopolysaccharide (LPS), R848, and CpG (TLR3, TLR4, TLR7, and TLR9 agonists, respectively). The mRNA levels of ISG20, CX3CL1/fractalkine, and CXCL10/IP-10 were measured by quantitative reverse transcription-polymerase chain reaction. ISG20 protein expression was assessed by Western blotting. RNA interference was used to knockdown IFN-β and ISG20 expression. CX3CL1 protein levels were assessed by enzyme-linked immunosorbent assay. We performed immunofluorescence to examine endothelial ISG20 expression in biopsy specimens from patients with lupus nephritis (LN). Results In GECs, the expression of ISG20 mRNA and protein was increased by polyIC, not by LPS, R848, or CpG treatment. Moreover, ISG20 knockdown prevented poly IC-induced CX3CL1 expression but had no effect on CXCL10 expression. Intense endothelial ISG20 immunoreactivity was observed in biopsy specimens obtained from patients with proliferative LN. Conclusion In GECs, ISG20 was regulated via TLR3 but not via TLR4, TLR7, or TLR9 signaling. Moreover, ISG20 was involved in regulating CX3CL1 production. In addition to regulating antiviral innate immunity, ISG20 may act as a mediator of CX3CL1 production, thereby inducing glomerular inflammation, particularly in patients with LN.

Merkel cell number and distribution, and CD200 expression in patients with lichen planopilaris and discoid lupus erythematosus.

Immune mechanisms are considered to be responsible for the pathogenesis of cicatricial alopecia in lichen planopilaris (LPP) and discoid lupus erythematosus (DLE) diseases. CD200 has an immunomodulatory function in hair follicles. The functions of Merkel cells (MCs) in hair follicles remain to be fully understood. This study aimed to determine the number and distribution of MCs as well as CD200 expression in patients with DLE and LPP. Using immunohistochemistry, the number and distribution of MCs (staining with CK20) and CD200 expression in biopsy specimens of LPP and DLE patients were compared with control group patients. The number of follicular MCs, total MCs, mean follicular MCs, and CD200 expression were significantly lower in the case groups compared to the control group. In CD200- cases, the number of follicular MCs and mean follicular MCs were significantly lower than in CD200+ cases. Retrospective design, lack of data regarding the history of alopecia in the control group, and unknown stage of disease in patients were the limitations. MC loss might play a role in immune privilege collapse in hair follicles. This study is novel in terms of investigating MCs in DLE and LPP patients.

Reduced Circulating Soluble Receptor for Advanced Glycation End-products in Chronic Hepatitis B Are Associated with Hepatic Necroinflammation.

The diagnosis and disease management of chronic hepatitis B (CHB) remain challenging due to the elusive assessment of disease severity. Recently, soluble receptor for advanced glycation end-products (sRAGE) has been implicated in the inflammatory-immune response initiated by liver injury. Nonetheless, its natural behavior and clinical importance in CHB remain elusive. One hundred and twenty CHB patients and forty healthy controls (HCs) were enrolled, and the serum sRAGE as well as RAGE expression in biopsy specimens from these subjects was analyzed, and correlation of sRAGE with clinical features as well as its potential predictive value for monitoring the CHB was also evaluated. Reduced serum sRAGE levels and decreased tissular RAGE expression were observed in CHB patients. sRAGE and RAGE were inversely correlated with gradually increased grades of hepatic necroinflammation as well as the routine indicator ALT. Furthermore, receiver operating characteristic (ROC) analysis showed that combination of ALT and sRAGE exerted better predictive power (area under the ROC curve (AUC) of 0.86) for hepatic necroinflammation than that of ALT (AUC of 0.82), sRAGE (AUC of 0.81), or sRAGE-to-ALT ratio (sRAGE/ALT) (AUC of 0.85) alone. More importantly, circulating sRAGE alone exerted valuable predictive power for hepatic moderate-to-severe necroinflammation in CHB patients but with normal ALT (AUC of 0.81) or minimally elevated ALT (AUC of 0.85). In conclusion, reduced serum sRAGE levels may imply an increased severity for necroinflammation, and it may serve as a potential alternative biomarker for monitoring hepatic necroinflammation in CHB.

Glomerular endothelial expression of type I IFN-stimulated gene, DExD/H-Box helicase 60 via toll-like receptor 3 signaling: possible involvement in the pathogenesis of lupus nephritis

Background Sustained type I interferon (IFN) activation via Toll-like receptor (TLR) 3, 7 and 9 signaling has been reported to play a pivotal role in the development of lupus nephritis (LN). Although type I IFN activation has been shown to induce interferon-stimulated genes (ISGs) expression in systemic lupus erythematosus, the implication of ISGs expression in intrinsic glomerular cells remains largely unknown. Methods We treated cultured human glomerular endothelial cells (GECs) with polyinosinic-polycytidylic acid (poly IC), R848, and CpG (TLR3, TLR7, and TLR9 agonists, respectively) and analyzed the expression of DExD/H-Box Helicase 60 (DDX60), a representative ISG, using quantitative reverse transcription-polymerase chain reaction and western blotting. Additionally, RNA interference against IFN-β or DDX60 was performed. Furthermore, cleavage of caspase 9 and poly (ADP-ribose) polymerase (PARP), markers of cells undergoing apoptosis, was examined using western blotting. We conducted an immunofluorescence study to examine endothelial DDX60 expression in biopsy specimens from patients with LN. Results We observed that endothelial expression of DDX60 was induced by poly IC but not by R848 or CpG, and RNA interference against IFN-β inhibited poly IC-induced DDX60 expression. DDX60 knockdown induced cleavage of caspase 9 and PARP. Intense endothelial DDX60 expression was observed in biopsy specimens from patients with diffuse proliferative LN. Conclusion Glomerular endothelial DDX60 expression may prevent apoptosis, which is involved in the pathogenesis of LN. Modulating the upregulation of the regional innate immune system via TLR3 signaling may be a promising treatment target for LN.

Prognostic significance of adipophilin expression in biopsy specimens of patients with triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is the most aggressive type of breast cancer. The present authors recently demonstrated that expression of the lipid-related protein adipophilin (ADP) in operative specimens is a significant poor prognostic factor in patients with TNBC. Using biopsy specimens is important in making clinical decisions for patients with breast cancer; however, the prognostic significance of ADP expression in biopsy specimens from TNBC patients remains unclear. The present study determined the prognostic significance of ADP expression in biopsy specimens from TNBC patients and compared ADP-expression status between biopsy and operative specimens. The present study analyzed ADP-expression profiles in biopsy specimens from 102 patients with TNBC using immunohistochemical staining and determined relapse-free survival and risk factors associated with ADP expression in these specimens, as well as the concordance of ADP expression between biopsy and operative specimens. The results identified ADP expression in 35.3% of biopsy specimens from TNBC patients. The Ki-67 labelling index was significantly higher in ADP-positive patients (P<0.001). Univariate analysis revealed that ADP expression in biopsy specimens was significantly associated with poor relapse-free survival in patients not administered neoadjuvant chemotherapy or adjuvant chemotherapy (P=0.026). Furthermore, the concordance rate of ADP expression between biopsy and operative specimens was 73.1%, with a Cohen's kappa coefficient of 0.385 (P=0.003). These findings suggested that ADP expression in biopsy specimens might be a useful prognostic marker for patients with TNBC and could potentially provide important information regarding treatment strategies for these patients.

The association between Fc gamma RIIb expression levels and chronic hepatitis B virus infection progression

BackgroundFc gamma receptor IIb (FcγRIIb) is an important inhibitory receptor that plays vital roles in regulating various immune response processes and the pathogenesis of many infectious diseases. The purpose of our research was to evaluate FcγRIIb expression in serum and liver biopsy specimens from hepatitis B virus (HBV)-infected patients and to explore the association of FcγRIIb with chronic HBV infection.MethodsEnzyme-linked immunosorbent assay (ELISA) was adopted to measure the serum FcγRIIb levels in 119 HBV-infected patients and 24 healthy controls. An immunohistochemical method was then employed to identify FcγRIIb expression in biopsy specimens from patients with chronic hepatitis B (CHB). The integrated optical density (IOD) value was measured to represent FcγRIIb expression levels.ResultsSerum FcγRIIb levels were decreased in CHB patients compared to controls (P < 0.001). The FcγRIIb levels in the CHB patient group were remarkably lower than those in the HBV carrier group (P < 0.001). In addition, FcγRIIb levels were negatively associated with AST and ALT (r = −0.3936, P = 0.0063; r = −0.3459, P = 0.0097, respectively). The IOD values of FcγRIIb expression in the moderate and severe CHB groups were significantly lower than those in the control group (P = 0.006 and P < 0.001, respectively). The FcγRIIb level tended to be lower with pathological changes related to hepatitis. Furthermore, correlation analysis revealed that FcγRIIb had negative correlations with AST and ALT (r = −0.688, P = 0.0016; r = −0.686, P = 0.0017, respectively) but a positive association with the platelet count (r = 0.6464, P = 0.0038).ConclusionsFcγRIIb levels are significantly related to chronic HBV infection and the progression of CHB. Changes in FcγRIIb may affect the progression of liver inflammation and fibrosis in CHB patients.

Role of MCP-1 in the development of tubulointerstitial fibrosis in patients with primary chronic glomerulonephritis

BACKGROUND. To date, the study of the factors involved in the glomerular-tubular pathological connections leading to damage to the tubulointerstitial tissue is one of the topical areas of nephrology. THE AIM: to study the effect of MCP-1 in the development of tubulointerstitial fibrosis as a factor in the irreversible progression of chronic renal failure. PATIENTS ANDMETHODS. Prospective observation and retrospective analysis of case histories were carried out, which included a total of 75 patients with primary chronic glomerulonephritis. RESULTS. The average age of the patients was 36.7 ± 12.3 years, of which 52 were males, 23 were women. The average length of service in a nephrological disease was 3.0 [1.0; 5.0] years. The calculated GFR values are 87.3 ± 31.2 ml / min / 1.73 m2. In the general population, the moderate degree of MCP-1 expression, estimated at 2 points, was 35 %, pronounced expression was found in 25 % of the respondents. In the mesangium of the glomeruli and in macrophages, the expressed degree of MCP-1 expression was 20 % and 16 %, respectively, which characterizes MCP-1 as a marker produced by resident cells. When studying the relationship of MCP-1 in blood with clinical parameters, a correlation was found with the values of total protein (Rs= –0.43; p &lt;0.05), with erythrocyturia (Rs= –0.28; p &lt;0.05), as well as with an albumin level (Rs= –0.5; p &lt;0.05), which indicates the role of MCP-1 in the development of nephritic forms of glomerulonephritis. Depending on the severity of MCP-1 expression in biopsy specimens, the incidence of focal tubulointerstitial fibrosis with MCP-1 expression estimated at 1 point was 13.3 %, 2 points – 14.3 %, 3 points – 44.0 %. The revealed significant correlation between the serum level of MCP-1 and the severity of tubulointerstitial fibrosis confirms the MCP-1-mediated mechanism of progression of CKD. CONCLUSION. The relationship of serum and tissue forms of MCP-1 with the progression of tubulointerstitial fibrosis in chronic glomerulonephritis has been demonstrated. MCP-1-induced mesangial cell plays a critical role in the development of renal tubular damage, and its increased expression is associated with progressive tubulointerstitial fibrosis and decreased renal function.

The prevalence of Sjögren’s syndrome and sicca symptoms in patients with systemic sclerosis and alpha-smooth muscle actin expression in biopsy specimens from minor salivary glands

This study aimed to investigate the prevalence of sicca symptoms and secondary Sjögren’s syndrome (SjS) in patients with systemic sclerosis (SSc). Also this study aimed to evaluate the expression of α-smooth muscle actin (α–SMA) in minor salivary gland (MSG) specimens, a possible marker of fibrosis responsible for myofibroblastic transformation. Patients with SSc who were followed in Rheumatology outpatient clinic at a university hospital evaluated. The questionnaire of sicca symptoms and classification of SjS were evaluated according to the American–European Consensus Group (AECG) criteria. Histopathologic evaluations were done in MSG specimens investigating the presence of focal lymphocytic sialadenitis and glandular fibrosis, also assessing the expression of α–SMA. This cross-sectional study included 102 patients with SSc [91 females (89%), mean age 52.5 ± 12 years]. In this cohort 76 (75%) patients had sicca symptoms and 36 (35.3%) patients fulfilled the AECG criteria for SjS; all with limited form. Having SjS found to be associated with older age and the presence of positive anti-SS-A antibodies. On histopathologic examinations, glandular fibrosis was observed in 67 (80%) and lymphocytic sialadenitis was detected in 38 (45%) patients; but only 7 samples were positive for α–SMA. This study suggested sicca symptoms were found to be very common among patients with SSc. Also secondary SjS was detected in nearly one-third of patients with SSc; especially in limited subtype. Anti SS-A positivity and older age were detected as predictors for SjS. Histopathologic evaluations showed significant glandular fibrosis but rare α-SMA staining in patients with SSc.

Expression of IFN-induced transmembrane protein 1 in glomerular endothelial cells.

Glomerular endothelial cells (GECs) are directly exposed to circulating viral particles in the glomerulus. Although viral infections may trigger the development of acute kidney injury or the worsening of pre-existing chronic kidney disease, the specific molecular mechanisms underlying antiviral reactions via the activation of endothelial Toll-like receptor 3 signaling in the kidney remain to be determined. Interferon (IFN)-induced transmembrane protein 1 (IFITM1), a member of interferon-stimulated gene protein family, is involved in the prevention of viral entry into cerebral vascular endothelial cells, respiratory epithelial cells, and endometrium. However, as far as we are aware, the implication of IFITM1 associated with viral infections in GECs has not been investigated to date. Cultured, normal human GECs were treated with polyinosinic-polycytidylic acid (poly IC), a synthesized viral double-stranded RNA, then the expression of IFITM1 was examined by quantitative real-time reverse transcription-polymerase chain reaction and western blotting. To further elucidate the poly IC-induced signaling pathway, the cells were applied to RNA interference against IFN-β, nuclear factor-κB p65, and IFN regulatory factor 3. We also conducted an immunofluorescence study to examine endothelial IFITM1 expression in biopsy specimens from patients with chronic kidney disease. We found that the activation of Toll-like receptor 3 induced endothelial expression of IFITM1, and that this involved IFN regulatory factor 3 and IFN-β, but not nuclear factor-κB. Intense endothelial IFITM1 immunoreactivity was observed in biopsy specimens from patients with lupus nephritis. Antiviral reaction-related endothelial expression of IFITM1 may be involved, at least in part, in the development of particularly in lupus nephritis. Further detailed studies of the implication of interferon stimulated genes, including IFITM1 in GECs are needed.

18F-Fluoroestradiol Tumor Uptake Is Influenced by Structural Components in Breast Cancer.

Estrogen receptor (ER) is expressed in the majority of invasive breast cancer and is an important prognostic indicator. The tumor stroma also plays an important role in disease progression. This study evaluated the effect of stromal components on 16α-[18F]-fluoro-17β-estradiol (18F-FES) uptake in breast cancer and proposed a partial-volume correction method for 18F-FES PET based on histopathological analyses. Fifteen patients with biopsy-confirmed breast cancer underwent preoperative 18F-FES PET. Estrogen receptor expression in biopsy specimens was assayed by immunohistochemistry, cellular components in surgical specimens were measured using hematoxylin-eosin staining, and nuclear components in surgical and biopsy specimens were measured using Azan-Mallory staining. The relationship between 18F-FES SUV of the primary tumor and histopathological findings including ER expression, the Allred score, ER-positive cellular component ratio, and ER-positive nuclear component ratio (NCR) was examined. The relationship between stroma-free 18F-FES SUV and ER expression was also examined. 18F-FES uptake was not significantly positively correlated with ER expression (r = 0.44, P = 0.10). 18F-FES uptake was significantly correlated with the Allred score, ER-positive cellular component ratio, and ER-positive NCR in surgical specimens (ρ = 0.60, P = 0.02; r = 0.55, P = 0.03; and r = 0.65, P = 0.01, respectively). 18F-FES uptake was predominantly correlated with ER-positive NCR in biopsy specimens (r = 0.84, P < 0.001). Stroma-free 18F-FES SUV was significantly correlated with ER expression (r = 0.78, P < 0.01). 18F-FES PET predominantly demonstrates the level of ER expression in breast cancer cell nucleus. Although tumor 18F-FES uptake is affected by the degree of stromal components, the partial volume effect on the uptake can be corrected by stroma-volume fraction in Azan-Mallory staining.

The detection value of PD-L1 expression in biopsy specimens and surgical resection specimens in non-small cell lung cancer: a meta-analysis.

BackgroundThe detection value of different types of specimens for programmed death ligand-1 (PD-L1) expression remains controversial. As such, the purpose of this meta-analysis was to compare the detection value of biopsy specimens and surgical resection specimens for PD-L1 expression in patients with non-small cell lung cancer (NSCLC).MethodsPubMed and Web of Science were searched prior to December 2020 to identify studies that compared the detection value of biopsy specimens and surgical resection specimens for PD-L1 expression in NSCLC. Quality Assessment of Diagnostic Accuracy Studies (QUADAS)-2 scale was used to evaluate the quality of the literature included. The detection value of different types of specimens for PD-L1 expression was then assessed. Besides, the relative risk (RR) with 95% CI were pooled using Review Manager 5.3 software and Stata 14.0 software.ResultsThe meta-analysis involved 12 articles and included 877 patients. There was no significant difference in the detection rate of PD-L1 at the 1% cutoff between biopsy specimens and surgical resection specimens (RR =0.89, 95% CI: 0.70–1.12, P=0.33). However, there was a significant difference between two groups when the cutoff is 50% (RR =0.69, 95% CI: 0.58–0.83, P<0.01). In addition, a subgroup analysis of the type of biopsy specimens and the PD-L1 qualitative immunohistochemistry (IHC) assays showed that the detection rate of PD-L1 in small biopsies and using the SP142 antibody were lower than in surgical specimens and using other antibodies for both the 1% and 50% cut-offs (P<0.01).ConclusionsCurrent evidence suggests that caution must be taken when using biopsy specimens from patients with advanced NSCLC to evaluate PD-L1 status eligible for immunotherapy, additional biopsy specimens sampling may be needed to minimize the risk of tumor misclassification. In addition, PD-L1 qualitative IHC assays and the type of biopsy specimens related to PD-L1 expression detection.

Aberrant Activation of NF-κB and Hypoxia Enhances Glycolytic Enzyme Hexokinase II Expression in B-Cell Lymphoma through Activation of HIF-1

Malignant lymphoma cells tend to use glycolysis for the production of ATP even with a substantial oxygen supply, a process known as aerobic glycolysis that is illustrated by a high uptake of FDG-glucose. Though elevated levels of glycolytic enzymes are responsible for aerobic glycolysis, the precise molecular mechanisms of this process are not well understood. In physiological conditions, the expression of glycolytic enzymes is mainly regulated by hypoxia inducible factor (HIF). Previously, we revealed that the aberrant activation of NF-κB causes the activation of HIF pathways in B-cell lymphoma (Exp.Hematology, 2010 &2013). It is widely accepted that abnormal activation of NF-κB is one of the hallmarks of B-cell lymphoma. Based on these notions, in the present study, we analyzed the possible link between aberrant activation of NF-κB, HIF and enhanced glycolysis in lymphoma cells. We also investigated the possibility that the inhibition of enhanced glycolysis could be a therapeutic target in lymphoma.For this study, we used a series of B-cell lymphoma cell lines. Under basal conditions, these cells display aberrant NF-κB activation and expression of the alpha subunit of HIF-1 (HIF-1α). Of the glycolytic enzymes, hexokinase II (HXKII) levels were specifically elevated in these cells. To confirm that aberrant activation of NF-κB was responsible for the elevation of HIF-1α and HXKII, we treated the cells with several NF-κB inhibitors. The NF-κB inhibitors clearly suppressed the expression of both HIF-1α and HXKII. Inhibition of HIF-1 by panobinostat also led to decreased HXKII levels. Panobinostat also suppressed hypoxia-induced HIF-1α and HXKII elevation. We further analyzed HXKII expression in biopsy specimens from newly diagnosed B-cell lymphoma cases after receiving approval from an institutional review board and acquisition of written informed consent. We analyzed 6 cases of diffuse large B-cell lymphoma (DLBCL), 3 cases of follicular lymphoma (FL) and 1 case of mantle cell lymphoma. HXKII expression was detected in 5 of the 6 DLBCL cases but not in the FL cases. Interestingly, according to Hans classification, the HXKII-positive DLBCL cases were non-GCB types.Finally, we explored the possibility that inhibition of HXKII could enhance the activity of anti-lymphoma chemotherapeutic drugs. We confirmed that hypoxia diminished the anti-lymphoma action of several chemotherapeutic drugs, including cisplatin. To investigate whether inhibition of HXKII could overcome the reduced anti-lymphoma action of these reagents, we treated the cells with either 2-deoxy-glucose or clotrimazole, which are both inhibitors for HXKII. We found that these reagents reversed the anti-lymphoma activity of cisplatin, even during hypoxia. Panobinostat also restored cisplatin-induced apoptosis in B-cell lymphoma cells during hypoxia.In conclusion, our present findings reveal that expression of glycolytic enzyme HXKII was elevated in B-cell lymphoma cells, and two distinct mechanisms, aberrant activation of NF-κB and a hypoxic microenvironment, induce HXKII elevation through activation of HIF-1. This work also suggests that the axis between HIF-1 and HXKII might be an attractive therapeutic target for B-cell lymphomas, especially those with aberrant NF-κB activity. DisclosuresKirito:Novartis Pharma KK: Honoraria.

Cyclooxygenase-2 expression is associated with chemoresistance through cancer stemness property in hypopharyngeal carcinoma.

Cyclooxygenase-2 (COX-2) is one of the two isoforms of COX, an enzyme that catalyzes the conversion of arachidonic acid to prostaglandins. COX-2 is associated with the progression in various types of cancer, and its expression has been associated with a poor prognosis in head and neck squamous cell carcinoma (HNSCC). Furthermore, COX-2 expression has been associated with resistance to anticancer drugs. However, the precise mechanism of COX-2 for chemoresistance in HNSCC has not been fully elucidated. The present study aimed to investigate the effect of COX-2 on cancer stem cell (CSC) property and to reveal its effect on chemoresistance using in vitro and clinicopathological assays in HNSCC cells and tissues. The current study analyzed the immunohistochemical expression levels of COX-2 and clinicopathological factors using matched samples of pretreatment biopsy and surgical specimens from patients with hypopharyngeal carcinoma who underwent tumor resection with preoperative chemotherapy, including docetaxel. Additionally, the chemoresistance to docetaxel with or without a COX-2 inhibitor (celecoxib) was examined in HNSCC cell lines by MTS assays. To evaluate the association of COX-2 expression with stemness property, the expression levels of CSC-associated genes after exposure to celecoxib were assessed by reverse transcription-quantitative PCR. A sphere formation assay was also performed using ultra-low attachment dishes and microscopic imaging. The immunohistochemical analysis of biopsy specimens revealed a negative association between COX-2 expression in biopsy specimens and the pathological effect of induction chemotherapy in surgical specimens. The cell survival rate under exposure to docetaxel was decreased by the addition of celecoxib. COX-2 inhibition led to downregulation of CSC-associated gene expression and sphere formation. The present findings suggested that COX-2 expression may be associated with chemoresistance through the cancer stemness property, and inhibition of COX-2 may enhance chemo-sensitivity in HNSCC. Therefore, COX-2 may be an attractive target for the treatment of HNSCC.

Overexpression of RPN2 suppresses radiosensitivity of glioma cells by activating STAT3 signal transduction

BackgroundRadiation therapy is the primary method of treatment for glioblastoma (GBM). Therefore, the suppression of radioresistance in GBM cells is of enormous significance. Ribophorin II (RPN2), a protein component of an N-oligosaccharyl transferase complex, has been associated with chemotherapy drug resistance in multiple cancers, including GBM. However, it remains unclear whether this also plays a role in radiation therapy resistance in GBM.MethodsWe conducted a bioinformatic analysis of RPN2 expression using the UCSC Cancer Genomics Browser and GEPIA database and performed an immunohistochemical assessment of RPN2 expression in biopsy specimens from 34 GBM patients who had received radiation-based therapy. We also studied the expression and function of RPN2 in radiation-resistant GBM cells.ResultsWe found that RPN2 expression was upregulated in GBM tumors and correlated with poor survival. The expression of RPN2 was also higher in GBM patients with tumor recurrence, who were classified to be resistant to radiation therapy. In the radiation-resistant GBM cells, the expression of RPN2 was also higher than in the parental cells. Depletion of RPN2 in resistant cells can sensitize these cells to radiation-induced apoptosis, and overexpression of RPN2 had the reverse effect. Myeloid cell leukemia 1 (MCL1) was found to be the downstream target of RPN2, and contributed to radiation resistance in GBM cells. Furthermore, STAT3 was found to be the regulator of MCL1, which can be activated by RPN2 dysregulation.ConclusionOur study has revealed a novel function of RPN2 in radiation-resistant GBM, and has shown that MCL1 depletion or suppression could be a promising method of therapy to overcome the resistance promoted by RPN2 dysregulation.

Isocitrate dehydrogenase 2 contributes to radiation resistance of oesophageal squamous cell carcinoma via regulating mitochondrial function and ROS/pAKT signalling

BackgroundAntioxidase alleviates the accumulation of radiation-induced reactive oxygen species (ROS) and therefore has strong connections with radioresistance. Isocitrate dehydrogenase 2 (IDH2) facilitates the turnover of antioxidase, but its role in radiotherapeutic efficiency in oesophageal squamous cell carcinoma (ESCC) still remains elusive.MethodsThe involvement of IDH2 in radiotherapeutic efficacy in ESCC was investigated in vitro and vivo by IDH2 knockdown. IDH2 expression in biopsy specimens of 141 patients was identified to evaluate its clinical significance.ResultsWe found that Kyse510 and Kyse140 cells were more radioresistant and had higher IDH2 expression. In these two cell lines, IDH2 knockdown intensified the radiation-induced ROS overload and oxidative damage on lipid, protein, and nucleic acids. In addition, IDH2 silencing aggravated the radiation-induced mitochondrial dysfunction and cell apoptosis and ultimately promoted radiosensitisation via inhibiting AKT phosphorylation in a ROS-dependent manner. Furthermore, IDH2 depletion facilitated the radiation-induced growth inhibition and cell apoptosis in murine xenografts. Finally, IDH2 expression was correlated with definite chemoradiotherapy (dCRT) efficacy and served as an independent prognostic factor for survival of ESCC patients.ConclusionsIDH2 plays a key role in the radioresistance of ESCC. Targeting IDH2 could be a promising regimen to improve radiotherapeutic efficiency in ESCC patients.

Expression pattern of p-Smad2/Smad4 as a predictor of survival in invasive breast ductal carcinoma.

The present study examined SMAD family member 4 (Smad4), SMAD family member 2 (Smad2) and phosphorylated (p-)Smad2 expression in biopsy specimens from patients with invasive breast ductal carcinoma, in order to assess their abilities as prognostic markers. A total of 126 tissue samples were selected, and the expression of Smad2, p-Smad2 and Smad4 in carcinoma tissues was detected by immunostaining, and the association between protein expression and clinicopathological variables was analyzed. Smad4 expression was negatively correlated with human epidermal growth factor receptor 2 in carcinoma tissues, and Smad4 expression was consistent with that of p-Smad2. Although multivariate analysis revealed that Smad2, p-Smad2 and Smad4 were not independent predictors, Kaplan-Meier curves demonstrated that Smad4 positivity was correlated with a longer overall survival (OS) and progression-free survival (PFS) time. However, upon analysis of combined markers, there was a significant difference between the p-Smad2/Smad4 co-positive and co-negative patients; the latter tended to exhibit a shorter OS and PFS time, and multivariate analysis revealed that the combined expression of p-Smad2 and Smad4 may be used as an independent prognostic factor. These results suggested that the assessment of p-Smad2 and Smad4 protein expression in breast ductal carcinoma biopsy specimens may provide additional prognostic information.

Expression of Ganglioside Disialosyl Globopentaosyl Ceramide in Prostate Biopsy Specimens as a Predictive Marker for Recurrence after Radical Prostatectomy.

Carbohydrate antigens are associated with carcinogenesis, cancer invasion, and metastasis and their expression reflect biological activities of various cancers. We previously reported that expression of disialosyl globopentaosyl ceramide (DSGb5), one of carbohydrate antigens, in radical prostatectomy specimens independently predicted biochemical recurrence (i.e., elevating serum prostate specific antigen without recurrent lesions in the image) after radical prostatectomy. However, it is important to evaluate the prognosis at the diagnosis. In this study we investigated DSGb5 expression in prostate biopsy specimens to develop a novel biomarker for providing appropriate management. Between 2005 and 2011, patients who underwent both prostate biopsy and radical prostatectomy in our institution were included. The median follow-up period was 88 months. DSGb5 expression was assessed by immunohistochemical staining and defined 116 patients as high DSGb5 expression (42 patients) or low DSGb5 expression (74 patients). High DSGb5 expression was significantly associated with lymphovascular invasion in radical prostatectomy specimens on both univariate and multivariable analyses (p = 0.028, 0.027). On multivariable analysis, Gleason Score in prostatectomy specimen, positive resection margin, and DSGb5 expression in the biopsy specimen were independently associated with biochemical recurrence-free survival following radical prostatectomy (p = 0.004, 0.008, 0.024). When targeting only patients with negative resection margin, DSGb5 expression was significantly associated with biochemical recurrence-free survival on both univariate and multivariable analyses (p = 0.006, 0.007). DSGb5 expression in prostate biopsy specimens is predictive of lymphovascular invasion and biochemical recurrence-free survival following radical prostatectomy. DSGb5 is a potential biomarker for preoperatively predicting oncological outcomes of prostate cancer.