Assessment of Utility of Immunohistochemical Marker Prostein for Evaluation of Primary and Metastatic Prostatic Carcinomas

Background: To assess utility of immunohistochemical marker prostein for evaluation of primary and metastatic prostatic carcinomas.Methods:Fifty- six samples of clinically suspected carcinoma prostate was included. Immunohistochemistry (IHC) was performed for assessment of Prostein (P501S). The intensity of positivity was scored from 0 to 3 as follows: score 0 = non-stained; score 1 = weak; score 2 = moderate; and score 3 = strong. The percentage of positively stained cells for each staining intensity was estimated in the respective lesions.Results:Age group 18-28 years comprised of 6 patients, 28-38 years had 12, 38- 48 years had 16 and >48 years had 22 cases. Type of cases were normal prostatic epithelium in 11, benign prostate hyperplasia in 23, HGPIN in 10, primary prostatic adenocarcinoma in 7 and metastatic prostatic adenocarcinoma in 5 cases. Prostein expression was seen in 100% in normal prostatic epithelium with intensity score of 1.8-2.1, benign prostate hyperplasia having 2-2.7, HGPIN with 2-2.3, primary prostatic adenocarcinoma having 1-1.6 and metastatic prostatic adenocarcinoma with 0.8-1.4 intensity score. Conclusion:Prostein is a new prostate specific marker which showed 100% sensitivity and specificity to identify normal and prostatic lesions.

Pediatric medulloblastoma express immune checkpoint B7-H3

Purpose Medulloblastomas (MB) are highly malignant brain tumors that predominantly occur in young infants. Immunotherapy to boost the immune system is emerging as a novel promising approach, but is often hampered by inhibitory immune checkpoints. In the present study, we have studied immune checkpoint B7-H3 expression in a tissue cohort of human pediatric MB. Methods Expression of B7-H3 was detected by immunohistochemistry and classified via B7-H3 staining intensity and percentage of B7-H3 positive tumor cells. Subsequently, B7-H3 protein expression was distinguished in MB molecular subtypes and correlated to immune cell infiltrates, patient characteristics, and survival. Results B7-H3 protein expression was found in 23 out of 24 (96%) human pediatric MB cases and in 17 out of 24 (71%) MB cases > 25% of tumor cells had any level of B7-H3 expression. B7-H3 protein expression was more frequent on Group-4 MB as compared with other molecular subtypes (p = 0.02). Tumors with high B7-H3 expression showed less influx of γδT cells (p = 0.002) and CD3+ T cells (p = 0.041). Conclusion Immune checkpoint B7-H3 is differentially expressed by the large majority of pediatric MB. This further warrants the development of novel B7-H3-directed (immuno)therapeutic methods for children with incurable, metastatic, or chemo-resistant MB.

Expression of OLFM4 and Lysozyme During Necrotizing Enterocolitis and Volvulus in Newborns: a Comparative Observational Research Study

Background: In neonatal patients with necrotizing enterocolitis (NEC) and volvulus the inflammatory response is mediated by a plurality of different proteins. The proteins olfactomedin 4 (OLFM4) and lysozyme (LYZ) are part of the intestinal mucosal defense and especially OLFM4 has rarely been evaluated in neonatal gastrointestinal diseases. The aim of this study was to compare the expression levels of OLFM4 and lysozyme during NEC and volvulus in neonates. Methods: Intestinal tissues of patients with NEC and patients with volvulus were examined using immunohistochemical staining of OLFM4 and lysozyme of formalin-fixed and paraffin-embedded sections of resected tissue. Staining-positive tissues were semi-quantitatively scored from 0 (no staining), 1 (weak staining), 2 (moderate staining) to 3 (highly intense staining) by two individual investigators.Results: Both applied antibodies against OLFM4 showed different staining patterns with higher staining intensity of the antibody OLFM4 (D1E4M). OLFM4 (median score of the antibody OLFM4 (D1E4M): 3.0) and lysozyme (median score: 3.0) are highly expressed in intestinal and immune cells during NEC. The expression of OLFM4 and lysozyme in tissue with intestinal volvulus was also observable (median score of the antibody OLFM4 (D1E4M): 1.25) and median score of the antibody against LYZ: 2.0), but lower levels could be seen in comparison to tissue with NEC (p=0.033 and p=0.037, respectively).Conclusions: Both proteins, OLFM4 and lysozyme, may play a role in the pathogenesis of NEC and volvulus in neonatal patients, but the exact mechanisms of OLFM4 and lysozyme function and their role in immunological responses have not yet been resolved. These observations add new insights as basis for further large-scale population research.

The Predictive Efficacy of Serum Exosomal microRNA-122 and microRNA-148a for Hepatocellular Carcinoma Based on Smart Healthcare

Objective. Hepatocellular carcinoma (HCC) remains a devastating tumor globally. Serum exosomes are reliable biomarkers for tumors, including HCC. Hence, this study explored the efficacy and mechanism of serum exosomes in HCC. Methods. microRNA (miR)-122 and miR-148a expressions in serum exosomes from HCC patients and healthy subjects and their predictive efficacy for HCC were detected. Correlation between serum exosomal miR-122/148a expressions with survival rate, clinical stage, lymph node metastasis, and tumor differentiation level and levels of HCC-related serum markers (CA199, FucAFP, ALD-A, and AFu) were detected. PAX2 staining intensity and expression in HCC were measured. PAX2 predictive efficacy for HCC and its correlation with clinical stage, lymph node metastasis, tumor differentiation level, and HCC-related serum marker levels were analyzed. The targeted binding relationship between miR-122 and miR-148a and PAX2 was predicted and verified. Results. Serum exosomal miR-122 and miR-148a expressions were downregulated in HCC, showing potent predictive efficacy for HCC, which was negatively related to clinical stage and lymph node metastasis and positively related to tumor differentiation level, patient survival rate, and HCC-related serum marker levels. PAX2 showed increased staining intensity and expression in HCC, together with high predictive efficacy for HCC. PAX2 expression showed a positive correlation with clinical stage and lymph node metastasis and a negative correlation with tumor differentiation level and HCC-related serum marker levels. miR-122 and miR-148a conjointly targeted PAX2 in HCC. Conclusion. We demonstrated that serum exosomal miR-122 and miR-148a played a predictive role and were linked to prognosis in HCC via interactions with PAX2.

Membrane attack complex (MAC) deposition in renal tubules is associated with interstitial fibrosis and tubular atrophy: a pilot study

IntroductionTreatment failures for lupus nephritis (LN) are high with 10%–30% of patients progressing to end-stage renal disease (ESRD) within 10 years. Interstitial fibrosis/tubular atrophy (IFTA) is a predictor of progression to ESRD. Prior studies suggest that tubulointerstitial injury secondary to proteinuria in LN is mediated by complement activation in the tubules, specifically through the membrane attack complex (MAC). This study aimed to investigate the associations between tubular MAC deposition with IFTA and proteinuria.MethodsIn this cross-sectional study, LN kidney biopsies were assessed for MAC deposition by staining for Complement C9, a component of the MAC. Chromogenic immunohistochemistry was performed on paraffin-embedded human renal biopsy sections using unconjugated, murine anti-human Complement C9 (Hycult Biotech, clone X197). Tubular C9 staining intensity was analysed as present versus absent. IFTA was defined as minimal (<10%), mild (10%–24%), moderate (25%–50%) and severe (>50%).ResultsRenal biopsies from 30 patients with LN were studied. There were 24 (80%) female sex, mean age (SD) was 33 (12) years old and 23 (77%) had pure/mixed proliferative LN. Tubular C9 staining was present in 7 (23%) biopsies. 27 patients had minimal-to-mild IFTA and 3 patients had moderate IFTA. Among the C9 + patients, 3 (43%) had moderate IFTA as compared with none in the C9- group, p=0.009. C9 + patients had higher median (IQR) proteinuria as compared with C9- patients: 6.2 g (3.3–13.1) vs 2.4 g (1.3–4.6), p=0.001 at the time of biopsy. There was no difference in estimated glomerular filtration rate (eGFR) between the C9 + and C9- groups.ConclusionThis study demonstrated that tubular MAC deposition is associated with higher degree of IFTA and proteinuria, which are predictors of progression to ESRD. These results suggest that tubular MAC deposition may be useful in classification of LN. Understanding the role of complement in tubulointerstitial injury will also identify new avenues for LN treatment.

Microtubule Integrity Is Associated with the Functional Activity of Mitochondria in HEK293

Mitochondria move along the microtubule network and produce bioenergy in the cell. However, there is no report of a relationship between bioenergetic activity of mitochondria and microtubule stability in mammalian cells. This study aimed to investigate this relationship. We treated HEK293 cells with microtubule stabilizers (Taxol and Epothilone D) or a microtubule disturber (vinorelbine), and performed live-cell imaging to determine whether mitochondrial morphology and bioenergetic activity depend on the microtubule status. Treatment with microtubule stabilizers enhanced the staining intensity of microtubules, significantly increased ATP production and the spare respiratory capacity, dramatically increased mitochondrial fusion, and promoted dynamic movement of mitochondria. By contrast, bioenergetic activity of mitochondria was significantly decreased in cells treated with the microtubule disturber. Our data suggest that microtubule stability promotes mitochondrial functional activity. In conclusion, a microtubule stabilizer can possibly recover mitochondrial functional activity in cells with unstable microtubules.

Early Initiation of Temozolomide Therapy May Improve Response in Aggressive Pituitary Adenomas

IntroductionAggressive pituitary adenomas (APAs) are, by definition, resistant to optimal multimodality therapy. The challenge lies in their early recognition and timely management. Temozolomide is increasingly being used in patients with APAs, but evidence supporting a favorable response with early initiation is lacking.MethodsThis was a single-center study of all patients with APAs who received at least 3 cycles of temozolomide (150–200 mg/m2). Their baseline clinico-biochemical and radiological profiles were recorded. Immunohistochemical evaluation for cell-cycle markers O6-methylguanine-DNA methyltransferase (MGMT), MutS homolog 2 (MSH2), MutS homolog 6 (MSH6), MutL homolog 1 (MLH1), and postmeiotic segregation increased 2 (PMS2) was performed, and h-scores (product of the number of positive cells and staining intensity) were calculated. Response was assessed in terms of radiological response using the RECIST criteria. Patients with controlled disease (≥30% reduction in tumor volume) were classified as responders.ResultsThe study comprised 35 patients (48.6% acromegaly, 37.1% prolactinomas, and 14.3% non-functioning pituitary adenomas). The median number of temozolomide (TMZ) cycles was 9 (IQR 6–14). Responders constituted 68.6% of the cohort and were more likely to have functional tumors, a lower percentage of MGMT-positive staining cells, and lower MGMT h-scores. There was a significantly longer lag period in the initiation of TMZ therapy in non-responders as compared with responders (median 36 vs. 15 months, p = 0.01). ROC-derived cutoffs of 31 months for the duration between diagnosis and TMZ initiation, low-to-intermediate MGMT positivity (40% tumor cells), and MGMT h-score of 80 all had a sensitivity exceeding 80% and a specificity exceeding 70% to predict response.ConclusionEarly initiation of TMZ therapy, functional tumors, and low MGMT h-score predict a favorable response to TMZ in APAs.

L-type amino acid transporter (LAT) 1 expression in 18F-FET-negative gliomas

Background O-(2-[18F]-fluoroethyl)-L-tyrosine (18F-FET) is a highly sensitive PET tracer for glioma imaging, and its uptake is suggested to be driven by an overexpression of the L-type amino-acid transporter 1 (LAT1). However, 30% of low- and 5% of high-grade gliomas do not present enhanced 18F-FET uptake at primary diagnosis (“18F-FET-negative gliomas”) and the pathophysiologic basis for this phenomenon remains unclear. The aim of this study was to determine the expression of LAT1 in a homogeneous group of newly diagnosed 18F-FET-negative gliomas and to compare them to a matched group of 18F-FET-positive gliomas. Forty newly diagnosed IDH-mutant astrocytomas without 1p/19q codeletion were evaluated (n = 20 18F-FET-negative (tumour-to-background ratio (TBR) < 1.6), n = 20 18F-FET-positive gliomas (TBR > 1.6)). LAT1 immunohistochemistry (IHC) was performed using SLC7A5/LAT1 antibody. The percentage of LAT1-positive tumour cells (%) and the staining intensity (range 0–2) were multiplied to an overall score (H-score; range 0–200) and correlated to PET findings as well as progression-free survival (PFS). Results IHC staining of LAT1 expression was positive in both, 18F-FET-positive as well as 18F-FET-negative gliomas. No differences were found between the 18F-FET-negative and 18F-FET-positive group with regard to percentage of LAT1-positive tumour cells, staining intensity or H-score. Interestingly, the LAT1 expression showed a significant negative correlation with the PFS (p = 0.031), whereas no significant correlation was found for TBRmax, neither in the overall group nor in the 18F-FET-positive group only (p = 0.651 and p = 0.140). Conclusion Although LAT1 is reported to mediate the uptake of 18F-FET into tumour cells, the levels of LAT1 expression do not correlate with the levels of 18F-FET uptake in IDH-mutant astrocytomas. In particular, the lack of tracer uptake in 18F-FET-negative gliomas cannot be explained by a reduced LAT1 expression. A higher LAT1 expression in IDH-mutant astrocytomas seems to be associated with a short PFS. Further studies regarding mechanisms influencing the uptake of 18F-FET are necessary.

Identification and expression analysis of the glycosyltransferase GT43 family members in bamboo reveal their potential function in xylan biosynthesis during rapid growth

Background Xylan is one of the most abundant hemicelluloses and can crosslink cellulose and lignin to increase the stability of cell walls. A number of genes encoding glycosyltransferases play vital roles in xylan biosynthesis in plants, such as those of the GT43 family. However, little is known about glycosyltransferases in bamboo, especially woody bamboo which is a good substitute for timber. Results A total of 17 GT43 genes (PeGT43–1 ~ PeGT43–17) were identified in the genome of moso bamboo (Phyllostachys edulis), which belong to three subfamilies with specific motifs. The phylogenetic and collinearity analyses showed that PeGT43s may have undergone gene duplication, as a result of collinearity found in 12 pairs of PeGT43s, and between 17 PeGT43s and 10 OsGT43s. A set of cis-acting elements such as hormones, abiotic stress response and MYB binding elements were found in the promoter of PeGT43s. PeGT43s were expressed differently in 26 tissues, among which the highest expression level was found in the shoots, especially in the rapid elongation zone and nodes. The genes coexpressed with PeGT43s were annotated as associated with polysaccharide metabolism and cell wall biosynthesis. qRT–PCR results showed that the coexpressed genes had similar expression patterns with a significant increase in 4.0 m shoots and a peak in 6.0 m shoots during fast growth. In addition, the xylan content and structural polysaccharide staining intensity in bamboo shoots showed a strong positive correlation with the expression of PeGT43s. Yeast one-hybrid assays demonstrated that PeMYB35 could recognize the 5′ UTR/promoter of PeGT43–5 by binding to the SMRE cis-elements. Conclusions PeGT43s were found to be adapted to the requirement of xylan biosynthesis during rapid cell elongation and cell wall accumulation, as evidenced by the expression profile of PeGT43s and the rate of xylan accumulation in bamboo shoots. Yeast one-hybrid analysis suggested that PeMYB35 might be involved in xylan biosynthesis by regulating the expression of PeGT43–5 by binding to its 5′ UTR/promoter. Our study provides a comprehensive understanding of PeGT43s in moso bamboo and lays a foundation for further functional analysis of PeGT43s for xylan biosynthesis during rapid growth.

Rational Targets in Systemic AL Amyloidosis

Introduction Dysproteinemia refers to a spectrum of gammopathies in which an aberrant clone(s) of late stage B lymphoid cells or plasma cells produces monoclonal immunoglobulins and/or fragments. The spectrum includes incidentally noted benign conditions like monoclonal gammopathy of undetermined significance (MGUS) to life threatening diseases such as multiple myeloma (MM) and systemic light chain (AL) amyloidosis. Exploitation of normal plasma cell biology forms the basis of treatments used in both MM and AL amyloidosis. In recent years, immunotherapeutic strategies targeting plasma cells have redefined the treatment landscape of MM and could be particularly useful in AL amyloidosis which is distinguished by a low burden of less proliferative disease and a paucity of high risk genetics. We sought to characterize the expression of potentially clinically relevant and/or immunotherapeutic targets in amyloidogenic plasma cells. Methods All patients diagnosed with systemic AL amyloidosis at Memorial Sloan Kettering Cancer Center, NY between January 1, 2012, and December 31, 2018, who had unstained bone marrow samples were identified. Decalcified formalin fixed paraffin embedded bone marrow biopsy sections were stained for G protein-coupled receptor, class C group 5 member D (GPRC5D), Cyclin D1 (CCND1), B-cell lymphoma 2 (BCL2), and B-cell maturation antigen (BCMA) expression using standard laboratory developed Immunohistochemistry (IHC) assays in a CLIA compliant setting. We scored the biopsies for percentage expression, and intensity of staining. We also obtained the demographic details, staging, and cytogenetic information for the patients from whom these samples were obtained. Results During the queried period, 32 unstained samples were available for testing from 27 unique patients. There were 27 diagnostic and 5 patients had additional samples from the time of relapse. The median age was 63 years (range 41-73). 64% of patients were male and 75% had lambda restricted plasma cells. Cytogenetic abnormalities using fluorescence in situ hybridization (FISH) were reviewed, t(11;14) was seen in 38% samples. The median clonal PC burden in BM at diagnosis was 10% (range 2-80%) and 36% had &gt;10% plasma cells. In clonal PCs, the median BCL2 expression was 100% (range 80-100%) with a staining intensity of 2 (range 1-3). 84% (27) samples had BCL2 expression meeting threshold used in the Bellini study. CCND1 expression could be tested in 16 samples median expression 100% (range 20-100%) and median staining intensity 2 (range 1-3). Patients with CCND1 expression also had 100% BCL2 staining. GPRC5D expression was available in 18 samples and all samples tested expressed GPRC5D with median 80% (range 30-100%) with median intensity 1 (range 1-3). BCMA expression was available for 25 samples, with median expression 80% (range 50-100%) with a median staining intensity 2 (range 1-3). Among the five patients with samples from diagnosis and relapse, samples retained their expression of BCL2, BCMA, and GPRC5D. Among samples with t(11;14) by FISH, 92% expressed BCL2 (per Bellini study) and 58% expressed CCND1. Conclusion Cell surface expression of novel targets has enabled the development of several efficacious therapeutic strategies in MM. Amyloidogenic plasma cells express GPRC5D, BCMA and BCL2. Our data provides the rationale for careful investigation and development of targeted agents (BCL2 inhibitors, e.g.venetoclax) and immunotherapeutic strategies directed at GPRC5D and BCMA in AL amyloidosis. Figure 1 Figure 1. Disclosures Dogan: Roche: Consultancy, Research Funding; Peer View: Honoraria; Seattle Genetics: Consultancy; Takeda: Consultancy, Research Funding; Physicians' Education Resource: Honoraria; EUSA Pharma: Consultancy. Hassoun: Celgene, Takeda, Janssen: Research Funding. Mailankody: Fate Therapeutics: Research Funding; Allogene Therapeutics: Research Funding; Bristol Myers Squibb/Juno: Research Funding; Plexus Communications: Honoraria; Legend Biotech: Consultancy; Evicore: Consultancy; Jansen Oncology: Research Funding; Physician Education Resource: Honoraria; Takeda Oncology: Research Funding. Giralt: SANOFI: Membership on an entity's Board of Directors or advisory committees; AMGEN: Membership on an entity's Board of Directors or advisory committees; JENSENN: Membership on an entity's Board of Directors or advisory committees; GSK: Membership on an entity's Board of Directors or advisory committees; JAZZ: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; PFIZER: Membership on an entity's Board of Directors or advisory committees; CELGENE: Membership on an entity's Board of Directors or advisory committees; Actinnum: Membership on an entity's Board of Directors or advisory committees. Landau: Takeda: Research Funding; Genzyme: Honoraria; Takeda, Janssen, Caelum Biosciences, Celgene, Pfizer, Genzyme: Membership on an entity's Board of Directors or advisory committees.