Staining Intensity Research Articles

Tumor- and host-derived heparanase-2 (Hpa2) attenuates tumorigenicity: role of Hpa2 in macrophage polarization and BRD7 nuclear localization

Little attention was given to heparanase 2 (Hpa2) over the last two decades, possibly because it lacks a heparan sulfate (HS)-degrading activity typical of heparanase. Emerging results suggest, nonetheless, that Hpa2 plays a role in human pathologies, including cancer progression where it functions as a tumor suppressor. Here, we examined the role of Hpa2 in cervical carcinoma. We report that high levels of Hpa2 correlate with prolonged survival of cervical carcinoma patients. Strong staining intensity of Hpa2 also correlates with low tumor grade. Overexpression of Hpa2 in SiHa cervical carcinoma cells resulted in tumor xenografts that were two-fold smaller than control tumors. Interestingly, even smaller tumor xenografts were developed by SiHa cells overexpressing the Pro140Arg and Asn543Ile Hpa2 missense mutations that were identified in patients diagnosed with urofacial syndrome (UFS). Utilizing the Ras recruitment system, we identified bromodomain-containing protein 7 (BRD7) to interact with Hpa2 and found that both BRD7 and the Hpa2 mutants are translocated to the cell nucleus in tumors developed by the Pro140Arg and Asn543Ile Hpa2 mutants. Utilizing our newly developed conditional Hpa2-KO mice, we further show that Hpa2 plays a critical role in macrophage polarization; in the absence of Hpa2, macrophages are shifted towards pro-tumorigenic, M2 phenotype. Notably, implanting SiHa cervical carcinoma cells together with Hpa2-KO macrophages promoted tumor growth. These results support, and further expand, the notion that Hpa2 functions as a tumor suppressor, co-operating with another tumor suppressor, BRD7.

Deployment of a Machine Learning Algorithm in a Real-World Cohort for Quality Control Monitoring of Human Epidermal Growth Factor-2-Stained Clinical Specimens in Breast Cancer.

Precise determination of biomarker status is necessary for clinical trial enrollment and endpoint analyses, as well as for optimal treatment determination in real-world practice. However, variabilities may be introduced into this process due to the processing of clinical specimens by different laboratories and assessment by distinct pathologists. Machine learning tools have the potential to minimize inconsistencies, although their use is not presently widespread. To assess the applicability of machine learning to the quality control process for biomarker scoring in oncology, we developed and validated an automated machine learning model to be applied as a quality control tool for monitoring the assessment of human epidermal growth factor-2 (HER2). The model was trained using whole slide images from multiple sources to quantify HER2 expression and measure immunohistochemistry stain intensity, tumor area, and the presence of artifacts or ductal carcinoma in situ across breast cancer phenotypes. The quality control tool was deployed in a real-world cohort of HER2-stained breast cancer sample images collected from routine diagnostic practice to evaluate trends in HER2 testing quality indicators and between pathology laboratories. Automated image analysis for HER2 scoring is consistent and reliable using this algorithm. Deployment of the HER2 quality control tool across 3 clinical laboratories revealed interlaboratory variability in HER2 scoring and inconsistencies in data reporting. These results support the future incorporation of quality control algorithms for real-time monitoring of clinical laboratories contributing to clinical trials in oncology and in the real-world setting of HER2 immunohistochemistry testing in local clinical laboratories and hospitals.

CD44 Immunohistochemical Expression in Central and Peripheral Parts of Prostatic Adenocarcinoma: An Institutional Study

Background and Objectives: Prostate cancer is one of the most commonly diagnosed cancers in the male population and the fifth leading cause of cancer death worldwide in men as of 2022. One of the potential biomarkers that can predict the progression of the disease is the transmembrane adhesion molecule CD44s. The aims of this study were to determine the expression of CD44s in prostate cancer in the central tumor mass and in the tumor periphery of the disease and to compare it with the clinicopathological parameters (PSA, Gleason score, surgical margins, and biochemical recurrence of the disease) in patients treated with radical prostatectomy. Materials and Methods: The research was randomized retrospectively during the period from 2001 to 2006. Tissue microarrays of 121 archival acinar prostate carcinoma samples were immunohistochemically evaluated for CD44s expression. The immunoexpression was determined semiquantitatively, taking into account the percentage (0 (0–5%), 1 (6–24%), 2 (26–75%), and 3 (76–100%) and intensity of the membranous staining of the tumor cells (0 absent; 1 weak at 400×; 2 intermediates at 100×; 3 strong at 40×) and calculated to obtain a final score (0–3 were regarded as negative; 4–6 were regarded as positive). Results: For statistical purposes, we divided the tumors into two categories: Gleason grade group 1 makes up 80.7% and grade group 2, which includes all the remaining Gleason grade groups (out of 2–5), accounts for 19.3% of the tumors. Grade group 1 had the highest incidence of score 4 (positive expression). There were statistically significantly more positive expressions in those tumors with negative prostatectomy margins (chi square: p = 0.001; Cramer V: 0.319). There was no correlation between CD44s expression and biochemical recurrence (p = 0.218), nor with the preoperative PSA values (p = 0.165). In the grade group 1 tumors, the CD44s immunoexpression and status of prostatectomy margin were statistically significantly related with negative margins (p = 0.028). An analysis of the expression of CD44s according to the localization in the central part of the tumor mass and on the periphery of the cancer in the group of tumors with a positive margin did not show a significant correlation because the sample was too small. Descriptively, it can be noted that the expression on the periphery was higher, and the central/peripheral expression ratio was higher in favor of the periphery. Conclusions: Our results provide insight into the possible value of CD44s expression for predicting the behavior of prostate tumors and the justification of therapy after a prostatectomy. Also hypothetically, they indicate a protective role of CD44s in a group of well-differentiated tumors at the periphery of the tumor mass. Therefore, it is useful to study the CD44s molecule further in this sense.

Changes in immunofluorescence staining during islet regeneration in a cystic fibrosis-related diabetes (CFRD) ferret model

ABSTRACT Background Knockout (KO) ferrets with the cystic fibrosis transmembrane conductance regulator (CFTR) exhibit distinct phases of dysglycemia and pancreatic remodeling prior to cystic fibrosis-related diabetes (CFRD) development. Following normoglycemia during the first month of life (Phase l), hyperglycemia occurs during the subsequent 2 months (Phase Il) with decreased islet mass, followed by a period of near normoglycemia (Phase Ill) in which the islets regenerate. We aimed to characterize islet hormone expression patterns across these Phases. Methods Immunofluorescence staining per islet area was performed to characterize islet hormone expression patterns in age matched CFTR KO and wild type (WT) ferrets, focusing on the first three phases. Results In Phase I, insulin staining intensity was higher in CF (p < 0.01) than WT but decreased in Phase III (p < 0.0001). Glucagon was lower in CF during Phases I and increased in Phase III, while proinsulin decreased (p < 0.0001) Phases II and III. CF sections showed lower proinsulin-to-insulin ratio in Phase I (p < 0.01) and in Phase III (p < 0.05) compared to WT. Conversely, glucagon-to-insulin ratio was lower in CF in Phase I (p < 0.0001) but increased in Phase III (p < 0.0001). Mender’s coefficient overlap showed higher overlap of insulin over proinsulin in CF sections in Phase II (p < 0.001) and Phase III (p < 0.0001) compared to WT. Mender’s coefficient rate was higher in CF sections during Phase II (p < 0.001). Conclusion CF ferret islets revealed significant immunofluorescent staining changes compared to WT during various phases of disease, providing insights into CRFD pathophysiology.

P53 marker expression in epithelial ovarian tumours in a centre in Nigeria – a descriptive study

Backgroundp53 is a tumor suppressor gene. p53 expression in epithelial ovarian tumors (EOTs) is correlated with their biological behavior and predicts patient overall survival. However, there is a dearth of knowledge regarding p53 expression in these tumors among women from southwest Nigeria. Our study aimed to determine the patterns of p53 expression in various types of epithelial ovarian tumours.MethodsWe conducted a retrospective study of epithelial ovarian tumours. We retrieved formalin-fixed, paraffin-embedded (FFPE) tissue blocks of previously diagnosed epithelial tumors from the departmental archive. We performed immunohistochemical analysis using p53 antibodies. We scored the expression and staining intensity of p53 as follows: negative (0), focal/weakly positive (1 +), and diffuse/strongly positive (2 +) on the basis of the recommended Cytomation scoring system.ResultsThe spectrum of p53 expression in the 51 histologically diagnosed cases revealed that 29 cases had no expression, consisting of 21 benign EOTs, two borderline EOTs, and six malignant EOTs. Nine cases exhibited wild-type expression, including six serous carcinomas, two mucinous carcinomas, and one signet ring cell carcinoma. p53 overexpression was observed in 13 patients overall, with 12 having serous carcinomas and one having endometrioid carcinoma. Among the 21 serous carcinoma patients, 28.6% (6 patients) presented with wild-type p53 expression, 57.1% (12 patients) presented with p53 overexpression, and 14.3% (three patients) presented negative p53 expression. There was a significant association between p53 expression and the histological grade of serous carcinoma.ConclusionMost epithelial ovarian carcinomas in our hospital are high grade, with many serous carcinomas showing either p53 overexpression or loss of expression. This may contribute to the poor patient survival rate.

The Potential Utility of RAS Q61R Immunohistochemistry as a Screening Tool in Pre-operative Fine Needle Aspirates of Medullary Thyroid Carcinoma.

Medullary thyroid carcinoma (MTC) can either be sporadic, often via mutually exclusive RET or RAS alterations, or inherited via a RET germline alteration. Germline testing is recommended for all patients diagnosed with MTC. RAS p.Q61R immunohistochemistry (RASQ61R-IHC) can identify a subset of RAS-mutated MTCs on resections, but whether this could be applied pre-operatively to cytology specimens remains unclear. Herein, we assessed RASQ61R-IHC in a tri-institutional cohort of cytologic and histologic MTC specimens with available molecular and germline data. Thirty-four fine needle aspirates with cell blocks were identified between three institutions from 2009 to 2024 with corresponding histology. Tumor sequencing and germline data were recorded, if available. RASQ61R-IHC was scored on staining intensity with documentation of membranous accentuation. Sensitivity, specificity, positive predictive (PPV), and negative predictive values (NPV) were calculated. Of the MTCs, 29% were germline-mutated, and 71% were sporadic. Among all sporadic MTCs (n = 22), 41% were RET-altered, 27% were RAS-altered, and 31.8% did not have available data. With any RASQ61R-IHC staining considered positive, sensitivity, specificity, PPV, and NPV for detecting RAS p.Q61R-mutated MTCs were 100%, 72.7%, 45.4%, and 100%, respectively. Requiring a stain score of > 1 and/or membranous accentuation for a true positive changed sensitivity, specificity, PPV, and NPV to 100%, 100%, 100%, and 100%, respectively. RASQ61R-IHC membranous staining was 100% predictive of RET-negative germline testing. RASQ61R-IHC, when requiring a score > 1 and/or membranous stain accentuation for true positive, had high sensitivity and specificity for RAS p.Q61R mutation in cytologic and surgical MTC specimens. Moreover, RASQ61R-IHC is a rapid and inexpensive modality that could potentially tailor which MTC patients undergo germline testing.

Expression of CD68+ Cells in Synovial Tissue from Patients with PsA and its Association with Disease Activity Indices: A Clinical Pilot Study.

Investigating CD68+ positive cells in the synovial tissue is crucial for understanding the pathogenesis of psoriatic arthritis (PsA) and developing targeted treatment strategies. The role of CD68+ positive cells in the synovial tissue of patients with PsA for joint destruction has not been fully studied. The objective of the study was to examine the presence of CD68+ cells in the synovial tissue of patients with PsA, particularly those with high inflammatory activity. Synovial tissue samples were collected during knee joint replacement surgeries from patients with PsA (16 patients) and gonarthrosis (25 patients). Immunohistochemical methods were employed to detect CD68+ cell expression in the tissue samples. The results were analyzed by histologists, and the staining intensity and percentage of positively stained cells were evaluated. The data were then divided into three groups for statistical analysis: negative, weakly positive, and strongly positive histological samples. Routine indices for disease activity, VAS, DAPSA, PASDAI, and mCPDAI were used to assess PsA activity in all patients and to assess correlations with CD68+ positive cells in the synovial tissue. Statistical analysis was performed using SPSS version 26.0 (SPSS Inc., Chicago, IL, USA). The expression of CD68+ positive cells was significantly higher in patients with PsA compared to those with activated gonarthrosis (p < 0.001). The indices for disease activity, VAS, DAPSA, PASDAI, mCPDAI, and mCPDAI showed a significant positive relationship with the expression of CD68 + cells on synovial tissue in patients with PsA (p < 0.01) Conclusion: The findings of the study confirm the increased numbers of CD68+ cells in PsA vs. gonathrosis synovium. This suggests the need to explore therapeutic approaches aimed at suppressing or blocking CD68+ cells to potentially mitigate joint damage.

Expression of Immune Checkpoint Regulator Cytotoxic T Lymphocyte Antigen 4 (CTLA-4) and Programmed Cell Death Protein Ligand 1 (PD-L1) in Invasive Ductal Carcinoma Breast.

Despite significant advancement in the diagnostic and therapeutic aspects of breast carcinoma, the prognosis remains dismal. Recently, with advances in its understanding, various immune system-based management strategies have been developed. CTLA-4 suppresses lymphocyte reactivity, IL-2 secretion, and IL-2 receptor expression and triggers cell cycle arrest. PD-L1 inhibits the proliferation and cytotoxicity of T cells and inhibits release of cytokines. Hence, we planned to evaluate the immunoexpression of CTLA-4 and PD-L1 in invasive ductal carcinoma breast and seek correlation between their immunopositivity and the clinicopathological parameters. This was a retrospective study conducted on archival material of 50 cases of breast carcinoma tissue microarrays. Clinicopathological details were recorded. All cases were evaluated for immunohistochemical expression of CTLA-4 and PD-L1. Cytoplasmic expression of CTLA-4 and membranous expression of PD-L1 were considered positive and staining intensity was recorded as mild, moderate, and intense. Data was recorded and analyzed. Immunopositivity for CTLA-4 was seen in 92% of cases of breast carcinoma. CTLA-4 staining intensity showed significant association with TNM staging of breast carcinomas (p = 0.036). Age group of the breast carcinoma cases showed a statistically significant correlation with PD-L1 immunoexpression (p = 0.002). No significant correlation was found between all other clinicopathological characteristics and CTLA-4 or PD-L1 immunostaining. Our study shows that CTLA-4 is a more important immune checkpoint regulator in breast carcinomas in comparison to PD-L1. Thus, anti-CTLA-4 immunotherapy might prove to be of immense help in the treatment of invasive ductal carcinoma breast showing overexpression of CTLA-4.

Characterization of Gastric/Gastrointestinal-Like Immunophenotypes in Endometrial Endometrioid Adenocarcinomas, Including Endometrioid Adenocarcinomas with Mucinous Differentiation.

Endometrial mucinous carcinoma of the gastric [gastrointestinal] type (MCG) is a rare, possibly aggressive subtype of endometrial cancer that should be distinguished from its potential mimics, including endometrioid carcinoma (EEC). Herein, we assess the frequency of gastric and gastrointestinal immunophenotypes in EEC without any discernible gastric/gastrointestinal-type morphology. Immunohistochemical analyses for KRT(CK)7, KRT20, CDX2, ER, SATB2, MUC6, PAX8, and HIK1083 were performed on 81 EEC, inclusive of consecutively archived low grade [with (n=22) and without (n=47) mucinous differentiation] and high grade (n=12) cases. None displayed gastric-type morphology or goblet cells. Expression levels were semi-quantified as H-scores (combining intensity and extent of staining) on a standardized 0-300 scale. Among the gastric/gastrointestinal-type markers, 56%, 62%, 23%, 25%, and 0% of cases expressed MUC6, CDX2, KRT20, SATB2, and HIK1083 respectively. The expression levels for positive cases were generally low, with average H-scores being 49.5 [range 1-250] for MUC6, 33.7 [1-285] for CDX2, 24.0 [1-270] for CK20, and 30.5 [2-220] for SATB2. Ten (12.35%) cases showed high expression (H ≥200) of at least 1 gastric/gastrointestinal-type marker, including 1, 2, 2 and 6 cases that were high positive for KRT20, SATB2, CDX2 and MUC6 respectively. Immunoreactive foci were generally indistinguishable from background at the morphologic level. There was no statistically significant correlation between the expression of any of the gastric/gastrointestinal-type markers and ER, KRT7 or PAX8 expression. In summary, gastric/gastrointestinal-type proteins are not uncommonly expressed at low levels in EEC. As such, positivity for these markers cannot be the sole basis for distinguishing EEC from MCG.

Prevalence of HER3 Expression in Pancreatic Cancer Patients Treated With Systemic Chemotherapy.

Although activation of human epidermal growth factor receptor 3 (HER3) is linked to resistance to targeted therapies in several cancer types, the HER3 expression profile during pancreatic cancer treatment remains unknown. We evaluated the HER3 expression status after chemotherapy for pancreatic cancer and its association with clinicopathological features and clinical outcomes. We included patients with pancreatic cancer who underwent chemotherapy and whose post-treatment archival tissue specimens were collected. HER3 expression was retrospectively assessed by immunohistochemistry scoring (0, 1+, 2+, and 3+) of the membranous staining intensity. HER3 expression after chemotherapy was evaluated in 41 patients, with matched-pair analysis in five patients before and after chemotherapy. HER3 expression was observed in most of the patients after chemotherapy, demonstrating IHC scores of ≥ 1+ and ≥ 2+ in 40 (98%) and 26 (63%) of 41 patients, respectively. Of the 38 patients with adenocarcinoma, the median overall survival in the HER3 (2+/3+) and HER3 (0/1+) groups was 21.0 and 17.1 months, respectively. The comparison of HER3 expression before and after chemotherapy performed in five cases revealed that scores changed from 2+/3+ to 0/1+ in one case, 0/1+ to 2+/3+ in another case, and remained at 2+/3+ in three cases. Cancer genome profiling tests in eight cases found no HER3 amplification or mutation, and seven of these cases had adenocarcinomas with KRAS and TP53 mutations. A high prevalence of HER3 expression was observed in pancreatic cancer patients after chemotherapy. Our findings indicate that HER3 is a potential therapeutic target for pancreatic cancer, deserving further clinical investigation.

R183Q GNAQ Sturge–Weber Syndrome Leptomeningeal and Cerebrovascular Developmental Mouse Model

Objective(s): Sturge–Weber syndrome (SWS), a rare neurovascular malformation disorder, is usually caused by the R183Q GNAQ somatic mosaic mutation enriched in brain endothelial cells. A developmental mouse model of SWS brain involvement is needed to investigate mutation impact upon brain vascular development and to facilitate preclinical drug studies. Methods: A new Tet-ON R183Q GNAQ transgenic mouse line was paired with rtTA tet transactivator mice under the Tie2 promoter to generate mice expressing endothelial R183Q GNAQ in the presence of doxycycline. Litters were perfused at P14-17; half received a subseizure dose (1.5 mg/kg; intraperitoneal) of kainate an hour before perfusion. A subset was perfused with Evans blue. Fixed mouse brains were stained with X-gal, DAPI, and antibodies for Gαq, Tie2, phosphorylated-S6, and claudin-5. Images were scored for vessel staining intensity. Results: X-gal staining was seen only in mutant mice; leptomeningeal endothelial X-gal staining was more frequent in kainate-treated mice (P &lt; 0.001). When perfused with Evans blue, only mutant brains showed severe staining (P = 0.028). Median phosphorylated-S6 vessel scores were significantly higher in the leptomeninges of mutant mice (P = 0.035). Mutant cortical microvessels demonstrated discontinuous claudin-5 and phosphorylated-S6 staining as well as increased vessel length in kainate-treated mice (P = 0.024). Conclusions: The new R183Q GNAQ Tet-ON developmental mouse brain model of SWS demonstrates endothelial expression of mutant Gαq associated with blood–brain barrier breakdown, altered vascular mammalian target of rapamycin activity, and abnormal cortical microvessel structure. This new translational model can be used to develop new drug targets and treatments for SWS.

The Efficacy and Safety of Mirvetuximab Soravtansine in FRα-Positive, Third-Line and Later, Recurrent Platinum-Sensitive Ovarian Cancer: The Single-Arm Phase 2 PICCOLO Trial

Mirvetuximab soravtansine-gynx (MIRV) is a first-in-class, folate receptor alpha (FRα)-targeting antibody-drug conjugate with US Food and Drug Administration approval for FRα-positive platinum-resistant ovarian cancer. PICCOLO is a phase 2, global, open-label, single-arm trial of MIRV as third-line or greater (≥3L) treatment in patients with FRα-positive (≥75% of cells with ≥2+ staining intensity) recurrent platinum-sensitive ovarian cancer (PSOC). Participants received MIRV (6 mg/kg adjusted ideal body weight every 3 weeks) until progressive disease (PD), unacceptable toxicity, withdrawal of consent, or death. Primary endpoint was investigator-assessed objective response rate (ORR). Key secondary endpoint was investigator-assessed duration of response (DOR). Additional endpoints included investigator-assessed progression-free survival (PFS), overall survival (OS), and safety. Analyses of subgroups by disease characteristics (eg, platinum-free interval) and treatment history (eg, prior bevacizumab and poly [ADP-ribose] polymerase inhibitor [PARPi] treatment), were exploratory. Seventy-nine participants were enrolled and efficacy evaluable. The primary endpoint was met; ORR was 51.9% (95% CI, 40.4-63.3). Median DOR was 8.25 months (95% CI, 5.55-10.78) and median PFS was 6.93 months (95% CI, 5.85-9.59). OS was not mature at data cutoff. ORR was 45.8% (95% CI, 32.7-59.2) in participants with PD while on/within 30 days of prior PARPi (n=59) and 60.0% (95% CI, 14.7-94.7) in those without PD with prior PARPi (n=5). No new safety signals occurred; most common treatment-emergent adverse events (TEAEs) were gastrointestinal, neurosensory, and resolvable ocular events. TEAEs led to discontinuation in 13 participants (16%) and death in 2 participants (3%). MIRV as ≥3L treatment in heavily pretreated recurrent FRα-positive PSOC demonstrated notable efficacy and tolerable safety, including among those with prior PD on or within 30 days of PARPi. (Funding, ImmunoGen, Inc; NCT05041257).

Immunohistochemical Expression of ROR2 in Gastrointestinal Stromal Tumor.

Gastrointestinal stromal tumors (GIST) are the most common mesenchymal tumors affecting the digestive tract, comprising approximately 0.1-3% of all gastrointestinal cancers. ROR2, a member of the receptor tyrosine kinase orphan receptor subfamily, functions as a signaling receptor for Wnt ligands. This study aims to assess the prognostic significance of ROR2 expression in GIST. A total of 56 paraffin-embedded blocks of GIST cases originating from different parts of the gastrointestinal tract (GIT) were included in this study. Immunohistochemistry was performed to detect ROR2 expression. ROR2 expression was observed in 71.4% of GIST cases, and strong intensity of ROR2 staining was significantly associated with prolonged overall survival of GIST patients (p = 0.048). Furthermore, ROR2 positivity and the percentage of immunostaining showed an inverse correlation with tumor progression. GIST cases displaying ROR2 positivity exhibit a reduced likelihood of disease progression and demonstrate favorable prognostic characteristics.

Physical and Mechanical Properties of Bulk-fill Resin Composites Submitted to Additional Polymerization for Use in Semi-direct Restorations.

Physical and mechanical properties of high-viscosity bulk-fill resin composites submitted to additional polymerization for semi-direct use were evaluated. Filtek Z350 XT, Aura Bulk Fill, Beautifil Bulk Restorative, Filtek One Bulk Fill Restorative, and Tetric N-Ceram Bulk Fill were submitted to additional polymerization to evaluate sorption, solubility, surface microhardness, surface roughness before and after simulated brushing, color stability after coffee staining, flexural strength, elastic modulus, and modulus of resilience. Filtek Z350 XT and Filtek One Bulk Fill Restorative showed higher sorption values, while Aura Bulk Fill showed higher solubility (p<0.0001). Microhardness values were significantly higher for Filtek Z350 XT (p<0.0001). Roughness increased after wear for all resins (p<0.05). All resins exhibited staining, with significantly higher ΔEab, ΔE00, and ΔWID values observed for Beautifil Bulk Restorative (p<0.0001). Flexural strength values were higher for Filtek Z350 XT and Filtek One Bulk Fill Restorative in comparison with the others (p<0.0001). Filtek One Bulk-Fill had higher elastic modulus and modulus of resilience values (p<0.0001). Physical and mechanical properties varied according to the composition. None of the bulk-fill resins showed surface microhardness and roughness properties after brushing similar to or superior to those of the conventional type. Color stability after staining depended on resin composition, with Beautifil Bulk Restorative showing more intense staining.

ROLE OF H3K27me3 AND Ki67 LABELLING INDEX IN ASSESSING THE BIOLOGICAL BEHAVIOUR OF MENINGIOMAS

Meningiomas are non-malignant neoplasms primarily originating from arachnoid cells and are classified into three grades [1, 2, and 3] based on histological features according to the WHO classification. However, this classification system is imperfect especially for grade 1 and 2 meningiomas as many grade 1 tumors recur. Meningiomas are hence a histologically diverse class of tumors exhibiting more unpredictable behaviour. Therefore, more improved classification is required, possibly using novel and more dependable biomarkers. In this study, we aim to investigate the role of the H3K27me3 and Ki67 labelling index in assessing the biological behaviour of meningiomas. The study was conceived, with the primary objective of examining the expression of H3k27me3 and Ki67 labelling index in grade 1/2 meningiomas with atypical features to ascertain if this potentially impacts patient prognosis. Upon obtaining clearance from the Institutional Ethical Committee (IEC), the authors studied 81 cases of meningiomas including 11 recurrent cases. The study used immunohistochemistry to evaluate the Ki67 index and H3K27me3 immunohistochemistry. The Ki67 labelling Index was determined by counting the positively stained MIB-1 cells and categorizing them into <5%, 5-10% and > 10%. The H3k27me3 staining was evaluated by finding the product of the tumor cells showing positive staining and the intensity of staining. Based on the product of the two, the cases were subdivided into Negative [0], Low [1-4] and high expression [5-9] of H3K27me3. The results showed that presence of atypical morphological features including necrosis and prominent nucleoli in grade 1 meningioma and low expression of H3K27me3 was significantly associated with higher grade, recurrence, and shorter progression-free survival (Kaplan Meier curves showed higher negative slope). The study also found that a higher Ki67 labelling Index was associated with recurrence and poor prognosis. This suggest that the H3K27me3 and Ki67 labelling Index can be useful prognostic markers in meningiomas, particularly in challenging grade 1 and 2 cases and recurrent meningiomas. The study highlights the importance of the H3K27me3 and Ki67 Labelling Index in assessing the biological behaviour of meningiomas. The findings provide valuable insights into the prognosis and treatment of meningiomas, emphasizing the need for further research to validate these markers and develop targeted therapeutic strategies.

Study of Microsatellite Instability by Immunohistochemistry in a Cohort of Patients With Melanoma.

Microsatellite instability (MSI) has prognostic value and impacts therapy strategies in several malignancies. Data regarding MSI in melanoma are scarce. The aim of this study was to assess MSI through the analysis of MMR protein expression in patients with melanoma. An observational retrospective single-center study was designed based on patients with primary melanoma. We assessed MSI through immunohistochemical staining with anti-MLH1, anti-MSH2, anti-MSH6, and anti-PMS2 on full-thickness excision tissue. Ninety-three patients were included in this study. The complete absence of nuclear staining in tumoral cells was extremely rare, with only one melanoma not expressing MSH6. Most melanomas showed an expression index for MLH1 (77.7%), MSH2 (87.2%), and PMS2 (78.6%) ≥ 75%. Most melanomas (57.8%) exhibited an MSH6 expression index in the range of 1%-74%. A lowMSH6 expression index and a reduced combined MMR protein expression index (MMR-e) were significantly associated with higher melanoma-specific survival. A mildPMS2 staining intensity was significantly associated with a higher melanoma-specific survival. The patients with high MMR-e who received immunotherapy progressed and died more frequently than those with reduced MMR-e (75% vs. 33.3%). More studies are needed to further define the role of MSI in melanoma prognosis and response to immunotherapy.

Clinical significance of Cyclin D1 by complete quantification detection in mantle cell lymphoma: positive indicator in prognosis

ObjectivesThe positive expression of Cyclin D1 in immunohistochemical (IHC) staining serves as the cornerstone for diagnosing mantle cell lymphoma (MCL). However, existing literature does not conclusively establish whether the expression ratio and staining intensity significantly influence diagnostic outcomes or patient prognosis. In this retrospective study, the correlation between comprehensive Cyclin D1 quantification and the prognosis of MCL patients was studied.MethodsThe Cyclin D1 protein level was assessed in 120 formalin-fixed paraffin-embedded samples from MCL patients using the quantitative dot blot (QDB) analysis technique. R language software was employed for statistical analysis to determine the optimal threshold with statistical significance. Additionally, Kaplan-Meier method was utilized to evaluate the relationship between the absolute level of Cyclin D1 protein and overall survival (OS) of patients. Furthermore, the Chi-square test was applied to analyze the causes of single and multiple fractures, with a significance level of p < 0.05. Finally, the Log-rank test was used to compare two survival curves, where a significance level of p < 0.05 was considered statistically significant.ResultsAt the optimized cutoff of 0.46 nmol/g, univariate analysis revealed a positive correlation between Cyclin D1 protein level and patient survival (OS). Specifically, in the subgroup with complete quantification of Cyclin D1 higher than the cutoff, the 5-year OS was 18%, whereas in the subgroup with complete quantification of Cyclin D1 lower than the cutoff, the 5-year OS was 4.8% (Log-rank test, P = 0.017). This indicates that patients with Cyclin D1 levels above the cutoff had significantly better overall survival compared to those below the cutoff. Additionally, in the Pearson distribution test, Ki-67 emerged as an independent prognostic factor for the complete quantification of Cyclin D1. Notably, Cyclin D1 complete quantification results remained unaffected by factors such as gender, age, LDH (Lactate Dehydrogenase) level, Ann Arbor stage(AAS), Ki-67, IPI(International prognostic index), MIPI(Mantle International prognostic index), and MIPI-c (MIPI Combined with Ki-67 Proliferation Index Chi-square test, p > 0.05).ConclusionsComprehensive Cyclin D1 quantification, especially above a threshold, significantly correlates with better overall survival in MCL. This highlights its prognostic importance in MCL management. Full quantification of CyclinD1 aids MCL prognosis, while QDB technology for biomarker quantification supports precise clinical prognostic stratification.

Regulator of G protein signalling 14 (RGS14) protein expression profile in the adult mouse brain.

Regulator of G protein signalling 14 (RGS14) is a multifunctional signalling protein that serves as a natural suppressor of synaptic plasticity in the mouse brain. Our previous studies showed that RGS14 is highly expressed in postsynaptic dendrites and spines of pyramidal neurons in hippocampal area CA2 of the developing mouse brain. However, our more recent work with monkey brain shows that RGS14 is found in multiple neuron populations throughout hippocampal area CA1 and CA2, caudate nucleus, putamen, globus pallidus, substantia nigra and amygdala. In the mouse brain, we also have observed RGS14 protein in discrete limbic regions linked to reward behaviour and addiction, including the central amygdala and the nucleus accumbens, but a comprehensive mapping of RGS14 protein expression in the adult mouse brain is lacking. Here, we report that RGS14 is more broadly expressed in mouse brain than previously known. Intense RGS14 staining is observed in specific neuron populations of the hippocampal formation, amygdala, septum, bed nucleus of stria terminalis and ventral striatum/nucleus accumbens. RGS14 is also observed in axon fibre tracts including the dorsal fornix, fimbria, stria terminalis and the ventrohippocampal commissure. Moderate RGS14 staining is observed in various other adjacent regions not previously reported. These findings show that RGS14 is expressed in brain regions that govern aspects of core cognitive functions such as sensory perception, emotion, memory, motivation and execution of actions and suggest that RGS14 may serve to suppress plasticity and filter inputs in these brain regions to set the overall tone on experience-to-action processes.

Glypican-3 and Cytokeratin-19 Expression in Pancreatic Cancer in a Canadian Population.

Background/Objectives: One study of pancreatic ductal adenocarcinoma has found expression of glypican-3 (GPC3) and cytokeratin-19 (CK19) determined by immunohistochemistry to be associated with higher stage and grade disease, with a more adverse prognosis. The reported 44% rate of GPC3 expression in pancreatic cancer raises the important possibility that targeted immunotherapies currently in development for hepatocellular carcinoma may also prove useful for GPC3-expressing pancreatic cancers. The present study aims to determine if a similar expression pattern of these markers and stage/grade/prognostic associations is present in our Canadian patient population. Methods: Patients with a pancreatic surgical resection for adenocarcinoma or neuroendocrine tumor (NET) were identified from pathology records over a 5-year period. Immunohistochemistry for GPC3 and CK19 was performed on archived tumor tissue and the proportion of positive cells and intensity of staining were recorded. Grade, stage, and overall survival were compared in patients with NETs that were CK19-positive versus -negative. Results: All 72 pancreatic adenocarcinomas and 20 NETs tested were negative for GPC3, apart from a single case of pancreatic adenocarcinoma. All 72 adenocarcinomas were positive for CK19 expression. Half of the NETs were positive for CK19. There was no correlation between CK19 expression in NETs and tumor grade, lymph node metastasis, distant metastasis, or overall survival. Conclusions: We are skeptical of the reported prognostic value of GPC3 and CK19 in pancreatic adenocarcinomas. CK19 as a prognostic marker in NETs has potential for further study. The results with our protocol for GPC3 immunohistochemistry suggest that pancreatic cancer may be a less promising target for GPC3-targeted immunotherapies than previously thought.

Quantitative proteogenomic characterization in MUC1 and MUC4 in oral squamous cell carcinoma, oral potentially malignant disorders, and normal oral mucosa in carcinogenesis.

Mucins are glycoproteins with a significant molecular weight that have a diverse range of biological functions. MUC1 & MUC4 are transmembrane mucin family members that are expressed in airway epithelial cells and body fluids. It is expressed excessively in numerous carcinomas in addition to their alteredexpression. A thorough review of the literature reveals very limited research about MUC1 & 4 expressionsin oral malignancies and oralpremalignant disorders. The aim of the research is to assess the pattern and quantity of gene expression in MUC1 & MUC4 individually in various grades of oral squamous cell carcinoma (OSCC), oral premalignant disorders (OPMDs)- oral epithelial dysplasia (OED), oral submucous fibrosis (OSF), oral lichen planus (OLP), and compared with normal oral mucosa (NOM). Immunohistochemistry and qRTPCR evaluation of MUC1 & 4 on sixty-three cases of OSCC, OPMD, and NOM was accomplished. The one-way ANOVA test and Chi-square test were done for statistical analysis. The overall immunoexpression of MUC1 & 4 increased significantly from NOM to OPMDs to OSCC. In the subgrades of OSCC and OPMDs, the staining intensity varied. For MUC4, the intensity was strongest in well-differentiated grades and MUC1, exhibiting a higher expression in poorly differentiated grades of OSCC and OPMDs. The study's results indicate that MUC1 & 4 individually are crucial formonitoringOSCC and OPMD pathogenesis as the former gives an idea of highly undifferentiated grades while the latter indicated more differentiated tumors and perhaps a better prognosis. Therefore, the two can be useful tumor markers for determining the severity and eliminating it in its early phases.