Including Immunohistochemistry Research Articles

A machine learning algorithm based on multi-omics biomarkers for the detection of tumor microsatellite instability.

1554 Background: Microsatellite instability (MSI) occurs via defects in mismatch repair (MMR) and is characterized by high tumor mutational burden (TMB) and a robust inflammatory response. Accurate MSI-high classification of immune-evasive tumors is vital due to the effectiveness of immune checkpoint inhibitor (ICI) therapy, such as pembrolizumab, which is FDA approved for all MSI-high tumors irrespective of anatomic origin. Detection methods of MSI-status vary widely, including immunohistochemistry (IHC), polymerase chain reaction (PCR), and next-generation sequencing (NGS). We developed a machine learning (ML) model to predict MSI status in patients with solid tumors using comprehensive genomic and immune profiling (CGIP), independent of direct sequencing data from microsatellite sites. Methods: We analyzed samples from 1,838 patients with colorectal cancer (CRC) by CGIP, which included DNA panel testing (523 genes) for pathogenic single nucleotide variants (pSNV) and determination of TMB (mut/Mb), and RNA sequencing (397 genes) for gene expression (GEX). The Boruta algorithm was used to select key genomic and GEX changes associated with MSI status. A distributed gradient boosting algorithm created a predictive model that was trained on 70% of the cohort. The model was tested on the remaining CRC cohort (Test), and in the PanCancer Atlas CRCs (TCGA), endometrial adenocarcinomas (EMCA), and CRC with indeterminate MSI testing by CGIP. Performance of the trained model was assessed using sensitivity, specificity, and positive (PPV) and negative (NPV) predictive value. Results: Feature selection identified 79 genes with pSNVs, 63 GEX changes, and TMB as informative for MSI prediction. The model showed high predictive accuracy in differentiating MSI-H tumors. Of the 39 cases that failed MSI component of CGIP (Indeterminate), 17 had MMR IHC results available, of which 1 demonstrated loss of MLH1/PMS2 and the other 16 showed intact (normal) expression. Testing of this cohort identified 3 cases as MSI-high, at least one of which was confirmed by IHC. No cases with intact MMR protein IHC were identified as MSI-high by the algorithm. Conclusions: Our ML-driven approach accurately assessed MSI status of CRC and EMCA using CGIP data. This approach also identified potential cases with MSI-high status where direct sequencing of microsatellites failed. This study highlights a method to identify patients with potential MSI-high status for orthogonal screening with the MSI component of a test fails. [Table: see text]

Immunophenotypic classification regarding prognosis in peripheral T cell lymphoma, NOS, and nodal T follicular helper T cell lymphoma, angioimmunoblastic-type.

This study aimed to determine the clinicopathological predictive factors of peripheral T-cell lymphoma, not otherwise specified (PTCL, NOS), and nodal T-follicular helper cell lymphoma, angioimmunoblastic-type (nTFH, AI-type). In this single-centered, retrospective study, medical records of 59 patients who were diagnosed with PTCL, NOS, or nTFH, AI-type from March 2007 to September 2022 were reviewed. The clinicopathological variables, including immunohistochemistry(IHC) subgroups, distinguishing TBX21 from the GATA3 subgroups were analyzed. Overall, 28 patients (75.7%) in the TBX21 group were PTCL, NOS. There were 9 (24.3%) patients in the GATA3 group. In univariable analyses, lymphoma subtype, age, and performance status were associated with progression-free survival (PFS), and overall survival (OS). In multivariable analyses, lymphoma subtype, and performance status were related to PFS and OS (P = 0.012, P < 0.001, P = 0.006, and P < 0.001, respectively). The GATA3 subgroup tended to have a worse prognosis in univariable analyses; however, it became more insignificant in multivariable when lymphoma subtype and performance status were adjusted (P = 0.065, P = 0.180, P = 0.972, and P = 0.265, respectively). The double-positive group showed variable prognoses of better PFS and worse OS. PD-1 and PD-L1 were associated with the EBV in situ hybridization (P = 0.027, and P = 0.005), and PD-1 was associated with CD30 expression (P = 0.043). This study demonstrated the potential of IHC classification to predict prognosis for PTCL, NOS, as well as nTFH AI-type, although further validation is necessary. Treatments targeting CD30, PD-1, and PD-L1 appear promising for lymphoma treatment.

Utility of gene expression–based cancer classification in diagnosis of malignant peritoneal mesothelioma (MPeM): Filling in the gaps in standard pathologic work-up for a rare cancer.

728 Background: Malignant peritoneal mesothelioma (MPeM) is a rare cancer (incidence of 0.12 per 100,000) and an arduous diagnostic challenge, requiring skillful evaluation by expert pathologists. Subjective and experiential immunophenotyping coupled with inattention to this rare entity causes delayed or misdiagnosis and leads to suboptimal treatment and outcomes. Gene-expression-based cancer classification offers an objective molecular assessment of the tissue of origin for cancers with uncertain diagnosis and improves diagnostic accuracy for MPeM compared to standard pathology. Methods: We retrospectively evaluated 23 patients (pts) with cancer of unknown primary (CUP) or uncertain diagnoses, with peritoneal/retroperitoneal carcinomatosis and reported as malignant mesothelioma using a 92-gene assay (CancerTYPE ID), a validated classifier for predicting tumor types, between January 2013 to December 2016. Pathology was verified by central review. Clinicopathologic data including immunohistochemistry (IHC) was collected using archived pathology specimens and reports. Results: Pts had a median age of 64 years (range: 39 – 93) at diagnosis, 57% were females and 74% had biopsy from omentum/peritoneum. Original histopathology was reported as poorly differentiated in 81% and as adenocarcinoma, carcinoma and malignant neoplasm in 10 (43%), 7 (30%) and 6 (27%) of cases, respectively. The number of IHC stains performed ranged between 0 and 28 (median: 10). Key IHC stains are shown in the table. Conclusions: In this cohort of CUP or uncertain diagnoses with a molecular cancer classification of malignant mesothelioma, we found huge variability and critical gaps in pathological work-up. Integration of molecular cancer classification using the 92-gene assay helped resolve this diagnostic uncertainty, further supporting its clinical utility to complement standard pathology. The impact of these findings goes beyond just MPeM and may benefit numerous pts with orphan cancers, who account for nearly 20% of all cancers diagnosed globally. [Table: see text]

CD56 polysialylation promotes the tumorigenesis and progression via the Hedgehog and Wnt/β-catenin signaling pathways in clear cell renal cell carcinoma

BackgroundCD56 has been observed in malignant tumours exhibiting neuronal or neuroendocrine differentiation, such as breast cancer, small-cell lung cancer, and neuroblastoma. Abnormal glycosylation modifications are thought to play a role in regulating tumour cell proliferation, migration, and invasion. Nevertheless, the exact roles and molecular mechanisms of CD56 and polysialylated CD56 (PSA-CD56) in the development and progression of clear cell renal cell carcinoma (ccRCC) remain elusive. Here we unveil the biological significance of CD56 and PSA-CD56 in ccRCC.MethodsIn this study, we employed various techniques, including immunohistochemistry (IHC), RT-qPCR, and western blot, to examine the mRNA and protein expression levels in both human ccRCC tissue and cell lines. Lentivirus infection and CRISPR/Cas9 system were utilized to generate overexpression and knockout cell lines of CD56. Additionally, we conducted several functional assays, such as CCK-8, colony formation, cell scratch, and transwell assays to evaluate cell growth, proliferation, migration, and invasion. Furthermore, we established a xenograft tumor model to investigate the role of CD56 in ccRCC in vivo. To gain further insights into the molecular mechanisms associated with CD56, we employed the Hedgehog inhibitor JK184 and the β-catenin inhibitor Prodigiosin.ResultsCD56 was significantly overexpressed in both human ccRCC tissues and renal cancer cell lines compared to adjacent normal tissues and normal renal epithelial cells. In vitro and in vivo experiments revealed that the knockout of CD56 inhibited the proliferation, migration, and invasion capabilities of ccRCC cells, whereas the overexpression of PSA-CD56 promoted these capacities. Finally, PSA-CD56 overexpression was found to activate both the Hedgehog and Wnt/β-catenin signaling pathways.ConclusionOur findings demonstrate that the oncogenic function of CD56 polysialylation plays a vital role in the tumorigenesis and progression of ccRCC, implying that targeting PSA-CD56 might be a feasible treatment target for ccRCC.

ROMO1 – a potential immunohistochemical prognostic marker for cancer development

Abstract Reactive Oxygen Species Modulator 1 (ROMO1) was first discovered in 2006, and its structural characteristics were elucidated by Lee et al. in 2018. This novel protein resides in the inner mitochondrial membrane and exerts control over the production of reactive oxygen species (ROS) by modulating membrane potential and permeability. ROS, in turn, plays a multifaceted role in cancer progression: at low concentrations, it serves as a critical player in cell signaling, influencing tumor suppression and immune system maintenance; at moderate concentrations, it promotes cancer progression, while high concentrations induce apoptosis. ROMO1, as a key regulator of intracellular ROS, significantly impacts cancer cell invasion and growth. Existing literature demonstrates that overexpression of ROMO1 is strongly associated with lymph node metastasis and a dismal prognosis in cancer patients, making it a promising prognostic factor for solid malignant tumors. ROMO1 can be investigated by various methods including immunohistochemistry (IHC) which is one very suitable method in our opinion.

The role of neuronal antibodies in cryptogenic new onset refractory status epilepticus.

Most cases with new onset refractory status epilepticus (NORSE) remain cryptogenic despite extensive diagnostic workup. The aim of this study was to analyze the etiology and clinical features of NORSE and investigate known or potentially novel autoantibodies in cryptogenic NORSE (cNORSE). We retrospectively assessed the medical records of adults with status epilepticus at a Swiss tertiary referral center between 2010 and 2021. Demographic, diagnostic, therapeutic, and outcome parameters were characterized. We performed post hoc screening for known or potentially novel autoantibodies including immunohistochemistry (IHC) on rat brain with cerebrospinal fluid (CSF) and serum samples of cNORSE. Twenty patients with NORSE were identified. Etiologies included infections (n = 4), Creutzfeldt-Jakob disease (n = 1), CASPR2 autoimmune encephalitis (n = 1), and carotid artery stenosis with recurrent perfusion deficit (n = 1). Thirteen cases (65%) were cryptogenic despite detailed evaluation. A posteriori IHC for neuronal autoantibodies yielded negative results in all available serum (n = 11) and CSF (n = 9) samples of cNORSE. Our results suggest that neuronal antibodies are unlikely to play a major role in the pathogenesis of cNORSE. Future studies should rather focus on other-especially T-cell- and cytokine-mediated-mechanisms of autoinflammation in this devastating disease, which is far too poorly understood so far.

An Optimized Melanin Depigmentation Method for Histopathological and Immunohistochemical Staining of Formalin-Fixed Paraffin-Embedded Tissues.

Introduction. The difficulty in diagnosis of severe melanocytic lesions is a problem to be overcome in pathological practice. Melanin bleaching is an effective approach to ameliorate melanin disturbances in severely pigmented lesions. Although various methods for improving melanin pigmentation in immunohistochemical staining have been reported, these depigmentation methods still need to be optimized and standardized. In this study, the coloring efficiency of 3,3'-diaminobenzidine (DAB) and alkaline phosphatase (AP) after melanin depigmentation was compared under the automatic immunohistochemical staining platform. Methods. The applicability of the optimized depigmentation method was validated in 10 formalin-fixed paraffin-embedded (FFPE) blocks of ocular melanoma tissues. Specimens were demelaninized with 10% hydrogen peroxide at 60°C for immunohistochemical staining (Melan-A and SOX10), and tissue chromogenic staining was performed with DAB and AP detection systems, respectively. Results. The optimized depigmentation method including immunohistochemistry (IHC) could be completed in 3 h, effectively preserving cell morphology and immunoreactivity. Among these, the color-rendering effect and contrast of AP are better than DAB. Conclusion. This optimized method can effectively remove melanin and improve the accuracy of IHC staining interpretation. AP staining has better visibility and readability without the interference of residual melanin. The comparison results showed that after melanin depigmentation, the immunohistochemical staining agent was replaced with red AP, which avoided the misjudgment caused by brown DAB when melanin depigmentation was incomplete. This improved method can be applied to future histopathological and immunohistochemical staining of melanin-deposited tissues.

Spatial genomics to reveal an immunosuppressive tumor microenvironment after chemoradiation in head and neck cancer patients.

6059 Background: Although chemoradiotherapy (CRT) remains the cornerstone of management for locoregionally advanced head and neck squamous cell carcinomas (HNSCC), recurrence occurs in ~50% of patients. We sought to characterize the impact of CRT on the tumor microenvironment (TME) and identify factors associated with progression. Emerging spatial biology technologies, such as NanoString GeoMx Digital Spatial Profiler (DSP), enable spatially defined whole-transcriptomic profiling of cells along the tumor-stromal interface in tumor biopsies to resolve immune cell identities, activation states and signaling pathways in adjacent tumor cells. We used a multimodal approach including immunohistochemistry (IHC), bulk tumor gene expression and spatial genomics to characterize paired pre- and post-CRT tumor samples from HNSCC patients. Methods: 20 patients with locoregionally advanced HNSCC who had undergone curative intent CRT and developed subsequent recurrence requiring salvage surgery from June 14, 2010 to June 14, 2020 were retrospectively identified at Duke University Medical Center. Matched formalin-fixed paraffin embedded tumor specimens collected prior to CRT and at salvage surgery were compared via IHC for markers of immune infiltration and PDL1 expression, bulk tumor whole transcriptomic profiling, and spatial transcriptomic analysis of tumor and stroma using DSP were further examined in “early” progressors ( &lt; 6 months after CRT) and “late” progressors (≥6 months after CRT). Results: IHC analyses demonstrated significant reductions in T-cell immune populations (total CD3+, CD8+, 4-1BB+ cell density [ p &lt; 0.001]) after CRT. PDL1+ expression was similar before and after CRT. Neither T-cell infiltrate nor tumor PDL1 levels at surgery were associated with progression. Bulk tumor gene expression analysis demonstrated that macrophage and fibroblast markers were increased post-CRT. In early progressors, molecular dissection of the tumor-stromal interface using DSP expression profiling revealed macrophage associated genes ( CD68, MRC1/CD206, CD163, F13A1) at salvage surgery had a more pronounced increase in macrophage markers in stroma with enriched M2 genes compared to late progressors. Conclusions: Integrated spatial analysis with high resolution transcriptomics and IHC suggest CRT induces an immunosuppressive environment characterized by decreased T-cell infiltration and an influx of putative immunosuppressive macrophages. We hypothesize that tumor associated macrophages and/or fibroblasts may contribute to immunosuppression due to “reprogramming” of the TME after CRT. This exploratory study suggests that patients with progressive disease after chemoradiation may benefit from additional immunoregulatory therapies that ameliorate the immunosuppressive TME. These findings will be further validated in a larger cohort of patients.

NTRK Gene Fusions in Non-Small-Cell Lung Cancer: Real-World Screening Data of 1068 Unselected Patients.

(1) Background: The main objectives of our study are (i) to determine the prevalence of NTRK (neurotrophic tyrosine kinase) fusions in a routine diagnostic setting in NSCLC (non-small cell lung cancer) and (ii) to investigate the feasibility of screening approaches including immunohistochemistry (IHC) as a first-line test accompanied by fluorescence in situ hybridization (FISH) and RNA-(ribonucleic acid-)based next-generation sequencing (RNA-NGS). (2) Methods: A total of 1068 unselected consecutive patients with NSCLC were screened in two scenarios, either with initial IHC followed by RNA-NGS (n = 973) or direct FISH testing (n = 95). (3) Results: One hundred and thirty-three patients (14.8%) were IHC positive; consecutive RNA-NGS testing revealed two patients (0.2%) with NTRK fusions (NTRK1-EPS15 (epidermal growth factor receptor pathway substrate 15) and NTRK1-SQSTM1 (sequestosome 1)). Positive RNA-NGS was confirmed by FISH, and NTRK-positive patients benefited from targeted treatment. All patients with direct FISH testing were negative. RNA-NGS- or FISH-positive results were mutually exclusive with alterations in EGFR (epidermal growth factor receptor), ALK (anaplastic lymphoma kinase), ROS1 (ROS proto-oncogene 1), BRAF (proto-oncogene B-Raf), RET (rearranged during transfection) or KRAS (kirsten rat sarcoma viral oncogene). Excluding patients with one of these alterations raised the prevalence of NTRK-fusion positivity among panTrk-(tropomyosin receptor kinase-) IHC positive samples to 30.5%. (4) Conclusions: NTRK fusion-positive lung cancers are exceedingly rare and account for less than 1% of patients in unselected all-comer populations. Both RNA-NGS and FISH are suitable to determine clinically relevant NTRK fusions in a real-world setting. We suggest including panTrk-IHC in a diagnostic workflow followed by RNA-NGS. Excluding patients with concurrent molecular alterations to EGFR/ALK/ROS1/BRAF/RET or KRAS might narrow the target population.

Synergy between oxaliplatin and adenovirus initiates immunogenic cell death in human colorectal carcinoma cells

Abstract Externalization of Calreticulin (CALR) is the critical first step in the initiation of Immunogenic Cell Death. Since Oxaliplatin (OX) cooperates with an oncolytic chimeric human adenovirus Ad5/3-NP2.ADP (ADP) to induce adaptive immunity as well as regression of established cancer, we sought to determine whether the cooperation was additive or synergistic. Methods include immunohistochemistry (IHC) for Calreticulin expression in mice 11 days after intratumoral injection of the OX-ADP mixture. In vitro assays with limiting dilutions of OX and ADP on human Clone A colorectal carcinoma cell monolayers were cultured for 3 days and cytotoxicity, viral infectivity by GFP, and the expression of externalized CALR (ectoCALR) on Clone A cells measured by static cytometry. Synergy was measured using SynergyFinder 3.0 (Ianevski et al., PMC9252834) that creates 2-D interactive surfaces to measure synergy and antagonism. Areas of synergy have mean scores &amp;gt; 10. IHC revealed strong CALR expression in OX-ADP treated tumors, weak expression in tumors injected with OX or ADP alone and minimal in PBS treated controls. Mean Viral Infectivity synergy score was 27.2 for the area of (Ox μM, ADP MOI): (0.12μM, 4 MOI) to (12.5μM, 100 MOI) while mean synergy score for ectoCALR expression was 29.2 for OX/ADP (0.01μM, 0.16) to (0.12μM, 20 MOI). The role of oxidative and endoplasmic reticulum stress as the basis of synergy is under study. Oxaliplatin and adenovirus are synergistic in their ability to initiate ICD which is important for vaccine development.

IMPACT OF PROGNOSTIC FACTORS ON SURVIVAL PATTERN OF BREAST CANCER PATIENTS: 5 YEAR SURVIVAL STUDY

Background:- Breast cancer is heterogeneous disease with various prognostic factors which play signicant role in deciding plan of management and further prognosis in terms of overall survival (OS) and disease free survival (DFS). The aim of this study was to assess the impact of tumor stage, tumor grade, molecular subtypes and lymph node metastasis on survival of carcinoma breast patients. Retrospective data of Methodology:- 216 post treatment breast cancer patients was collected from hospital records and analyzed. These patients were treated at Department of Radiation Oncology at B.Y.L. Nair Ch. Hospital, Mumbai. Tumor stage, tumor grade, molecular subtypes including immunohistochemical (IHC) parameters like hormonal receptor status and lymph node metastasis were observed. For statistical analysis, chi square test and Kaplan Meier tests were used for signicant correlation and survival analysis respectively. Among 216 patient Results:- s , majority of patients (45.59%) belong to pathological stage II with predominant T2 stage, 93.51 % patients diagnosed with IDC (Invasive Ductal Carcinoma) as common histological type. Most common subtype on basis of immunohistochemistry (IHC) was basal (TNBC) subgroup with 62 (29%) patients. Overall survival was 68.51% with 148 patients survived at end of 5 years and 68 (31.48%) patients died. Stage wise 5 year overall survival rates were 79.41%, 75%, 57.77% for stage I, Stage II, and stage III respectively. Tumor Stage, Nodal Stage and molecula Conclusion:- r subtypes of Breast Cancer were having statistically signicant impact on 5 year survival of patients. Multidisciplinary treatment modalities played important role in complete cure of breast cancer patients which is considered as very complex disease.

The essential role of forkhead box P4 (FOXP4) in thyroid cancer: a study related to The Cancer Genome Atlas and experimental data.

Thyroid cancer (THCA) is the most common endocrine cancer in the world. Although most patients with THCA have a good prognosis, the prognosis of those with THCA who have an extra-glandular invasion, vascular invasion, and distant metastasis is poor. Therefore, it is very important to find potential biomarkers that can effectively predict the prognosis and progression of highly aggressive THCAs. It has been identified that forkhead box P4 (FOXP4) may be a new biomarker for the proliferation and prognosis for tumor diagnosis. However, the expression and function of FOXP4 in THCA remain to be determined. In the present study, the function of FOXP4 in cells was investigated through the comprehensive analysis of data in The Cancer Genome Atlas and combined with experiments including immunohistochemistry (IHC), colony formation, Cell Counting Kit-8 assay, wound scratch healing, and transwell invasion assay. In the present study, relevant bioinformatic data showed that FOXP4 was highly expressed in THCA, which was consistent with the results of the IHC and cell experiments. Meanwhile, 10 FOXP4-related hub genes were identified as potential diagnostic genes for THCA. It was found in further experiments that FOXP4 was located in the nucleus of THCA cells, and the expression of FOXP4 in the nucleus was higher than that in the cytoplasm. FOXP4 knockdown inhibited in vitro proliferation of the THCA cells, whereas overexpression promoted the proliferation and migration of THCA cells. Furthermore, deficiency of FOXP4 induced cell-cycle arrest. FOXP4 might be a potential target for diagnosing and treating THCA.

HPB P60 Pancreatic Carcinosarcoma as a rare cause of upper gastrointestinal bleeding requiring resection

Abstract Background Pancreatic carcinosarcoma is a rare malignant neoplasm and has a very poor prognosis. Diagnosis is mainly by pathological examination including immunohistochemistry (IHC) of the biopsy or resected specimen. Due to the rarity of the cancer, information regarding clinical presentation, pathology and management is limit to a few case reports. Methods We report a 61-year-old fit and healthy male, who presented to the emergency department with melaena and syncope. Initial CT scan showed a pancreatic body/tail tumour, likely pancreatic cancer without nodal or intra-abdominal metastasis. Upper GI endoscopy revealed a lesion in the second part of the duodenum, with a visible vessel, and evidence of recent bleeding . Two clips were applied, and a biopsy of the lesion showed ulcer slough only. Tumour markers and IgG4 levels were normal. An MRI pancreas showed no definite pancreatic mass, features of chronic pancreatitis and irregular bile ducts. HPB MDT consensus was to proceed for EUS, which confirmed a hypoechoic, vascular, 3cm mass lesion in D1-D2. Biopsies again, showed ulcer slough. A PET CT scan showed avidity in the second part of the duodenum and pancreatic head, neck and distal body. The patient presented to the surgical emergency unit twice during this time with symptoms of anaemia requiring IV iron and blood transfusions, and with early signs of obstruction, which settled with conservative management. Interval MRI showed increase in size of the duodenal mass, and stable appearance of the lesion in the head of the pancreas. Eventually, semi-emergency total pancreaticoduodenectomy and splenectomy was undertaken. Results Intraoperatively, a large D2 tumour was found extending into the pancreatic head posteriorly, compressing but not infiltrating the IVC. The rest of the pancreas was hard in consistency and nodular. Soft lymph nodes were found at the aorto-caval, common hepatic and around the SMA. There was no obvious intra-abdominal metastasis. The final histopathology showed a malignant, biphasic tumour favouring pancreatic carcinosarcoma staged pT3 N0 (0/29) L1 V1 R1 (posterior and SMV). On molecular cytogenetic analysis there was no evidence of rearrangement associated with synovial sarcoma in the cells examined. He recovered well post operatively and has commenced adjuvant chemotherapy of modified Folfirinox. Conclusions Pancreatic carcinosarcoma is a rare entity, which due to its unusual presentation, can be challenging to manage. Prompt MDT discussion is instrumental in such cases. The assessment of different radiological modalities, showing progressive increase in the size of the lesion within a short time period, raised the suspicion of an aggressive mitotic lesion, with the repeat biopsies unlikely to yield diagnosis in an otherwise operable lesion. Ultimately, the patient underwent a semi-emergency, total pancreaticoduodenectomy and splenectomy due to ongoing bleeding and the rapidly growing tumour size. In this case, surgery followed by adjuvant chemotherapy has been adopted as the optimum management strategy, for both bleeding control, and oncological benefit.

Immunohistochemical Detection of BRAFV600E in Cutaneous Melanocytic Neoplasms in a Cohort of Egyptian Population

Objective: Melanocytic neoplasia shows different pathways that have been implicated in tumorigenesis. One of the key genes involved in the BRAFV600E gene, can be detected through DNA-based or protein-based tests including immunohistochemistry (IHC). To check its reproducibility, this study was conducted to evaluate BRAFV600E IHC by different observers in a range of melanocytic neoplasms. Methods: The present study was performed to evaluate immunohistochemical staining (IHC) using the RM-08 to detect BRAF V600E in 50 formalin-fixed paraffin-embedded (FFPE) blocks of different melanocytic neoplasms after bleaching using Tris-HCL buffer. IHC was considered positive when more than 90% of tumour cells showed cytoplasmic staining. IHC assessment by a team of two pathologists and a third pathologist to check the interobserver variability. IHC scores were compared among nevi and melanoma. Results: IHC evaluation revealed good agreement by different observers (ĸ-coefficient=0.691, p=0.00). BRAFV600E showed a statistically significant difference between nevi and melanoma. Conclusions: These findings suggest that BRAFV600E IHC assessment shows good reproducibility among pathologists, however, more strict criteria should be adopted in interpretation, especially in bleached samples.

Microsatellite Instability: From the Implementation of the Detection to a Prognostic and Predictive Role in Cancers.

Microsatellite instability (MSI) has been identified in several tumors arising from either germline or somatic aberration. The presence of MSI in cancer predicts the sensitivity to immune checkpoint inhibitors (ICIs), particularly PD1/PD-L1 inhibitors. To date, the predictive role of MSI is currently used in the selection of colorectal cancer patients for immunotherapy; moreover, the expansion of clinical trials into other cancer types may elucidate the predictive value of MSI for non-colorectal tumors. In clinical practice, several assays are used for MSI testing, including immunohistochemistry (IHC), polymerase chain reaction (PCR) and next-generation sequencing (NGS). In this review, we provide an overview of MSI in various cancer types, highlighting its potential predictive/prognostic role and the clinical trials performed. Finally, we focus on the comparison data between the different assays used to detect MSI in clinical practice.

Challenges and limitation of MTAP immunohistochemistry in diagnosing desmoplastic mesothelioma/sarcomatoid pleural mesothelioma with desmoplastic features

AimGenomic-based ancillary assays including immunohistochemistry (IHC) for BRCA-1 associated protein-1 (BAP1) and methylthioadenosine phosphorylase (MTAP), and fluorescence in situ hybridization (FISH) for CDKN2A are effective for differentiating pleural mesothelioma (PM) from reactive mesothelial proliferations. We previously reported a combination of MTAP and BAP1 IHC effectively distinguishes sarcomatoid PM from fibrous pleuritis (FP). Nevertheless, cases of sarcomatoid PM with desmoplastic features (desmoPM) are encountered where the IHC assessment is unclear. Methods and resultsWe evaluated assessment of MTAP IHC, BAP1 IHC, and CDKN2A FISH in 20 desmoPM compared to 24 FP. MTAP and BAP1 IHC could not be assessed in 11 (55 %) and 10 (50 %) cases, respectively, due to loss or faint immunoreactivity of internal positive control cells, while CDKN2A FISH could be evaluated in all cases. The sensitivities for MTAP loss, BAP1 loss, and CDKN2A homozygous deletion in desmoPM were 40 %, 10 %, and 100 %. A combination of MTAP loss and BAP1 loss yielded 45 % of sensitivity. ConclusionsMTAP IHC is a useful surrogate diagnostic marker in differentiating ordinary sarcomatoid PM from FP, but its effectiveness is limited in desmoPM. CDKN2A FISH is the most effective diagnostic assays with 100 % sensitivity and specificity in discriminating desmoPM from FP in the facilities where the FISH assay is available.

Identification of CXCR4 Upregulation in Diffuse Large B-Cell Lymphoma Associated with Prognostic Significance and Clinicopathological Characteristics.

Background Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous malignant lymphoma with distinct characteristics. Patients with treatment failure after the standard immunochemotherapy have worse prognosis, which implies the necessity to uncover novel targets. The C-X-C chemokine receptor 4 (CXCR4) overexpression has been identified in several hematopoietic malignancies. However, the expression signatures and prognostic significance of CXCR4 in DLBCL associated with clinicopathological features remain unclear. Methods Gene expression profiles of DLBCL were obtained from the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. Then, a meta-analysis with an integrated bioinformatic analysis was performed to assess the relationship between CXCR4 expression and clinicopathological features of DLBCL. Finally, experimental verification including immunohistochemical (IHC) staining and real-time quantitative PCR (qPCR) was carried out using patient samples. In vitro cell line viability tests were conducted using CXCR4 inhibitor WZ811. Results DLBCL patients with activated B-cell-like (ABC) subtype have higher expression level of CXCR4 with worse survival. Differential expressed genes in the CXCR4-upregulation group were enriched in canonical pathways associated with oncogenesis. DLBCL with CXCR4 upregulation had lower degree of CD8+ T cell infiltration. TIMER analysis demonstrated that the CXCR4 expression was positively correlated with the expression of CD5, MYC, NOTCH1, PDCD1, CD274, mTOR, FOXO1, and hnRNPA2B1 in DLBCL. IHC study in patient samples showed the positive correlation between CXCR4 and nongerminal center B-cell (non-GCB) subtype and mTOR expression. Meanwhile, quantitative polymerase chain reaction results revealed that high CXCR4 mRNA level was correlated to double-hit DLBCL. Finally, cell viability test showed that WZ811 exerted antiproliferation effect in DLBCL cell lines in a dose-dependent manner. Conclusion CXCR4 was upregulated in ABC-DLBCL associated with worse prognosis. Our analysis predicted CXCR4 as a potential target for DLBCL treatment, which may serve as an inhibitor both on BCR signaling and nuclear export warranting further investigation in clinical trials.

Abstract 5271: Correlative analysis of RNA biomarkers for adjuvant capecitabine benefit in the CIBOMA/2004-01phase III clinical trial of triple negative breast cancer patients

Abstract Introduction: Predictive biomarkers for capecitabine benefit in triple negative breast cancer (TNBC) have been recently identified using samples from phase III clinical trials, including immunohistochemical (IHC) non-basal phenotype and RNA biomarkers related to angiogenesis, stroma and capecitabine activation genes. We aimed to validate these findings on the larger phase III CIBOMA clinical trial. Experimental Design: Tumor tissues from TNBC patients randomized to standard (neo)adjuvant chemotherapy with capecitabine vs. observation were analyzed using a 164 gene NanoString custom nCounter codeset. A prespecified statistical plan sought to verify the predictive capacity of PAM50 non-basal molecular subtype previously found by IHC, and tested the hypotheses that breast tumors with increased expression of (meta)genes for cytotoxic T cells, mast cells, endothelial cells, PDL2 and 38 individual genes benefit from adjuvant capecitabine for distant recurrence free survival (DRFS, primary endpoint) and overall survival. Exploratory analyses investigated (a) predictive capacity of categorical expression of biomarkers, and continuous expression of additional genes included in the codeset; (b) the prognostic capacity of continuous biomarker expression. Results: Of the 876 women enrolled in the CIBOMA trial, 658 (75%) were evaluable for analysis (337 with capecitabine and 321 without) with similar baseline characteristics relative to the intention-to-treat population. 553 (84%) cases were profiled as PAM50 basal-like while 105 (16%) were PAM50 non-basal. PAM50 non-basal subtype was the most significant predictor for capecitabine benefit (HRcapecitabine=0.19; 95%CI, 0.07-0.54; p=0&amp;lt;0.001) when compared to PAM50 basal-like (HRcapecitabine=0.9; 95%CI, 0.63-1.28; p=0.55) (p-interaction&amp;lt;0.001, adjusted p-value=0.01). Analysis of biological processes related to PAM50 non-basal subtype revealed its enrichment for mast cells, extracellular matrix, angiogenesis and features of the mesenchymal stem-like TNBC subtype. Multivariate analysis showed a significantly lower DRFS on the observation arm for the mast cell metagene (HRobservation=1.35; 95%CI, 1.12-1.62; p=0.002, adjusted p-value=0.006), particularly among PAM50 non-basal tumors (HRobservation=2.70; 95%CI, 0.99-7.35; p=0.01, p-interaction=0.08). Tumors above the median for genes involved in immune response (PDL2, CCR5), capecitabine metabolism (CES1) and angiogenesis (STC1) were significantly associated with favorable survival rates on the capecitabine arm (HRcapecitabine ranged between 0.51-0.60; p=0&amp;lt;0.05). Conclusions: In this prespecified correlative analysis of the CIBOMA trial, PAM50 non-basal status and the mast cell metagene identified early-stage TNBC patients most likely to benefit from adjuvant capecitabine. Citation Format: Karama Asleh, Ana Lluch, Angela Goytain, Carlos Barrios, Xue Q. Wang, Jesus Herranz, Dongxia Gao, Rosalia Caballero, Samuel Leung, Federico Rojo, Torsten O. Nielsen, Miguel Martin. Correlative analysis of RNA biomarkers for adjuvant capecitabine benefit in the CIBOMA/2004-01phase III clinical trial of triple negative breast cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5271.

Characterization of Axon Guidance Phenotypes in Wnt/PCP Mutant Mice.

Our lab showed that the Wnt family proteins can function as axon guidance molecules and the planar cell polarity (PCP) pathway mediates the function of Wnts in axon guidance. One of the key evidences was by identifying the axon guidance defects in knockout or conditional knockout animals. We utilized a variety of axon tracing and labeling techniques, including immunohistochemistry (IHC), DiI, BDA, and fluorescent reporters (GFP or tdTomato). These studies have primarily been conducted in spinal cord commissural axons, but have been applied to retinal ganglion cell axons, corticospinal tract axons, dopaminergic and serotonergic projections.

Glyoxal fixation: an immunohistochemistry assay evaluation

ABSTRACT Analysis of surgical pathology specimens by histological techniques including immunohistochemistry (IHC) assays is a mainstay of disease diagnosis in humans. Neutral buffered formalin (NBF) is currently the primary fixative used, but its use is not without risks due to toxicity and carcinogenicity. Several glyoxal-based fixatives have been commercially produced, are considered safer alternatives to NBF, and produce histochemical staining results comparable to that of tissues fixed in NBF. However, previous studies evaluating IHC assay results in tissues fixed in NBF and glyoxal solutions have indicated mixed results. This study demonstrated that while tissues fixed in NBF were slightly superior to tissues fixed in glyoxal solutions among the 34 antibodies assayed with IHC, all fixative solutions produced results compatible for use in an anatomic pathology laboratory.