IHC Studies Research Articles

Potential for tumor aggressiveness and its association with vasculogenic mimicry marker in colorectal carcinomas in Indian population- A tertiary care hospital level study.

Vasculogenic mimicry (VM) is the ability of cancer stem cells of aggressive phenotype to mimic embryonic vascular network by forming channels lined by tumor cells, thereby, promoting tumor growth. These channels are periodic acid-schiff positive and CD34 negative and have been associated with tumor invasion, metastasis, and poor prognosis. It is a promising target for developing novel anticancer therapeutics and contributes to personalized medicine. Our study aims to look for an association of vasculogenic mimicry marker (VMM) with the aggressiveness of colorectal carcinomas (CRCs). All retrospective and prospective cases of colorectal adenocarcinomas received over period of five years, from 2017 to 2021 were included in this study and a case-control study was planned. The study population was divided into two comparison groups based on TNM staging (a) those with non-aggressive tumors (controls; TNM stage I and II) and (b) those with aggressive tumors (cases; TNM stage III and IV). A total of 36 controls and 27 cases were studied. Hematoxylin and eosin stain, PAS, and CD34 immunohistochemistry were conducted. PAS positivity with negative CD34 in the same case was designated as VMM positive. VMM status was correlated with tumor stage, grade, and other pathological markers of prognosis. The Chi-square test was used to find an association between the aggressiveness of CRCs and VM positivity. The P value < 0.05 was considered to be significant. It was found that VMM was significantly associated with the aggressiveness of CRCs. VMM is a promising marker for its clinical utility in day-to-day routine IHC studies and its positivity correlates with tumor aggressiveness.

A clinical, cyto-histopathological and immunohistochemical study of spindle cell lesions of breast in a tertiary care hospital

Spindle cell lesions in the breast are a diverse group of entities that can be either benign or malignant. Spindle cell lesions often pose a challenge to the diagnosing clinician because of the vast differential diagnoses, the rarity of these lesions and the histological similarities. We aim to conduct this study to profile these rare lesions of the breast and correlate the cyto-histopathological features on FNAC with immunohistochemical characteristics to determine its efficacy.A total 260 patients, suffering from spindle cell lesions of the breast, attending the outpatient and in-patient services were included in this 5 year study (3 years of retrospective cases and 2 years of prospective cases) conducted at Department of Pathology, JNMCH, AMU, Aligarh.: Only Diagnosed cases of Spindle cell lesions of breast were included in the study. The study sample comprised of 260 cases (105 cytological cases and 220 histopathological cases with 65 cases that are common to both) Apart from diagnosed cases of Spindle cell lesions of breast all other breast lesions either reactive benign or malignant were excluded.Total number cases of breast lesions received in the Department of Pathology during 5 year study period were 15,270 out of which 9700 cases were of Histopathology and 5570 were of Cytopathology. Among 15,270 cases, a total number of 15,010 (98.29%) cases belonged to non-spindle cell lesions which were excluded from the study and the remaining cases i.e., 260 (1.7%) cases diagnosed as spindle cell lesions of breast constituted the study group. Of the total260 cases, 105 cases were of cytopathology and 220 cases were of histopathology and cases common to both i.e., those cases which had cyto-histopathological correlation were 65 cases (20%). The highest incidence of spindle cell lesions occurred in 4 decade of life comprising of 126/260 cases (48.46%).Spindle cell lesions are not so common in breast, accounts for 1.7% of cases only. The workup involves thorough histopathological examination due to considerable morphological overlap and an elaborate IHC studies since no single Immunohistochemical marker is sufficient/specific to clinch the final diagnosis. Hence, it is difficult to make the final diagnosis of spindle cell lesions of breast on histopathology alone in majority.

An insight into immunohistochemical profile of triple negative breast carcinoma in a tertiary care center

Introduction: Triple Negative Breast Cancer (TNBC) is a type of breast cancer that fails to express Estrogen Receptor (ER), Progesterone Receptor (PR) and Human Epidermal Growth Factor Receptor 2 (HER2) in Immunohistochemical analysis. TNBC has a unique molecular profile, aggressive nature and lack of efficient targeted therapies. The aim of the study was to evaluate the expression of basal markers like Epidermal Growth Factor Receptor (EGFR) and Cytokeratin 5/6 (CK 5/6) and to correlate their expression with the clinico-pathological parameters, thus enabling the identification of the aggressive basal like phenotype of TNBC in resource-constrained settings. Methodology: The study was conducted at a South Indian tertiary care centre for 2 years. Data such as age of presentation, tumour measurements, margins and necrosis, numbers, levels and sizes of lymph nodes were collected. Data on histological type and grade, in situ component, lymphovascular and perineural invasion, stromal reaction and lymph node status were recorded. Primary Immunohistochemical studies were performed for the markers ER,PR and HER2.Patients with negative expression for all the three markers were identified. IHC studies for EGFR and Cytokeratin 5/6(CK5/6) were done on tissue blocks and the expression of these two markers were equated with the clinico-pathological parameters. Results: Among the 94 cases, 44 cases (46.8%) expressed CK5/6 and 46 (48.9%) expressed EGFR. A plausible association was noted between the expression of the two markers with age of the patient, tumour size, histological grade and tumour necrosis. Conclusion: Combination of the time honoured gross and histopathological examinations along with the application of basal markers like CK 5/6 and EGFR, helps in identifying the basal-like phenotype with dismal clinical outcome among the triple-negative breast cancers in resource-constrained settings. An insight into such an aggressive tumour, provided by these markers may be critical in designing personalized treatment strategies for such patients.

Abstract 5911: In vivo efficacy of a novel peptide-conjugated drug in patient-derived xenograft models of breast cancer

Abstract Introduction: Antibody and peptide-conjugated drugs are rapidly entering the clinic for the treatment of patients with early and advanced breast cancer. These drugs are designed to achieve more targeted delivery of cytotoxic agents to tumor cells with robust clinical activity. ARB-1-6, a sortilin (SORT1) targeted peptide conjugated to the cytotoxic agent MMAE, has demonstrated pre-clinical activity and tolerability in cell line xenografts of breast cancer. To further characterize its spectrum of antitumor activity and biomarker correlates we evaluated ARB-1-6 in a collection of clinically and genomically annotated breast cancer patient-derived xenografts (PDX). Methods: Breast cancer PDX models were generated and propagated under IRB approved protocols [14-8358]. ARB-1-6 was dosed in tumor-bearing SCID mice by tail vein injection at 3 mg/kg weekly x 4 doses. Tumor volume was measured twice weekly until day 35 or until models reached humane endpoints, after which tumors were collected. Animals were weighed regularly and assessed for treatment-related toxicity. Results: 7 models (5 TN and 2 HR) have completed efficacy assessment to date. 3/7 models were derived from treatment naïve patients while 4/7 were derived from pre-treated patients (Table 1). Complete response (mRECIST) was seen in 5/7 (71.4%) models with stable disease occurring in 2/7 (28.6%). ARB-1-6 was well tolerated with no weight loss observed. Complete tumor regression was seen in a PDX derived from a patient with clinical resistance to a TROP2 antibody drug conjugate (ADC). Antitumor activity was observed in models with low SORT1 RNA expression; SORT1 IHC and other correlatives studies are underway. Conclusion: ARB-1-6 demonstrates substantial preclinical activity in PDX models at doses that are well tolerated in mice. Treatment of additional models is ongoing. Full results and correlative analyses including relationships between antitumor activity and target protein expression will be presented. PDX Model Characteristics and Patient Treatment PDX Subtype Pt. Treatment 1 TN AC-T 2 TN FEC-D, Gem+Carbo 3 TN AC-T 4 TN No 5 TN No 6 HR No 7 HR TC, ET, Cape, Gem+Cis, TROP2-ADC Citation Format: Mitchell J. Elliott, Meghan Mcguire, Jennifer Silvester, Chantal Tobin, Samah El Ghamrasni, Francine Lui, Andrew Zhai, Ozge Yoluk, Aron Broom, Tracy Stone, Glenn Butterfoss, Serban Popa, Tianyu Lu, Chris Ing, David White, David W. Cescon. In vivo efficacy of a novel peptide-conjugated drug in patient-derived xenograft models of breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5911.

Upregulation of Enhancer of Zeste Homolog 2 (EZH2) with Associated pERK Co-Expression and PRC2 Complex Protein SUZ12 Correlation in Adult T-Cell Leukemia/Lymphoma

EZH2, a subunit of the polycomb repressive complex 2 (PRC2), is an important methyltransferase that catalyzes the trimethylation of histone H3 at lysine 27 (H3K27me3). EZH2 is overexpressed in various malignancies. Here, we investigated EZH2 expression and potential signaling molecules that correlate with EZH2 expression in ATLL and other T-cell neoplasms. Immunohistochemical staining (IHC) was performed for EZH2, pERK, MYC, and pSTAT3 on 43 ATLL cases and 104 cases of other T-cell neoplasms. Further IHC studies were conducted for Ki-67, SUZ12, and H3K27me3 on ATLL cases. All ATLL cases showed EZH2 overexpression. In other T-cell neoplasms, a high prevalence of EZH2 overexpression was identified (86%), except for T-PLL (33%). In ATLL, EZH2 overexpression correlated with pERK co-expression (86%), while only a small subset of cases showed MYC (7%) or pSTAT3 (14%) co-expression. In the other T-cell neoplasms, there was a variable, but higher, co-expression of EZH2 with pERK, MYC, and pSTAT3. In ATLL, enhanced EZH2 expression correlated with higher Ki-67 staining, SUZ12 (another PRC2 subunit), and H3K27me3 co-expression. In conclusion, EZH2 is overexpressed in ATLL and is associated with pERK expression. It correlates with an increased proliferation index, indicating an aggressive clinical course. EZH2 also correlates with SUZ12 and H3K27me3 co-expression, suggesting its PRC2-dependent catalytic activity through trimethylation. Additionally, EZH2 is overexpressed in most T-cell neoplasms, suggesting that EZH2 could function as an oncogenic protein in T-cell tumorigenesis. EZH2 and pERK could serve as potential therapeutic targets for treating aggressive ATLL. EZH2 could also be targeted in other T-cell neoplasms.

Fine Needle Aspiration and Core Biopsy of Liver Mass as First Time Diagnosis of Sarcomatoid Anaplastic Thyroid Carcinoma, A Rare Malignancy Presenting at an Unexpected Body Site

Abstract Introduction/Objective Anaplastic thyroid carcinoma (ATC) is a fatal malignancy. We report an unusual case of a patient with ATC whose chief complaint and clinical history were listed as “jaundice and liver mass” without mention of disease in the neck. Methods/Case Report A 66-year-old female presented to the emergency department with worsening “yellow-green skin discoloration” for a few weeks. She also reported worsening dyspnea and recent dysphagia with solid foods, fatigue, abdominal pain and recent hemoptysis. Physical examination revealed conjunctival icterus, jaundice, a tender neck mass, right upper chest wall tenderness and palpable lymphadenopathy in the lower neck. Assessment notes indicated inability to carry out self-care and being bedridden, even though she reported having been active, swimming and cycling, up to one month before her presentation. Computed tomography studies confirmed findings compatible with widespread malignancy with mass lesions in her liver, lungs, adrenals, kidneys, neck, pancreas, gallbladder, and brain. Mild anemia, hemoglobin 10.5g/dL, mild leukocytosis, leukocytes 17,000/mm3, were confirmed. Bilirubin was 15µmol/L, and hepatic enzymes were elevated. An ultrasound-guided liver biopsy was performed and evaluated for cytologic features, histologic features and pattern of immunoreactivity. The combined cytomorphologic and histologic features were those of a pleomorphic sarcomatoid high-grade malignancy with variably spindled to epithelioid morphology and associated necrosis. Immunohistochemical studies performed on sections from the accompanying core biopsy confirmed the malignant cells to be positive with CK-AE1/AE3, CK 7, TTF-1, GATA3, PAX8, SATB2, and BRAF V600E. The malignant cells were nonreactive with CK 20, thyroglobulin, calcitonin, napsin, CDX2, PCEA, and ER. The combined clinical setting, imaging findings, cytomorphology, histomorphology and ancillary testing results together allowed for a specific diagnosis of metastatic anaplastic thyroid carcinoma. The patient was referred for palliative care services and died within a few days of diagnosis. Results (if a Case Study enter NA) NA Conclusion Metastatic disease from a sarcomatoid ATC, as diagnosed in this case, is an important differential diagnosis to keep in mind when an elderly patient presents with multisite metastases from an undefined primary source. Clinical, radiological and laboratory correlations are of paramount importance in correlating with cytomorphology/histomorphology and in helping to determine the best panel of IHC studies to consider.

Not all hematological malignancies in the gastrointestinal tract are lymphoma

Abstract Introduction/Objective Myeloid sarcoma (MS) is a rare entity that may develop in patients with acute myeloid leukemia (AML). Presentation of MS in the gastrointestinal (GI) tract is uncommon. We describe two cases of MS involving unusual sites: the stomach and appendix. Methods/Case Report A 64-year-old female consulted for fatigue and abdominal pain. A CT scan of abdomen/pelvis revealed appendicitis and CBC showed 76% blasts. An appendectomy was performed. Histologic examination demonstrated the appendiceal wall infiltrated by malignant cells, ultimately demonstrated to be positive for MPO and weak/variable CD117. In the second case, a 50-year-old male presented to the emergency department for nausea, vomiting, and elevated amylase. Esophagogastroduodenoscopy with endoscopic ultrasound demonstrated pancreatic inflammatory changes and a subepithelial nodule in the gastric fundus. Biopsy of the nodule revealed gastric epithelium infiltrated by sheets of malignant cells, ultimately demonstrated to be positive for CD34, myeloperoxidase and CD43. Results (if a Case Study enter NA) NA Conclusion MS is defined as a tumor of myeloid blasts forming outside the bone marrow. Overall, it is an uncommon entity that can occur in patients with known AML, and rarely, as the first sign of AML. Presentation of MS in the GI tract is infrequent (10%), involving primarily the small intestine (63%). The differential diagnosis includes lymphoma, poorly differentiated carcinomas, and melanomas. A review of the clinical history for AML or other myeloproliferative neoplasms should raise suspicion of MS. Expression of lymphoid markers and scant tissue for IHC studies can be diagnostic challenges, rendering B cell lymphoma as the most common misdiagnosis of MS in up to 40-47% of cases. Our two MS cases presented exclusively with GI symptoms without previously known AML and involved unusual GI sites. IHC for myeloid lineage, monocytic or myelomonocytic differentiation should be considered in specific cases. In patients without prior AML diagnosis, especially without hematologic findings, MS is a diagnostic challenge.

Clinical and cytohistomorphological evaluation of peripheral nerve sheath tumours with special reference to immunohistochemical diversity in a tertiary care hospital

Peripheral Nerve Sheath Tumours (PNST), though have a frequent prevalence, are diagnostically challenging. There is a wide spectrum of lesions ranging from benign entities like neurofibroma, Schwannoma, perineurioma to malignant entities like malignant peripheral nerve sheath tumours (MPNST), malignant granular cell tumour and malignant perineurioma. Since there is an overlap with other spindle and round cell tumours, IHC and molecular studies are needed to provide a clear distinction between different tumour types. It was a 5 years observational study (3 years retrospective and 2 years prospective) conducted in the Department of Pathology, JNMCH, AMU from 2015-2020. . History and relevant clinical findings were retrieved from the archives of both the Histopathology and Cytopathology Lab. Imprints as well as FNA smears were included from Cytopathology Lab and biopsies and resection specimen from Histopathology Lab. Immunohistochemistry was applied wherever necessary. A total of 82 cases were diagnosed over a 5 year period out of which 70 were benign and 12 malignant. Male preponderance was seen. Benign tumours comprised mostly of Neurofibroma and Schwannoma and there were 12 cases of MPNST. Correlation was established between Cytopathological and Histopathological findings and IHC, mostly S100 proved useful in differentiating it from other malignant spindle cell tumours. The differential diagnoses of these tumours has always posed a problem given their monotonous appearance and clinical overlap. This study, therefore, aims to analyse the diverse morphological features of Peripheral Nerve Sheath Tumours in the light of cytopathological and histopathological findings and immunohistochemistry.

Targeted inhibition of eIF5Ahpu suppresses tumor growth and polarization of M2-like tumor-associated macrophages in oral cancer

Eukaryotic initiation factor 5A2 (eIF5A2) is overexpressed in many types of cancer, and spermidine-mediated eIF5A hypusination (eIF5Ahpu) appears to be essential to most of eIF5A’s biological functions, including its important role in regulating cancer cell proliferation, epithelial–mesenchymal transition (EMT), and cancer stem cell (CSC) properties as well as immune cell functions. Here we investigated the role of eIF5Ahpu in the growth of oral squamous cell carcinoma cells (OSCCs) and OSCC-induced polarization of M2-like tumor-associated macrophages (TAMs). TCGA dataset analysis revealed an overall upregulation in the mRNA expression of eIF5A2 and several key enzymes involved in polyamine (PA) metabolism in HNSCC, which was confirmed by Western blot and IHC studies. Blocking eIF5Ahpu by GC-7 but not the upstream key enzyme activities of PA metabolism, remarkably inhibited cell proliferation and the expression of EMT- and CSC-related genes in OSCC cells. In addition, blocking eIF5Ahpu robustly inhibited OSCC-induced M2-like TAM polarization in vitro. More Importantly, blocking eIF5Ahpu dramatically retarded tumor growth and infiltration/polarization of M2-like TAM in a syngeneic orthotopic murine tongue SCC model. Thus, eIF5Ahpu plays dual functions in regulating tumor cell growth and polarization of M2-TAMs in OSCC.

Clinical Evaluation of IDH Mutation Status in Formalin-Fixed Paraffin-Embedded Tissue in Gliomas.

Determination of isocitrate dehydrogenase (IDH) 1/2 mutational status is crucial for a glioma diagnosis. It is common for IDH mutational status to be determined via a two-step algorithm that utilizes immunohistochemistry studies for IDH1 R132H, the most frequent variant, followed by next-generation sequencing studies for immunohistochemistry-negative or immunohistochemistry-equivocal cases.The objective of this study was to evaluate addinga rapid real-time polymerase chain reaction (RT-PCR) assay to the testing algorithm. METHODS: We validated a modified, commercial, qualitative, RT-PCR assay with the ability to detect 14 variants in IDH1/2 in formalin-fixed paraffin-embedded glioma tumor specimens. The assay was validated using 51 tumor formalin-fixed paraffin-embedded specimens. During clinical implementation of this assay, 48 brain tumor specimens were assessed for IDH result concordance and turnaround time to result. Concordance between the RT-PCR and sequencing and IHC studies was 100%. This RT-PCR assay also showed concordant results with IHC for IDH1 R132H for 11 of the 12 (92%) tumor specimens with IDH mutations. The RT-PCR assay yielded faster results (average 2.6 days turnaround time) in comparison to sequencing studies (17.9 days), with complete concordance. In summary, we report that this RT-PCR assay can reliably be performed on formalin-fixed paraffin-embedded specimens and has a faster turnaround time than sequencing assays and can be clinically implemented for determination of IDH mutation status for patients with glioma.

Молекулярно-генетические критерии малигнизации эпителия слизистой оболочки рта

Introduction. When examining the oral mucosa (OM) in the presence of an inflammatory process, the initial signs of malignancy are not always visible. Amid an inflammatory infiltrate, benign changes such as epithelial hyperplasia (EH), epithelial dysplasia (ED) of varying severity, and squamous cell carcinoma (SCC) can be hidden. The aim of the paper was to study the proliferative activity of epithelial cells of the OM, TERT expression, and amplification of telomerase TERC gene in the development of epithelial hyperplasia, dysplasia, and SCC. Materials and methods. We divided 60 patients into four groups: Group 1 with EH (n=16, 26.6%); Group 2 with low-grade epithelial dysplasia (LGED) [n=16, 26.6%]; Group 3 with high-grade epithelial dysplasia (HGED) [n=13, 21.6%]; and Group 4 with SCC (n=15, 25%). IHC and histological studies were carried out using standard methods; expression to Ki-67 protein and telomerase activity were evaluated. The FISH method was used with LSP TERC and CCP3 DNA probes. Results. High-cell proliferation and telomerase activity were revealed by IHC study in all groups. Amplification of TERC RNA site in chromosome 3 by FISH in Group 1 was almost absent and showed a 2:2 signal ratio in 1.3% of cases; in other groups, the amplification of TERC gene was positive: in groups 2, 3, and 4 it was in 83.9%, 97.3%, and 89.8% of cases, respectively. Conclusion. Amplification of TERC site signals in chromosome 3 is detected during malignization which may be the main criterion for early diagnosis of malignization of OM epithelium. Keywords: squamous cell carcinoma, epithelial dysplasia, TERC amplification, telomerase

Contrast-Free FLIM Reveals Metabolic Changes in Pathological Islets of Langerhans.

FLIM (Fluorescence Lifetime Imaging Microscopy) is a powerful tool that could be used in the future to diagnose islet cell recovery after therapy. The identification of appropriate FLIM parameters is required to determine islet quality and islet cell metabolism throughout the organ under various conditions of insulin deficiency. The aim of the work was to identify key FLIM parameters, changes of which are characteristic of pancreatic pathologies. The τm, τ1, τ2, α1, α2 and α1/α2 of free and bound forms of NAD(P)H of the islet cells of animals (rats and pigs) and of humans with and without pathologies were measured and analyzed. The data were confirmed by IHC and histological studies. We identified three FLIM parameters in islet cells from animals with streptozotocin (STZ)-induced diabetes mellitus (DM) and from humans with chronic pancreatitis + type 2 diabetes (T2D), which differ in the same way: τm and α2 take lower values compared to the nonpathological islet cells, while α1/α2 takes higher values. In islet cells from patients with adenocarcinoma (PDAC) and chronic pancreatitis, these parameters had reverse tendency relative to the norm or did not differ. Thus, minimally invasive and non-contrast FLIM methods may, in the future, be used to diagnose pathological islet cells.

Primary Nasal Mucosal Melanoma: A Case Report

Nasal mucocal melanoma is a rare and aggressive neoplasm. In most cases, it arises in the lateral wall of the nasal cavity. As it shows nonspecific clinical symptoms and risk factors, anterior nasofibroscopy and imaging plays a crucial role in the diagnosis. The final diagnosis is based on histological and immunohistochemical findings. The wide surgical excision is the mainstay of the treatment and the adjuvant radiotherapy is often used to prevent local recurrence and metastasis. We report a case of a 61 years old female patient with obstruction and epsistaxis. The first facial magnetic resonance imaging was suggestive of an inverted papilloma misleading the diagnosis. The patient had undergone nasal endoscopy with a biopsy. The histological and immunohistochemical were consistent with the diagnosis of a mucosal melanoma. A facial computed tomography revealed a solid mass filling the entire right nasal cavity with extension to the homolateral ethmoidal cells and the maxillary sinus and erosion of the middle and lower turbinates and the nasal septum. A second facial magnetic resonance imaging showed a neoformation of the right nasal cavity. The lesion was initially removed using and endonasal endoscopic approach. Local recurrence occurred one month later, which leaded to perform a lateral rhinotomy approach. The histopathological aspect was consistent with the diagnosis of malignant melanoma and the patient received a postoperative 3D radiation therapy. The diagnosis is based on histological and IHC studies. Wide surgical excision with negative margins is the cornerstone of any treatment regimen for sinonasal mucosal melanoma followed by adjuvant radiotherapy even if there is no clear consensus. In spite of a well conducted treatment, the melanoma of the nasal cavity remains an aggressive tumor with a non-negligent risk of local recurrence, with a poor prognosis and a survival rate not exceeding 35% after 5 years.

COVID-19 lung disease shares driver AT2 cytopathic features with Idiopathic pulmonary fibrosis.

SummaryBackgroundIn the aftermath of Covid-19, some patients develop a fibrotic lung disease, i.e., post-COVID-19 lung disease (PCLD), for which we currently lack insights into pathogenesis, disease models, or treatment options.MethodsUsing an AI-guided approach, we analyzed > 1000 human lung transcriptomic datasets associated with various lung conditions using two viral pandemic signatures (ViP and sViP) and one covid lung-derived signature. Upon identifying similarities between COVID-19 and idiopathic pulmonary fibrosis (IPF), we subsequently dissected the basis for such similarity from molecular, cytopathic, and immunologic perspectives using a panel of IPF-specific gene signatures, alongside signatures of alveolar type II (AT2) cytopathies and of prognostic monocyte-driven processes that are known drivers of IPF. Transcriptome-derived findings were used to construct protein-protein interaction (PPI) network to identify the major triggers of AT2 dysfunction. Key findings were validated in hamster and human adult lung organoid (ALO) pre-clinical models of COVID-19 using immunohistochemistry and qPCR.FindingsCOVID-19 resembles IPF at a fundamental level; it recapitulates the gene expression patterns (ViP and IPF signatures), cytokine storm (IL15-centric), and the AT2 cytopathic changes, e.g., injury, DNA damage, arrest in a transient, damage-induced progenitor state, and senescence-associated secretory phenotype (SASP). These immunocytopathic features were induced in pre-clinical COVID models (ALO and hamster) and reversed with effective anti-CoV-2 therapeutics in hamsters. PPI-network analyses pinpointed ER stress as one of the shared early triggers of both diseases, and IHC studies validated the same in the lungs of deceased subjects with COVID-19 and SARS-CoV-2-challenged hamster lungs. Lungs from tg-mice, in which ER stress is induced specifically in the AT2 cells, faithfully recapitulate the host immune response and alveolar cytopathic changes that are induced by SARS-CoV-2.InterpretationLike IPF, COVID-19 may be driven by injury-induced ER stress that culminates into progenitor state arrest and SASP in AT2 cells. The ViP signatures in monocytes may be key determinants of prognosis. The insights, signatures, disease models identified here are likely to spur the development of therapies for patients with IPF and other fibrotic interstitial lung diseases.FundingThis work was supported by the National Institutes for Health grants R01- GM138385 and AI155696 and funding from the Tobacco-Related disease Research Program (R01RG3780).

IHC characteristics of stem cell markers in PDAC with different degrees of differentiation

Introduction. Cancer stem cells (CSCs) play a key role in the development, progression and metastasis of cancer. Cell surface markers, in particular CD44 and EpCAM, are used to identify CSCs. Marker expression patterns depend on the stage of progression and differentiation. Materials and methods. Histopathological and IHC studies of 49 cases of the pancreas ductal adenocarcinoma (PDAC) of G2, G3 degrees of differentiation. Results of IHC reactions with antibodies against CD44 and EpCAM were estimated by photo digital morphometry and were expressed in immunostained cells relative area (%). Results. Adenocarinoma of the pancreas is characterized by a moderate level of expression of cell surface markers CSC - CD44 and EpCAM. The expression level of CD44 in the І gr. - Me = 34.11% (Q1 = 31.03; Q3 = 38.39), in the II gr. - Me = 43.17% (Q1 = 39.83; Q3 = 47.04), significantly higher than in the I gr. (Kruskal-Wallis test p&lt;0.05). The expression level of EpCAM in the I gr. - Ме=26,10% (Q1=20,42; Q3=31,89), in the II gr. - Ме=31,07% (Q1=27,03; Q3=34,00) (p&gt;0,05). In both groups, the relative area of EpCAM was significantly lower than CD44 (Kruskal-Wallis test p&lt;0.05). Correlation analysis (Pearson's ratio) showed a weak negative relationship between markers at G2 (r = -0.17, p&gt;0.05), at G3 - a weak positive (r = + 0.06, p&gt;0.05).

Combination of Ribociclib and Gemcitabine for the Treatment of Medulloblastoma.

Group3 (G3) medulloblastoma (MB) is one of the deadliest forms of the disease for which novel treatment is desperately needed. Here we evaluate ribociclib, a highly selective CDK4/6 inhibitor, with gemcitabine in mouse and human G3MBs. Ribociclib central nervous system (CNS) penetration was assessed by in vivo microdialysis and by IHC and gene expression studies and found to be CNS-penetrant. Tumors from mice treated with short term oral ribociclib displayed inhibited RB phosphorylation, downregulated E2F target genes, and decreased proliferation. Survival studies to determine the efficacy of ribociclib and gemcitabine combination were performed on mice intracranially implanted with luciferase-labeled mouse and human G3MBs. Treatment of mice with the combination of ribociclib and gemcitabine was well tolerated, slowed tumor progression and metastatic spread, and increased survival. Expression-based gene activity and cell state analysis investigated the effects of the combination after short- and long-term treatments. Molecular analysis of treated versus untreated tumors showed a significant decrease in the activity and expression of genes involved in cell-cycle progression and DNA damage response, and an increase in the activity and expression of genes implicated in neuronal identity and neuronal differentiation. Our findings in both mouse and human patient-derived orthotopic xenograft models suggest that ribociclib and gemcitabine combination therapy warrants further investigation as a treatment strategy for children with G3MB.

Abstract 316: Carbohydrate-dependent protein binding antibodies with superior tumor-specificity

Abstract Background: Highly potent therapeutic approaches require very clean targets. However, the majority of antibodies in clinical development or approved for cancer therapy address protein targets that are only overexpressed on cancer cells and yet often show significant expression in healthy organs. Glycans tend to elicit superior tumor specificity as compared to proteins, since glycosylation is strongly altered in cancer, reflecting the drastic changes in tumor metabolism. The changes in glycosylation are mostly due to mutated or mislocated glycosyltransferases and glycosidases, giving rise to highly fucosylated, highly sialylated and truncated glycans. Therefore, proteins expressed in cancer cells can carry tumor-associated carbohydrates like the Thomsen-Friedenreich (TF) and the Thomsen novelle (Tn) antigen as well as their respective sialylated forms sTF and sTn. To increase the tumor-specificity of protein-targeting antibodies, we have developed antibodies against several different protein/carbohydrate combined epitopes (GlycoTargets). Targeting these specific antigens offers reduced on-target/off tumor toxicity, which is key for highly potent therapies. Methods: The specificity and glycosylation dependency of our antibodies were shown using differentially glycosylated proteins or carbohydrate PAA-conjugates in an ELISA format. Binding to several tumor cell lines and healthy human leukocytes was analyzed via flow cytometry. Binding to different cancer indications as well as related healthy tissues was analyzed by immunohistochemistry. Results: We have generated antibodies that bind to their target protein only if a specific tumor-associated carbohydrate is present and do not cross-react with the non-glycosylated protein itself, or the carbohydrate structure on different carrier-proteins. Due to the carbohydrate-dependent binding, our antibodies show markedly decreased on-target/off-tumor binding. We will show that our antibodies mediate no binding of different healthy immune cell subsets in comparison to simple protein binding antibodies in flow cytometric analyses. Further, IHC studies reveal that our antibodies stain tumor tissue of different cancer indications but do not stain related normal tissues. Conclusions: We successfully used the fact that glycosylation is strongly altered in cancer for the generation of highly tumor-specific antibodies that target neither glycan nor protein alone, but the potent combination thereof - a tumor-specific protein/carbohydrate epitope. We could show that by using this approach we were able to reduce on-target/off-tumor binding towards different healthy tissues and healthy immune cell subsets making these antibodies ideal candidates for a therapeutic development. Citation Format: Patrik Kehler, Anika Jaekel, Antje Danielczyk, Christoph Goletz, Timo Lischke, Evelyn Hartung, Lisa Weiß, Manon Weis, Stephanie Gurka, Sophie Marinoff, Naomi Kast, Manon Weiske, Marianne Morche, Johanna Gellert, Theresa Neumann. Carbohydrate-dependent protein binding antibodies with superior tumor-specificity [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 316.

SARS-CoV-2-Induced Pathology-Relevance to COVID-19 Pathophysiology.

In spite of intensive studies of different aspects of a new coronavirus infection, many issues still remain unclear. In a screening analysis of histopathology in l200 lethal cases, authors succeeded in performing a wide spectrum of immune histochemical reactions (CD2, CD 3, CD 4, CD 5, CD 7, CD 8, CD14, CD 20, CD 31, CD 34, CD 56, CD 57, CD 68, CD 163, collagen 1,3, spike protein SARS-CoV-2, caspase-3, MLCM; ACE2 receptor, occludin, and claudin-1 and -3) and electron microscopy. The results of the histological and IHC studies of deceased people with varying degrees of severity of coronavirus infection confirmed the ability of these pathogens to cause cytoproliferative changes, primarily in epithelial and endothelial cells. Lesions of various organs are possible, while the reasons for significant differences in organotropy remain unclear. Severe respiratory failure in COVID-19 in humans is associated with a very peculiar viral pneumonia. In the pathogenesis of COVID-19, the most important role is played by lesions of the microcirculatory bed, the genesis of which requires further study, but direct viral damage is most likely. Endothelial damage can be associated with both thrombosis in vessels of various calibers, leading to characteristic complications, and the development of DIC syndrome with maximal kidney damage. Such lesions can be the basis of clinically diagnosed septic shock, while usually there are no morphological data in favor of classical sepsis caused by bacteria or fungi. A massive infiltration of the lung tissue and other organs, mainly by T lymphocytes, including those with suppressor properties, makes it necessary to conduct a differential diagnosis between the morphological manifestation of the protective cellular immune response and direct viral lesions but does not exclude the hypothesis of an immunopathological component of pathogenesis. In many of the deceased, even in the absence of clear clinical symptoms, a variety of extrapulmonary lesions were also detected. The mechanism of their development probably has a complex nature: direct lesions associated with the generalization of viral infection and vascular disorders associated with endothelial damage and having an autoimmune nature. Many aspects of the pathogenesis of coronavirus infection require further comprehensive study.

Morphological features of complicated gastroduodenal ulcers

The objective of the study was to evaluate structural changes in the margin of gastroduodenal ulcers complicated by perforation, bleeding or penetration in terms of the features of ulcer healing.Methods and materials. Histological and IHC studies were performed on 25 patients of the main group with perforated gastroduodenal ulcers and 23 patients of the control group with chronic recurrent ulcers complicated by bleeding and penetration. Histological sections were stained with hematoxylin and eosin, and Van Gieson's picrofuchsin. Immunohistochemical reactions were performed with antibodies to Ki-67, CD68, CD138, and CD34.Results. On macroscopic evaluation, a clear even margin of the perforation was noted. Histological examination with the background of acute dyscirculatory changes clearly shows a zone of necrosis and a weakly expressed layer of granulation tissue, which in majority of the cases was practically absent. The margin of a perforated ulcer consists of a minimally presented, clearly delimited zone of tissue detritus and a practically unchanged muscle layer. Plasma cells dominated in the cell infiltrate, and the index of proliferative activity in the epithelium of the ulcer margins was about 45 %, distributed fairly evenly.Conclusions. Perforated ulcers can be considered as a special group of ulcers with the most pronounced changes of infiltration by plasmatic cells and eosinophils, edema and acute dyscirculatory changes, uniform and fairly high proliferative activity of the epithelium. Changes in the margins of the perforated ulcer allow for complete regeneration without excessive scarring with precise suturing of the perforation.

The use of photodynamic therapy in patients with severe forms of leukoplakia

The aim the study is to increase the effectiveness of the treatment of severe forms of leukoplakia with the use of photodynamic therapy. 120 patients with severe forms of leukoplakia were examined. 30 patients were diagnosed with Cr and carcinoma in situ, and therefore they were excluded from the study. In 90 patients, the diagnosis was confirmed by histological and IHC studies. Before the use of photodynamic therapy, the degree of saturation of pathological foci with a photosensitizer was determined by the method of fluorescent diagnostics using the LED device «RFS-400». As a result of treatment with photodynamic therapy using a 2nd generation photosensitizer («Photoditazine» 0.5% gel penetrator), a significant improvement in the epithelization of lesion elements was observed: in 92% of patients with verrucous leukoplakia and in 83% of patients with erosive leukoplakia. The use of photodynamic therapy for the treatment of patients with complicated forms of leukoplakia is an effective method of treatment for this pathology.