Immunohistochemical Panel Research Articles

PAX1 expression in thymic epithelial neoplasms and morphologic mimics

Thymic epithelial neoplasms are morphologically diverse and can pose a diagnostic challenge that is complicated by a lack of immunohistochemistry (IHC) markers that are entirely sensitive and specific for thymic epithelium. Polyclonal PAX8 is often used in this context, but it is not a specific marker. The PAX1 transcription factor shares significant homology with PAX8 and plays an integral role in thymic development in humans and murine models. This study evaluated the role of PAX1 IHC in differentiating thymic epithelial neoplasms from morphologic mimics on whole slide tissue sections. The PAX1 antibody stained all 74 thymoma cases; however, there was wide variability in staining intensity within each subtype. The antibody was less sensitive in thymic carcinomas and thymic neuroendocrine tumors compared to thymomas and demonstrated weak staining in a subset of morphologic mimics (21 squamous cell carcinomas, 6 pulmonary neuroendocrine tumors, 1 mesothelioma, 1 lymphoblastic lymphoma, and 1 granulosa cell tumor). With a H-score positive threshold of 75, the antibody had 100% specificity, and sensitivities of 92%, 56%, and 47% in thymomas, thymic neuroendocrine tumors, and thymic carcinomas respectively. The PAX1 antibody showed frequent geographic reduction in staining consistent with compromised antigenicity from variable formalin fixation. PAX1 IHC has a moderate-to-high sensitivity for thymic epithelial neoplasms; however, the wide staining variability and fixation effects may lead to difficulty with consistent interpretation. This marker is unlikely to supplant the role of PAX8 in diagnostic practice, but it may be a useful addition to immunohistochemistry panels when evaluating for thymic primary tumors.

HLF promotes ovarian cancer progression and chemoresistance via regulating Hippo signaling pathway

Hepatic leukemia factor (HLF) is aberrantly expressed in human malignancies. However, the role of HLF in the regulation of ovarian cancer (OC) remains unknown. Herein, we reported that HLF expression was upregulated in OC tissues and ovarian cancer stem cells (CSCs). Functional studies have revealed that HLF regulates OC cell stemness, proliferation, and metastasis. Mechanistically, HLF transcriptionally activated Yes-associated protein 1 (YAP1) expression and subsequently modulated the Hippo signaling pathway. Moreover, we found that miR-520e directly targeted HLF 3′-UTR in OC cells. miR-520e expression was negatively correlated with HLF and YAP1 expression in OC tissues. The combined immunohistochemical (IHC) panels exhibited a better prognostic value for OC patients than any of these components alone. Importantly, the HLF/YAP1 axis determines the response of OC cells to carboplatin treatment and HLF depletion or the YAP1 inhibitor verteporfin abrogated carboplatin resistance. Analysis of patient-derived xenografts (PDXs) further suggested that HLF might predict carboplatin benefits in OC patients. In conclusion, these findings suggest a crucial role of the miR-520e/HLF/YAP1 axis in OC progression and chemoresistance, suggesting potential therapeutic targets for OC.

Complementary value of molecular analysis to expert review in refining classification of uncommon soft tissue tumors.

The classification of many soft tissue tumors remains subjective due their rarity, significant overlap in microscopic features and often a non-specific immunohistochemical (IHC) profile. The application of molecular genetic tools, which leverage the underlying molecular pathogenesis of these neoplasms, have considerably improved the diagnostic abilities of pathologists and refined classification based on objective molecular markers. In this study, we describe the results of an international collaboration conducted over a 3-year period, assessing the added diagnostic value of applying molecular genetics to sarcoma expert pathologic review in a selected series of 84 uncommon, mostly unclassifiable mesenchymal tumors, 74 of which originated in soft tissues and 10 in bone. The case mix (71% historical, 29% contemporary) included mostly unusual and challenging soft tissue tumors, which remained unclassified even with the benefit of expert review and routine ancillary methods, including broad IHC panels and a limited number of commercially available fluorescence in situ hybridization (FISH) probes. All cases were further tested by FISH using a wide range of custom bacterial artificial chromosome probes covering most of known fusions in sarcomas, whereas targeted RNA sequencing was performed in 13 cases negative by FISH, for potential discovery of novel fusion genes. Tumor-defining molecular alterations were found in 48/84 tumors (57%). In 27 (32%) cases the tumor diagnosis was refined or revised by the additional molecular work-up, including five cases (6%), in which the updated diagnosis had clinical implications. Sarcoma classification is rapidly evolving due to an increased molecular characterization of these neoplasms, so unsurprisingly 17% of the tumors in this series harbored abnormalities only very recently described as defining novel molecularly defined soft tissue tumor subsets.

Expression of TLE1 in a Carcinoid Tumor of the Lung With Spindle Cell Morphology-A Potential Diagnostic Pitfall.

Tumors of the lung with a spindle cell morphology require consideration of many entities in the differential diagnosis, including metastases. Ancillary immunohistochemical stains but also molecular studies are typically required to arrive at the proper diagnosis. We present a case of a 71-year-old woman with multiple lung nodules, mediastinal lymphadenopathy, and a history of uterine cancer who underwent endobronchial ultrasound-guided fine needle aspiration and biopsy of the lung and mediastinal lymph nodes. A sampling of the lung lesion showed a cytologically bland neoplasm with spindle cell morphology, lacking necrosis or brisk mitotic activity. In conjunction with the cytomorphology, strong and diffuse Transducin-like enhancer of split 1 (TLE1) reactivity in the tumor cells initially raised the diagnosis of synovial sarcoma; however, subsequent results of additional testing showed strong and diffuse expression with AE1/AE3, CK 8/18, TTF-1, synaptophysin and chromogranin and focal or negative staining with a large number of other antibodies. This warranted a diagnosis of a carcinoid tumor. This is the first report of TLE1 staining in a carcinoid tumor of the lung. Therefore, when evaluating tumors of the lung with spindle cell morphology in which the differential diagnosis may include both carcinoid tumor and synovial sarcoma, TLE1 expression should be interpreted with caution and in conjunction with an expanded immunohistochemical staining panel.

Microsatellite instability (MSI) and p16/p53 protein status in different subtypes of endometrial carcinoma: with emphasis on tumor aggressiveness

Objective: We investigated microsatellite instability (MSI) in endometrial cancer (EC) and correlated results with traditional markers (p16, p53, Ki-67) to predict tumor aggressiveness. 
 Materials and Methods: Records of patients admitted with EC between 2010 and 2022 were reviewed, and the widest immunohistochemical (IHC) panel including (1) estrogen or progesterone receptors (ER, PR), (2) mismatch repair (MMR) proteins (MLH1, PMS2, MSH2, MSH6), (3) Ki-67, (4) p16 and (5) p53 proteins were recorded. Chi square test was used for statistical analysis. 
 Results: Total of 44 female patients with pathology reports containing all five IHC panel markers were included. Mean age was 64.1±12.51 years. Type I EC was the most common pathology (72%). ER or PR positivity were very prominent in type I tumors in comparison with non-endometrioid (type II) tumors (84% vs 16%, respectively; p

First report of primary testicular leiomyosarcoma in two dogs

BackgroundTesticular tumours are common in dogs and, among them, interstitial cell tumours, seminomas and sustentacular cell tumours are the most reported. Mesenchymal testicular tumours are rarely reported in humans as in veterinary medicine where only three cases of sarcomas (leiomyomas and leomyosarcomas) have been described in two stallions and in a ram.Case presentationThe present cases regarded a 12-year-old mixed-breed dog and a 10-year-old American Staffordshire Terrier that underwent bilateral orchiectomy. Formalin fixed testes were referred for histopathological diagnosis. At gross examination, in one of the testes of both dogs, a white, firm and variably cystic testicular mass, effacing and replacing the testicular parenchyma was detected. Samples were collected from both neoplastic and contralateral testes, routinely processed for histology and serial sections were also examined immunohistochemically with primary antibodies against cytokeratins, vimentin, Von Willebrand factor, inhibin-α, α-smooth muscle actin, smooth muscle myosin and desmin. Histopathological features as well as the immunohistochemical results, positive for vimentin, actin, myosin and desmin, confirmed the mesenchymal origin and the myoid phenotype of both testicular tumours supporting the diagnoses of leiomyosarcoma.ConclusionsTo the authors knowledge these are the first cases of primary testicular sarcoma reported in the canine species. However, even rare, these tumours deserve to be considered in routine diagnosis when a testicular spindle cell tumour is observed. The immunohistochemical panel applied was useful to distinguish the present tumours from undifferentiated Sertoli cell tumours confirming the diagnosis of leiomyosarcoma.

Assessment of endometrial carcinoma on biopsy as a predictor of final surgical pathology: Are we doing it right? A completed audit cycle and recommendations.

Typing and grading of endometrial carcinomas (ECs) on small biopsy specimens is crucial to determine the need for full surgical staging. Histological subtype and grade are key factors available for risk stratification before surgery. However, this can be diagnostically challenging on small biopsy specimens, especially when morphologic features are subtle or overlapping. The aims of this audit were to assess concordance of endometrial carcinomas on biopsy specimens with hysterectomy specimens and to determine if the immunohistochemistry (IHC) panel being used in our practice was adequately subtyping ECs. The audit was approved by the Clinical Effectiveness Team of the Royal College of Pathologists (UK) as meeting all the criteria and standards set out by the College. Biopsies from 67 cases of EC were compared for histological subtype and grade of endometrioid carcinoma with resection specimens. A re-audit was carried out on 59 cases after implementation of changes recommended by the initial audit. Two of 35 (6%) tumours defined as G1 on biopsy were upgraded (to G2) on final pathology, as was one of 7 (14%) G2 tumours (to G3). One of these cases had solid areas just amounting to more than 6% on resection. In the second case, a comment was made that assessment had been difficult as the specimen was suboptimally fixed, but nuclei appeared atypical. Of seven G2 biopsies, one case was upgraded to grade 3 on final pathology based on proportion of solid areas. Our data show lower rates of discordance as compared to previous studies and on re-audit, the concordance between endometrioid and nonendometrioid serous carcinoma improved with the addition of immunohistochemistry (IHC) for Phosphatase and tensin homolog (PTEN) to biopsies. PTEN IHC can complement other stains and aid in the distinction of grade 3 endometrioid carcinoma from serous carcinoma on endometrial biopsies.

Plurihormonal Pituitary Neuroendocrine Tumours - A Single Centre Experience.

Introduction. Plurihormonal pituitary neuroendocrine tumours (PitNET)/adenomas are pituitary neuroendocrine tumours composed of monomorphous cell populations expressing anterior pituitary transcription factors and/or hormones belonging to more than one cell lineage. Studies dedicated to plurihormonal tumours are rare and quite heterogenous with their results, bearing in mind changes in diagnostic criteria and inconsistent use of antibodies for anterior pituitary transcription factors in the diagnostic immunohistochemical panel. Material and Methods. We retrospectively analysed all patients surgically treated for PitNETs from 2016 to July 2022 in a tertiary healthcare institution. All tumours previously diagnosed PitNETs with the word "plurihormonal" were re-examined and potentially re-classified, according to 2022 WHO classification of endocrine tumours. Results. Among 721 patients surgically treated for PitNET in 5.5 years period, the diagnosis of plurihormonal PitNET was established in 11 tumours (1.3%). All tumours showed diffuse and intensive positivity for anterior pituitary transcription factors PIT1 and SF1. Clinically, all patients presented with acromegaly. Conclusions. Retrospective studies related to newly defined plurihormonal PitNETs with a reassessment of diagnoses are necessary due to their rarity and ambition to investigate their origin and biological behaviour. The fact that the majority of plurihormonal PitNETs are clinically presented with acromegaly and show simultaneous positivity to PIT1 and SF1 transcription factors deserve special attention and need for further research in larger cohorts of these exceptional tumours.

Trichorhinophalangeal Syndrome Type 1 Is a Highly Sensitive and Specific Marker for Diagnosing Triple-Negative Breast Carcinomas on Cytologic Samples.

Definitive diagnosis of metastatic triple-negative breast carcinoma (TNBC) is challenging on cytologic samples. Recent studies demonstrated that trichorhinophalangeal syndrome type 1 (TRPS1) is a highly sensitive and specific marker for diagnosing breast carcinomas, including TNBC, on surgical specimens. To evaluate TRPS1 expression in TNBCs on cytologic samples and a large series of nonbreast tumors on tissue microarray sections. Immunohistochemical (IHC) analysis of TRPS1 and GATA-binding protein 3 (GATA3) was performed on 35 TNBC cases on surgical specimens, and 29 consecutive TNBC cases on cytologic specimens. IHC analysis of TRPS1 expression was also performed on 1079 nonbreast tumors on tissue microarray sections. Of the surgical specimens, 35 of 35 TNBC cases (100%) were positive for TRPS1, all with diffuse positivity, whereas 27 of 35 (77%) were positive for GATA3, with diffuse positivity in 7 cases (26%). Of the cytologic samples, 27 of 29 TNBC cases (93%) were positive for TRPS1, with diffuse positivity in 20 cases (74%), whereas 12 of 29 (41%) were positive for GATA3, with diffuse positivity in 2 cases (17%). Of the nonbreast malignant tumors, TRPS1 expression was seen in 9.4% (3 of 32) of melanomas, 10.7% (3 of 28) of small cell carcinomas of the bladder, and 9.7% (4 of 41) of ovarian serous carcinomas. Our data confirm that TRPS1 is a highly sensitive and specific marker for diagnosing TNBC cases on surgical specimens as reported in the literature. In addition, these data demonstrate that TRPS1 is a much more sensitive marker than GATA3 in detecting metastatic TNBC cases on cytologic samples. Therefore, inclusion of TRPS1 in the diagnostic IHC panel is recommended when a metastatic TNBC is suspected.

Immunohistochemical phenotype of colorectal carcinoma in patients with KRAS mutation and mismatch repair status.

Aberrant expression of CK7/CK20/CDX2 is reported in percentage of colorectal carcinomas (CRC).

High-grade Serous Carcinoma can Show Squamoid Morphology Mimicking True Squamous Differentiation.

Tubo-ovarian high-grade serous carcinoma (HG-SC) and ovarian endometrioid carcinoma (EC) can show overlapping morphologic features, such as glandular and solid patterns. The differential diagnosis of these subtypes is thus sometimes difficult. The existence of "squamous differentiation" tends to lead to a diagnosis of EC rather than HG-SC. We noticed that HG-SC can contain a "squamoid component," but its nature has been poorly investigated. This study was thus established to clarify the nature of this "squamoid component" in HG-SC by investigating its frequency and immunohistochemical features. We reviewed hematoxylin and eosin-stained slides of 237 primary untreated cases of tubo-ovarian HG-SC and identified 16 cases (6.7%) of HG-SC with "squamoid component." An immunohistochemical staining panel (CK5/6, CK14, CK903, p40, p63, WT1, ER, and PgR) was used to analyze all of these 16 cases. We also selected 14 cases of ovarian EC with "squamous differentiation" as a control. The "squamoid component" in HG-SC was completely p40-negative and showed significantly lower expression of CK5/6, CK14, CK903, and p63 than the "squamous differentiation" in EC. The immunophenotype of the "squamoid component" in HG-SC was concordant with the conventional HG-SC component (WT1-positive/ER-positive). Furthermore, all 16 tumors were confirmed to be truly "HG-SC" by the findings of aberrant p53 staining pattern and/or WT1/p16 positivity, and the lack of mismatch repair deficiency and POLE mutation. In conclusion, HG-SC can on rare occasions show a "squamoid component" mimicking "squamous differentiation." However, the "squamoid component" in HG-SC does not represent true "squamous differentiation." The "squamoid component" is one part of the morphologic spectrum of HG-SC, which should be interpreted carefully for the differential diagnosis of HG-SC and EC. An immunohistochemical panel including p40, p53, p16, and WT1 is a useful adjunct to achieve a correct diagnosis.

BI19 Cutaneous post-transplant lymphoproliferative disorder in an immunosuppressed patient

Abstract Post-transplant lymphoproliferative disorders (PTLDs) are a common form of de novo neoplasia developing in patients with immunosuppression following a transplant. The majority are associated with Epstein–Barr virus (EBV) infection. Cutaneous PTLD manifestations are rare and not well documented in literature. We present a case of systemic PTLD with secondary extensive cutaneous involvement 4 months after allogenic stem cell transplant for JAK2-mutated myelofibrosis. A 68-year-old man was admitted to the haematology department with fevers, diarrhoea, cytopenia, EBV viraemia and a nodular rash behind his ears. Biopsies from colonoscopy confirmed monomorphic PTLD with subsequent extensive ileocalceal lymphadenopathy area detected on computed tomography. Asymptomatic, large, hardened violaceous nodules on the head and neck worsened further. Skin biopsy showed an infiltrate of atypical lymphocytes in the dermis, composed predominantly of large lymphoid cells with visible prominent nucleoli. A limited immunohistochemical panel showed positivity for BCL-2 but not CD20. EBV-encoded RNA in situ hybridization was also positive. Features were compatible with cutaneous involvement by the known monomorphic PTLD. Unfortunately, the patient deteriorated with haemodynamic instability due to sepsis, despite two further infusions of rituximab, and was directed to the palliative team. He died soon after the discontinuation of all treatments. PTLD has a wide spectrum of clinical manifestations. It is classified into three nondestructive types: polymorphic, monomorphic and classic Hodgkin lymphoma type. It can have a poor prognosis so it should always be considered as a diagnosis in transplant patients. Monomorphic PTLDs tend to be more aggressive and rarely respond to immunosuppression reduction. On the contrary, primary cutaneous involvement is rare and seems to have a favourable outcome. Treatment depends on the severity of the disease, and both localized and systemic therapies have been used. Transplant patients should have long-term follow-up to diagnose and promptly treat possible complications.

Molecular classification of muscle-invasive bladder cancer based on a simplified immunohistochemical panel using GATA3, CK5/6 and p16.

The choice of therapy for muscle-invasive bladder cancer (MIBC) could be influenced by the tumor's molecular subtype. Currently, well-defined consensus subtypes are based on tumor microarray mRNA data. Clearly defined and easy-to-use surrogate molecular subtypes, based on immunohistochemistry (IHC) performed on whole slides, are needed to make subtyping cost-effective and useful in routine work and future research. To aid in the development of a simple immunohistochemical classifier, a retrospective single-center series of 92 cases of localized bladder cancer was identified. Routine IHC for GATA3, cytokeratins 5 and 6 (CK5/6), and p16 was performed on whole tissue blocks containing muscle-invasive disease. Electronic medical records were retrieved and searched for clinical variables, treatment, and survival data. The mean age was 69.6 years, and 73% were males. Conservative treatment was used in 55% of cases, while cystectomy with chemotherapy was used in 45%. GATA3 and CK5/6 expression divided cases into broad luminal and basal subtypes, respectively, while p16 expression was used to subclassify luminal cases into luminal papillary and luminal unstable types according to the consensus molecular classification. When subtyped in this way, GATA3 and CK5/6 negative cases showed worse overall survival. Molecular subtyping of MIBC on whole slides containing muscle-invasive tumor using only three commonly used, consensus-based antibodies, is a feasible and cost-effective method for detecting subtypes of invasive bladder cancer. Future work combining morphological analysis and IHC is needed to fully translate the consensus molecular classification into a comprehensive, cost-effective subtyping strategy.

Claudin-4 Expression in the Distinction of Mammary and Extramammary Paget Disease From Morphologic Mimics: Diagnostic Utility and Pitfalls.

Claudin-4 Expression in the Distinction of Mammary and Extramammary Paget Disease From Morphologic Mimics: Diagnostic Utility and Pitfalls.

LOW-GRADE MYOFIBROBLASTIC SARCOMA OF THE ORAL CAVITY

Low-grade myofibroblastic sarcoma (LGMS) represents a distinct atypical myofibroblastic tumor of deep soft tissues, with a head and neck predilection. It has a variety of appearances, from fasciitis-like or fibromatosis-like to fibrosarcoma-like. Here, a series of four cases from two different institutions is shown, represented by 2 men and 2 women presenting a nodular lesion, with equal distribution in maxilla and mandibula. Microscopically, all cases showed neoplasms formed by oval to spindle cells in a fibrous stroma with myxoid and dense areas, some atypical mitoses, and prominent nucleoli. These findings suggested a mesenchymal tumor. An immunohistochemical panel was performed, which showed positivity for smooth muscle actin (3 of 4 cases), HHF35 (2 of 4 cases), β-catenin (3 of 3 cases), calponin (2 of 4 cases), and Ki-67 (range 5-40%). H-Caldesmon was negative for all cases, demonstrating myofibroblastic and smooth muscle differentiation. The diagnosis of LGMS was confirmed in all cases. Low-grade myofibroblastic sarcoma (LGMS) represents a distinct atypical myofibroblastic tumor of deep soft tissues, with a head and neck predilection. It has a variety of appearances, from fasciitis-like or fibromatosis-like to fibrosarcoma-like. Here, a series of four cases from two different institutions is shown, represented by 2 men and 2 women presenting a nodular lesion, with equal distribution in maxilla and mandibula. Microscopically, all cases showed neoplasms formed by oval to spindle cells in a fibrous stroma with myxoid and dense areas, some atypical mitoses, and prominent nucleoli. These findings suggested a mesenchymal tumor. An immunohistochemical panel was performed, which showed positivity for smooth muscle actin (3 of 4 cases), HHF35 (2 of 4 cases), β-catenin (3 of 3 cases), calponin (2 of 4 cases), and Ki-67 (range 5-40%). H-Caldesmon was negative for all cases, demonstrating myofibroblastic and smooth muscle differentiation. The diagnosis of LGMS was confirmed in all cases.

Utility of a PAX2, PTEN, and β-catenin Panel in the Diagnosis of Atypical Hyperplasia/Endometrioid Intraepithelial Neoplasia in Endometrial Polyps.

The diagnosis of atypical hyperplasia/endometrioid intraepithelial neoplasm (AH/EIN) within endometrial polyps (EMPs) often poses a diagnostic conundrum. Our previous studies demonstrated that a panel of immunohistochemical (IHC) markers consisting of PAX2, PTEN, and β-catenin can be effectively utilized for the identification of AH/EIN. A total of 105 AH/EIN within EMP were analyzed using the 3-marker panel. We also evaluated these cases for the presence of morules. Benign EMP (n=90) and AH/EIN unassociated with polyp (n=111) served as controls. Aberrant expression of PAX2, PTEN, or β-catenin was observed in AH/EIN in EMP in 64.8%, 39.0%, and 61.9% of cases, respectively. At least 1 IHC marker was abnormal in 92.4% of cases. Overall, 60% of AH/EIN in EMP demonstrated abnormal results for≥2 IHC markers. The prevalence of PAX2 aberrancy was significantly lower in AH/EIN in EMP than in nonpolyp AH/EIN (64.8% vs. 81.1%, P =0.007), but higher than in benign EMP (64.8% vs. 14.4%, P <0.00001). The prevalence of β-catenin aberrancy was significantly higher in AH/EIN in EMP than in nonpolyp AH/EIN (61.9% vs. 47.7%, P =0.037). All control benign EMP demonstrated normal expression of PTEN and β-catenin. Morules were present in 38.1% of AH/EIN in EMP versus 24.3% in nonpolyp AH/EIN, and absent in benign EMP. A strong positive association was found between β-catenin and morules (Φ=0.64). Overall, 90% cases of atypical polypoid adenomyoma (n=6) and mucinous papillary proliferation (n=4) showed IHC marker aberrancy. In conclusion, the 3-marker IHC panel (PAX2, PTEN, and β-catenin) is (1) a useful tool in the diagnosis of AH/EIN in EMP; (2) PAX2 loss should be interpreted with caution and in combination with morphology and other markers.

Gestational trophoblastic neoplasia of intermediate trophoblasts: Epithelioid trophoblastic tumor and placental site trophoblastic tumor, a study of morphologic, immunohistochemical, and next generation sequencing.

Gestational trophoblastic tumors are very rare neoplasms. We determined the distinctive morphological, immunohistochemical, and clinical features of placental site trophoblastic tumors (PSTT) and epithelioid trophoblastic tumors (ETT) in our cohort. Nine cases of PSTT and four cases of ETT were retrieved from the archives. Histomorphologic, immunohistochemical, and clinical features were noted. A molecular study was performed on one PSTT and one ETT case using next-generation sequencing. While the nodular pattern, geographic necrosis, and extracellular eosinophilic globules were peculiar to ETTs, vessel wall affinity, marked pleomorphism, intranuclear pseudoinclusion, spindle tumor cell, and vacuolar degeneration were more specific for PSTTs in our series. An immunohistochemical panel of p63, hPL, and CD146 were helpful for the exact typing of the tumor. p63 positivity supports the ETT and diffuse staining of hPL and CD146 supports the PSTT diagnosis. Three of the patients with metastatic disease (lung and brain metastasis) except one have a high mitotic count (12 and 8) and a long interval between (8 and 10 years) antecedent pregnancy and diagnosis. While KIT and TP53 mutations were observed only in PSTT, amino acid changes in KDR, APC, and SMAD4 genes were detected both in the ETT and PSTT cases. In the prediction of metastasis, the long intervals between antecedent pregnancy and diagnosis, deep myometrial invasion, mitotic count, and Ki67 proliferation index were involved rather than other histomorphological parameters, but none of the parameters is an absolute predictor of the metastasis.

The immune microenvironment in hormone positive, HER2 negative breast cancer: A focus on tumour infiltrating lymphocytes, spatial phenotypes and tertiary lymphoid structures.

2522 Background: Hormone positive, HER 2 negative breast cancer (ER+/HER2-) has traditionally been considered an immune cold tumour subtype. Research to date indicates that TILs in ER+/HER2- breast cancer may be a negative prognostic marker. However, there is a paucity of data in this area regarding immune subpopulations and spatial phenotypes. Tertiary lymphoid structures (TLS) have been associated with improved prognosis and response to immunotherapy in other solid organ malignancies. We aim to explore the immune microenvironment in early stage ER+/HER2- breast cancer with a focus on tumour infiltrating lymphocytes, spatial phenotypes and tertiary lymphoid structures (TLS). Methods: Full face sections of archival tissue samples from patients with early stage ER+/HER2- breast cancer were stained with CD45 antibody (DAKA clone M0701) via the automated Leica Bond III system. Open source digital pathology analysis software, Qupath, was utilised to identify positive cells and calculate the percentage of positive CD45 staining cells (CD45%) within the tumour area. Spatial analysis was done to divide tumour area into three spatial phenotypes; Immune cold -lacking immune infiltrate;Immune excluded- immune cells restricted to the tumour periphery and immune hot - immune cells infiltrating the tumour core. A tissue micro array (TMA) was created and stained manually with a multiplex chromogenic immunohistochemistry panel for CD20/CD3/DCLamp to identify immune subpopulations associated with tertiary lymphoid structures. Results: A total of 409 patient samples were included in the analysis. 89% (n=367) of samples had a CD45% of 0-10%. CD45% stratification by 21-gene-recurrence score(RS) into Low, Intermediate and High showed a statistically higher CD45% in those with a High RS compared to Low RS (p value= 0.0015). We identified a trend towards worse disease free survival (DFS) in the CD45%high group which did not reach statistical significance. The immune hot spatial phenotype was associated with a worse DFS (p value = 0.034) with no statistically significant difference in DFS between immune cold and immune excluded phenotypes. The majority of samples had minimal immune infiltrate. Staining with a TLS panel revealed that samples with a significant infiltrate could broadly be divided into T cell only infiltrate or those with the presence of TLS, the significance of this is unclear. Conclusions: The majority of ER+/HER2- breast cancer samples had low numbers of TILs, however there is clear heterogeneity within this subgroup. Spatial elements and immune cell subpopulations may further guide the value of TILs in prognosis in this breast cancer subtype.

MIOEPITHELIAL CELLS: STRUCTURE, FUNCTION, AND THEIR ROLE IN NORMAL AND NEOPLASTIC TRANSFORMATION OF THE BREAST

Abstract. Myoepithelial cells (MECs) are characteristic of exocrine glands and are responsible for synthesizing the extracellular matrix, particularly the structural components of the basal membrane. MECs are of epithelial origin and exhibit contractile function, which aids in milk ejection from the ducts during lactation. Scientific literature suggests that MECs act as suppressors of tumors and play an important diagnostic role in mammary gland pathology, especially in distinguishing diff erent neoplastic processes.Research objective: T o elucidate the histological features and immunohistochemical verifi cation of myoepithelial cells in normal breast tissue and their role in neoplastic transformation of the mammary gland.Materials and Methods. The histological examination of myoepithelial cells of the mammary gland was conducted on archival slides from the Department of Histology, Cytology, and Embryology, Danylo Halytsky Lviv National Medical University. Histological sections, stained with hematoxylin and eosin, as well as immunohistochemical markers p63 (Clone 4A4, Masterdiagnostica) and cytokeratin 5/6Ab-2 (D5/16 B4, Thermoscientifi c) for confi rming the myoepithelial origin of the cells, were analyzed under a light microscope (UlabXSP-137TLED) with 10x and 40x objectives and a 10x eyepiece. Photographs were taken using an XCAM 1080P camera.Research Results. The structural units of the mammary gland (MG) are ductal-l obular formations, the terminal portions of which have an alveolar shape and are formed by secretory exocrine cells with underlying myoepithelial cells – basket-like cells. Identifi cation of myoepithelial cells using conventional histological staining techniques such as hematoxylin and eosin does not always provide a clear understanding of their topography. All myoepithelial cells are located on the basal membrane and contribute to the synthesis of its components (laminin, entactin, etc.). The myoepithelial cell layer, which is consistently present in the normal mammary gland, can appear diff erently: resembling smooth muscle or epithelioid cells, having a stellate or spindle- shaped form, or not being visualized at all. It has been shown that p63 is a sensitive and specifi c nuclear myoepithelial marker, expressed in myoepithelial cells of the normal mammary gland, in residual lobular acini, sclerosing adenosis, carcinoma in situ, and not detected in invasive carcinomas. It can be included in immunohistochemical panels aimed at identifying myoepithelial cells in diagnostically challenging breast pathologies.Conclusions. Myoepithelial cells in the mammary glands play a key role in their function, facilitating the process of milk secretion. However, in certain cases, they also play an important role in the development of neoplastic transformations. Intercellular connections between secretory and myoepithelial cells indicate that the last ones suppress both the progression of carcinoma in situ to invasive breast cancer and the carcinomainduced angiogenesis. The use of the immunohistochemical marker p63 is mandatory in the panel of antibodies to identify myoepithelial cells in the mammary gland and diff erentiate between benign and malignant lesions.

Endometrium Kanseri Alt Tiplerinde Mikrosatellit İnstabilite (MSI) ve p16/p53 Gen Durumu: Tümör Saldırganlığı ile İlişkisi

Aim: Microsatellite instability (MSI) has recently emerged as one of the most important pathways in endometrial carcinogenesis, especially in endometrioid subtype (type I). However, non-endometrioid subtypes, like serous, papillary or mucinous tumors, have long been known to overexpress p16 and/or p53 genes. In the present study, we investigated the immunohistochemical (IHC) panel in all subtypes of endometrial cancer (EC), and correlated the results with nuclear mitosis index (Ki-67) as a marker of tumoral mitosis rate. Methods: Medical records of patients admitted with EC and underwent surgery between 2010 and 2022 were reviewed. IHC panel results of estrogen and progesterone receptors (ER, PR), mismatch repair (MMR) proteins MLH1, PMS2, MSH2, MSH6, Ki-67 and p16/p53 status were recorded. Chi square test was used for statistical analysis. Results: Total of 44 female patients (with pathology reports including all of IHC panel markers) were included. Mean age was 64.1±12.51 years. Type I EC was the most common pathology (72%). ER and PR positivity were very prominent in type I tumors in comparison with non-endometrioid (type II) tumors (84% vs 16%, respectively; p