Positive Immunohistochemistry Research Articles

Abstract A007: Circulating tumor DNA and tissue staining analyses reveal heterogeneous ERBB2/HER2 status in urothelial cancer

Abstract Human epidermal growth factor receptor 2 (HER2) is encoded by the ERBB2 gene, and frequently mutated, amplified, and/or overexpressed in urothelial cancer (UC). Promising antibody-drug conjugates have led to new interest in HER2 as a UC clinical target. Patient selection for HER2-targeted therapy typically relies on immunohistochemistry (IHC) and fluorescence in-situ hybridization (FISH) of tumor tissue, however DNA sequencing can identify ERBB2 genomic alterations. Accurate biomarker-driven patient selection will be critical to optimize the clinical benefit of HER2-targeted therapy. Therefore, we evaluated plasma circulating tumor DNA (ctDNA) for ERBB2/HER2 status determination in UC, as compared to IHC and FISH of metachronous tissue. 411 plasma samples from 236 metastatic UC patients were profiled with targeted sequencing across >50 genes frequently altered in UC, including dense coverage flanking the ERBB2 locus. Using an established bioinformatics workflow, we determined the presence of ERBB2 alterations (mutations, amplifications, and/or structural variants) in the 181 patients with evidence of ctDNA in ≥1 sample. Clinical records were reviewed for the availability of archival tumor tissue; in total, 81 formalin-fixed paraffin-embedded tissue specimens were retrieved from 23 patients with ctDNA ERBB2 alterations and 20 ERBB2-wildtype controls. HER2 IHC was performed with a polyclonal rabbit anti-human Her2 antibody (Dako) and scored according to gastric cancer guidelines. HER2 FISH was performed with the PathVysion HER-2 DNA Probe Kit. Protein-altering ERBB2 mutations were identified in 14% of evaluable patients, with two-thirds at known oncogenic hotspots. ERBB2 copy gain was detected in 8% of patients overall, 9% when excluding low tumor fraction samples. IHC results were assessable for 82 tissue samples from 43 patients, and 33 patients had at least one sample with positive HER2 staining (2+/3+). IHC scores varied in 16/23 patients with ≥2 tissue samples, with the variation leading to a change in classification (HER2-negative [0/1+] versus positive [2+/3+]) in half (8/16). In two patients with mixed variant histology, the urothelial component showed IHC 3+ while the scores were 2+ (plasmacytoid) and 0 (squamous) in the variant regions. Frequent focal staining patterns were observed with both IHC and FISH. ERBB2 alterations in ctDNA were correlated with IHC positivity – when considering the most recent tissue sample per patient, 79% of ctDNA ERBB2-altered patient’s tumors were also positive by IHC. Conversely, 55% of ctDNA ERBB2-wildtype cases were positive by IHC. FISH results were available from 24 samples (20 positive, 4 negative), all from patients with ctDNA ERBB2 amplification. 18/20 samples with FISH positivity were HER2 IHC 2+/3+; the remaining two samples were IHC 1+. Our results demonstrate significant spatial and/or temporal heterogeneity in ERBB2/HER2 status, with implications for the rational implementation of biomarker-directed (HER2-targeted) therapy in UC. Citation Format: Gillian Vandekerkhove, Andrew J. Murtha, David C. Müller, Kimia Rostin, Carlos Vasquez-Rios, Jussi Nikkola, Maria Stephenson, Emily Fung, Jaskirat Atwal, Karan Parekh, Cecily Q. Bernales, Gráinne Donnellan, Gang Wang, Tilman Todenhöfer, Piet Ost, Peter C. Black, Kim N. Chi, Bernhard J. Eigl, Alexander W. Wyatt. Circulating tumor DNA and tissue staining analyses reveal heterogeneous ERBB2/HER2 status in urothelial cancer [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr A007.

High-Risk HPV CISH Detection in Cervical Biopsies with Weak and/or Focal p16 Immunohistochemical Positivity.

In cervical biopsies, for diagnosis of Human Papilloma Virus (HPV) related conditions, the immunohistochemical staining for p16 has a diagnostic value only if diffusely and strongly positive, pattern named "block-like". "Weak and/or focal (w/f) p16 expression" is commonly considered nonspecific. In our previous study, we demonstrated the presence of high-risk HPV (hrHPV) DNA by LiPa method in biopsies showing w/f p16 positivity. The aim of the present study was to investigate the presence of hrHPV-DNA by CISH in the areas showing w/f p16 expression. We assessed the presence of hrHPV16, 18, 31, 33, 51 by CISH in a group of 20 cervical biopsies showing w/f p16 expression, some with increased Ki67, and in 10 cases of block-like expression, employed as control. The immunohistochemical p16 expression was also assessed by digital pathology. hrHPV-CISH nuclear positivity was encountered in 12/20 cases of w/f p16 expression (60%). Different patterns of nuclear positivity were identified, classified as punctate, diffuse and mixed, with different epithelial distributions. Our results, albeit in a limited casuistry, show the presence of HPV in an integrated status highlighted by CISH in w/f p16 positive cases. This could suggest the necessity of a careful follow-up of the patients with "weak" and/or "focal" immunohistochemical patterns of p16, mainly in cases of increased Ki67 cell proliferation index, supplemented with molecular biology examinations.

Relative Value of Immunohistochemistry in Detection of Mycobacterial Antigen in Suspected Cases of Tuberculosis in Tissue Section.

Due to its infectious nature, complex immunological response, chronic progression, and the necessity for long-term treatment, tuberculosis has always been a major health burden. Immunohistochemistry (IHC) has the capacity to highlight the occurrence of mycobacterial antigens for tissue diagnosis. This study was conducted to understand the advantage of immunostaining over culture of Mycobacterium tuberculosis. A cross-sectional study was conducted on 30 samples of suspected cases of tuberculosis. Specimens received were fixed in 10% formalin and processed; 3-5 µm thick sections were made from paraffin block, stained with hematoxylin and eosin, Ziehl-Neelsen stain, and immunohistochemistry. Culture was done using Lowenstein-Jensen medium. Immunohistochemistry was interpreted as fine granular brownish cytoplasmic, coarse granular brownish cytoplasmic, and bacillus staining. Out of the 30 samples studied, 12 (40.0%) were culture positive while 20 (66.7%) of them were IHC positive. Immunohistochemistry showed 17 granulomatous lesions of which 11 (55.0%) were well-formed granulomas. The sensitivity and negative predictive value were found to be high with immunohistochemistry, while specificity and positive predictive value were found to be on the lower side. Among the 20 positive IHC cases, the degree of staining was fine granular cytoplasmic staining in 13 cases (65.0%) and coarse granular staining in 7 cases (35.0%). Immunohistochemistry is a reliable test with high sensitivity as well as high negative predictive value which can be done rapidly for establishing an etiological diagnosis of tuberculosis in histologic specimens.

Abstract PO3-15-07: False-negative results of hyperexpression of HER2 receptor in breast cancer at a public tertiary hospital in Brazil

Abstract Introduction: Breast cancer (BC) is the second leading cause of cancer death in women worldwide. One of the major advances in BC management was its molecular classification, especially regarding the Human Epidermal growth factor Receptor 2 (HER2). HER2 positive tumors are at greater risk of visceral metastasis and are associated with worse survival rates. Thus, the use of specific drugs to target this pathway is essential and false-negative have a high impact on patient care. HER2 status is usually accessed using immunohistochemistry (IHC), but false-negatives results occur. When IHC results are inconclusive, in-situ hybridization (ISH) testing is necessary. Objective: to determine if there is variability between the positivity rates between laboratories that performed the ISH, according to the IHQ, and to evaluate if there are false-negative results and possible prognostic impact. Methods: retrospective, observational study in a public hospital in the city of Curitiba (Brazil), followed by cross-sectional analysis of histological samples. All cases of BC that underwent HER2 assessment by IHC and ISH between January 2008 and December 2018 were included. Participants classified as HER2 negative by IHC and patients whose medical records were not available were excluded. A new analysis of all IHC slides and of cases with negative ISH was performed. Results: We identified 205 people with an average age of 53.52 (± 11.89) years. Most cases were classified as clinical stage I and II (68.3%), and the most prevalent histological features were Invasive Ductal Carcinoma (82.4%) and luminal molecular subtype (49.8%). Initial ISH testing was performed by four laboratories. There was a significant difference in HER2 positivity rates between these laboratories, even after individual reanalysis of all IHC slides. Of the 114 cases with negative ISH, it was possible to obtain histological material in 82 of them to perform a new standardized ISH. The false-negative rate for HER2 in this 82 patients was 41.46%, with 10 cases out of 24 for positive IHC (3+) and 22 out of 58 for inconclusive IHC (2+). In this paper, false-negative results did not impact in survival, most likely to differences between groups. Conclusion: our work showed different rates of positivity for HER2 among laboratories that performed confirmatory test with ISH. The performance of a second standardized ISH proved that the difference between the laboratories was due to false-negative results. There was no difference in recurrence and cancer-specific survival in this false-negative sample. Keywords: breast cancer. Biomarkers. Health technology assessment. HER2 receptor. DIfferences between laboratories reagarding HER2 false negatives –insert figrue image– Citation Format: Bruno Batista, Sergio Ioshii, Sergio Padilha, Jacqueline Nabhen, Tayza Ostroski, Caroline Batista. False-negative results of hyperexpression of HER2 receptor in breast cancer at a public tertiary hospital in Brazil [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-15-07.

Abstract PO2-21-01: Granular Cell Tumors of the Breast: A Case Series

Abstract Objective: Granular cell tumors (GCT) of the breast are rare, Schwann-cell derived tumors that are largely benign in nature, but may present with malignant characteristics in 1-2% of cases. There are few reports highlighting the clinical presentation, cytologic differences, operative management and clinical outcomes of patients with these benign versus malignant GCTs. Here, the authors report the clinical course of 9 patients identified with either benign or malignant GCT of the breast. Methods: The medical records for a total of 9 patients treated for histologically confirmed GCT of the breast between March 2015 and May 2023 at our institution were retrospectively reviewed. Clinical data, imaging findings, immunohistochemical markers, surgical interventions, and clinical follow-up data were collected and analyzed. Results: All patients were female, with a mean age of 46 years (Range: 29-61). 5 (56%) patients presented with left-sided lesions with an average maximum dimension of 1.1 cm (SD: 0.7 cm). All 9 (100%) tumors were positive for S100, 4 (44%) were positive for CD68, and 3 (33%) were positive for Inhibin. 4 (44%) patients underwent local mass excision, 3 (33%) underwent lumpectomy, 1 (11%) underwent lumpectomy with sentinel lymph node biopsy, and 1 (11%) underwent partial mastectomy with sentinel lymph node biopsy. There were no perioperative complications nor complications noted at postoperative follow up visits. 1 patient (11%) had 2 recorded recurrences of GCT after the initial breast mass excision, with each tumor exhibiting different positive immunohistochemical markers. 1 (11%) patient had a previous history of breast cancer at time of GCT diagnosis, and 1 (11%) patient had concurrent breast malignancy with the granular cell tumor being in the lymph node of the same breast. Conclusions: Granular cell tumors of the breast are rare Schwann cell derived tumors that often present near breast scar tissue as spiculated, ill-defined masses with microcalcifications on radiologic imaging, mimicking breast malignancy, leading to overtreatment. This study found that for GCTBs that present in lymph nodes, regardless of low malignant potential seen histologically, there was a higher risk of recurrence, and may require further workup and close follow up after wide local excision. For all other granular cell tumors presenting with normal appearing lymph nodes on imaging, a local mass excision was adequate in management, with low risk of recurrence or malignant potential. Future studies should be performed analyzing long-term follow up for these patients to determine risk of recurrence over longer periods of time, and risk for breast malignancy. Granular Cell Tumor Characteristics Tumor characteristics including laterality, tumor location, maximum dimension in centimeters, and positive immunohistochemistry markers were shown for each patient. Citation Format: Shwetha Kochi, Madeline Karsten, Monika Masanam, Teagan Thorson, Roshanak Derakhshandeh, Patricia Wehner, Lucy De La Cruz, Jennifer Son. Granular Cell Tumors of the Breast: A Case Series [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-21-01.

Abstract 1032: Development of a novel companion diagnostic immunohistochemistry antibody for claudin 18.2-targeted therapies

Abstract Background: CLDN18.2, the isoform 2 of CLDN18, is physiologically confined to gastric mucosa tight junctions, the epitopes of which would be exposed on the cancer cell surface upon malignant transformation and is highly expressed in a significant proportion of cancers, e.g., gastric and pancreatic adenocarcinomas, which makes it a promising drug target for cancer therapy. Currently, multiple drug modalities, such as monoclonal antibodies, ADCs, and CARTs targeting CLDN18.2 are being evaluated in clinical trials. A substantial correlation exists between the expression of CLDN18.2 and the efficacy of the these therapies. However, to date, no companion diagnostic (CDx) antibodies that are specific to CLDN18.2 have been approved. Therefore, it is important to promptly develop an anti-CLDN18.2 CDx antibody with a high specificity, sensitivity, and suitability for immunohistochemistry (IHC). In this study, we present the discovery and validation of a novel, highly sensitive IHC antibody that selectively identifies CLDN18.2. Methods: 3 CLDN18.2 specific peptide fragments from extracellular domain of human CLDN18.2 were conjugated with KLH. Antibodies were discovered by immunizing BALB/c and SJL mice with these peptides-KLH conjugates. Screening and selection process was relied on positive ELISA results against the immunogen, as well as FACS and Acumen Explorer Microplate staining of CLDN18.2-experssing cells. Hit antibodies were evaluated for IHC on formalin-fixed paraffin-embedded CHOK1 cells expressing full-length CLDN18.2 or CLDN18.1. The antibodies were further validated on human gastric cancer tissue samples to establish the analytical sensitivity and specificity of the IHC assay. Results: The mAb clone 43F11 exhibited binding to a fusion polypeptide comprising the first extracellular loop of human CLDN18.2 (aa32-53) at a half maximal effective concentration (EC50) of 0.212 nM. 43F11 showed positive cell surface IHC staining on CLDN18.2-experssing cells after fixation but demonstrated no staining on CLDN18.1-experssing cells. The IHC test method was established by utilizing clinical tumor tissues and PDX samples that had predetermined expression levels of CLDN18.2. The 43F11 antibody accurately identified the expression level of CLDN18.2 in the IHC assay, with a minimum concentration of 0.25 μg/mL. When compared to the commercial CLDN18.2 IHC antibody EPR19202, 43F11 demonstrated greater sensitivity, allowing for positive staining on cancer tissues that have significantly lower levels of CLDN18.2 expression. Conclusion: 43F11 exhibits specific recognition of human CLDN18.2 in IHC assays, while showing no reactivity towards CLDN18.1. It demonstrated superior sensitivity compared to the benchmark antibody. These data suggest that 43F11 has the capacity to serve as an effective diagnostic tool for identifying patients who positively express CLDN18.2. Citation Format: Jishun Chen, Suya Bai, Yu Bai, Zaoshun Hu, Peng Chen, Jay Mei, Bo Shan, Bing Hou. Development of a novel companion diagnostic immunohistochemistry antibody for claudin 18.2-targeted therapies [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 1032.

Abstract 4808: Small intestinal leiomyosarcoma in a young adult

Abstract Objectives: • Outline the epidemiology of leiomyosarcoma•Identify the immunohistochemistry of a leiomyosarcoma Introduction: Primary small intestinal malignancies are rare malignancies as compared with other malignancies of the gastrointestinal tract. Here, we present a case of a jejunal leiomyosarcoma in a young adult. Case Presentation: A 23-year-old Caucasian male with GERD presented with five months of abdominal pain, intermittent nausea with non-bloody emesis, and a 20-pound weight loss. Presenting vital signs were within normal limits. Physical exam revealed conjunctival pallor with epigastric tenderness to palpation. Serology demonstrated iron deficiency anemia, hemoccult positive stool, negative CEA, CA19-9, and AFP tumor markers. HIV antigen antibody and EBV-PCR were non-reactive. CT of the abdomen and pelvis revealed markedly thickened heterogenous jejunal loop in the left hemiabdomen with associated mesenteric adenopathy and small volume pelvic ascites. EGD with push enteroscopy revealed a single, malignant appearing and ulcerated mass in the proximal jejunum. PET-CT showed a hypermetabolic, multilobulated, ill-defined mass involving multiple loops of jejunum. He underwent exploratory laparotomy with resection of a proximal jejunal mass (10x8 x7 cm), mesenteric lymphadenectomy, and a jejunojejunostomy creation. Pathology demonstrated leiomyosarcoma (pT2aN0MoG3), and he was initiated on cyclophosphamide and doxorubicin. Discussion: Primary small intestinal malignancies are rare malignancies with an incidence of less than 5% of all GI malignancies. 56 cases of GI leiomyosarcoma in adults were identified between 2000-2012 with a peak incidence between 55-59 years of age (Aggarrwal 2012). Leiomyosarcomas are soft tissue sarcomas arising from bones, tendons, blood vessels, or muscles with a spectrum of disease ranging from low-grade cutaneous lesions to aggressive lesions of internal organs with significant potential for metastasis (Pipe 2002). Diagnosis is based on immunohistochemical positivity for SMA, desmin, and h-caldesmon, and negativity for CD117, CD34, and DOG1.1, notably lacking KIT and PDGFRA mutations (Aggarwal 2012). Treatment involves surgical resection and a cost benefit analysis of adjuvant chemotherapy with (Reynolds, 2014). This case of a 23 year old, without significant risk factors, diagnosed with a jejunal leiomyosarcoma presents as one of the youngest cases ever to be reported and emphasizes the importance of recognizing the cancer in a broader range of the adult population. Citation Format: Alyssa A. Guo, Blake H. Bentley, Abigail L. Ellington, Haiyan Lu, William C. Lippert. Small intestinal leiomyosarcoma in a young adult [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 4808.

Gingival squamous cell carcinoma in 2 lions under managed care.

Neoplasia is one of the main causes of euthanasia in geriatric captive nondomestic felids. However, few studies have examined oral tumors in these animals. We describe here the clinicopathologic features of gingival squamous cell carcinoma (SCC) in 2 lions (Panthera leo) from separate zoologic collections. In both cases, the lions had a history of sialorrhea, bloody oral discharge, and anorexia. Autopsy findings in both lions were similar and were characterized by poorly circumscribed, friable, and bloody gingival masses with grossly apparent invasion of the mandibular bone; a pathologic fracture was observed in 1 case. Histologically, the masses consisted of poorly circumscribed, unencapsulated, densely cellular proliferations of neoplastic epithelial cells arranged in irregular islands, cords, and anastomosing trabeculae with formation of keratin pearls, which, coupled with positive immunohistochemistry for pancytokeratin, were diagnostic for SCC. Although no metastases were found in either animal, both lions were ultimately euthanized because of poor prognosis.

Alteration of Neurotrophic Factors and Innervation in Extraocular Muscles of Individuals With Concomitant Esotropia.

To determine whether neurotrophic factors and innervation in extraocular muscles (EOMs) were altered in different types of concomitant esotropia, and to explore the possible association between neurotrophic factors and innervation of EOMs in humans. Patients with concomitant esotropia who required strabismus surgery were recruited from January to December 2022. Lateral rectus EOMs were obtained from patients, and controls were obtained from deceased organ donors. Immunofluorescence (IF) was performed to detect innervation of EOMs (neurofilament and synaptophysin), and immunohistochemistry (IHC) was used to detect the neurotrophic factors insulin-like growth factor-1 (IGF-1), brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), and neurotrophin-3 (NT-3). The positive IHC results were further verified using western blotting (WB). One-way ANOVA followed by a Dunnett's multiple comparison post hoc test was used for continuous variables and the χ2 test for categorical variables. Spearman correlation analysis was used for the correlation analysis. We collected lateral rectus EOM samples from acute and chronic types of concomitant esotropia and controls. Consistent with IHC, WB showed that IGF-1 was significantly increased in patients with acute acquired comitant esotropia or essential infantile esotropia compared with controls. In IF, synaptophysins were significantly increased only in acute acquired comitant esotropia compared with controls. Furthermore, Spearman correlation analysis showed that the correlation between IGF-1 and synaptophysin was borderline (P = 0.057) for patients with acute acquired comitant esotropia. Our study highlights the role of IGF-1 and altered innervation of EOMs in acute acquired comitant esotropia, suggesting that an effect of increased IGF-1 on nerve innervation may temporarily cause a compensatory increase in the strength of lateral rectus muscles.

High serum alpha-fetoprotein and positive immunohistochemistry of alpha-fetoprotein are related to poor prognosis of gastric cancer with liver metastasis

Liver metastasis in gastric cancer is incurable. Alpha-fetoprotein-producing gastric cancer has a poor prognosis and is prone to liver metastasis. We investigated the association between preoperative serum alpha-fetoprotein levels, liver metastasis, and expression of primitive enterocyte phenotype markers. We reviewed the medical records of 401 patients with gastric cancer who underwent curative surgical resection and immunohistochemically evaluated the primitive phenotype markers. The preoperative serum alpha-fetoprotein levels were elevated and normal in 8 and 393 patients, respectively. Liver metastasis was more frequent in patients with higher preoperative alpha-fetoprotein levels. The 5-year postoperative recurrence-free survival and overall survival rates were significantly worse in patients with higher preoperative serum alpha-fetoprotein levels. Although alpha-fetoprotein and Glypican3 and Spalt-like transcription factor 4 tended to be stained with high preoperative serum alpha-fetoprotein levels, these markers were also positive in some patients with normal alpha-fetoprotein levels. In summary, patients with gastric cancer and high preoperative serum alpha-fetoprotein levels have a poor prognosis and high incidence of liver metastasis. Alpha-fetoprotein can help detect liver metastasis relating to the primitive enterocyte phenotype.

Spatial expression of claudin 18.2 in matched primaries and metastases of tubo-ovarian carcinoma of all subtypes.

Physiologically, claudin 18 splice variant 2 (CLDN18.2) expression is restricted to the gastric epithelium, but its expression has been detected in solid cancers. Zolbetuximab, a chimeric IgG1 antibody targeting CLDN18.2, has demonstrated promising effects in patients suffering from CLDN18.2-positive, HER2-negative locally advanced gastric cancer and is currently being studied further. To date, little is known about CLDN18.2 expression in other histological subtypes of tubo-ovarian carcinoma (TOC) and their matching metastases.Using a cohort of all histological TOC subtypes, we investigated the immunohistochemical (IHC) CLDN18.2 expression in both TOCs (n = 536), their matching metastatic tissue (n = 385) and in 93 metastases without primary. Tissue microarrays comprised both the tumor center and periphery. IHC positivity was defined as biomarker expression of ≥ 75% in tumor cells with moderate-to-strong membranous staining.Overall CLDN18.2 positivity was 4.1% (21/515) in the TOC centers and 3.6% (18/498) in their peripheries. In primaries of mucinous tubo-ovarian carcinoma (MTOC), CLDN18.2 positivity rates were 45% (18/40) and 36.6% (15/41), respectively. Positivity rates for the corresponding metastases were 33% (4/12, center) and 27% (3/11, periphery). The expression was relatively homogenous throughout all tumor sites. With no expression in 99.5% of nonmucinous tumors, CLDN18.2 positivity was almost exclusively seen in the mucinous subtype.In tubo-ovarian carcinoma, CLDN18.2 expression was, with rare exceptions, restricted to the mucinous subtype. Among them, 33% of metastasized MTOCs presented with CLDN18.2 positivity. Hence, CLDN18.2 might display a promising target for personalized therapy in patients with advanced MTOC.

Comparison of S100A8 and PRAME as biomarkers fordistinguishing melanoma from melanocytic naevus: acase-control analysis.

S100A8 is a melanoma biomarker expressed in the melanoma-associated epidermal keratinocytes, but its diagnostic utility has not been compared with other biomarkers, including PRAME. To compare the utility of S100A8 and PRAME immunohistochemistry (IHC) in the differential diagnosis of melanoma and naevi in a case-control study. A previously described cohort of 209 melanomas (case samples) and naevi (control samples) dual-immunostained for S100A8 and PRAME were included. For S100A8, previously reported scores indicating the proportion of tumour-associated epidermis stained (0 = indeterminate; 1 = 0-4%; 2 = 5-25%; 3 = 26-50%; 4 = 51-75%; 5 = > 75%) were utilized. PRAME IHC was reviewed by at least two reviewers and a consensus score assigned, with score indicating the proportion of tumour stained (0 = indeterminate; 1 = 0%; 2 = 1-50%; 3 = > 50%). A positive test was defined as > 50% staining. The area under the receiver operating characteristic curves for S100A8 (0.833) and PRAME (0.874) were not significantly different from each other (P = 0.22). The diagnostic sensitivity and specificity were 42.4% [95% confidence interval (CI) 32.6-52.8%] and 98.2% (95% CI 93.6-99.8%) for S100A8, and 79.8% (95% CI 70.5-87.2%) and 87.3% (95% CI 79.6-92.9%) for PRAME, respectively. A combined test requiring both S100A8 and PRAME IHC positivity had a sensitivity of 39.4% (95% CI 29.7-49.7%) and specificity of 99.1% (95% CI 95.0-100.0%). S100A8 and PRAME have utility in the diagnostic workup of melanoma, with S100A8 being more specific and PRAME being more sensitive when using this threshold. Our findings suggest that these two immunohistochemical markers may favourably complement one another to improve the detection of melanoma.

Prognostic value of O6-methylguanine-DNA methyltransferase (MGMT) status in pancreatic neuroendrocrine tumors with capecitabine and temozolomide (CAPTEM).

598 Background: Loss of MGMT expression has been suggested as a predictor of response to alkylating agents in pancreatic neuroendocrine tumors (pNET), however, its determination is controversial. We aim to analyze the prognostic value of MGMT status assessed by immunohistochemistry (IHC) or pyrosequencing (PSQ) in patients with pNET receiving CAPTEM. Methods: Retrospective and unicentric analysis in patients (p) with pNET treated with CAPTEM from April 2008 to February 2023. MGMTdeficiency was determined by IHC and MGMT promoter methylation by PSQ. We analyzed patients’ characteristics, progression-free survival (PFS) and overall survival (OS) according to MGMT status. Results: 20p were included. Age (median): 62 [46-77]. Males (45%). Tumor grade (G): G1 (15%), G2 (55%) and G3 (20%). MGMT deficiency was detected in 12p (60%) and promoter methylation in 10p (50%). Results by IHC were consistent with PSQ in 10p (50%), with mismatches in 4p (methylated MGMT and positive IHC) and 6p (unmethylated MGMT and negative IHC). We reported a higher response rate in MGMT-deficient and MGMT-methylated p (Table) . In MGMT-deficient p we observed significantly better PFS (88m vs 11.1m; p=0.02, HR=0.21) and OS (141.9m vs 35.8m; p=0.03, HR=0.20). Also, in MGMT-methylated p we detected better PFS (88m vs 17.7m; p=0.20, HR=0.44) and OS (188.7m vs 141.9m; p=0.11, HR=0.28). A subanalysis was performed based on G, showing better PFS (88m vs 6.2m; p=0.03, HR=0.11) and OS (188.7m vs 35.8m; p<0.01, HR=0.06) in those with G2 vs G3. There was a strong negative correlation between G and MGMT by PSQ; the higher G, the lower probability of promoter methylation r(16)= -0.62, p<0.01. Conclusions: Our results support MGMT status, deficiency by IHC or promoter methylation by PSQ, as a potential prognostic biomarker in pNET treated with alkylating agents. However, it is necessary to evaluate its prognostic value in prospective studies. [Table: see text]

Evaluation of NTRK expression and fusions in a large cohort of early-stage lung cancer

Tropomyosin receptor kinases (TRK) are attractive targets for cancer therapy. As TRK-inhibitors are approved for all solid cancers with detectable fusions involving the Neurotrophic tyrosine receptor kinase (NTRK)-genes, there has been an increased interest in optimizing testing regimes. In this project, we wanted to find the prevalence of NTRK fusions in a cohort of various histopathological types of early-stage lung cancer in Norway and to investigate the association between TRK protein expression and specific histopathological types, including their molecular and epidemiological characteristics. We used immunohistochemistry (IHC) as a screening tool for TRK expression, and next-generation sequencing (NGS) and fluorescence in situ hybridization (FISH) as confirmatory tests for underlying NTRK-fusion. Among 940 cases, 43 (4.6%) had positive TRK IHC, but in none of these could a NTRK fusion be confirmed by NGS or FISH. IHC-positive cases showed various staining intensities and patterns including cytoplasmatic or nuclear staining. IHC-positivity was more common in squamous cell carcinoma (LUSC) (10.3%) and adenoid cystic carcinoma (40.0%), where the majority showed heterogeneous staining intensity. In comparison, only 1.1% of the adenocarcinomas were positive. IHC-positivity was also more common in men, but this association could be explained by the dominance of LUSC in TRK IHC-positive cases. Protein expression was not associated with differences in time to relapse or overall survival. Our study indicates that NTRK fusion is rare in early-stage lung cancer. Due to the high level of false positive cases with IHC, Pan-TRK IHC is less suited as a screening tool for NTRK-fusions in LUSC and adenoid cystic carcinoma.

TLE1 Expression in NUT Carcinoma: A Case Report Highlighting a Potential Diagnostic Pitfall for the Pathologist.

NUT carcinoma is a rare, aggressive malignancy defined as a carcinoma with a chromosomal rearrangement affecting the nuclear protein in testis (NUTM1) gene. This small round blue cell tumor classically exhibits focal abrupt keratinization and immunohistochemical positivity for keratin and squamous markers. However, keratinization is not always present and reports of positivity for other markers that may obscure the diagnosis are increasing. It is also noteworthy that gene fusions involving NUTM1 are not restricted to NUT carcinoma. Herein, we report a NUT carcinoma arising in the mediastinum of a male patient in his 40 s with morphological and immunohistochemical overlap with Ewing family sarcoma and poorly differentiated synovial sarcoma given a round cell morphology, diffuse strong immunoreactivity for CD99, and patchy strong immunoreactivity for TLE1. Squamous differentiation by morphology and p40 expression were notably absent in this case. Classification as NUT carcinoma was ultimately possible when the morphological and immunohistochemical findings were considered in the context of a BRD4::NUTM1 gene fusion identified by next-generation sequencing. While the patient initially responded to palliative radiotherapy, he died approximately one month later. To our knowledge, this is the first report of TLE1 immunoreactivity in NUT carcinoma. This case highlights a potential diagnostic pitfall and emphasizes the need for molecular confirmation in equivocal situations.

RC48-ADC combined with tislelizumab as neoadjuvant treatment in patients with HER2-positive locally advanced muscle-invasive urothelial bladder cancer: a multi-center phase Ib/II study (HOPE-03).

Bladder cancer with high expression of human epidermal growth factor receptor-2 (HER2) is related to pathological malignancy and poor prognosis. The standard care for muscle-invasive urothelial bladder cancer (MIBC) is neoadjuvant chemotherapy (NAC) followed by radical cystectomy (RC) with pelvic lymph node dissection. For HER2-positive MIBC, the efficacy of cisplatin-based NAC is unsatisfactory, and adverse reactions are inevitable or even intolerable. New regimens with higher efficiency and lower toxicity need to be explored in the neoadjuvant setting for this population. HOPE-03 is a multi-center, open-label, single-arm, phase Ib/II study aiming to evaluate the safety and efficacy of RC48-ADC (disitamab vedotin (DV)), a humanized anti-HER2 antibody conjugated with monomethyl auristatin E, and tislelizumab (PD-1 antibody) as a novel neoadjuvant treatment combination in patients with HER2-positive locally advanced urothelial MIBC. Fifty-one patients with cT2-4bN0-3M0-1a pathology- and imaging-diagnosed HER2 positive (immunohistochemistry status 3+ or 2+ or 1+) MIBC were recruited. Of these patients, six were enrolled in the dose-escalation phase (three patients in the RC48-ADC 1.5 mg/kg group and three patients in the 2.0 mg/kg group), and 45 patients were enrolled in the phase II study (the expected recommended phase II dose for RC48-ADC was 2.0 mg/kg). Patients without disease progression received radical cystectomy or bladder-sparing therapies as their preference after neoadjuvant treatment. The primary endpoints were clinical complete remission rate (cCR rate; T0/Ta/Tis), pathological complete remission rate (pCR rate), and safety. The secondary endpoints were overall survival (OS), local recurrence-free survival (LRFS), distant metastasis-free survival (DMFS), and quality of life. The HOPE-03 trial provides a description of the safety profile of RC-48 and tislelizumab combination in the neoadjuvant treatment of HER2-positive locally advanced urothelial MIBC, and the efficacy is explored as well in this population. https://www.chictr.org.cn/showproj.html?proj=137111, identifier ChiCTR2200060153.

High-grade Solid Pseudopapillary Neoplasms of the Pancreas: Distinct Clinicopathological Malignant Features With Intriguing Gene Alterations through a Comparison With the Conventional Type.

Pancreatic solid pseudopapillary neoplasm (SPN) is a low-grade malignant neoplasm with a good prognosis. Clinically aggressive SPNs have rarely been reported but have not been analyzed in detail. In this study, we referred to this highly malignant type of SPN as high-grade SPN (HG-SPN) and compared its clinicopathological and genetic characteristics with conventional SPN (C-SPN) using immunohistochemistry and gene panel analyses. Five HG-SPNs and 15 C-SPNs were evaluated in this study. HG-SPNs share many pathologic characteristics: macroscopically, solid/cystic appearances, microscopically, pseudopapillary/pseudorosette pattern (100%), tumor cell loose cohesiveness (100%), thin/delicate vasculature (100%), tumor cell cytoplasmic vacuolization (100%), immunohistochemical positivity for β-catenin (nuclear expression) (100%), CD10 (80%), CD56 (80%), and vimentin (100%). Conversely, HG-SPNs showed distinct malignant features compared with C-SPNs: mean tumor size (11.7 vs. 2.9 cm, P <0.001); true necrosis (100% vs. 0%, P <0.001); high-grade nuclear atypia (100% vs. 0%, P <0.001); lymphatic and/or venous invasion (100% vs. 20%, P =0.004); mean mitotic count (4.38 vs. 0.05/high-power field, P <0.001); and mean Ki-67 labeling index (33.9% vs. 3.4%, P <0.001). All HG-SPN patients died of primary disease 3 to 36 months after surgery, while all C-SPN patients were alive without disease. Genetic studies have shown that all analyzed HG-SPNs have CTNNB1 mutations. Two HG-SPN cases showed RB1 mutations with altered immunohistochemical findings for RB1 and p16. Two HG-SPN cases had TP53 mutation and/or p53 overexpression. In conclusion, HG-SPNs show distinct malignant features and some genetic alterations that differ from C-SPNs, indicating the importance of differentiating between these 2 subtypes.

Spontaneously Regressing Retropharyngeal Neuroblastoma – A Rare Cause of Upper Airway Obstruction in a Neonate: A Case Report with Review of Literature

Abstract Background: Retropharyngeal masses may rarely cause persistent respiratory distress, stridor, and feeding difficulties in a neonate. We report a rare case of a primary cervical neuroblastoma presenting as a retropharyngeal mass, which underwent spontaneous regression. Clinical Description: A 29-day-old male baby was referred for recurrent vomiting since day 10 of life followed by severe difficulty in breathing since day 14 of life. At admission, the baby had stridor, cyanosis with respiratory failure, without any facial dysmorphism or cardiovascular involvement. Management and Outcome: After initial stabilization, investigations showed pneumonia in chest X-ray, with no evidence of sepsis. On intubation, a retropharyngeal bulge was noticed which was later confirmed by direct laryngoscopy. Later, computed tomography delineated the soft-tissue mass extending from C2–C5. Endoscopy with marsupialization of the tumor was done, and on histopathological examination, it was diagnosed as a small round blue cell tumor, suggestive of neuroblastoma, along with positive immunohistochemistry. According to the International Neuroblastoma Staging System, the tumor belonged to Stage I. As the baby had started improving clinically, further management by a multidisciplinary team was collectively decided to continue conservative observation. On follow-up, there was spontaneous regression in size of tumor with no recurrence of symptoms till the last follow-up at 11 months. Conclusion: Neuroblastoma, the most common solid tumor malignancy in children, may rarely have a primary cervical presentation, causing breathing and feeding difficulties. Depending on the course of symptoms, a strategy of observation and close monitoring by a multidisciplinary team can be taken, as such tumors may regress spontaneously.

Transcriptomic analysis of GITR and GITR ligand reveals cancer immune heterogeneity with implications for GITR targeting.

Glucocorticoid-induced tumor necrosis factor related protein (GITR) is a transmembrane protein expressed mostly on CD25+CD4+ regulatory T-cells (Tregs) and upregulated on all T-cells upon activation. It is a T-cell co-stimulatory receptor and has demonstrated promising anti-tumor activity in pre-clinical studies. To date, however, the efficacy of GITR agonism has been discouraging in clinical trials. This study explores GITR and GITR ligand (GITR-L) ribonucleic acid (RNA) expression in solid tumors in an attempt to delineate causes for variable responses to GITR agonists. RNA expression levels of 514 patients with a variety of cancer types were normalized to internal housekeeping gene profiles and ranked as percentiles. 99/514 patients (19.3%) had high GITR expression (defined as ≥ 75th percentile). Breast and lung cancer had the highest proportion of patients with high GITR expression (39% and 35%, respectively). The expression of concomitant high GITR and low-moderate GITR-L expression (defined as <75th percentile) was present in 31% and 30% of patients with breast and lung cancer respectively. High GITR expression also showed a significant independent association with high RNA expression of other immune modulator proteins, namely, PD-L1 immunohistochemistry (IHC) ≥1 (odds ratio (OR) 2.15, P=0.008), CTLA4 (OR=2.17, P=0.05) and OX40 high RNA expression (OR=2.64, P=0.001). Overall, these results suggest that breast and lung cancer have a high proportion of patients with a GITR and GITR-L RNA expression profile that merits further investigation in GITR agonism studies. The association of high GITR expression with high CTLA4 and OX40 RNA expression, as well as positive PD-L1 IHC, provides a rationale for a combination approach targeting these specific immune modulator proteins in patients whose tumors show such co-expression.

Periarticular histiocytic sarcoma in a French Bulldog

Histiocytic sarcoma is a neoplasm originating from the dendritic cell lineage and presents an aggressive biological behavior and poor prognosis due to its increased metastatic rate. It can be localized, such as the articular and periarticular forms, or disseminated to several organs. Histopathological examination associated with immunohistochemistry can lead to a definitive diagnosis. The treatment of choice is surgical excision, chemotherapy, radiotherapy, or combination. Here, we report a case of periarticular histiocytic sarcoma involving the left knee joint in a female French Bulldog. The animal showed signs of lameness, a mass in the left pelvic limb, and radiographic findings suggestive of neoplasia. After cytological examination suggesting malignant mesenchymal neoplasia, the limb was amputated, and histiocytic sarcoma was diagnosed by histopathological examination and positive immunohistochemistry for CD18 and IBA1. After amputation, multimodal chemotherapy was instituted, and the animal survived for nine months from the start of chemotherapy treatment. However, the patient presented metastasis to the lungs, right pelvic limb, and superficial inguinal and popliteal lymph nodes.