Tumor Staining Research Articles

Precise identification of bladder tumors utilizing mucoadhesive thiolated hollow mesoporous silica nanoparticles.

Non-muscle invasive bladder cancer (NMIBC) poses significant challenges due to its high recurrence rates and the difficulty in accurately distinguishing tumor lesions. Effective and economical methods for identifying cancerous tissues are urgently needed. In this study, we employed thiolated hollow mesoporous silica nanoparticles loaded with Evans blue (EB@HMSN(E)-SH), a traditional tumor staining dye, in conjunction with white light cystoscopy (WLC) to enhance the detection of bladder tumors. We observed that EB@HMSN(E)-SH exhibited mucoadhesive properties, demonstrating significant aggregation upon interaction with mucin, as assessed by the mucin-particle method using Dynamic Light Scattering (DLS). The permeation-enhancing capability of EB@HMSN(E)-SH was evaluated using tumor spheroid models. Despite repeated flushing, EB@HMSN(E)-SH adhered effectively to the mice bladder mucosa, aiding in the differentiation of tumor tissue from normal and inflammatory lesions, facilitated by the disordered structure of tumor tissue. Tissues stained with EB@HMSN(E)-SH showed co-localization with NBT-2 tumor cells expressing GFP, confirmed by confocal microscopy, which revealed deeper penetration of EB released from HMSN(E)-SH into bladder tumors compared to free EB. The combined use of WLC and EB@HMSN(E)-SH enabled precise identification of tumor-like tissues, corroborated by histopathological examination using H&E staining. The mucoadhesive properties and extended retention time of EB@HMSN(E)-SH complement WLC effectively in identifying NMIBC, suggesting its potential as a promising diagnostic tool.

Efficacy of Magnifying Texture and Color Enhancement Imaging With Indigo Carmine as an Alternative to Crystal Violet for Colorectal Tumors.

Pit pattern diagnosis using crystal violet staining for colorectal tumors is useful for qualitative and depth diagnosis. However, due to its reported carcinogenic potential, the use of crystal violet has been restricted. This study was aimed at investigating the efficacy of texture and color enhancement imaging (TXI) magnification in pit pattern diagnosis. This study enrolled consecutive patients with consent obtained and with colonic tumors indicated for magnifying endoscopy in which all modalities' images (magnified observation with crystal violet staining (CV), magnified white light observation with indigo carmine (IC-WLI), and magnified TXI observation with indigo carmine (IC-TXI)) were evaluable between July 2020 and January 2023. The visibility of the pit pattern using a 5-point Likert scale and its diagnostic accuracy were compared (IC-TXI vs. IC-WLI, reference: CV, by three experts). A total of 145 colorectal tumors from 145 patients were included. Visibility scores for the pit pattern were significantly higher with IC-TXI compared to IC-WLI (all three experts, p < 0.0001). The pit pattern match rate (Type II/III/IV/V) of IC-TXI compared to CV was also superior to IC-WLI (72.9% vs. 59.7%; p = 0.02). IC-TXI provided reasonably good and higher visibility and diagnostic accuracy than IC-WLI for pit pattern diagnosis of colorectal tumors compared to CV, suggesting it as an alternative to CV.

Tumor-infiltrating plasma cells are a prognostic factor in penile squamous cell carcinoma.

Penile cancer (PeCa) is a rare disease with poor prognosis in the metastatic stage. Neither effective adjuvant nor palliative therapeutic options are available. Research efforts in this field have so far failed to establish robust predictors of survival. To identify prognostic targets in PeCa, the current project focused on characterizing the tumor microenvironment (TME). A study cohort of 93 men with PeCa was used for the construction of a tissue microarray and immunohistochemical staining for CD3, CD4, CD8, CD20, CD56, CD138, FoxP3, and PD-L1. The quantity and spatial distribution of tumor-infiltrating immune cells were analyzed using digital image analysis. PD-L1 staining of tumor and immune cells was manually scored (combined positivity score (CPS)). T cells, T helper cells, cytotoxic T cells (CTLs), and regulatory T cells were detected in > 90% of PeCa and B cells in 88%, plasma cells in 85%, and NK cells in 23%. Approximately 50% of the PeCa samples were PD-L1 positive. In the univariate survival analysis, high PD-L1 CPS, plasma cells, CTLs, and B cells were significantly associated with favorable overall survival (OS), and the latter two with adverse recurrence-free survival. In multivariate analysis, plasma cells remained a significant factor for favorable OS (p = 0.04). In this study, the immune cells in the TME, especially plasma cells, were favorably associated with patient survival compared to other established prognostic factors in PeCa. Contemporarily, plasma cells have been discussed in the light of contributing to responses to modern immunotherapies. The results of this study support this notion.

CSIG-25. MAPK/ERK SIGNALING IN GLIOMAS MODULATES INTERFERON RESPONSES, PROMOTES TUMOR-MICROGLIA CROSSTALK, AND CONFERS SUSCEPTIBILITY TO ANTI-PD-1 AND ANTI-CTLA-4 IMMUNOTHERAPY

Abstract While immune checkpoint blockade (ICB) is not effective in unselected glioblastoma (GBM) patients, we previously established that MAPK/ERK signaling is associated with overall survival following anti-PD-1 and anti-CTLA-4 treatment in recurrent GBM. However, the causal relationship between MAPK/ERK signaling and susceptibility to ICB, as well as the mechanisms underlying this association, remain to be determined. We conducted in vivo kinome-wide CRISPR/Cas9 screenings in murine gliomas to identify key regulators of susceptibility to anti-PD-1 and CD8+ T-cell response and performed survival studies to validate the most relevant hits. Additionally, paired scRNA-seq with p-ERK staining, spatial transcriptomics, and ex-vivo slice culture of BRAFV600E mutant GBM tumors treated with BRAFi/MEKi were used to determine the causal relationship between MAPK signaling, tumor cell immunogenicity, and modulation of microglia phenotype. Our CRISPR/Cas9 screening results identified the MAPK pathway, particularly the RAF-MEK-ERK pathway, as the most critical modulator of glioma susceptibility to anti-tumoral immunity across all kinases. Survival studies showed that mice bearing high p-ERK gliomas exhibited prolonged survival with anti-PD-1 treatment, which was not seen when ERK phosphorylation diminished. MAPK activation in murine gliomas led to durable anti-tumoral immunity upon implant re-challenge, with memory T cell infiltration in long-term survivors. Elevated p-ERK in glioma cells was associated with interferon responses and antigen presentation. Moreover, in BRAFV600E human GBM cells with ERK1/2 knockout and in slice cultures of human BRAFV600E GBM tissue analyzed by spatial transcriptomics, we observed modulation of interferon responses by the MAPK/ERK pathway. Notably, in slice cultures from BRAFV600E mutant GBM, BRAFi/MEKi treatment disrupted the interaction between tumor cells and tumor-associated macrophages/microglia. In conclusion, the MAPK/ERK pathway is a critical driver of GBM susceptibility to anti-tumoral immunity, modulating the interferon responses and antigen-presenting machinery in glioma cells, as well as tumor cell interaction with the microenvironment.

Tumour assessment of ROR1 levels in various adult leukaemia and lymphoma types.

Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is a tumour target currently used for the development of novel therapeutic modalities, such as antibody-drug conjugates, chimeric antigen receptor T-cell therapies, and others. Success of these new drugs depends on the selection of relevant indications based on ROR1 tumour prevalence, staining heterogeneity, and subcellular localization, among other parameters. We investigated ROR1 immunophenotype using validated antibody clones for immunohistochemistry (IHC) and flow cytometry (FC), analyzing 292 tumour specimens from 7 haematological malignancies and triple negative breast cancer (TNBC) as a reference solid tumour indication. ROR1 prevalence varied significantly across distinct tumour types, showing 100% of ROR1 positivity in all chronic lymphocytic leukaemia (n = 48) and hairy cell leukaemia (n = 14) specimens analyzed via FC with ranges between 1.1-99.8% and 0.8-62.1%, respectively. Samples analysed via IHC showed ROR1 membrane/cytoplasmic positivity in 44% of mantle cell lymphoma tumour samples (n = 27; H-score range: 10-285 in positive cases); 30% in TNBC (n = 46; H-score range: 1-200); 15% in diffuse large B-cell lymphoma (n = 45; H-score: 40-250); and 11% in follicular lymphoma (n = 34; H-score: 2-300). Finally, all acute myeloid leukaemia (n = 52) and most T-cell non-Hodgkin lymphoma (n = 31/32) tested samples were negative for ROR1 via IHC. In conclusion, ROR1 shows a heterogeneous tumour cell expression profile across multiple leukaemias and lymphomas, making it a tumour target that would require different patient selection strategies to develop novel therapeutic modalities.

Is Immunohistochemical Galectin-3 Expression Associated with the Epithelial-Mesenchymal Transition in High- and Low-Grade Invasive Urothelial Carcinomas of the Bladder?

Background/Objectives: Bladder cancer, predominantly urothelial carcinoma, is an important malignancy of the urinary system. Despite the same histologic grade and stage, some patients seem to have a worse prognosis. In this context, the epithelial-mesenchymal transition (EMT), characterized by the loss of E-cadherin and gain of vimentin expression, is an important process in tumor progression. Galectin-3, a lactose-binding protein involved in various cellular processes, has been associated with increased tumor cell migration, invasion, and treatment resistance. Methods: In this study, 223 bladder cancer cases were examined, and E-cadherin, vimentin, and galectin-3 expression was evaluated by immunohistochemical staining in tumor budding areas and invasive components. These markers were also correlated with clinicopathological parameters, including tumor grade and stage. Results: The results indicated a significant decrease in E-cadherin expression and an increase in vimentin staining in higher-grade and higher-stage tumors, supporting EMT involvement. Galectin-3 expression was notably higher in T1 high-grade tumors but decreased in T2 stage tumors. Despite this, no significant correlation was found between galectin-3 and E-cadherin or vimentin, suggesting a complex role of galectin-3 in EMT. Conclusions: High galectin-3 expression in T1 high-grade tumors highlights its potential role in early tumor progression and as a therapeutic target. However, the decrease in its expression in advanced stages underscores the need for further research to understand its multifaceted involvement in bladder cancer. These findings suggest that while galectin-3 may contribute to the EMT and early tumor progression, its exact role and potential as a therapeutic target require more detailed investigation.

7550 Case Report On Concurrent Metastases To The Pituitary And Adrenal From A Primary Breast Cancer

Abstract Disclosure: M.V. David-santos: None. M.C. Isidro: None. Background: Metastatic Cancer to the pituitary gland is a rare condition accounting for 0.5% of all pituitary tumors. It rarely manifests clinically and is often found incidentally. Endocrine dysfunction usually arise, with 38.4% of cases presenting with Diabetes Insipidus, while 37.7% as Panhypopituitarism. On the other hand, prevalence of adrenal metastases is at 0.5 to 3.8% with 43% occurring bilaterally. Primary adrenal insufficiency occurs at 12.4% among those with bilateral metastases. Case: A 32-year old Chinese woman was diagnosed with Stage 4 breast adenocarcinoma ER PR Her2 Neu positive with metastases to the axillary lymph nodes, bone and right adrenal in July 2021. She underwent chemotherapy with Paclitaxel and Trastuzumab then radical mastectomy of the left breast in April 2022.5 months post op, PET scan was done which showed a 11 x 18 x 21 mm pituitary mass. In light of the primary malignancy, lesion was considered a metastatic focus. At that time, patient did not exhibit any neurologic symptoms such as blurring of vision or headaches. No polyuria, polydipsia, nocturia, easy fatigability, nausea or vomiting. Physical examination findings were normal. A hormonal panel showed the following: elevated Prolactin at 150 ng/ml (NV 3-20), normal plasma ACTH IRMA 10 pg/ml (NV &amp;lt; 50 pg/ml), low 8AM plasma cortisol at 1.55 ug/dL (Ref 5- 25 ug/dL), TSH was low at 0.15iIU/ml (0.27-3.75) while FT4 was normal at 0.87 ng/dl (NV 0.68 - 2.56).ACTH stimulation showed low cortisol at the 30th minute and 1st hour post ACTH infusion. Serum sodium and urine Osmolality were both normal. The hyperprolactinemia was attributed to Stalk effect, while the decreased cortisol was considered as central adrenal insufficiency. She was then started on Prednisone 5mg tab daily. Repeat TSH and FT4 5 months after showed normal TSH at 1.209, low FT4 at 5.97 pmol/L (NV 9.01-19.05). She was started on Levothyroxine 50mcg daily for the central hypothyroidism.Repeat Cranial MRI 4 months post diagnosis of the pituitary mass showed increased in its size. An endoscopic transsphenoidal resection and biopsy of the pituitary mass showed metastatic carcinoma of the pituitary gland. Staining of the sellar tumor were compatible with a metastatic carcinoma of breast origin. Intensity-modulated radiation therapy (IMRT) at 400 gray was done for 5 fractions and Trastuzumab was continued. Cranial MRI done 6 months post IMRT showed decreasing size of the pituitary mass to its latest size of 6 x 9 x 13 mm as of November 2023. Latest Whole abdominal CT scan of the adrenals showed clearing of the right adrenal Nodule. Conclusion: Breast cancer may also metastasize to endocrine organs causing hormonal dysfunction. Although curative treatment is no longer the goal for Metastatic breast cancer, prolongation of the quality of life is an important aspect to consider. Hormonal work up, surveillance and hormonal replacement can help in attaining this goal. Presentation: 6/3/2024

8169 Bilateral Inferior Petrosal Sinus Sampling Using Metyrapone as a Reliable Alternative to Differentiate Between Cushing Disease and Ectopic ACTH Syndrome- A Single Center Experience

Abstract Disclosure: M. Azmath: None. S. Faisal: None. C. Caetano: None. T. Vedere: None. H. Aftab: None. B. Tendler: None. C. Malchoff: None. Introduction: Bilateral IPSS is a crucial test in accurately diagnosing Cushing disease (CD) and differentiating it from ectopic ACTH syndrome (EAS). [1] CRH stimulated IPSS is the gold standard technique but cannot be performed due to current shortage of CRH. Metyrapone is a safe and reliable alternative to CRH that can stimulate corticotrophs to secrete ACTH and hence used for IPSS. [2] We would like to share a single center experience using a novel low dose metyrapone protocol for IPSS that successfully helped differentiate CD and EAS. Methods: This was a retrospective analysis of patients presenting with Cushing syndrome. Initial diagnosis was confirmed based on standard testing including 24-hour urinary free cortisol (UFC), late night saliva concentrations (LNSC) and MRI. Metyrapone 500 mg every 4 hours for 3 doses was administered orally starting midnight prior to IPSS performed in the morning. ACTH levels drawn during initial evaluation for Cushing syndrome was compared to ACTH values post metyrapone from the peripheral vein sample to calculate a gradient. Ultimately, pituitary CD or EAS was confirmed based on cure following surgery and /or positive ACTH of tumor staining on pathology and/or concordant results with CRH stimulated IPSS. Results: There were 9 patients, out of which 5 had pituitary CD, and 4 had EAS. Pre ACTH levels for patients ranged between 14.5 and1164 pg/ml (normal reference range 6-50). Following metyrapone, ACTH level rose to 40-4705 pg/ml (normal reference range 6-50). The increase in ACTH levels ranged from 81-547% pre and post metyrapone. An average of 5-6 ACTH samples were drawn during IPSS. Catheterization was successful in all patients and was confirmed by IR. The mean central to peripheral (C/P) ACTH ratio for patients with confirmed CD was 20.53 in comparison with a ratio of 1.34 in patients with EAS. IPSS was confirmed to be diagnostic for pituitary CD if the peak ratio after metyrapone administration was more than 3. No adverse effects were observed during or after the procedure and no hospitalization was required pre-procedure. Discussion: We present single center experience with a novel low dose metyrapone protocol for IPSS. Our protocol was successful in increasing ACTH/cortisol secretion by tumorous cells as evidenced by increase in ACTH levels post metyrapone. It is an easy oral protocol which proved to be safe without the risk for adrenal insufficiency that can occur with higher doses. It also mitigates the small thromboembolic risk that can occur with DDAVP administration. A clear distinction in C/P ACTH ratios was observed in patients with CD versus EAS as early as the 3rd sample. In conclusion, our low dose metyrapone protocol was safe and reliable in accurately differentiating between CD and EAS in small sample of patients at our institution. Presentation: 6/1/2024

The branched N-glycan of PD-L1 predicts immunotherapy responses in patients with recurrent/metastatic HNSCC

Immunotherapy has revolutionized cancer treatment, but the lack of a reliable predictive biomarker for treatment response remains a challenge. Alpha-1,6-Mannosylglycoprotein 6-β-N-Acetylglucosaminyltransferase 5 (MGAT5) is a key regulator of complex N-glycan synthesis, and its dysregulation is associated with cancer progression. The lectin Phaseolus vulgaris leukoagglutinin (PHA-L) specifically binds to mature MGAT5 products. Previous studies have indicated elevated PHA-L staining in head and neck squamous cell carcinoma (HNSCC), which implies increased activity of MGAT5. However, the specific role of MGAT5 in HNSCC remains unclear. In this study, we found significantly higher PHA-L staining and MGAT5 expression in HNSCC tumors compared to adjacent non-tumor tissues. Using a mass spectrometry (MS)-based glycoproteomic approach, we identified 163 potential protein substrates of MGAT5. Functional analysis revealed that protein substrates of MGAT5 regulated pathways related to T cell proliferation and activation. We further discovered that PD-L1 was among the protein substrates of MGAT5, and the expression of MGAT5 protected tumor cells from cytotoxic T lymphocyte (CTL) killing. Treatment of nivolumab alleviated the protective effects of MGAT5 on CTL activity. Consistently, patients with MGAT5-positive tumors showed improved responses to immunotherapy compared to those with MGAT5-negative tumors. Using purified PD-L1 from HNSCC cells and a glycoproteomic approach, we further deciphered that the N35 and N200 sites carry the majority of complex N-glycans on PD-L1. Our findings highlight the critical role of MGAT5-mediated branched N-glycans on PD-L1 in modulating the interaction with the immune checkpoint receptor PD-1. Consequently, we propose that MGAT5 could serve as a biomarker to predict patients’ responses to anti-PD-1 therapy. Furthermore, targeting the branched N-glycans at N35 and N200 of PD-L1 may lead to the development of novel diagnostic and therapeutic approaches.

A Recombinant Lentiviral Vegfr2-Silencing Vector Attenuates Roxarsone-Promoted Growth of Rat Vascular Endothelial Cells and Angiogenesis in Matrigel Plug and B16F10 Xenograft Models.

Roxarsone (ROX) is widely used as a feed addictive for livestock and poultry. ROX promotes angiogenesis, which can lead to health problems, and it is necessary to identify methods to counter this angiogenic effect of ROX. The VEGF/VEGFR2 signaling pathway is involved in the growth and reconstruction of new blood vessels during angiogenesis. In this study, a recombinant lentiviral vector encoding Vegfr2 shRNA was transfected into rat vascular endothelial cells and used in mouse matrigel plug and melanoma xenograft models to investigate its potential to regulate ROX-induced angiogenesis and tumor growth. Treating endothelial cells with ROX increased cell proliferation, migration, and a tube-like structure of growth relative to the control group. The addition of the lentiviral Vegfr2-silencing vector significantly attenuated the effects of ROX on endothelial cells. The hemoglobin content of mouse matrigel plugs treated with ROX was increased significantly. This effect was dramatically attenuated by the co-administration of shRNA targeting Vegfr2. The volume, weight and CD34 staining of the melanoma xenograft tumors increased by ROX were also attenuated by Vegfr2 silence. These results indicate that the down-regulation of VEGFR2 protein plays an inhibitory role in the ROX-promoted angiogenesis in vivo and in vitro. These data support the targeting of Vegfr2 gene as an effective means to treat ROX-induced angiogenesis and tumor growth.

Case report: response to immunotherapy and association with the fh gene in hereditary leiomyomatosis and renal cell cancer-associated renal cell cancer

Hereditary leiomyomatosis and renal cell cancer (HLRCC) is a rare autosomal dominant syndrome caused by a germline mutation in the fumarate hydratase (FH) gene that manifests with cutaneous leiomyomas, uterine fibroids, and renal cell cancer (RCC). Patients with HLRCC-associated RCC (HLRCC-RCC) have aggressive clinical courses, but there is no standardized therapy for advanced HLRCC-RCC. In this study, we described a case of aggressive HLRCC in a 33-year-old female who exhibited a novel heterozygous germline insertion mutation in exon 8 of the FH gene (c.1126 C > T; p.Q376*). The patient underwent laparoscopic resection of the right kidney, but metastases appeared within 3 months after surgery. Histological staining of the resected tumor revealed high expression levels of programmed cell death-ligand 1 (PD-L1). Therefore, the patient was treated with immunotherapy. The patient achieved a partial response to immunotherapy, and the treatment of metastatic lesions has continued to improve. A thorough literature review pinpointed 76 historical cases of HLRCC-RCC that had undergone immunotherapy. From this pool, 46 patients were selected for this study to scrutinize the association between mutations in the FH gene and the effectiveness of immunotherapy. Our results indicate that immunotherapy could significantly improve the overall survival (OS) of patients with HLRCC-RCC. However, no influence of different mutations in the FH germline gene on the therapeutic efficacy of immunotherapy was observed. Therefore, our study suggested that immunotherapy was an effective therapeutic option for patients with HLRCC regardless of the type of FH germline mutation.

Primary lung chordoma: a case report

BackgroundChordoma, a rare malignant tumor arising from notochordal tissue, usually occurs along the spinal axis. Only a few published reports of primary lung chordomas exist. Herein, we present a case of primary lung chordoma and discuss important considerations for diagnosing rare chordomas.Case presentationWe report a case of primary lung chordoma in a 39-year-old male with a history of testicular mixed germ-cell tumor of yolk sac and teratoma. Computed tomography revealed slow-growing solid lesions in the left lower lobe. We performed wedge resection for suspected germ-cell tumor lung metastasis. Histologically, large round or oval cells with eosinophilic cytoplasm were surrounded by large cells with granular, lightly eosinophilic cytoplasm. Tumor cells were physaliphorous. Immunohistochemistry was positive for brachyury, S-100 protein, epithelial membrane antigen, vimentin, and cytokeratin AE1/AE3, suggesting pulmonary chordoma. Re-examination of the testicular mixed germ-cell tumor revealed no notochordal elements. Although some areas were positive for brachyury staining, hematoxylin and eosin (HE) staining did not show morphological features typical of chordoma. Complementary fluorescence in situ hybridization (FISH) of the lung tumor confirmed the absence of isochromosome 12p and 12p amplification. Thus, a final diagnosis of primary lung chordoma was established.ConclusionsIn patients with a history of testicular mixed germ cell tumors, comparison of histomorphology using HE and Brachyury staining of lung and testicular tumors, and analyzing isochromosome 12p and 12p amplification in lung tumors using FISH is pivotal for the diagnosis of rare lung chordomas.

Folate Receptor Immunohistochemical Staining and Gynecologic Tumors: Initial Experience With 216 Cases.

Folate receptor alpha has been shown to have possible mechanisms of tumorigenesis in malignancies, becoming a potential target for therapy. Mirvetuximab soravtansine is an antifolate receptor alpha monoclonal antibody, with an approved FOLR1-2.1 immunohistochemical biomarker. After IRB approval, a retrospective review of gynecologic pathology cases was performed to identify cases in which FOLR1 immunohistochemistry (IHC) was performed at our institution over a period of 9 months as part of clinical care for therapy eligibility. Clinical data collected included patients' age, tumor histotype, tumor grade, primary tumor site, FIGO stage, dates of recurrence/progression, and use of mirvetuximab therapy. FOLR1 IHC data were recorded, including the date specimen obtained, date IHC was performed, site tested, case type, percentage tumor staining, and intensity. Cases were deemed positive or negative according to current recommendations (75%, 2-3+intensity). Two hundred sixteen cases were identified. Patient ages ranged from 25 to 83 years old (median: 59yr). Staining intensity was reported as 0 in 15 (6.9%) cases, weak (1+) in 8 (3.7%), moderate (2+) in 27 (12.5%), strong (3+) in 27 (12.5%), weak-to-moderate (1-2+) in 15 (6.9%), and moderate-to-strong (2-3+) in 99 (45.8%); intensity was not provided in 25 (11.6%). Percentage of tumor staining ranged from 0 to 100, with a median of 60. The IHC was overall deemed positive in 98 (45.4%) cases and negative in 118 (54.6%). By histotype, 5 of 17 (29.4%) low-grade serous carcinomas, 88 of 162 (54.3%) high-grade serous carcinomas, 3 of 5 (60%) of carcinosarcomas, and 2 of 6 (33.3%) of mixed carcinomas were positive. No case of clear cell CA, endometrioid CA, Mullerian CA NOS, serous borderline, mucinous CA, or granulosa cell tumor was positive. The primary site of disease was tubo-ovarian in 192 (88.9%) cases, peritoneal in 8 (3.7%) cases, uterine in 3 (1.4%) cases, and unknown in 13 (6%) cases. By site on which immunohistochemical stain was performed: primary site positive in 53 of 96 (55.2%) cases, metastatic site at time of diagnosis/debulking positive in 23 of 41 (52.1%) cases, and metastatic/recurrent cases positive in 22 of 79 (27.8%) cases. There was a statistically significant correlation when comparing the positivity rates between these sites (P = 0.0004). Survival data were examined with high-grade serous carcinoma, with no statistically significant difference between positive and negative cases in overall survival (P = 0.622) or progression-free survival (P = 0.711). Biopsy specimens were positive in 17 (25%) cases, while negative in 51 (75%), whereas resection specimens were positive in 81 (54.7%) and negative in 67 (45.3%), a statistically significant difference (P < 0.0001). Cases that were <19 months old had 38 (36.2%) positive and 67 (63.8%) negative, compared with cases ≥19 months old that had 60 (54.1%) positive and 51 (45.9%) negative, a statistically significant difference (P = 0.0084). Significant differences in FOLR1 staining were noted between histotypes, age of the specimen, type of case tested, and site of disease tested. Further testing is needed to help determine the best tissue to be utilized for this new biomarker.

Unfolding the role of cell state on stress granule heterogeneity and function in pancreatic cancer.

e16357 Background: A major impediment to cancer therapy is the reality that cancer is a heterogeneous disease. A single tumor can consist of many genotypic or phenotypic profiles, which are marked by degrees of sensitivity to different therapeutics. Identifying and targeting origins of cancer cell heterogeneity is critical for predicting patient outcome and enhancing treatment efficacy. We observe a novel source of cancer cell heterogeneity that involves one of the largest stress-sensing platforms described to date: the stress granule (SG). Stress granules are non-membranous, cytoplasmic organelles that assemble in response to stress, and promote cell survival. Deregulation of SGs has been linked to several pathological conditions, including cancer, neurodegeneration, ischemia, and viral infections. SGs have been shown to promote tumor growth and metastasis, as well as contribute to mechanisms of chemo-resistance. Thus, these signaling hubs are emerging as a potential target in cancer. Methods: To induce SG formation, human pancreatic ductal adenocarcinoma (PDAC) cell lines were treated with sodium arsenite, thapsigargin, or exposed to hypoxic conditions. Immunoflourescent staining of core SG proteins was then conducted. To investigate cell cycle-driven mechanisms of SG heterogeneity, cell cycle stage was identified by expression of a Fluorescent ubiquitination-based cell cycle indicator (Fucci) lentiviral system. To investigate cPLA2’s role in SG formation, the chemical inhibitor Pyrrophenone, an shRNA targeting cPLA2, and an exogenous full-length or cleaved form of cPLA2, were used. In vivo, ES149 cells expressing Luciferase and the Fucci system were orthotopically implanted into C57BL/6 mice. Established tumors were treated every 3 days with Vehicle, Oxaliplatin, Pyrrophenone, or a combination of the latter. Tumor size was tracked via IVIS Spectrum. Fucci flourescence was used to identify cell cycle phase. SGs and cell death were assessed by immunoflourescent staining of tumors. Results: Cancer cells show a high degree of heterogeneity in their production of SGs. Cell cycle phase dictates SG heterogeneity. G2 phase cells have significantly higher levels of SG formation as compared cells in G1/S. This process is regulated by cleavage of the cPLA2 protein. Oxaliplatin administration increases G2 phase content in tumors. Co-treatment with Oxaliplatin and Pyrrophenone significantly decreases SG formation, increases cell death, and leads to tumor regressions in vivo. Conclusions: G2 phase cancer cells develop higher levels of SGs when exposed to stressful stimuli, as compared to G1/S phase counterparts. The cPLA2 protein is cleaved into a catalytically dead form that is present in G1/S phases, but absent in G2 phase. Oxaliplatin treatment in vivo leads to an increase in G2 phase tumor cells. When paired with cPLA2 inhibition, this results in increased cancer cell death and decreased tumor growth.

Prevalence of “HER2 ultra-low” among patients with advanced breast cancer with historical IHC0 status.

e13156 Background: Breast Cancer (BC) patients are classified as HER2-positive (IHC3+ or IHC2+/ISH+) or HER2-negative (IHC0, IHC1+ and 2+/ISH-). Within the HER2-negative category, patients who meet HER2-low (IHC1+ or IHC2+/ISH-) criteria can benefit from HER2 targeted therapy like trastuzumab deruxtecan. Patients who meet IHC0 criteria with incomplete and faint membrane staining in &gt;0 but ≤10% of tumor cells — defined as “HER2 Ultra-low”, may also benefit from HER2 targeted therapy. This study aimed to assess the prevalence of “HER2 Ultra-Low” expression based on re-scored HER2 IHC biopsy slides. Methods: This study utilized EHR data from all three US Mayo Clinic campuses. A total of 300 patients with advanced BC (stages III-IV), and clinically documented HER2 IHC0 status between Jan 2017 and Jan 2023, were identified. One biopsy slide per patient (IHC assay: VENTANA Pathway anti-HER2/neu (4B5) rabbit monoclonal primary antibody) was digitized and scanned at 40x magnification. Two Mayo Clinic pathologists independently scored and reported HER2 status according to 2023 ASCO-CAP guidelines and included tumor stain percentage for each slide. Patients were considered “HER2 Ultra-Low” if their slide was scored as IHC0 with &gt;0% but ≤10% staining. Cohen’s κ was used to quantify agreement between pathologists. Results: The re-scored patients (n=300) had a mean age of 57.8 years (SD=13.5), with ~97% having IHC0 documentation within 30 days of the reviewed biopsy slide. A total of 285 (95%) patients remained classified as IHC0 by at least one pathologist. Of these, 60% (n=171) were determined as “HER2 Ultra-low” by at least one pathologist. The rate of “HER2 Ultra-Low” per pathologist ranged from 43% (n=121) to 45% (n=104). The overall inter-pathologist concordance was 57%; patients with no observable HER2 IHC staining accounted for majority of the concordant patients (Table). Conclusions: About 3 in 5 advanced BC patients originally classified as HER2 IHC0 met the “HER2 Ultra-Low” criteria by at least one pathologist, implying that significant number of additional patients may benefit from HER2 targeted therapy. The relatively low concordance between HER2 expert pathologists in identifying lower levels of HER2 expression suggests the need for increased precision enabled by digital tools, leveraging AI, training for community practice pathologists, and continuing to follow best practice recommendations in determining HER2 IHC status. [Table: see text]

The Combination of Auranofin and Celecoxib Suppresses Local Synovial Sarcoma Progression In Vivo.

Synovial sarcoma (SS) is a rare malignant tumor with a poor survival rate. We previously reported that a combination of auranofin (AUR), a thioredoxin reductase inhibitor, and celecoxib (CE), an anti-inflammatory drug, significantly impedes the local progression of osteosarcoma (OS). However, the role of redox regulation in SS remains to be elucidated. This study aimed to investigate the efficacy of combined treatment of AUR and CE on the local progression of SS in vivo. Nu/nu mice were implanted with the human SS cell line, Aska-SS, and treated with vehicle control, AUR, or a combination of AUR and CE (AUR-CE). Primary tumor size and weight were evaluated for the study duration and upon resection, respectively. Hematoxylin and eosin (H&E) and Ki-67 staining were performed to assess the local progression of SS. A statistically significant reduction in tumor size and weight was observed in the AUR- and AUR-CE-treated groups upon excision compared to that in the vehicle-treated group. The AUR-CE-treated group showed synergistic inhibition of local tumor growth. H&E staining of local SS tumors revealed decreased cell density and nuclear deformation in the AUR- and AUR-CE-treated groups compared to those in the vehicle-treated group. Immunohistochemical staining revealed a statistically significant decrease in Ki-67-positive cells in the AUR-CE-treated group compared to the vehicle-treated group. The combination of AUR and CE showed significant potential for delaying the local progression of SS. These findings support the repurposing of AUR and CE as early treatment options for SS.

GLI Transcriptional Targets S100A7 and KRT16 Show Upregulated Expression Patterns in Epidermis Overlying the Tumor Mass in Melanoma Samples.

Although not completely understood, the role of the Hedgehog-GLI (HH-GLI) signaling pathway in melanoma and epithelial skin tumors has been reported before. In this study, we confirmed in various melanoma cell line models that keratin 16 (KRT16) and S100 Calcium-Binding Protein A7 (S100A7) are transcriptional targets of GLI Family Zinc Finger (GLI) proteins. Besides their important role in protecting and maintaining the epidermal barrier, keratins are somehow tightly connected with the S100 family of proteins. We found that stronger expression of KRT16 indeed corresponds to stronger expression of S100A7 in our clinical melanoma samples. We also report a trend regarding staining of GLI1, which corresponds to stronger staining of GLI3, KRT16, and S100A7 proteins. The most interesting of our findings is that all the proteins are detected specifically in the epidermis overlying the tumor, but rarely in the tumor itself. The examined proteins were also not detected in the healthy epidermis at the edges of the sample, suggesting that the staining is specific to the epidermis overlaying the tumor mass. Of all proteins, only S100A7 demonstrated a statistically significant trend regarding tumor staging and staining intensity. Results from our clinical samples prove that immune infiltration is an important feature of melanoma. Pigmentophages and tumor-infiltrating lymphocytes (TIL) demonstrate a significant association with tumor stage, while mononuclear cells are equally present in all stages. For S100A7, we found an association between the number of TILs and staining intensity. Considering these new findings presented in our study, we suggest a more detailed examination of the possible role of the S100A7 protein as a biomarker in melanoma.

CCK Receptor Inhibition Reduces Pancreatic Tumor Fibrosis and Promotes Nanoparticle Delivery.

The poor prognosis for pancreatic ductal adenocarcinoma (PDAC) patients is due in part to the highly fibrotic nature of the tumors that impedes delivery of therapeutics, including nanoparticles (NPs). Our prior studies demonstrated that proglumide, a cholecystokinin receptor (CCKR) antagonist, reduced fibrosis pervading PanIN lesions in mice. Here, we further detail how the reduced fibrosis elicited by proglumide achieves the normalization of the desmoplastic tumor microenvironment (TME) and improves nanoparticle uptake. One week following the orthotopic injection of PDAC cells, mice were randomized to normal or proglumide-treated water for 3-6 weeks. Tumors were analyzed ex vivo for fibrosis, vascularity, stellate cell activation, vascular patency, and nanoparticle distribution. The histological staining and three-dimensional imaging of tumors each indicated a reduction in stromal collagen in proglumide-treated mice. Proglumide treatment increased tumor vascularity and decreased the activation of cancer-associated fibroblasts (CAFs). Additionally, PANC-1 cells with the shRNA-mediated knockdown of the CCK2 receptor showed an even greater reduction in collagen, indicating the CCK2 receptors on tumor cells contribute to the desmoplastic TME. Proglumide-mediated reduction in fibrosis also led to functional changes in the TME as evidenced by the enhanced intra-tumoral distribution of small (<12 nm) Rhodamine-loaded nanoparticles. The documented in vivo, tumor cell-intrinsic anti-fibrotic effects of CCK2R blockade in both an immunocompetent syngeneic murine PDAC model as well as a human PDAC xenograft model demonstrates that CCK2R antagonists, such as proglumide, can improve the delivery of nano-encapsulated therapeutics or imaging agents to pancreatic tumors.

Abstract PO2-08-06: Metformin may improve the outcome of patients with breast cancer and type 2 diabetes mellitus through the effect of tumor immune microenvironment

Abstract Metformin, an anti-diabetic drug, is known to have anti-tumor effects. We examined the outcome of 177 patients with type 2 diabetes mellitus (T2DM) who received surgery for breast cancer. Among them, 49 patients were treated with drugs including metformin. In those patients, recurrence in distant organs was less frequent and postoperative disease-free survival (DFS) tended to be better than those without metformin intake. In patients who received preoperative systemic therapy (PST), rate of pathological complete response (pCR) was significantly more frequent in patients with metformin treatment (p &amp;lt; 0.05). Multiplex immunohistochemical staining of resected tumors revealed that the density of tumor associated macrophages (TAM), especially of CD68(+)CD163(+) M2-type TAM, was significantly lower in tumors with metformin treatment. In contrast, the rate of CD8(+) phenotype in CD3(+) tumor infiltrating lymphocytes (TILs) was significantly higher in metformin group. The results suggest that metformin can change the immune microenvironment from pro-tumorigenic to anti-tumorigenic status, which leads to the favorable outcome of the patients with breast cancer and T2DM. Citation Format: Satomi Shiba, Michiko Harao, Kasumi Ogihara, Takayo Fukuda, Masako Sakuragi, Kitayama Joji, Naohiro Sata. Metformin may improve the outcome of patients with breast cancer and type 2 diabetes mellitus through the effect of tumor immune microenvironment [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 PO2-08-06.

Abstract PO1-02-11: Characterization of MammaPrint UltraLow Risk tumors in more than 1400 patients from the real-world evidence FLEX study

Abstract BACKGROUND: MammaPrint is a gene expression signature used to determine the risk of distant recurrence for patients with early-stage breast cancer. Previous studies from MINDACT, FOCUS, IKA, and STO-3 trials have demonstrated that postmenopausal node-negative patients with MammaPrint UltraLow Risk tumors are considered indolent and patients have excellent long-term survival outcomes up to 20 years with little to no endocrine treatment. These studies suggest that patients with UltraLow Risk tumors are ideal candidates for treatment optimization which has resulted in the inclusion of the UltraLow Risk result in the NCCN guidelines. Here, we characterized patients with UltraLow Risk tumors from the real-world evidence FLEX study. METHODS: FLEX (NCT03053193) is a prospective, observational registry study with 99 sites open in the United States, Canada, Greece, and Israel. Patients enrolled in FLEX have early-stage breast cancer and receive MammaPrint testing as standard of care (with or without BluePrint molecular subtyping), and consent to full transcriptome analysis and clinical data collection. MammaPrint is a gene expression signature of 70 genes that classifies tumors as having a Low Risk or High Risk of distant recurrence. Within Low Risk tumors, a subcategory of UltraLow Risk (MammaPrint Index &amp;gt; 0.355) has been defined in previous studies. Clinical data for this analysis was locked February 2023. Clinical risk was defined as low risk or high risk based on Adjuvant Online criteria. RESULTS: Among the 12,328 patients enrolled in FLEX, 1465 (11.9%) have UltraLow Risk tumors. All tumors with available BluePrint molecular subtyping were classified as Luminal-Type. Of the patients with detailed clinical data, the majority had clinically low risk tumors (78.8%; Table 1). Notably, 34.6% of patients with UltraLow Risk tumors had Ki67 tumor staining of greater than 10%, 16.7% were pre- or peri-menopausal, 43.6% had grade 2-3 tumors, and 20.1% had tumors &amp;gt; 2 cm (Table 1). Among patients with treatment information, 6.5% received a combination of chemotherapy with or without endocrine and HER2-targeted therapy, 82.4% received endocrine therapy, 4.8% received other treatment (endocrine with targeted therapy (CDK4/6), radiation alone, novel treatment regimen, or supporting agents), and 6.3% did not receive treatment (Table 1). CONCLUSIONS: Previous trials have demonstrated the utility of MammaPrint UltraLow Risk in patients with clinically low risk features. In this large real-world evidence study of patients with early-stage breast cancer, UltraLow Risk tumors can be identified across all clinical categories. Overall, we report a substantial number of premenopausal patients with UltraLow Risk tumors, and these young women would be good candidates for treatment optimization. Future studies will assess long-term outcomes in this cohort further stratified by treatment. Table 1. Clinical and genomic characteristics of UltraLow Risk tumors Data are presented as n (%). Patients with unknown or missing data for each category have not been included. Citation Format: Cathy Graham, Kent Hoskins, Vijayakrishna K. Gadi, Suzanne Hoekstra, Gordon Srkalovic, Douglas Marks, Beth Sieling, Patricia Dauer, Andrea Menicucci, William Audeh, Joyce O'Shaughnessy, FLEX Investigators' Group. Characterization of MammaPrint UltraLow Risk tumors in more than 1400 patients from the real-world evidence FLEX study [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 PO1-02-11.