Squamous Cell Carcinoma Specimens Research Articles

739. SIGNIFICANCE OF COX2 INHIBITOR FOR ESOPHAGEAL CANCER FROM THE CLINICAL AND EXPERIMENTAL ESTIMATE

Abstract (Aims) We report experimental and clinical evidence that COX2 represent a potential molecular target for the treatment and prevention of esophageal cancer. (Methods) (A)Experiment: Thirty 8-week male wistar rats were exposed to duodenal content esophageal reflux. All animal underwent an esophagoduodenal anastomosis (EDA) with total gastrectomy in order to produce chronic esophagitis. In ten rats the sham (Control). They were sacrified at 40th week. Their esophagi were examined for HE, COX2,PGE2, PCNA and total bile acids in the esophageal lumen was measured. (B)Clinical: The expression of COX2 was examined for 68 specimens of esophageal squamous cell carcinoma (SCC) and the correlation of COX2 expression with clinicopatholigic features was examined. (Results) (A) After 40 weeks of reflux, columnar dysplasia, squamous cell carcinoma and adenocarcinoma were found. Total bile acid in the esophageal lumen was significantly increased in the EDA group compared with the sham operated rats. PCNA LI and esophageal tissue PGE2 were higher in dysplastic and cancer tissue than that of control. Overexpression of COX2 was shown in dysplastic and cancer tissue. COX2 may play an important role in esophageal cancer by duodenal content reflux.(B) COX2 immunoreactivity was weak in 27(40%) and strong in 41(60%) of the carcinomas. The proportion of poorly differentiated SCC among tumors with a strong expression of COX2 (34%) was significantly higher than among tumors with a weak expression of COX2 (19%, p=0.02). The depth of tumors (p=0.004), lymph node metastasis (p=0.0009) and the stage of the tumors (p=0.001) were advanced significantly more progressively in ECCS with a strong COX2 expression. Moreover, survival was significantly reduced (p=0.02) among patients with strong COX2 expression when compared with the COX2 weak group. This study showed that strong expression of COX2 was correlated with tumor progression and poor differentiation in ESCC. (Conclusion) In this experimental study, we demonstrate that bile reflux of duodenal contents induce COX2 and increase prostaglandin synthesis in dysplastic and cancer tissue. Our study suggests that COX2 may play a role in esophageal carcinoma development and progression, COX2 inhibitors may be potential targets for the prevention or treatment of esophageal carcinoma.

Transmission of Genetic Material and Malignant Cells of Cutaneous Origin via Surgical Sutures.

Seeding of skin cancer cells following diagnostic or therapeutic surgical procedures can occur and might cause local recurrences. As current preferred therapy for skin malignancy is surgical excision, seeding of tumour cells by manipulating malignant tissue or suturing can be another factor leading to recurrences. To evaluate whether genetic material and malignant cells adhere to standard suture materials. This prospective study included patients who underwent excision of skin lesions. Monofilament and braided sutures were examined. Sutures were passed through the observed tumour or healthy skin margins and were examined for DNA material and cells by cytological analysis, cell culture and characterization, and DNA analysis. Twenty-two patients and 148 sutures were included. DNA quantification showed DNA material on all sutures, with no significant difference between braided and monofilament sutures. Cytological analysis showed that all slides prepared from cell blocks contained normal squamous and atypical cells. Cell culture and characterization showed viable cells adhering to the sutures under direct light microscopy. Cell cultures showed rapid proliferation of epithelial cells from squamous cell carcinoma specimens. Suture materials carry DNA material and cells, including malignant cells of cutaneous origin and may seed them at distant sites.

Biomarker Turnaround Times and Impact on Treatment Decisions in Patients with Advanced Non-Small Cell Lung Carcinoma at a Large Canadian Community Hospital with an Affiliated Regional Cancer Centre.

Background: Timely reporting of molecular biomarkers is critical in guiding optimal treatment decisions in patients with advanced non-small cell lung carcinoma (NSCLC). Any delays along the tissue or treatment pathway may be associated with suboptimal treatment/outcomes and a reduced quality of life. For many centres, biomarkers are tested off-site. Methods: A retrospective chart review of 123 patients with advanced NSCLC seen between 1 June 2021 and 30 June 2022 was conducted. With a focus on core biomarkers (PD L1, EGFR, and ALK), the outcome variables were as follows: total turnaround time (total TAT), divided into pre-laboratory, laboratory, and post-laboratory time intervals, as well as time to treatment decision (TOTD) and time to optimal systemic therapy decision (TOTSD). Results: At first consult, only 20.3% of patients had all core biomarker results available. The median total TAT was significantly longer for non-squamous (non-SCC) than squamous cell carcinoma (SCC) specimens (36.5 versus 22 days, p < 0.001). The median pre-laboratory time for the entire cohort was 5 calendar days. The median laboratory testing time was greater for non-SCC compared to the SCC specimens (23 versus 12 days, p < 0.001). The median time from consult to TOTD was 19 calendar days for the entire cohort. Conclusions: This study emphasizes the need for the expansion of regional resources to meet the clinical needs of advanced NSCLC patients treated at a regional cancer centre which uses an off-site molecular laboratory.

Tracing human papillomavirus in skin and mucosal squamous cell carcinoma: a histopathological retrospective survey.

Worldwide, squamous cell carcinoma (SCC) incidence is rising. The literature debates the human papillomavirus (HPV)'s role in cutaneous SCC development. We examined HPV histopathology in SCC samples in this study. Retrospective study at tertiary referral skin center in 2020. Histopathological features of HPV, including koilocytosis, hyperkeratosis, acanthosis, hypergranulosis, parakeratosis, solar elastosis, papillomatosis, and tumor grade, were examined in SCC specimens. Two dermatopathologists independently reevaluated all samples. We examined 331 SCC cases (male:female ratio = 3.9:1). The mean age was 68.1, with 15.1 standard deviation. Lesions were most common on the face (40.5%), scalp (22.7%), and extremities (20.8%). Koilocytes were found in 50 (15.1%) lesions. Nail (38.1%, p=0.007), oral cavity (36.8%, p=0.014), and genitalia (60.0%, p=0.026) lesions had higher koilocytosis rates. SCCs were found in 6.6% of specimens, but in situ tumors had the highest koilocytosis (64.7%), significantly higher than other grades (p<0.001). SCC pathology often shows HPV and specific koilocyte histopathology. Well-differentiated SCC has a stronger association with nail, oral, and genital lesions.

Role of asprosin and meteorin-like peptide in progression of actinic keratosis to squamous cell carcinoma

ABSTRACT Squamous cell carcinoma (SCC) often develops from an underlying premalignant lesion. Factors that affect the progression of actinic keratosis (AK) to invasive SCC are not fully known. Asprosin (ASP) and meteorin-like peptide (METRNL) are adipokines that are involved primarily in glucose metabolism. We investigated the expression of ASP and METRNL in AK and SCC to evaluate the role of these adipokines in the development of SCC. We used 15 SCC specimens, 12 AK specimens and 12 healthy control skin specimens. ASP and METRNL protein expression in tumor and surrounding tissue was evaluated using immunohistochemistry. ASP expression in tumor tissue was significantly greater in the SCC group than in the control and AK groups, but it did not differ significantly between the AK and control groups. A positive correlation was observed for both ASP and METRNL expressions between tumor tissue and adjacent epidermis, hair follicles, sebaceous gland, eccrine gland, inflammatory cells and vascular structures. ASP and METRNL may exert pro-tumor effects toward development of invasive SCC. The expression intensity of ASP and METRNL can be used as a biomarker of risk of progression to SCC.

Study of Expression of P16 in Premalignant and Malignant Lesions of Penis and Their Significance.

Penile squamous cell carcinoma (SCC) is an extremely rare malignancy. It is usually caused by chronic human papillomavirus (HPV) 16 and HPV 18 infections. This study was conducted to investigate the immunohistochemical overexpression of p16, a surrogate marker for HPV, and to evaluate its usefulness as a potential diagnostic biomarker. In this cross-sectional prospective and retrospective cohort study, 56 penile squamous cell carcinoma (SCC) specimens and five penile premalignant specimens were evaluated in Kasturba Medical College, Mangalore, India, from January 2013- December 2018 in terms of clinical and histopathological features. Immunohistochemical expression for p16 in cases and controls was evaluated. Statistical comparison of p16 expression among clinical features, histological subtype, grade, and stages of tumor were done. Analysis of the pattern of p16 staining showed diffuse and strong nuclear and cytoplasmic expression in 32.8% of the cases. There was a highly significant association (P<0.001) of pattern of p16 expression among the HPV and non-HPV subtypes of penile carcinoma. p16 expression was not significantly associated with other prognostic parameters like site of the lesion, lymphovascular invasion, perineural invasion, histologic grade, and pathologic stage. Expression of p16 would be a useful tool in differentiation between the HPV-associated and non-HPV-associated subtypes of penile SCC that may be helpful in prediction of aggressiveness and invasive potential of the respective histologic subtypes.

Correlation of PD-1 and PD-L1 expression in oral leukoplakia and oral squamous cell carcinoma: an immunohistochemical study

The programmed cell death protein (PD-1)/programmed cell death protein ligand (PD-L1) pathway and cytotoxic T lymphocyte antigen are the most important co-stimulatory molecules that play a key role in the negative regulation of T cells during carcinogenesis. We aimed to evaluate the immunohistochemical expression of PD-1 and PD-L1 in oral leukoplakia and squamous cell carcinoma compared with normal oral mucosa. Twenty-five cases of oral squamous cell carcinoma, oral leukoplakia and normal oral mucosa tissue specimens were immunohistochemically stained to assess PD-1 and PD-L1 expression. The PD-L1 positivity of subepithelial TAFs (p < 0.001) increased with increasing grades of oral leukoplakia. Pearson’s correlation indicated a high positive correlation between the PD-L1 labelling index of epithelial tumour cells and the PD-1 labelling index of tumour infiltrating lymphocytes (p value: 0.005) in OSCC. A high positive correlation was noted between the H-score of PD-L1 positive tumour epithelial cells and the H-score of PD-1 positive tumour infiltrating lymphocytes in OSCC (p value: 0.001). PD-L1 positivity increased in dysplastic epithelial cells from premalignant lesions to malignancy. The sub-epithelial PD-L1 positive TAFs were higher in oral leukoplakia compared to OSCC inferring that PD-L1 positivity in TAFs decreased with malignant transformation. The PD-1 positivity in TILs was higher in oral leukoplakia than in OSCC.

Perilipin1 Expression as a Prognostic Factor in Patients with Squamous Cell Carcinoma of the Lung.

Perilipin (PLIN) is a major structural protein located on the surface of lipid droplets. PLIN plays an important role in human metabolism and is associated with metabolic diseases, such as obesity, diabetes, hypertension, and endocrine disorders. The dysregulation of lipid metabolism is one of the most prominent metabolic changes observed in cancers. Therefore, the PLIN protein family has recently attracted attention owing to its role in lipid metabolism and cancer. To date, no studies have addressed the association between the prognosis of lung cancer and PLIN1 expression. For the first time, we found that high PLIN1 expression was significantly correlated with worse disease-free survival (DFS) in lung squamous cell carcinoma (SCC). We examined PLIN1 expression by the immunohistochemical analysis of surgical lung SCC specimens obtained from 94 patients. We analyzed the correlation between PLIN1 expression, clinicopathological data, and patient survival, using a chi-squared test, Kaplan-Meier analysis with log-rank tests, and the multivariate Cox proportional hazards regression test. High PLIN1 expression was significantly correlated with lower DFS in the Kaplan-Meier analysis and the multivariate Cox proportional hazards regression model. High PLIN1 expression was significantly correlated with worse prognosis in lung SCC.

Oral Cavity Cancer Tissues Differ in Isotopic Composition Depending on Location and Staging.

The aim of this paper was to characterise the isotopic composition of oral squamous cell carcinoma (OSCC) specimens of different areas of the oral cavity. Secondly, we assessed whether there was a correlation between clinical stages of OSCC and isotopic abundance. The IRMS procedure was performed on 124 samples derived from 31 patients with OSCC of 15 N and 13 C to assess the isotopic composition. From each individual, four samples from the tumour, two from the margins, and two samples of healthy oral mucous membranes were derived. The two samples from the tumour and two samples from the margin were additionally subjected to histopathological assessment. Then, statistical analysis was conducted. Tumour infiltration tissues of the lower lip were characterised by higher mean δ13C values compared to samples derived from cancers of the other oral cavity regions (-23.82 ± 1.21 vs. -22.67 ± 1.35); (p = 0.04). The mean percentage of nitrogen content in tumour tissues was statistically higher in patients with the most advanced cancers (11.89 ± 0.03%) versus the group of patients with II and III stage cancers (11.12 ± 0.02%); (p = 0.04). In patients at stage IV, the mean δ13C value in the cancer samples equalled -22.69 ± 1.42 and was lower than that in patients at less severe clinical stages (p = 0.04). Lower lip cancer tissues differed in the isotopic abundance of carbon in comparison with tissues derived from the group of combined samples from other locations. Values of δ13C observed in specimens derived from lower lip cancers were similar to those observed in healthy oral mucous membranes. Cancer tissues obtained from patients in the last stage of OSCC had a different isotopic composition in comparison with those obtained from earlier stages. To confirm these observations, further research on larger groups of patients is needed.

Optimization of proteinase K incubation protocol duration during DNA extraction from oral squamous cell carcinoma FFPE samples

Background: Formalin-fixed paraffin-embedded (FFPE) specimen archives are a valuable source of sample material for molecular biological analysis. However, the DNA isolated from FFPE samples is usually low in concentration and fragmented. Thus, it is necessary to optimize the FFPE DNA extraction protocol to obtain the best results. Proteinase K incubation is undoubtedly crucial in DNA extraction procedures, but this step is often not well explained in the manufacturer’s manual. Purpose: This study aimed to find the optimal duration for proteinase K incubation protocols to achieve the highest DNA yields. Methods: Fifteen paraffin blocks of Oral Squamous Cell Carcinoma (OSCC) specimens were obtained, and the cancerous areas were microdissected into smaller cuts for DNA extraction. The samples were randomly divided into three groups (n=5) and subjected to three different proteinase K incubation protocols: one-hour incubation at 56ºC as per the manufacturer’s instructions (Group I), 24-hour incubation at 56ºC (Group II), and 48 hours at room temperature with an additional four hours at 56ºC (Group III). The extracted DNA was then quantified using a Nanodrop spectrophotometer. The recorded data were analyzed using ANOVA-LSD. Results: The highest DNA concentration was found in Group III (107.74 ± 41.92), which was significantly higher compared to Group II (59.46 ± 30.32) and Group I (6.46 ± 1.97) (p&lt;0.05). Conclusion: In conclusion, modifying the duration of proteinase K incubation protocols can lead to different DNA yield results. In this study, the most optimized protocol for proteinase K incubation, resulting in the highest DNA yields, was 48 hours at room temperature with an additional four hours at 56ºC.

A Prospective Observational Study on the Role of Immunohistochemical Expression of Orphanin in Laryngeal Squamous Cell Carcinoma Recurrence.

To date, histological biomarkers expressed by laryngeal cancer are poorly known. The identification of biomarkers associated with laryngeal squamous cell carcinoma (SCC), would help explain the tumorogenesis and prevent the possible recurrence of the lesion after treatment. For this reason, the aim of this study is to investigate, for the first time, the Orphanin expression in 48 human specimens of laryngeal SCC and evaluate its possible correlation with patients prognosis. We analyzed pathological specimens from 48 patients with laryngeal SCC to detect the presence of Orphanin by using an immunohistochemistry test. We compared the findings with healthy tissue acquired from patients who underwent surgery for mesenchymal benign tumours of the larynx. The specimens were stained with anti-Orphanin monoclonal antibodies. Results were processed through a computerised image analysis system to determine a scale of staining intensity. All the tumoural specimens examined showed a significant immunoreaction for Orphanin when compared with healthy tissues (p < 0.05) but with a different immune reactivity related to clinical-pathological features. A high Orphanin expression was not significantly related to Histological Grading (HG), TNM, and stage (p > 0.05). In the multivariate analysis, the Orphanin expression was significantly related only to the malignant recurrence (p < 0.05). Our study suggests that Orphanin could have a role in tumorigenesis by increasing the recurrence of cancer; therefore, it should be further explored as a possible biomarker for laryngeal cancer.

Inducible nitric oxide synthase (iNOS)-activated Cxcr2 signaling in myeloid cells promotes TGFβ-dependent squamous cell carcinoma lung metastasis

Transforming growth factor beta (TGFβ) activity is linked to metastasis in many cancer types, but whether TGFβ activity is necessary for squamous cell carcinoma (SCC) lung metastasis has not been studied. Here we used a lung metastatic SCC model derived from keratin 15 (K15). KrasG12D.Smad4−/− SCC and human SCC specimens to identify metastasis drivers and test therapeutic interventions. We demonstrated that a TGFβ receptor (TGFβR) inhibitor reduced lung metastasis in mouse SCC correlating with reduced CD11b+/Ly6G+ myeloid cells positive for inducible nitric oxide synthase (iNOS). Further, TGFβ activity and iNOS were higher in primary human oral SCCs with metastasis than SCCs without metastasis. Consistently, either depleting myeloid cells with anti-Gr1 antibody or inhibiting iNOS with L-N6-(1-iminoethyl)-l-lysine (L-NIL) reduced SCC lung metastasis. L-NIL treated tumor-bearing mice exhibited reductions in tumor-infiltrating myeloid cells and in plasma Cxcl5 levels, and attenuated primary tumor growth with increased apoptosis and decreased proliferation. Blocking Cxcl5 with an antagonist of its receptor Cxcr2, SB225002, also reduced SCC lung metastasis.

Lung adenocarcinoma and squamous cell carcinoma difficult for immunohistochemical diagnosis can be distinguished by lipid profile

In patients with unresectable non-small cell lung cancer, histological diagnosis is frequently based on small biopsy specimens unsuitable for histological diagnosis when they are severely crushed and do not retain their morphology. Therefore, establishing a novel diagnostic method independent of tissue morphology or conventional immunohistochemistry (IHC) markers is required. We analyzed the lipid profiles of resected primary lung adenocarcinoma (ADC) and squamous cell carcinoma (SQCC) specimens using liquid chromatography-tandem mass spectrometry. The specimens of 26 ADC and 18 SQCC cases were evenly assigned to the discovery and validation cohorts. Non-target screening on the discovery cohort identified 96 and 13 lipid peaks abundant in ADC and SQCC, respectively. Among these 109 lipid peaks, six and six lipid peaks in ADC and SQCC showed reproducibility in target screening on the validation cohort. Finally, we selected three and four positive lipid markers for ADC and SQCC, demonstrating high discrimination abilities. In cases difficult to diagnose by IHC staining, [cardiolipin(18:2_18:2_18:2_18:2)-H]− and [triglyceride(18:1_17:1_18:1) + NH4]+ showed the excellent diagnostic ability for ADC (sensitivity: 1.00, specificity: 0.89, accuracy: 0.93) and SQCC (sensitivity: 0.89, specificity: 0.83, accuracy: 0.87), respectively. These novel candidate lipid markers may contribute to a more accurate diagnosis and subsequent treatment strategy for unresectable NSCLC.

Determination of esophageal squamous cell carcinoma and gastric adenocarcinoma on raw tissue using Raman spectroscopy

Cancer detection is a global research focus, and novel, rapid, and label-free techniques are being developed for routine clinical practice. This has led to the development of new tools and techniques from the bench side to routine clinical practice. In this study, we present a method that uses Raman spectroscopy (RS) to detect cancer in unstained formalin-fixed, resected specimens of the esophagus and stomach. Our method can record a clear Raman-scattered light spectrum in these specimens, confirming that the Raman-scattered light spectrum changes because of the histological differences in the mucosal tissue.To evaluate the use of Raman-scattered light spectrum for detecting endoscop-ically resected specimens of esophageal squamous cell carcinoma (SCC) and gastric adenocarcinoma (AC).We created a Raman device that is suitable for observing living tissues, and attempted to acquire Raman-scattered light spectra in endoscopically resected specimens of six esophageal tissues and 12 gastric tissues. We evaluated formalin-fixed tissues using this technique and captured shifts at multiple locations based on feasibility, ranging from six to 19 locations 200 microns apart in the vertical and horizontal directions. Furthermore, a correlation between the obtained Raman scattered light spectra and histopathological diagnosis was performed.We successfully obtained Raman scattered light spectra from all six esophageal and 12 gastric specimens. After data capture, the tissue specimens were sent for histopathological analysis for further processing because RS is a label-free methodology that does not cause tissue destruction or alterations. Based on data analysis of molecular-level substrates, we established cut-off values for the diagnosis of esophageal SCC and gastric AC. By analyzing specific Raman shifts, we developed an algorithm to identify the range of esophageal SCC and gastric AC with an accuracy close to that of histopathological diagnoses.Our technique provides qualitative information for real-time morphological diagnosis. However, further in vivo evaluations require an excitation light source with low human toxicity and large amounts of data for validation.

Radiotherapy upregulated immune checkpoints contribute to the development of second primary OSCC.

Radiation injury is common after radiotherapy for head and neck cancer. Radiotherapy can reshape the immune microenvironment and cause immunosuppression, including dysregulation of immune checkpoints (ICs). However, the relationship between oral ICs expression after radiation and the development of second primary tumors is unclear. Clinical specimens of second primary oral squamous cell carcinoma after radiotherapy (s-OSCC) and primary OSCC (p-OSCC) were collected. The expression and prognostic value of PD-1, VISTA, and TIM-3 were analyzed using immunohistochemistry. To further clarify the relationship between radiation and ICs alteration, a rat model was constructed to explore the spatiotemporal changes of ICs in the oral mucosa after radiation. In carcinoma tissue, the expression of TIM-3 was higher in s-OSCC than in p-OSCC, while the expression of PD-1 and VISTA was similar between the groups. In para-carcinoma tissue, the expression of PD-1, VISTA, and TIM-3 was higher in s-OSCC. High ICs expression was associated with poor survival. In the rat model, ICs were locally upregulated in the irradiated tongue. Moreover, there was a bystander effect, in which the ICs were also upregulated in the unirradiated site. Radiation may upregulate ICs expression in oral mucosa and contribute to the development of s-OSCC.

Abstract 6656: Expression pattern and clinicopathological implication of B7 family immune checkpoints, VTCN1 and HHLA2, in non-small cell lung cancer

Abstract Background: Efforts are needed to discover novel immunotherapeutic targets in non-responders of current immunotherapies. VTCN1 (B7-H4/B7x/B7S1) and HHLA2 (B7-H7/B7y) are B7 family co-inhibitory molecules reported to be expressed in PD-L1-negative or EGFR-mutant lung adenocarcinoma (LUAD). To elucidate the clinicopathological implications of these two immune checkpoints, we checked their detailed expression patterns including intratumoral heterogeneity. Methods: VTCN1 and HHLA2 expression were evaluated in public single-cell RNA sequencing (scRNAseq) and The Cancer Genome Atlas (TCGA) dataset. Their immunohistochemistry (IHC) was performed on tissue microarrays of 413 LUAD and 382 lung squamous cell carcinoma (LUSC) specimens. Intratumoral heterogeneity was evaluated by IHC on representative sections of 59 surgically resected LUAD and 49 LUSC specimens. Results: In LUAD scRNAseq, VTCN1 and HHLA2 were predominantly expressed on epithelial cells and were not co-expressed with PD-L1. However, IHC showed their expression in both tumor and immune cells. In TCGA analysis, there was a negative correlation between VTCN1 and PD-L1 (LUAD, r = -0.094, p = 0.033; LUSC, r = -0.227, p &amp;lt;0.001). VTCN1 and HHLA2 showed positive correlations in both TCGA (LUAD, r = 0.211, p &amp;lt;0.001; LUSC, r = 0.182, p &amp;lt;0.001) and IHC analysis (LUAD, p &amp;lt;0.001; LUSC, p = 0.016). They were more frequently expressed in EGFR-mutant LUAD (52.9% vs. 40.3%, p = 0.015 for VTCN1 in EGFR-mutant vs. wildtype; 59.8% vs. 40.5%, p &amp;lt;0.001 for HHLA2). CD8+ T-cell infiltration was significantly higher in VTCN1- or HHLA2-positive EGFR-mutant LUAD, but there was no significant relationship in EGFR-wildtype. In 60% of cases expressing both VTCN1 and HHLA2, the two showed spatially concordant expression (15 out of 24 LUAD; 3 out of 6 LUSC). However, VTCN1 and PD-L1 showed concordant expression in 37.2% of cases expressing both (6 out of 16 LUAD; 10 out of 27 LUSC). HHLA2 expression was associated with better overall survival in LUSC, but there was no significant difference in survival according to VTCN1 expression. Conclusions: VTCN1 and HHLA2 are frequently co-expressed in PD-L1-negative NSCLC, especially EGFR-mutant LUAD rich in CD8+ T-cells and can serve as potential novel immunotherapeutic targets. Citation Format: Sojung Lim, Jaemoon Koh, Seung Geun Song, Jeemin Yim, Bogyeong Han, Young A Kim, Doo Hyun Chung, Yoon Kyung Jeon. Expression pattern and clinicopathological implication of B7 family immune checkpoints, VTCN1 and HHLA2, in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6656.

Investigating miR-9 as a mediator in laryngeal cancer health disparities.

For several decades, Black patients have carried a higher burden of laryngeal cancer among all races. Even when accounting for sociodemographics, a disparity remains. Differentially expressed microRNAs have been linked to racially disparate clinical outcomes in breast and prostate cancers, yet an association in laryngeal cancer has not been addressed. In this study, we present our computational analysis of differentially expressed miRNAs in Black compared with White laryngeal cancer and further validate microRNA-9-5p (miR-9-5p) as a potential mediator of cancer phenotype and chemoresistance. Bioinformatic analysis of 111 (92 Whites, 19 Black) laryngeal squamous cell carcinoma (LSCC) specimens from the TCGA revealed miRNAs were significantly differentially expressed in Black compared with White LSCC. We focused on miR-9-5 p which had a significant 4-fold lower expression in Black compared with White LSCC (p<0.05). After transient transfection with either miR-9 mimic or inhibitor in cell lines derived from Black (UM-SCC-12) or White LSCC patients (UM-SCC-10A), cellular migration and cell proliferation was assessed. Alterations in cisplatin sensitivity was evaluated in transient transfected cells via IC50 analysis. qPCR was performed on transfected cells to evaluate miR-9 targets and chemoresistance predictors, ABCC1 and MAP1B. Northern blot analysis revealed mature miR-9-5p was inherently lower in cell line UM-SCC-12 compared with UM-SCC-10A. UM -SCC-12 had baseline increase in cellular migration (p < 0.01), proliferation (p < 0.0001) and chemosensitivity (p < 0.01) compared to UM-SCC-10A. Increasing miR-9 in UM-SCC-12 cells resulted in decreased cellular migration (p < 0.05), decreased proliferation (p < 0.0001) and increased sensitivity to cisplatin (p < 0.001). Reducing miR-9 in UM-SCC-10A cells resulted in increased cellular migration (p < 0.05), increased proliferation (p < 0.05) and decreased sensitivity to cisplatin (p < 0.01). A significant inverse relationship in ABCC1 and MAP1B gene expression was observed when miR-9 levels were transiently elevated or reduced in either UM-SCC-12 or UM-SCC-10A cell lines, respectively, suggesting modulation by miR-9. Collectively, these studies introduce differential miRNA expression in LSCC cancer health disparities and propose a role for low miR-9-5p as a mediator in LSCC tumorigenesis and chemoresistance.

Immunohistochemical analysis of B7-H3 expression in patients with lung cancer following various anti-cancer treatments.

B7 homolog 3 protein (B7-H3), an immune checkpoint molecule belonging to the B7 family, has been studied as a target for the development of anti-cancer treatment; however, changes in B7-H3 expression during the clinical course remain unknown. This retrospective study aimed to investigate changes in B7-H3 expression of lung cancer specimens in patients with advanced lung cancer following various anti-cancer treatments. The immunohistochemistry (IHC) score was evaluated on a 0-3 scale, and B7-H3 expression was considered positive for grade ≥ 2. The difference in IHC scores before and after anti-cancer treatment was defined as the change in B7-H3 expression. Among 160 patients with lung cancer who received anti-cancer treatment, 88 (55%) and 101 (63%) had B7-H3 expression before and after anti-cancer treatment, respectively. Before treatment, B7-H3 expression was significantly more common in squamous cell carcinoma specimens than in adenocarcinoma specimens (95% vs. 49%, P < 0.001). Of the 19 patients with squamous cell carcinoma, 18 (95%) continued to have high (IHC score: 3) B7-H3 expression following treatment. In contrast, of the 130 patients with adenocarcinoma, 46 (35%) and 17 (13%) showed an increased and a decreased expression, respectively. Patients who received targeted therapy had a significant increase in B7-H3 expression compared with those who received chemotherapy alone (P = 0.015). Overall, squamous cell carcinoma specimens maintained high B7-H3 expression during the clinical course, whereas adenocarcinoma specimens showed changes in expression following anti-cancer treatments. Our results provide the basis for further studies on the development of anti-cancer treatments targeting B7-H3.

CORRELATION BETWEEN BRANDWEIN-GENSLER'S HISTOLOGIC RISK MODEL AND BRODER'S HISTOLOGICAL GRADING IN ORAL SQUAMOUS CELL CARCINOMAS

Introduction: Several clinicopathological and molecular factors have been used to prognosticate and predict outcomes in the patients of oral squamous cell carcinomas (OSCC). Many histological grading systems have been described, however there is a lack of agreement as to a satisfactory robust predictive model. The Brandwein-Gensler risk model has been validated in various multicentre studies as a reliable prognostic model for advanced as well as early disease. The Broders grading system, still recommended by WHO, is probably the best-known attempt at prognostication with subjective assessment of key histological features. This study was conducted to evaluate various histological prognostic markers in case of Oral Squamous Cell Carcinomas (OSCC) and understand their correlation with each other. Total 66 Materials and Methods: excised specimens of Oral squamous cell carcinomas were retrospectively analysed and Histologic risk scoring was done according to the Histologic risk model (HRM) given by Brandwein-Gensler et al. Broder's histological grading was done on the basis of differentiation of tumor. Results: Histological grading of tumor based on differentiation of tumor shows poor correlation with Brandwein-Gensler's Histologic risk model with a sensitivity and specicity of 34.7% and 52.9% respectively; inferring that differentiation status of tumor is not a good parameter for prognostic evaluation of Oral Squamous Cell Carcinomas. However, owing to the fact that no poorly differentiated tumor was noted in the current study, further evaluation and large sample size is required to conrm. Researc Conclusion: h on predictive and prognostic biomarkers has ignited hope for individualizing therapy to facilitate better survival and reduce overtreatment in the patients of oral SCC. There is no signicant association between differentiation of tumor and WPOI. No signicant correlation exists between differentiation of tumor and Histologic risk model scoring thereby inferring that histological differentiation of tumor is not a good prognostic parameter.

266 Decoding the role of polyamine metabolism on anti-tumor immunity in head and neck cancer

OBJECTIVES/GOALS: The effect of immunosuppressive metabolites on anti-tumor immunity in human papillomavirus (HPV)-associated vs carcinogen-driven head and neck cancer is unknown. The objective of this study is to define the extent to which metabolites impair this response and identify novel metabolic targets for enhancing anti-tumor immunity. METHODS/STUDY POPULATION: HPV-associated and carcinogen-driven head and neck squamous cell carcinoma specimens were frozen following surgical excision, and tumor sections were cut onto glass slides. Slides were coated in alpha-cyano-4-hydroxy-cinnamic acid (CHCA) matrix and subjected to mass spectrometry imaging using matrix-assisted laser desorption ionization (MALDI) on a Bruker SolariX XR 12T Hybrid QqFT-ICR mass spectrometer run in positive mode. Slides were then stained for immunohistochemistry (IHC) using markers of CD8 T cells, macrophages (CD163), B cells (CD20), and tumor cells (panCK). Mass spectrometry imaging and IHC spatially resolved data will be co-registered and metabolite intensity in regions of interest (cell types) quantified. RESULTS/ANTICIPATED RESULTS: A total of seven HPV-associated (three metastatic lymph nodes and four primary tumors) and six carcinogen-driven (primary tumors) HNSC specimens were subjected to MALDI and IHC. Metabolites significantly enriched in HPV-associated HNSC relative to carcinogen-driven HNSC include 2,3-diphosphoglyceric acid, xanthine, 2,3,5-Trichloromaleylacetate, and indole-3-carboxyaldehyde. Metabolites significantly enriched in carcinogen-driven HNSC relative to HPV-associated HNSC include hesperetin 3'-O-sulfate, hypoxanthine, phosphorylcholine, and L-homocysteine sulfonic acid. In ongoing analyses, we anticipate identifying a relationship between CD8+ T cell enriched vs depleted regions and immunosuppressive metabolites (e.g., kynurenine, adenosine monophosphate). DISCUSSION/SIGNIFICANCE: Defining the extent to which CD8+ T cells interact with the metabolic milieu of the microenvironment will provide a foundation for metabolic Precision Medicine. Strategically targeting metabolic pathways to enhance the anti-tumor immune response will be leveraged for the design and implementation of immune modulatory metabolic therapy.