Dysplastic Epithelium Research Articles

Cancer Stem Cell Markers, CD44 and ALDH1, for Assessment of Cancer Risk in OPMDs and Lymph Node Metastasis in Oral Squamous Cell Carcinoma.

Tumour heterogeneity in oral cancer is attributed to the presence of cancer stem cells (CSCs). CSCs are the most migratory and metastatic cellular subpopulation within tumours. Assessment of CSC markers as significant predictors of lymph node metastasis may prove valuable in the clinical setting. Furthermore, analysis of this panel of putative stem cell markers in oral dysplasia may additionally inform of the likelihood for oral potentially malignant disorders (OPMDs) to progress to oral squamous cell carcinoma (OSCC). The present study aims to assess the significance of CSC markers in the progression of OPMDs to OSCC and assessment of lymph node metastasis in OSCC. CD44 and ALDH1 were assessed immunohistochemically in 25 normal, 30 OPMDs, and 24 OSCCs. CD44 is a membranous marker and ALDH1 is a cytoplasmic marker. The immunohistochemical expression of these markers were compared between OPMDs with and without dysplasia, as well as between low-risk and high-risk dysplasias. Similarly, expression was compared between OSCC with and without lymph node metastasis and among grades of OSCC. Positive CD44 expression was seen in all normal mucosal tissues. The expression decreased from normal epithelium to OPMDs but increased in OSCC. CD44 expression was positive in 21 cases of OSCC (87.5%) and reduced from well-differentiated to poorly differentiated OSCC. CD44 staining index was higher in OSCC without lymph node metastasis (3.59) when compared with OSCC with lymph node metastasis (1.33). There was a statistically significant difference observed in the ALDH1 staining index among three groups (p < 0.05), with highest expression seen in OSCC. Within OPMDs, the ALDH1 staining index was statistically higher in OPMDs with dysplasia as compared to OPMDs without dysplasia. Furthermore, the expression was higher in OPMDs with high-risk dysplasia when compared with low-risk dysplasia, but this was not statistically significant (p = 0.82). In conclusion, The CD44 positive population possesses properties of CSCs in head and neck carcinoma, and continuous shedding could be found after CD44 down-regulation. The present study reports differences in ALDH1 expression between OPMDs with and without dysplasia, dysplastic and non-dysplastic epithelia, and low-risk and high-risk dysplasia. These findings may suggest ALDH1 as a specific marker for dysplasia. CD44 demonstrated a difference in staining index in OSCC without lymph node metastasis versus OSCC with lymph node metastasis. These findings may suggest CD44 as a marker for lymph node metastasis. Both proteins may play key roles in the tumorigenicity of CSCs in OPMDs and OSCC.

Immunohistochemical evaluation of D2-40, Galectin-3, Maspin and MCM7 expression in palate squamous cell carcinomas.

Squamous cell carcinoma (SCC) is the most frequent cancer in oral cavity and its prognosis has exhibited little improvement in the last decades. Although much less common palate SCCs manifests a higher local aggression invading very quickly the adjacent muscles and jawbones, thus being able frequently to lead to dysfunctions in chewing, swallowing, and speech. To elucidate what underlies such local aggression, we investigated the immunohistochemical expression in palate SCCs of Podoplanin (D2-40), Galectin-3 (Gal-3), mammary serine protease inhibitor (Maspin) and minichromosome maintenance complex component 7 (MCM7), markers that are known to be involved in tumor invasiveness. We found a progressive increase in reactivity for D2-40 and MCM7 from the normal epithelium toward dysplastic epithelium and respectively to SCC, which suggests the intervention of these markers in the early stages of squamous cell carcinogenesis in the palate. The highest D2-40, Gal-3 and MCM7 reactivity was observed in basaloid and in poorly differentiated (G3) palate SCCs, while for Maspin the well-differentiated (G1) palate SCCs were the most reactive. The first three markers mentioned above were most intensely expressed at the invasion front, while the Maspin reactivity was low or absent at this level. Statistically, we found significant stratification on localization, grading, muscle invasion, and survival for all investigated markers, but with very high direct correlations between D2-40, Gal-3, and MCM7 immunoreactive score (IRS) values, while between the Maspin and each of the previous markers there were very high inverse correlations. Overall, all these investigate markers proved to be responsible for the local invasiveness and regional lymph node metastasis, thus allowing a prognostic and therapeutic stratification of patients with palate SCCs.

Primary Sclerosing Cholangitis-Associated Cholangiocarcinoma Demonstrates High Intertumor and Intratumor Heterogeneity.

INTRODUCTION:Intertumor and intratumor heterogeneity may explain the diagnostic challenge and limited efficacy of chemotherapy for primary sclerosing cholangitis–associated cholangiocarcinoma (PSC-CCA). In this study, tumor heterogeneity was assessed through p53 and p16 protein expression analysis and next-generation sequencing (NGS) of TP53 and CDKN2A genetic alterations in PSC-associated CCA.METHODS:Formalin-fixed paraffin-embedded tissue samples from resection material of patients with PSC-CCA or patients with PSC diagnosed with biliary dysplasia were selected. Sections with CCA and foci with dysplastic epithelium were identified by 2 independent gastrointestinal pathologists. Immunohistochemical evaluation of p53 and p16 protein expression and NGS of TP53 and CDKN2A genetic alterations were performed.RESULTS:A total of 49 CCA and 21 dysplasia samples were identified in the resection specimens of 26 patients. P53 protein expression showed loss of expression, wild type, and overexpression in 14%, 63%, and 23% CCA and in 19%, 62%, and 19% dysplasia samples, respectively. P16 protein expression showed negative, heterogeneous, and positive results in 31%, 57%, and 12% CCA and in 33%, 53%, and 14% dysplasia samples, respectively. NGS showed high intertumor and intratumor heterogeneity of TP53 mutations and CDKN2A loss. Nearly 70% of the samples with a TP53 missense mutation demonstrated p53 overexpression, whereas all samples with a TP53 nonsense mutation demonstrated loss of p53 protein expression.DISCUSSION:PSC-associated CCA is characterized by high intertumor and intratumor heterogeneity of both p53/p16 protein expression and genetic alterations in TP53/CDKN2A, indicating that these tumors consist of multiple subclones with substantially different genetic makeup. The high intertumor and intratumor heterogeneity in PSC-CCA should be acknowledged during the development of diagnostic and therapeutic strategies.

A comparative study of the oral epithelia of inflammatory hyperplasias and the dysplastic epithelia of oral squamous cell carcinoma by transmission electron microscopy

ObjectiveThis study aims to compare the atypical epithelial changes of oral inflammatory lesions with the dysplastic epithelial changes of oral squamous cell carcinomas and to examine the underlying pathogenetic mechanisms that cause the changes by transmission electron microscopy (TEM). MethodsEight cases of parulides (oral inflammatory lesions) and 10 cases of oral squamous cell carcinomas were studied. Each specimen was cut into two pieces: one piece was fixed in a neutral buffered 10 % formaldehyde solution and prepared for light microscopy, and the other was fixed in a cold, 2.5 % glutaraldehyde solution and prepared for TEM. ResultsBased on the six histopathologic criteria in this study, both types of epithelia showed similar ultrastructural features, but some significant differences are observed as follows: In parulides, the epithelial cells with features similar to abnormal mitoses showed nuclear fragments with chromatin condensation (karyorrhexis), and no discernible organelles in the cytoplasm, indicating that these are necrotic cells. In contrast, in oral squamous cell carcinomas, the epithelial cells with abnormal mitotic figures showed clumping of chromosomes with abnormal mitotic spindles, indicating that these are abnormally dividing cells. Abnormal keratinization was also seen. The nuclei of the abnormally keratinized cells were irregular with coarse and clumped chromatin. ConclusionsThe atypical epithelial changes in oral inflammatory lesions are a result of necrotic changes in the epithelia due to underlying inflammatory processes and are not true epithelial dysplasia. In contrast, the dysplastic epithelial changes in oral squamous cell carcinomas are dysplastic changes due to premalignant neoplastic processes.

Epithelial Dysplasia at Excision Margins of Oral Squamous Cell Carcinoma: A Review on Relationship to Clinicopathological Parameters and Prognosis.

Background:Epithelial dysplasia (ED) at oral cancer excision margins is a frequent finding. Dysplastic epithelium at excision margins may not be similar to dysplasia in Oral potentially malignant disorders (OPMD) as malignant transformation has already taken place. Therefore, management of ED at excision margins should be different to that of OPMD. ED creates a dilemma in relation to further management of cancer patients, since there are no accepted guidelines. Therefore, the objective of this review is to analyze existing literature and to arrive at evidence based recommendations for the management of ED at excision margins. Methods:A comprehensive string was run on PubMed, Medscape and Medline. The final outcome included 113 studies. Finally, the most relevant 10 articles were critically assessed for inclusion and exclusion criteria against various parameters. Results and Conclusions: Severe and Moderate ED need re-excision in order to improve prognosis. There is not enough sound evidence for the management of Mild ED at excision margins of oral squamous cell carcinoma. Guidelines for the management of ED at excision margins should be formulated after comprehensive multi center studies using lager cohorts of patients.

The mRNA expression levels of calpains and their activity in malignant and dysplastic epithelium of the upper respiratory tract

Background. The calpain proteolytic system plays an important role in the development of cancer. Detection of early cancer in the upper respiratory tract is often challenging, as symptoms are largely non-specific, and most cases are diagnosed at an advanced stage. Aim . To identify candidate markers of transition from premalignant lesions to invasive carcinoma, we studied mRNA expression levels of CAPN1 and CAPN2 and the total activity of calpains in the tumor tissues of patients with head and neck squamous cell carcinoma (HNSCC) and in the epithelial dysplasia-affected tissues of patients with chronic diseases of the upper respiratory tract. Materials and methods. The study included 32 patients with HNSCC (Т1-3N0-1М0) and 12 patients with chronic diseases of the upper respiratory system associated with epithelial dysplasia. The expression levels of CAPN1 and CAPN2 were assessed using real-time polymerase chain reaction (PCR). The calpain activity was determined by hydrolysis of the fluorogenic Suc-LLVY-AMC oligopeptide. Results. The mRNA expression levels of CAPN1 and CAPN2 were respectively 3 and 4 times higher in the tumor tissue of patients with HNSCC than in the tissue of patients with endothelial dysplasia in the upper respiratory tract. The level of calpain activity was 4.4 times higher in patients with HNSCC than in patients with epithelial dysplasia of different severity. Conclusion. The elevated mRNA expression levels of CAPN1 and CAPN2 and their activity in the tumor tissues of patients with HNSCC compared to patients with chronic respiratory diseases associated with epithelial dysplasia are likely to characterize a high potential for transition from precancerous lesion to cancer. To clarify the role of calpains in the carcinogenesis of HNSCC, further studies of intact tissues using animal models are required.

Abstract 2117: Immunomodulatory functions of SMYD3 in HPV-negative head and neck squamous cell carcinoma

Abstract Background: Head and neck squamous cell carcinoma (HNSCC) causes approximately 10,000 deaths annually in the United States, with human-papilloma-virus (HPV)-negative patients having a particularly poor prognosis. Pembrolizumab, a programmed-death-1 (PD-1) antibody, was recently approved as a first-line therapy for patients with relapsed disease, however response rates are as low as 19%, underscoring the need for an improved understanding of immunotherapy resistance mechanisms. We have previously shown that HPV-negative HNSCC tumors with higher mRNA levels of SET and MYND Domain Containing 3 (SMYD3), a chromatin modifier known to trimethylated H3K4 (H3K4me3), have significantly lower mRNA levels of CD8 and CD8+ T-cell attracting chemokines, and that SMYD3 depletion in HNSCC cells in vitro leads to upregulation of CD8+ T-cell attracting chemokines. The goal of this work was to elucidate epigenetic mechanisms of immune evasion mediated by SMYD3 in HPV-negative HNSCC. Methods: siRNA- mediated SMYD3 knockdown was performed for 3 days to investigate immuno-modulatory epigenetic mechanisms mediated by SMYD3. RT-qPCR for immune-related genes, Western blotting for histone marks, ChIP-sequencing for H3K4me3, ChIP-qPCRs to determine genomic interactions of SMYD3 and ATAC-sequencing were performed. SMYD3 and CD8 immunohistochemical staining (IHC) of normal squamous, dysplastic epithelium and HPV-negative HNSCC tumors was also performed. Results: siRNA mediated knockdown of SMYD3 resulted in enrichment of inflammatory pathways and upregulation of type 1 interferon response and antigen presentation machinery genes in HNSCC cell lines. Immunoblotting for histone marks showed no reduction in H3K4me3 following SMYD3 depletion for 3 days. ChIP-sequencing for H3K4me3 showed differential distribution pattern with an increase of H3K4me3 peaks on immune-related genes, and a decrease of H3K4me3 on UHRF1, a reader of the repressive histone mark H3K9me3. RNA-seq, RT-PCR and ChIP-qPCR showed that SMYD3 may regulate the transcription of UHRF1 by binding to its promoter. SMYD3 depletion also led to decreased protein levels of UHRF1. ChIP for UHRF1 and H3K9me3 showed that UHRF1 binds directly on the promoters of CXCL9 and CXCL10 which are enriched for H3K9me3. IHC on Normal Squamous Epithelium and Squamous Cell Carcinoma tissues of increasing cancer stages showed increase in SMYD3 levels with the higher stage of cancer, and an inverse correlation with CD8+ T cell infiltration within the tumors. Conclusion: This is the first study reporting that the chromatin modifier SMYD3 may attenuate type I IFN responses in HPV-negative HNSCC by regulating the expression of a major epigenetic regulator, UHRF1, which may silence the expression of immune-related genes through H3K9me3. Citation Format: Nupur Nigam, Benjamin Jonah Bernard, Kyunghee Burkitt, Sohyoung Kim, Yvette Robbins, Richard L. Bennett, Jonathan D. Licht, Vassiliki Saloura. Immunomodulatory functions of SMYD3 in HPV-negative head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2117.

HUMAN PAPILLOMAVIRUS (HPV)-ASSOCIATED ORAL INTRAEPITHELIAL NEOPLASIA: A CASE REPORT

<h3>Background</h3> Human papillomavirus (HPV)-associated oral intraepithelial neoplasia is a subset of oral dysplastic lesions with the presence of transcriptionally active high-risk oncogenic HPV subtypes 16 and 18. There is limited evidence in the literature regarding the potential rate of transformation of this entity to oral squamous cell carcinoma. <h3>Case Report</h3> A 51-year-old male patient with a heavy smoking (35 pack-year) and alcohol consumption (6 standard drinks a day) history presented with widespread oral leukoplakia. Initial biopsies demonstrated the following:SiteHistopathology findings1.Floor of mouthKeratosis with moderate to focally severe epithelial dysplasia2.Left lower lipActinic keratosis with mild to moderate epithelial atypiaSubsequent mapping biopsies (sites 3, 4) and wide local excision (site 5) demonstrated:3.Left ventral tongueHPV-associated intraepithelial neoplasia4.Lower lipcarcinoma in situ5.Right anterior ventral tongue/floor of mouthHPV-associated intraepithelial neoplasia, with excision margins free of dysplasia <h3>Investigations</h3> For sites 3 and 5, immunoperoxidase stains demonstrated diffuse, positive p16 staining of dysplastic epithelium and HPV-16 in situ hybridization showed positive nuclear staining. Both sites demonstrated hyperkeratosis, basal cell hyperplasia, increased numbers of mitoses (including suprabasal), and interspersed apoptotic cells. <h3>Follow-Up</h3> Wide local excision of the anterior ventral tongue and floor of mouth demonstrated HPV-associated intraepithelial neoplasia with involved margins (p16 and HPV-16-ISH positive). The lower lip vermilionectomy showed carcinoma in situ with clear margins. There is ongoing 3 monthly mucosal surveillance. <h3>Discussion</h3> Further studies are required to evaluate the behavior and prognosis of this subset of HPV-related oral dysplastic lesions.

Cytokeratins (CK7 and CK20) Genes Expression Association with Clinicopathological Indices in Oral Squamous Cell Carcinoma and Dysplastic Oral Epithelium.

High prevalence of oral squamous cell carcinoma (OSCC) demands the additional novel biological markers. Due to the established roles of cytokeratin in the prognosis of metastasis evaluation the relation of expression of both CK7 and CK20 in OSCC compared to the dysplastic oral epithelium biopsies with clinicopathological factors were investigated. We examined the coordinate mRNA expression of cytokeratin 7 (CK7) and cytokeratin 20 (CK20) using quantitative reverse transcription polymerase chain reaction (qRT-PCR) in 110 biopsies of oral squamous epithelium samples including 72 tumoral and 38 dysplastic biopsies. We also collected demographic and pathological data including tumor stage and grade from our patients. There was a significant difference in CK7 and CK20 gene expression between OSCC and dysplastic samples (p< 0.001). Further, their mean expression in OSCC samples was significantly higher compared to dysplastic samples. Relative mRNA levels of CK7 and CK20 showed that their mean expression in OSCC grade I was significantly lower than other grades (p< 0.01). The relationship between CK7 and CK20 mRNA expression and age or gender was not significant (p> 0.05). Samples in the advanced stage of disease had significantly higher CK7 and CK20 expression compared to early-stage samples of OSCC specimens (p= 0.001). We found an increase in CK7 and CK20 mRNA levels in grade III OSCC samples compared to other grades. This finding suggests a potential role for CK7 and CK20 in oral mucosal carcinogenesis and OSCC prognosis.

Nondysplastic Crypts in Fission in Nonpolypoid Adenomas and in the Adjacent Mucosa Support Field Cancerization in the Colon

We recently noticed in nonpolypoid adenomas (NPA) and the adjacent normal mucosa, nondysplastic crypts in symmetric and asymmetric fission (NDCSAF). All NDCSAF found in 80 small NPA and in the adjacent mucosa were registered. A total of 178 NDCSAF (mean, 2.2) were found: 12 (6.7%) interspersed between adenomatous glands, 36 (20.2%) partially replaced by dysplastic epithelium, and 130 (73%) underneath the adenomatous tissue. Of the 61 cases with normal mucosa adjacent to NPA, 40 (65.6%) disclosed NDCSAF, and the remaining 21 (34.4%) normal crypts, exclusively. The accruing of NDCSAF within NPA and surrounding mucosa, are outstanding findings. Given that colonic crypts may undergo only one fission every 30-40 years, the accruing of NDCSAF in and about small NPA reveals mucosal hubs with pathological aberrations of cryptogenesis, probably conveyed by somatic mutations. The findings support the existence of field cancerization in the colonic mucosa.

Comparative Evaluation of EGFR Expression in Oral Normal Epithelium, Dysplastic Epithelium and Squamous Cell Carcinoma – An IHC Study

Comparative Evaluation of EGFR Expression in Oral Normal Epithelium, Dysplastic Epithelium and Squamous Cell Carcinoma – An IHC Study

Chloride Intracellular Channel Protein 1 (CLIC1) Ιs Over-expressed in Muscle Invasive Urinary Bladder Cancer.

Invasive bladder cancer mortality remains high despite progresses made in early diagnosis and surgical procedures. Thus, there is a need to define new markers for bladder cancer. CLIC1 has not been previously studied in bladder cancer and thus, we aimed to assess its immunohistochemical expression in relation to different stages of bladder cancer development. Immunohistochemistry for CLIC1 was applied in 50 cases of muscle invasive bladder cancer. CLIC1 was not expressed in the normal urothelium, but a strong reaction was observed in dysplastic urothelium, carcinoma in situ and in 94% of the cases with invasive urothelial carcinoma; however, it was not expressed in squamous cell carcinoma cases. No correlation was found between the immunohistochemical expression of CLIC1 and the stage and grade of the tumour. CLIC1 was overexpressed in urinary bladder dysplastic epithelium, carcinoma in situ and invasive carcinoma. CLIC1 constitutes a new potential marker of invasive bladder cancer.

The Effect of Alcohol, Cigarette Smoking and Their Cessation for Risk of Field Cancerization of Esophagus and Head and Neck

Background: Multiple development of dysplastic squamous epithelium and squamous cell carcinoma (SCC) in the esophagus and head/neck region was associated with alcohol and cigarette use and have been explained by field cancerization phenomenon. Second primary cancer developed after curative treatment have harmful effect on patients’ quality of life and survival, however, until recently there have been no evidence-based assessment of the effects of these consumption and cessation on field cancerization. Methods: We conducted a multicentre cohort study at 16 hospitals in Japan. Patients with superficial esophageal SCC (ESCCs) who treated by endoscopic resection were registered. Dysplastic squamous epithelium in the background esophageal mucosa was evaluated by Lugol chromoendoscopy and was classified into 3 groups based on the number of Lugol-voiding lesions (LVLs) per endoscopic view: grade A, 0; grade B, 1–9; or grade C, ³10 LVLs. Lifestyle surveys were conducted using a self-administered questionnaire. Patients were counselled on the need for ceasing alcohol and cigarette consumption by physicians, and were evaluated endoscopical surveillance every 6 months. This study conforms to the STROBE guidelines Findings: Between September 12, 2005 and April 9, 2010, 331 patients with superficial ESCCs were registered. Pooled data from 1,022 healthy subjects were used for comparison. Grades of LVL were positively associated with alcohol drinking intensity, flushing reactions, smoking, high-temperature food and negatively with eating green/yellow vegetables and fruit. Secondary ESCCs and HNSCCs were significantly more prevalent in the grade C LVL group (cumulative 5-y incidences 47.1%, 95% confidence interval [CI], 38.0 to 57.2, and 13.3%, 95% CI, 8.1 to 21.5, respectively). Ceasing alcohol and cigarette use significantly reduced the development of secondary ESCCs (adjusted hazard ratios 0.47, 95% CI, 0.26 to 0.85 and 0.49, 95% CI, 0.26 to 0.91, respectively) Interpretation: Alcohol and cigarette use were closely associated with field cancerization and cessation of them significantly reduced the risk of development of second primary cancer. Funding Statement: Grant from National Cancer Center Research and Development Fund 36 by the Ministry of Health, Labour and Welfare of Japan. Declaration of Interests: The authors declared no conflict of interest. Ethics Approval Statement: This study was approved by the institutional review board at each hospital, and we obtained written informed consent from all patients. (UMIN Clinical Trials Registry ID:UMI01676).

Inactivation of homeodomain-interacting protein kinase 2 promotes oral squamous cell carcinoma metastasis through inhibition of P53-dependent E-cadherin expression.

Homeodomain‐interacting protein kinase 2 (HIPK2), a well‐known tumor suppressor, shows contradictory expression patterns in different cancers. This study was undertaken to clarify HIPK2 expression in oral squamous cell carcinoma (OSCC) and to reveal the potential mechanism of HIPK2 involvement in OSCC metastasis. Two hundred and four OSCC tissues, together with paired adjacent normal epithelia, dysplastic epithelia, and lymph node metastasis specimens, were collected to profile HIPK2 expression by immunohistochemical staining. High throughput RNA‐sequencing was used to detect the dysregulated signaling pathways in HIPK2‐deficient OSCC cells. Transwell assay and lymphatic metastatic orthotopic mouse model assay were undertaken to identify the effect of HIPK2 on tumor invasion. Western blotting and luciferase reporter assay were used to examine the HIPK2/P53/E‐cadherin axis in OSCC. Nuclear delocalization of HIPK2 was observed during oral epithelial cancerization progression and was associated with cervical lymph node metastasis and poor outcome. Depletion of HIPK2 promoted tumor cell invasion in vitro and facilitated cervical lymph node metastasis in vivo. According to mRNA‐sequencing, pathways closely related to tumor invasion were notably activated. Homeodomain‐interacting protein kinase 2 was found to trigger E‐cadherin expression by mediating P53, which directly targets the CDH1 (coding E‐cadherin) promoter. Restoring P53 expression rescued the E‐cadherin suppression induced by HIPK2 deficiency, whereas rescued cytoplasmic HIPK2 expression had no influence on the expression of E‐cadherin and cell mobility. Together, nuclear delocalization of HIPK2 might serve as a valuable negative biomarker for poor prognosis of OSCC and lymph node metastasis. The depletion of HIPK2 expression promoted OSCC metastasis by suppressing the P53/E‐cadherin axis, which might be a promising target for anticancer therapies.

S2832 Mucinous Signet-Cell Adenocarcinoma of the Ileum: A Diagnostic Challenge

INTRODUCTION: Malignancies of the small intestine are rare, and signet-ring cell carcinoma is one of the rarest forms of adenocarcinoma that can arise in the small bowel. We present a rare case of a patient who originally presented with abdominal pain and radiographic findings suggestive of ileal congestion and question of an infective process concerning for Crohn’s disease (CD). After an extensive work-up including multiple colonoscopies and computerized tomography scans (CT), the patient underwent an exploratory laparotomy and resection of an ileocolic mass was positive for signet ring cell adenocarcinoma. CASE DESCRIPTION/METHODS: A 68-year-old woman with a new diagnosis of Crohn’s disease presented as a transfer for recurrent severe lower-abdominal pain and weight loss. The patient had initially presented to an outside hospital. Family history was positive for CD. The initial CT scan revealed wall thickening and fat stranding of the terminal ileum with right lower quadrant mesenteric lymph nodes. An initial colonoscopy showed a normal colon with superficial, linear erosions in the distal rectum. The patient’s bloodwork was pertinent for an elevated C-reactive protein and calprotectin. Given these findings, the patient was diagnosed with CD and treated. After multiple readmissions for similar pain and a repeat CT scan showing a possible abscess or phlegmon, the patient was transferred. Upon arrival, the patient was hemodynamically stable with unchanged labs. A repeat colonoscopy was performed and a biopsy of the terminal ileum showed mild inflammatory changes and congestion. The pathological analysis confirmed the presence of SRCC. The patient underwent an esophagogastroduodenoscopy (EGD) to assess for malignancy, which was negative. An exploratory laparotomy was performed along with the resection of an ileocolic mass. Pathology confirmed mucinous adenocarcinoma with signet-ring cell differentiation. The patient was started on FOLFOX regimen. She is currently on her third cycle of chemotherapy and doing well with an aim to cure. DISCUSSION: The aforementioned case illuminated the diagnostic challenge of signet-cell adenocarcinoma of the ileum. This case report is important because it highlights the importance of considering malignancy in the differential diagnosis when newly diagnosed inflammatory bowel disease does not respond to treatment. Despite its rarity, clinicians should maintain a suspicion of SRCC in the absence of histological confirmation.Figure 1.: Tubulovillous adenoma. Cecal polyp showing finger-like projections formed by fibrovascular cores and lined by dysplastic epithelium. Invasive carcinoma is noted identified (H&E stain, 20x).Figure 2.: Invasive signet ring cell carcinoma of the colon with extracellular mucin pools. The tumor is seen invading the muscularis propria (arrow). A ruptured abscess is noted within the muscularis next to the invasive carcinoma (arrowhead) (H&E stain, 20x).Figure 3.: Invasive carcinoma showing signet ring cell morphology, at higher magnifications (H&E stain, Figure D: 400x).

Abstract 1123: Simultaneous p16/ki-67 expression in detection of high-grade squamous intraepithelial lesions, in women with ASC in cervical smears

Abstract Background Immunocytochemical staining for overexpression of the cell-cycle regulatory protein p16INK4a has been shown to be an efficient approach to triage to colposcopy women with cervical smears categorized as Atypical Squamous Cells (ASC). Overexpression of p16 is associated with the transforming activity of the HR-HPV-E7 oncoprotein in cervical dysplasia. Ki-67 is a marker of cell proliferation of dysplastic epithelium. The efficiency of p16 and Ki-67 separately has been widely described. In high-grade squamous intraepithelial lesion (H-SIL) and cancer they can be co-expressed and the identification of cells with double staining reactivity (p16/Ki-67) can be used as an indicator of H-SIL. We aim to evaluate the diagnostic performance of double p16/Ki-67 staining in the detection of underlying H-SIL in cytological smears of women referred to colposcopy due to an ASC cervical smear result. Design Prospective diagnostic test study. It took 18 months to complete the representative number of cases considering the prevalence of ASC in the colposcopy consultation. Samples were included from patients referred to colposcopy with a cytological diagnosis of ASC. Liquid based cytology was taken and processed to obtain a blank cytological preparation. The p16/Ki-67 staining was performed using CINtec Plus™. The immunoreactivity for both markers evidenced by one or more cervical epithelial cells with brown cytoplasmic immunostaining for p16, and nuclear red for Ki-67, was evaluated by two pathologists blind to colposcopy or biopsy results. Biopsies and/or endocervical curettage were obtained when the colposcopist considered necessary and they were considered as gold standard. In cases where there was no visible lesion and no biopsy was taken, colposcopy was considered gold standard and it was classified as: satisfactory/unsatisfactory, negative/normal or positive/abnormal. A high-grade biopsy lesion (≥ CIN2) with H&amp;E was considered as a final diagnosis. Results There were 120 cases included of ASC (114 ASCUS and 6 ASC-H), from women between 18 and 82 years (mean=38.6). 90 (75.0%) cases had histological study: 62 (68.9%) biopsies and 28 (31.1%) endocervical curettages, of which 17 (14.28%) cases had ≥CIN2, and none were cases of invasive cancer. p16/Ki-67 staining was positive in 21 (17.5%) cases, (64.7% sensitivity for ≥CIN2). All the ASC-H cases (3, 100%), and 57.1% of ASCUS cases with ≥CIN2 as final diagnosis were positive to p16/Ki-67. The specificity of p16/Ki-67 dual staining was greater than 90%. Conclusions There was a p16/Ki-67 specificity that exceeded the values ​​published in other studies on this double staining, but, the positivity and sensitivity estimates were lower than reported previously. The variability of p16/Ki-67 staining in HSILs would reflect the heterogeneity of these lesions. Citation Format: Lia Barrios, Luis Alvis, Ines Benedetti. Simultaneous p16/ki-67 expression in detection of high-grade squamous intraepithelial lesions, in women with ASC in cervical smears [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1123.

Predicting transition from oral pre-malignancy to malignancy via Bcl-2 immuno-expression: Evidence and lacunae

Bcl-2 (B cell Lymphoma −2) family comprises of both anti-apoptotic and pro-apoptotic proteins whose altered expression or change in ratio inhibits apoptosis, and promotes tumor progression. The aim of this study is to assess the usefulness of Bcl-2 in distinguishing dysplastic or malignant epithelium from non-dysplastic or normal epithelium to aid in prediction of malignant transformation potential. Material and methodStudy group comprised of 30 cases of clinically diagnosed leukoplakia (OPMD), 15 cases of Oral Squamous Cell Carcinoma (OSCC) and 5 normal tissue samples. The labeling index of Bcl-2 was analyzed in immunohistochemically stained sections. Different statistical tools were used to analyze the data and to compare Bcl-2 expression qualitatively and quantitatively among all the groups. ResultsAn increasing trend of Bcl-2 immunoexpression was observed from normal epithelium to non-dysplastic and from non-dysplastic to dysplastic lesions. In OSCC, the peripheral cells in the differentiating epithelial islands (within the connective tissue) showed Bcl-2 immuno-reactivity, which gradually decreased towards the center. In contrast, intense and diffuse Bcl-2 immuno-reactivity was seen in poorly differentiated carcinoma. But the overall Bcl-2 positivity was less in OSCC as compared to dysplastic lesions. ConclusionIncreased expression of Bcl-2 oncoprotein in sequentially progressing epithelial dysplasia and down-regulation in differentiating carcinoma (well and moderately differentiating OSCC) unveils the clinical relevance of Bcl-2 in early stages of OSCC tumorigenesis. The heterogenous expression of Bcl-2 in carcinoma with different grades of differentiation renders them unable to be used as an independent tool for predicting transition from oral pre-malignancy to malignancy.

Zeb2 drives invasive and microbiota-dependent colon carcinoma.

Colorectal cancer (CRC) is highly prevalent in Western society, and increasing evidence indicates strong contributions of environmental factors and the intestinal microbiota to CRC initiation, progression and even metastasis. We have identified a synergistic inflammatory tumor-promoting mechanism through which the resident intestinal microbiota boosts invasive CRC development in an epithelial-to-mesenchymal transition-prone tissue environment. Intestinal epithelial cell (IEC)-specific transgenic expression of the epithelial-to-mesenchymal transition regulator Zeb2 in mice (Zeb2IEC-Tg/+) leads to increased intestinal permeability, myeloid cell-driven inflammation and spontaneous invasive CRC development. Zeb2IEC-Tg/+ mice develop a dysplastic colonic epithelium, which progresses to severely inflamed neoplastic lesions while the small intestinal epithelium remains normal. Zeb2IEC-Tg/+ mice are characterized by intestinal dysbiosis, and microbiota depletion with broad-spectrum antibiotics or germ-free rederivation completely prevents cancer development. Zeb2IEC-Tg/+ mice represent the first mouse model of spontaneous microbiota-dependent invasive CRC and will help us to better understand host-microbiome interactions driving CRC development in humans.

Ciliated Muconodular Papillary Tumor of the Lung: a Surgical Case Report

We describe a surgical case of ciliated muconodular papillary tumor (CMPT) of the lung. A 48-year-old man presented to our hospital complaining of right chest pain. Computed tomography (CT) revealed a part-solid grand glass nodule (GGN) in the superior segment of the lower lobe (S6) of the right lung. The maximum diameter of the GGN was 7 mm, with the solid portion having a 5 mm diameter. On follow-up CT, performed 14 months after the first examination, the size of the mass was stable, with almost the same extent of pleural indentation. A CT scan obtained 12 years previously had shown no evidence of a tumor. Considering the continued growth of the lesion, an adenocarcinoma was suspected, and a wedge resection of the right lower lobe was performed. The intra-operative pathological diagnosis by frozen section revealed a dysplastic epithelium. Thus, we did not proceed with lobectomy and lymph node dissection. Postoperative pathological diagnosis of CMPT was confirmed. For comparison, we included an overview of 41 cases of CMPT previously published in 17 studies. In these cases, computed tomography was performed over the follow-up period in 13 cases (31.7%). Tumor growth, which mimicked malignancy, was observed in 9 of 13 cases (69.2%). Since it is usually difficult to make a diagnosis pre- and intra-operatively, most cases of CMPT are pathologically diagnosed postoperatively. During surgery for small peripheral pulmonary tumor like lung cancer, which is not definitive in intra-operative pathological diagnosis, CMPT should be considered as one of differential diagnoses.

Amplification of 3q26.2, 5q14.3, 8q24.3, 8q22.3, and 14q32.33 Are Possible Common Genetic Alterations in Oral Cancer Patients.

The lack of clinical biomarkers for head and neck cancer subtypes limits early diagnosis and monitoring of disease progression. This study investigates genetic alterations in clinically identical tumor, tumor-adjacent dysplastic epithelium (TADE) and normal epithelium (NE) in five oral cancer patients to identify differences and commonalities between oral cancer, TADE and NE. A VELscope®Vx device was used to identify TADE and NE surrounding a clinical tumor for analysis of genetic alterations using the OncoScan® assay. One of the tumor samples examined was an “M” class tumor with a high confidence BRAF:p.G469A:c.1406G>C somatic mutation, which is the first to be reported in oral cancer. Another tumor showed mosaicism in genetic alterations, indicating the presence of multiple clones. Overall, each patient's tumor, TADE and NE showed a distinct genetic profile which indicates intertumoral clonal/genetic diversity. Interestingly, four tumors showed gain of 3q26.2, 5q14.3, 8q24.3, 8q22.3, 14q32.33 and loss/LOH in 9p21.3 while all TADE had LOH on 22q11.23. In addition, some genetic alterations progressed from NE through TADE into tumor in individual patients. Furthermore, no molecular event was identified that is common to all NE and/or TADE that progressed into tumor. This pilot study demonstrates the presence of genetic heterogeneity in oral tumorigenesis, and suggests that there might exist some common genetic alterations between tumors and TADE. However, this observation would need to be further investigated and validated in a larger cohort of oral cancer patients for its potential role in oral tumorigenesis.