Pan Cytokeratin Research Articles

Assessment of Tumor Budding in Different Grades of Oral Squamous Cell Carcinoma

Introduction: Oral squamous cell carcinoma (OSCC) is the most common malignancy affecting the oral cavity in which patient prognosis and treatment primarily depend upon histological grading and clinical staging. Tumor budding has been recognized as a morphological marker of tumor invasion, which represents an aggressive feature of epithelial malignancies. This study was done to assess the activity of tumor budding in different grades of OSCC. Materials and Methods: An immunohistochemical study was performed on tissue sections obtained from 30 paraffin-embedded blocks of clinically and histologically diagnosed cases of OSCC. The sections were stained with pan cytokeratin and observed under high-power magnification. Tumor budding activity and cell nest size were assessed in different grades of OSCC. Results: A significant correlation was observed between tumor budding and cell nest size in OSCC. Conclusion: This study suggests the importance of using tumor buds as an additional parameter in the diagnosis and grading of OSCC.

Spatial profiling of ovarian clear cell carcinoma (OCCC) and identification of immune-hot features.

e17543 Background: OCCC is an ovarian cancer subtype with high incidence in Asia. Despite its frequent occurrence as an early-stage disease, optimal therapeutic options for high-risk patients are limited. Recently, immune-hot features have been proposed as an indicator for poor prognosis for early-stage OCCC. Specific patterns of intra-tumoral heterogeneity (ITH) associated with these immune-hot features have yet to be defined. Methods: Formalin-fixed paraffine embedded (FFPE) tumor sections from 10 early-stage primary OCCC patients were included. Digital Spatial Profiling (DSP) of 18 protein targets from the Human Protein Core Panel was conducted by using the nanoString GeoMx system to profile selected regions of interest (ROIs) based on the reference H&E staining morphology. Areas of illumination (AOIs) were defined according to ROI segmentation by the fluorescence signals of pan-cytokeratin (PanCK), CD45, or DNA. The digital signals were quantified by the nanoString nCounter system. Results: Unsupervised hierarchical clustering of 252 AOIs from 229 ROIs showed 5 distinct clusters. PanCK+ AOIs distributed in 5 clusters were found to express combinations of markers suggestive of immune mimicry: granzyme B (GZMB) high epithelial cells (C1-a), immune signal high epithelial cells (C1-b), immune-like cells (C1-c), fibronectin-high epithelial cells (C2-a), and signal-cold epithelial cells (C2-b). CD45+ AOIs were mostly clustered in C1-c. Tumor cells with C1-c and C2-a features were associated with recurrence while those in C1-a were associated with no recurrence (Chi-square p=2.8E-04). Tumor infiltrating immune cells (TIIs), which were found to be more enriched of the macrophage lineage (stronger CD68 expression, p=4.5E-03), were associated with recurrence (66.7% in recurrence vs 14.3% in no recurrence, Chi-Square p=0.098). TIIs appeared in PanCK segments of C1-b and C1-c with higher frequencies (45% in C1-b, 25% in C1-c, Chi-square p=0.046) and in PanCK segments of C1-a with extremely low frequency (0%). Correlating with morphology, tumors with recurrence showed higher frequency of papillary pattern (54.7%, Chi-Square p=1.01E-04) and lower frequency of tubulocystic pattern (18.9%, Chi-Square p=7.21E-03). Plus, TIIs were found with high frequency in ROIs with papillary pattern (65.2%, Chi-Square p=4.99E-04) and low frequency in ROIs with tubulocystic pattern (8.7%, Chi-Square p=6.43E-03). Papillary ROIs, which showed macrophage lineage immune mimicry, high CD68 intensity and low PanCK intensity, were found to have very low frequency of PanCK segments of C1-a (6.2%, Chi-Square p=1.26E-04), but extremely high frequency (100%) of PanCK segments of C1-c. Conclusions: Spatial profiling of early-stage OCCC tumors revealed immune-hot tumor features associated with recurrence. These features included immune mimicry of tumor cells, the presence of TIIs, and a papillary pattern in morphology.

High expression of STAT3 within the tumour-associated stroma predicts poor outcome in breast cancer patients.

Triple-negative breast cancer (TNBC) patients have the poorest clinical outcomes compared to other molecular subtypes of breast cancer. IL6/JAK/STAT3 signalling is upregulated in breast cancer; however, there is limited evidence for its role in TNBC. This study aimed to assess the expression of IL6/JAK/STAT3 in TNBC as a prognostic biomarker. Tissue microarrays consisting of breast cancer specimens from a retrospective cohort (n = 850) were stained for IL6R, JAK1, JAK2 and STAT3 via immunohistochemistry. Staining intensity was assessed by weighted histoscore and analysed for association with survival/clinical characteristics. In a subset of patients (n = 14) bulk transcriptional profiling was performed using TempO-Seq. Nanostring GeoMx® digital spatial profiling was utilised to establish the differential spatial gene expression in high STAT3 tumours. In TNBC patients, high expression of stromal STAT3 was associated with reduced cancer-specific survival (HR = 2.202, 95% CI: 1.148-4.224, log rank p = 0.018). TNBC patients with high stromal STAT3 had reduced CD4+ T-cell infiltrates within the tumour (p = 0.001) and higher tumour budding (p = 0.003). Gene set enrichment analysis (GSEA) of bulk RNA sequencing showed high stromal STAT3 tumours were characterised by enrichment of IFNγ, upregulation of KRAS signalling and inflammatory signalling Hallmark pathways. GeoMx™ spatial profiling showed high stromal STAT3 samples. Pan cytokeratin (panCK)-negative regions were enriched for CD27 (p < 0.001), CD3 (p < 0.05) and CD8 (p < 0.001). In panCK-positive regions, high stromal STAT3 regions had higher expression of VEGFA (p < 0.05). High expression of IL6/JAK/STAT3 proteins was associated with poor prognosis and characterised by distinct underlying biology in TNBC.

Long term follow-up results of enucleation as the definitive treatment for melanocytic neuroectodermal tumour of infancy and a review on its treatment.

Melanotic Neuroectodermal tumour of Infancy (MNTI) is a rare entity of pigmented neoplasms of head and neck region. It predominantly occurs within the first year of life. The authors present enucleation as the definitive surgical treatment, with reference to the five departmental cases of MNTI with no recurrence at 5years and 1year of follow-up of other 4 cases. Five cases of MNTI (age group of 2.5months-7 months) presented to our department as a large bluish-brown non tender swelling protruding into the oral cavity. Radiologic imaging revealed a well-circumscribed solid-cystic enhancing lesion causing elevation of orbit and nasal obliteration in maxillary region and causing buccolingual expansion in mandible. The tumor was enucleated without any bony margin. Histopathological and immunohistochemical evaluation (EMA, Pan Cytokeratin, HMB45, S100, p53, ki67) were done. Patients were followed up at regular intervals and had no recurrence at mean 3years follow-up. A detailed mention of surgical pearls, differential diagnosis and a brief literature review are also done. MNTI is a pigmented neoplasm that occurs in infants and in head and neck region mostly involves the upper alveolus and maxilla, followed by skull and mandible. Incisional biopsy is needed to confirm the tumour and rule out other malignant round cell tumours. Enucleation of the lesion is necessary without the need for any extra bony margin removal. Close long term follow up is necessary. Conservative surgical approach is usually the best first choice for MNTI treatment.

Radiation-Induced Undifferentiated Pleomorphic Sarcoma after Breast Conserving Therapy: Case Report

Introduction: Breast sarcoma is a rare form of malignancy that arises from connective tissue, comprising less than 5% of all sarcomas. Undifferentiated pleomorphic sarcoma (UPS) of the breast is a rare and aggressive subtype of radiation-induced sarcoma that can occur in treated breast cancer patients. The diagnosis is challenging and often missed due to the low incidence, long latency period, unspecific imaging finding, and difficulties in clinical and histological detection. Case report: A 56-year-old woman was diagnosed with early-stage triple-negative breast cancer in 2013 and underwent breast-conserving therapy (BCT). After five years follow-up, she developed mastalgia and breast induration, and after mammography and ultrasound without suspicious lesions, a magnetic resonance imaging (MRI) was performed and showed a highly suggestive malignancy mass measuring 8.0 cm and invading the chest wall. The core biopsy revealed a spindle cells malignant tumor, negative for pan cytokeratin and most of immuno-histochemical markers, suggesting sarcoma, but requiring investigation of surgical specimen to exclude metaplastic carcinoma. She underwent Halsted radical mastectomy, full-thickness left anterior chest wall resection contemplating segments of the 4th and 5th ribs and reconstruction with synthetic mesh. The surgical specimen evidenced a UPS with clear margins. The patient had good postoperative recovery and remains in follow-up with the mastology team. Conclusion: This report shows that radiation-induced sarcomas of the breast can be difficult to diagnose, and how later treatment can demand a major surgery with higher morbidity. Prognosis may improve if detected early.

Abstract 1000: Associations of parity and body mass index with NanoString digital spatial protein profiling in early onset breast cancer

Abstract Purpose: Young women’s breast cancers (YBCs), defined herein as breast cancer (BC) diagnosed at age ≤ 40 years, are etiologically and biologically distinct from postmenopausal BCs, demonstrating a higher proportion of estrogen receptor (ER) negative subtypes and a worse prognosis. Studies have not assessed whether risk factors such as recent parity and body mass index (BMI) are linked to protein expression profiles in YBCs. Accordingly, we examined protein expression assessed with GeoMx® Digital Spatial Profiling (DSP) in 640 YBCs prepared as tissue microarrays (TMAs) in the Young Women’s BC Study (YWBCS). Methods: The YWBCS is a multi-institutional cohort of 1,297 women with incident BC diagnosed at age &amp;lt;40 years identified through medical record review, with risk factor data collected via questionnaires. TMAs including 1,505 cores of 640 YBCs that were collected prior to therapy and were suitable for molecular analysis were evaluated for 72 markers with NanoString GeoMx DSP. Associations of log-transformed biomarker expression with BMI and time from last live birth to BC diagnosis were carried out using linear mixed modeling approaches, accounting for multiple tumor tissue cores per individual. Results: Mean age at BC was 35.5 years (range 17-40). Tumor subtypes based on clinical immunostaining were the following: luminal B 46%; luminal A 36%; triple negative 12% and HER2+ 6%. Of 72 biomarkers tested, 53 (74%) were evaluable after QC and normalization. GeoMx ER and PR protein levels were higher in luminal A and luminal B YBCs than in HER2+ and TNBC (p=2x10-94 and 2x10-52, respectively), supporting the accuracy of the GeoMx assay. Compared to nulliparas or those with most recent birth &amp;gt; 3 years before BC, those who gave birth ≤ 3 years prior to BC had tumors with higher expression of PR (8x10-6) and ER (p=0.006), and lower expression of BCL-2 (p=0.006). In contrast, women with last birth ≥ 6 years prior to BC had higher expression of CD8 (p=0.006) and PD-1 (p=0.001) than nulliparas and those with more recent births. Compared to women with BMI between 18.5 and 29.9, those with higher or lower BMIs had higher expression of CD34 (p=0.0003), CD4 (p=0.0009) and CD14 (p=0.001). BCL-2 expression was higher among women with BMI &amp;lt; 18.5 compared to those with higher values (p=0.005). Women with BMI of 30+ had higher β2-microglobulin levels than those with lower BMIs (p=0.003). Inverse dose-response associations (with higher BMI related to lower levels) were observed for expression of ER (p=0.01), pan cytokeratin (p=0.004) and four proteins in the p13K/AKT signaling pathway (p ≤ 0.01 for each). Conclusions: Preliminarily, we identify differences in hormonal and immune markers in YBCs in relation to timing of births and BMI, suggesting that these factors may contribute to etiologic heterogeneity. Multivariate modeling is ongoing to incorporate molecular subtype and other risk factors. Citation Format: Robert A. Vierkant, Laura M. Pacheco-Spann, Stacey J. Winham, Jennifer M. Kachergus, Ji Shi, Craig Snow, Fergus J. Couch, Janet E. Olson, Chen Wang, Derek C. Radisky, Amy C. Degnim, E. Aubrey Thompson, Laura C. Collins, Kathryn J. Ruddy, Ann H. Partridge, Mark E. Sherman. Associations of parity and body mass index with NanoString digital spatial protein profiling in early onset breast 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 1000.

Abstract 2203: Circulating rare events inform disease progression in patients with upper tract urothelial carcinoma undergoing surgery

Abstract The liquid biopsy presents potential as a non-invasive cancer screening tool, and its role in diagnosing and monitoring the progression of primary upper tract urothelial carcinoma (UTUC) to reveal the tumor’s kinetics remains to be fully understood. This study includes 22 patients diagnosed with UTUC, from which we collected 1 peripheral blood (PB) sample prior to surgery (pre-op; n = 22) and 1 sample at follow-up (post-op; n = 11). Each patient’s sample was analyzed via our enrichment-free third generation comprehensive High-Definition Single Cell Assay (HDSCA3.0) workflow to detect rare events in the PB. Rare events include circulating tumor cells (CTCs) and large extracellular vesicles (LEVs) and are identified via a 4-channel immunofluorescence assay that captures the following morphometric expressions: pan cytokeratin (CK), vimentin (V), CD45/CD31 (CD), and cellular nucleation (which is absent in LEVs) through DAPI (D). Through a matched sample analysis approach via a Wilcoxon signed-rank test, pre-op and post-op liquid biopsy profiles were compared to each other as well as to samples from 50 normal donors (NDs) who had no known pathology. The HDSCA3.0 analysis discovered a highly heterogeneous spread of rare events in PB samples collected from patients with UTUC in both pre-op and post-op time periods and in ND samples. For pre-op vs ND samples, significant differences were found in total rare events, total rare cells, events expressing D|V, D|CK|V|CD, D|V|CD, total LEVs, total CK+ cells, CK LEVs, and events expressing D and D|CK. For post-op vs ND samples, significant differences were in total rare events, total rare cells, events expressing D|V, D|CK|V|CD, total CK+ cells, and events expressing D|CK|V. For pre-op vs post-op samples, significant differences manifested in total LEVs, CK LEVs, and CK|CD LEVs. At follow-up, 3 patients had an increase in rare events, and 8 patients had a decrease in rare events, 4 of the 8 presenting with ND rare event levels. This study has the potential to inform how primary UTUC evolves in patients over time as well as elucidate if rare event expressions may correlate with specific disease states. By analyzing circulating rare events through channel-type classifications, different frequencies at various time points compared to ND samples can assist in clinical decision making for treating UTUC. Citation Format: Salmaan Sayeed, George Courcoubetis, Alireza Ghoreifi, Amy Huang, Jeremy Mason, Inderbir S. Gill, Peter Kuhn, Hooman Djaladat, Stephanie N. Shishido. Circulating rare events inform disease progression in patients with upper tract urothelial carcinoma undergoing surgery [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 2203.

Abstract 3354: Isolation and characterization of circulating tumor cells from ductal carcinoma in situ patients using label free technology

Abstract Ductal carcinoma in situ (DCIS) is an early form of breast cancer that has traditionally been characterized by neoplastic cells that are still confined to the mammary ducts. The 20-year breast cancer mortality rate following a DCIS diagnosis, with or without treatment, remains at 3.3%. Despite the overall success in DCIS treatments preventing local invasive breast cancer recurrence, there are still a substantial number of women who, after a DCIS diagnosis, die from metastatic breast cancer. We currently lack predictive tools to identify the high-risk patients who would benefit most from treatment. Emerging clinical and experimental evidence suggest that breast cancer cells may disseminate throughout the body very early in carcinogenesis. These circulating tumor cells (CTCs), rare cancer cells that are shed from the primary tumor into blood, may offer insights into early cancer progression and help identify high-risk patients. Because CTCs can extravasate from blood into tissue and form secondary tumor sites or dormant niches, they are potential biomarkers of metastasis and recurrence. The labyrinth, a high-throughput inertial microfluidic device, has previously demonstrated success in isolating CTCs through inertial separation due to their larger size when compared to white blood cells. Compared to antigen-based isolation technology, inertial microfluidic devices can isolate heterogeneous CTCs with a variety of surface proteins. Using this technology, we isolated and identified CTCs (DAPI+/Pan cytokeratin+/CD45- cells) in 63.2% (12/19) of DCIS patients with an average of 1.27 CTCs per five mls of blood compared to 0.259 CTCs per five mls of blood found in healthy controls (p =0.0078, unpaired t-test). The majority of healthy controls (15/19) had no detectable CTCs. Additionally, immunofluorescence staining showed that 10.5% of CTCs were EpCAM (Epithelial cell adhesion molecule) positive and 26.3% were positive for vimentin, a mesenchymal marker. Single-cell qPCR revealed potential CTCs with no gene expression of traditional white blood cell markers such as CD45, CD20, CD11B, and CD3D. The majority of these cells expressed proliferation (PKM2, TIMP1) and mesenchymal (SPARC, TGFβ1) markers, which can contribute to the cells’ ability to migrate and metastasize. Preliminary targeted DNA sequencing of eight CTC-enriched blood samples identified copy number variations previously associated with higher grade and poorly differentiated DCIS, and single nucleotide variations previously found in DCIS samples (NOTCH1 c.7541_7542del). Ultimately, this study suggests that DCIS generates CTCs prior to microinvasion. CTC isolation and detection may serve as a biomarker for high-risk DCIS and may also be used to explore mechanisms of early cancer dissemination. Citation Format: Brittany Rupp, Brooke Thanasiu, Neha Nagpal, Yan Hong, Dean E. Brenner, Kirsten Tuck, Kirk Herman, Fariba Behbod, Justin Colacino, Max Wicha, Sunitha Nagrath. Isolation and characterization of circulating tumor cells from ductal carcinoma in situ patients using label free technology [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 3354.

Abstract 4322: AI assisted detection and characterization of tertiary lymphoid structures in patients with colorectal cancer

Abstract Tertiary Lymphoid Structures (TLSs) are ectopic immune cell aggregates that form in response to abnormal physiology and inflammation. TLS presence in the tumor environment has been associated with positive clinical outcomes and response to immune checkpoint inhibition therapy. Using a fluorescent multiplex panel consisting of Pan-cytokeratin (PanCK), Programmed Death Ligand-1 (PD-L1), cluster of differentiation (CD) 8, CD3, and CD68 we developed a HALO-based artificial intelligence (AI) classifier to detect TLSs in colorectal cancer (CRC) tissue. In conjunction with cell-type and spatial analyses, we used the AI TLS classifier to characterize several features of the TLS and peri-TLS environment. We trained the AI classifier using 51 examples of TLSs from 8 patients, and applied it to images of 21 CRC patient tissues. TLSs were detected in 9/21 patient samples. We then used cellular detection and spatial analysis to characterize features of the TLS, including the germinal center (GC), T cell zone and peri-TLS area. We detected a higher cell density (Mean=12250 ±2039 SD; cells/mm2) within the interior of AI-detected GCs relative to the 1st 200 μm outside of the AI-defined GC border (8005±1870). In addition, we examined the spatial distribution of CD3+ and CD8+ T cells relative to the TLS GC. We found elevated CD3+ cell density in the interior of the GC (5784±1649) and within 200 μm of the GC border (5126±2239) which decreased at distances &amp;gt;200 μm from the GC border. We found CD8+ density to be elevated within the 1st 200 μm of the GC border (1903±1182) and declined 400 μm away from the GC border (963±828). The spatial distribution of T cells surrounding an area of high cell density is indicative of a T cell zone surrounding germinal center. Further analysis of the AI-detected TLS GCs indicated that PD-L1+ cell density was highest within the germinal center (355± 534;9) relative to the exterior of the germinal center (1st 150μm outside germinal center:276± 441). We created an AI classifier that can detect TLS(s) within patient tissue in the absence of a B-cell marker and confirmed that our AI classifier was detecting TLSs by characterizing canonical features of TLSs. Further exploration of the TLS and peri-TLS area may offer insights into immune-tumor interaction within the tumor microenvironment, and paired detection of TLSs in a clinical cohort with outcome data may predict patient response to immune checkpoint inhibitor therapy. Citation Format: Samantha Burg, Nathaniel Hart, Astin Powers, Wenjun Zhang. AI assisted detection and characterization of tertiary lymphoid structures in patients with colorectal 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 4322.

Abstract 5592: Expansion and characterization on ALK positive NSCLC circulating tumor cells isolated using a size based inertial microfluidic Labyrinth device

Abstract Introduction Lung cancer (LC) is the leading cause of cancer deaths. 85% of LC are non-small cell lung cancer (NSCLC). Fusions involving anaplastic lymphoma kinase (ALK) are oncogenic drivers in ~ 7% of NSCLC. Previous studies have shown that drug resistance develops within ~2 years after ALK tyrosine kinase inhibitor targeted therapy. Due to resistance mechanisms and the biological heterogeneity that evolves over the treatment of ALK+ NSCLC, it is important to monitor and study those dynamic changes through the clinical treatment course to understand cancer progression. Circulating tumor cells (CTCs) are rare cells, shed from cancer lesions into the bloodstream. They can be isolated from a patient's peripheral blood sample (~20ml) to enable study of tumor characteristics at multiple time points. They are promising biomarkers that not only offer phenotypic and genotypic information, but also present a potential to expand into patient derived ex vivo tumor models. In this study, we use a high throughput label-free microfluidic device, Labyrinth, developed in the Nagrath lab for cell size based CTC enrichment, followed by expansion of CTCs in organoid models, further molecular characterization, and growth in patient-derived xenograft (PDX) models. Methods Blood samples were collected from patients (n=22), three with known ALK status. CTC isolation was performed using Labyrinth at 2.0 mL/min. A part of the isolated CTCs were immunofluorescently stained for pan cytokeratin (pan CK), CD45, DAPI, epithelial cell adhesion molecule (EpCAM), and vimentin markers for enumeration. Others were processed for expansion in ultralow attachment plates with three types of organoid culture media. The rest of the CTCs were used for molecular characterizations. Growth curves were produced on expanded samples on Day 0, 7, 14, and 21 in different culture conditions. A live/dead assay and in situ hybridization (FISH) were also performed. On Day 7, selected samples were injected into NSG™ mice. Bulk RNA sequencing was conducted on the original tumor, Day 0 and 7 CTCs, and PDX samples. Results The median CTC concentration in this cohort (n=22) was 316.2 CTCs/ml of blood. Isolated CTCs from ALK+ NSCLC patients showed ALK fusions by FISH. We observed that the number of ALK CTCs was reduced over time consistent with decreased tumor size in CT scans. There were significant differences (p&amp;lt;0.05) for vimentin positive CTCs compared to the other three types of CTCs, which indicated a higher prevalence of a mesenchymal state of NSCLC CTCs. One of the two samples successfully developed into PDX after two months. In summary, we demonstrated a successful workflow to enrich and expand CTCs from ALK+ NSCLC patients. The expanded CTCs can be further characterized and can generate tumors in PDX models. Citation Format: Yuru Chen, Shamileh Fouladdel, Harrison Ball, Xu Cheng, Liwei Bao, Habib Serhan, Albert Liu, Bryce Vandenburg, Laura Goo, Nathan Merrill, Aaron Udager, Angel Qin, Sofia D. Merajver, Sunitha Nagrath. Expansion and characterization on ALK positive NSCLC circulating tumor cells isolated using a size based inertial microfluidic Labyrinth device. [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 5592.

Establishment and preliminary application of organoids in ovarian cancer

Objective: To explore the establishment and application of ovarian cancer organoids. Methods: Fresh ovarian tumor tissues, obtaining from patients underwent surgery in the First Affiliated Hospital of Nanjing Medical University between October 2021 and March 2022, were collected, enzymatic degraded, digested, and embedded into matrigel to establish organoids. A total of 32 ovarian cancer samples were collected. Hematoxylin eosin (HE) staining and immunofluorescence (IF) procedure were used to verify the morphological structure of organoids and their expression of molecular markers. 3D cyto-live or dead assay was used to detecte the live or dead cells in organoids. Carboplatin with a concentration ranging from 5 to 80 μmol/L (5, 10, 20, 40, 80 μmol/L) was added to organoids to calculate the 50% inhibitory concentration (IC50) in different organoids. Results: (1) Organoids from a total of 32 patients were established, of which 18 cases could be passaged stably in the long term in vitro, while 14 could be passaged in the short time. The average amplification time of long-term passage in vitro was over 3 months, and the longest reached 9 months. (2) In HE staining, significant nuclei atypia and local micropapillary structures were observed in organoids. IF staining revealed that ovarian cancer organoids expressed molecular markers similar to primary tumor tissues, such as Pan cytokeratin (Pan-CK), p53, paired box gene 8 (PAX8), and Wilms tumor gene 1 (WT1). (3) In 3D cyto-live or dead assay, a large number of apoptotic cells were observed inside and around the organoids after added carboplatin. The sensitivity to carboplatin varied in 18 organoids could amplify in the long term, with an average IC50 of (29.5±15.8) μmol/L. Moreover, IC50 values of 4 organoids derived from patients received neoadjuvant chemotherapy were much higher than the 14 organoids which did not received neoadjuvant chemotherapy [(48.7±11.3) μmol/L vs (24.0±12.1) μmol/L; t=3.429, P=0.022]. Conclusions: Organoids recapitulate ovarian cancers in vitro and could be stably passaged. Organoids derived from patients received neoadjuvant chemotherapy have higher resistance to carboplatin.

Spatially restricted tumour-associated and host-associated immune drivers correlate with the recurrence sites of pancreatic cancer

ObjectiveMost patients with pancreatic ductal adenocarcinoma (PDAC) will experience recurrence after resection. Here, we investigate spatially organised immune determinants of PDAC recurrence.DesignPDACs (n=284; discovery cohort) were classified according to recurrence...

Accurate prostate cancer detection based on enrichment and characterization of prostate cancer specific circulating tumor cells.

The low specificity of serum PSA resulting in the inability to effectively differentiate prostate cancer from benign prostate conditions is a persistent clinical challenge. The low sensitivity of serum PSA results in false negatives and can miss high-grade prostate cancers. We describe a non-invasive test for detection of prostate cancer based on functional enrichment of prostate adenocarcinoma associated circulating tumor cells (PrAD-CTCs) from blood samples followed by their identification by immunostaining for pan-cytokeratins (PanCK), prostate specific membrane antigen (PSMA), alpha methyl-acyl coenzyme-A racemase (AMACR), epithelial cell adhesion molecule (EpCAM), and common leucocyte antigen (CD45). Analytical validation studies were performed to establish the performance characteristics of the test using VCaP prostate cancer cells spiked into healthy donor blood (HDB). The clinical performance characteristics of the test were evaluated in a case-control study with 160 known prostate cancer cases and 800 healthy males, followed by a prospective clinical study of 210 suspected cases of prostate cancer. Analytical validation established analyte stability as well as acceptable performance characteristics. The test showed 100% specificity and 100% sensitivity to differentiate prostate cancer cases from healthy individuals in the case control study and 91.2% sensitivity and 100% specificity to differentiate prostate cancers from benign prostate conditions in the prospective clinical study. The test accurately detects PrAD-CTCs with high sensitivity and specificity irrespective of stage, serum PSA or Gleason score, which translates into low risks of false negatives or overdiagnosis. The high accuracy of the test could offer advantages over PSA based prostate cancer detection.

Complete remission of an unusual form of unspecified peripheral T-cell lymphoma: a case report and review of the literature

Introduction: Peripheral T-cell lymphomas have a poor prognosis with less common sustainable remissions after standard anthracycline-based chemotherapy. We report a case of isolated skin involvement of an unspecified peripheral T-cell lymphoma with an unremarkable outcome under chemotherapy. Observation: A 72-year-old hypertensive patient under treatment was seen in a hematology consultation for the management of a postero-lateral mass on theleft-hand first metacarpal, ulcerative-bulging with a clean background, rounded, firm, mobile, painless, measuring around 12 cm in length, and evolving for 4 months. Peripheral lymph nodes and other organs were unremarkable on examination. The patient underwent a biopsy of the mass.Histological examination of the specimen showed a malignant proliferation made up of medium-sized rounded cell layers with reduced cytoplasm and irregular nuclei with nucleated patterns sometimes. The stroma was densely fibrous and mitoses were quite frequent. On immunohistochemistry, the cells were CD3 positive, CD5, CD4, CCD8, CD7, CD25, CD20 and melan A negative. Perforin, FOXP3, CD30, and Pan cytokeratins were also negative. Ki67 was 80%. The hand X-raydidn’tidentify bone involvement. The biological work-up was unremarkable. The thoracic-abdominal-pelvic CT scan was normal. The diagnosis of nonspecific T-lymphoma stage IE Ann Arbor and IPI 2 was set. After 6 cycles of Cyclophosphamide-Doxorubicin-OncovinPrednisone chemotherapy, we obtainedcomplete tumor remission. MRI of the left hand aftermanagement showed a focal subcutaneous thickening opposite the 1st ray, without any underlying mass syndrome. After 3 months, the patient had a good general condition with no clinical recurrence. Conclusion: This case illustrates an unusual location of an unspecified peripheral T-cell lymphoma with a complete response after standard anthracycline-based chemotherapy. The short-term prognosis is unremarkable.

Analysis of clinical features, treatment, and prognosis of primary Xlymphoepithelioma-like carcinoma of the lung.

Primary pulmonary lymphoepithelioma-like carcinoma (PPLELC) is a rare lung malignancy occurring most frequently in young non-smokers from Southeast Asia. Given its low incidence, PPLELC clinical features, treatment methods, and the factors affecting its prognosis remain elusive. To date, PPLELC data are mainly derived from clinical case reports, and no cohort studies are available. Therefore, we retrospectively analyzed a group of PPLELC cases and summarized the clinical features of patients, treatment responses, and the factors affecting patient prognosis. A total of 91 patients having primary pulmonary lymphoepithelioma-like carcinoma were recruited in this study. These included sex, age, place of birth, smoking history, pre-treatment symptoms, tumor location, tumor markers, maximum tumor diameter, treatment regimen, lymph node presence metastasis after an operation, pathological picture, immunohistochemistry, genetic findings, and tumor stage grading. We determined the overall survival (OS), progression-free survival (PFS), basic clinical characteristics, treatment option, treatment response, and recurrence pattern among the patients. In addition, we understood the influence of sex, age, tumor, nodes, and metastases (TNM) stage, tumor size, and surgery over patient prognosis. Primary pulmonary lymphoepithelioma-like carcinoma is more common among young non-smokers, with a slightly higher incidence in women than in men. The expression of Epstein-Barr virus-encoded small RNA (EBER), pancytokeratin (PCK), Cytokeratin 5/6 (CK5/6), and tumor protein 63 (P63) was positive in immunohistochemistry. Serum cytokeratin 19 fragment antigen (CYFRA21-1) and Epstein-Barr DNA (EB-DNA) could be used as markers to diagnose primary pulmonary lymphoepithelioma-like carcinoma. TNM stage and surgery were independent prognostic factors. Primary pulmonary lymphoepithelioma-like carcinoma is rare, showing a good prognosis.

The eyes don't see, what the mind doesn't know

Abstract Introduction/Objective Calcifying fibrous tumor is a rare benign mesenchymal neoplasm. The etiology and pathogenesis of this tumor are uncertain. It has wide anatomical distribution. The tumor is most commonly found in the soft tissues of the extremities in younger individuals. However, in middle aged patients it tends to affect the visceral locations more commonly. In visceral location it can mimic aggressive lesions clinically. The purpose of this report is to describe a case of calcifying fibrous tumor in a 71-year-old female with a history of breast carcinoma who was found to have an incidental small bowel mass on her follow up. Clinically and radiologically and the main diagnostic consideration were metastatic disease and gastrointestinal stromal tumor. Methods/Case Report The patient underwent an open small bowel resection, and a 6.5 cm segment of small bowel was sent to pathology. Grossly, a 2.0 cm tan-pink smooth round submucosal polyploid mass protruding into the lumen, mimicking gastrointestinal stromal tumor was identified. The tumor was hard and serially sectioned to reveal a white, calcified cut surface. Microscopically, the tumor appeared hypocellular and composed of scant spindle cells embedded in a dense, hyalinized and calcified, collagenous stroma. Immunohistochemical stains for pan cytokeratin, DOG1, desmin, S100, CD34, and MUC4 were negative, and diagnosis of calcifying fibrous tumor was rendered. Results (if a Case Study enter NA) NA. Conclusion This case highlights the importance of knowledge of rare entities in providing accurate diagnosis for lesions that can clinically mimic common tumors.

Sekretuar Meningiom: Radyolojik ve Patolojik Olarak Malign Tümörlerle Karıştırılabilen Nadir Bir Antite

AbstractMeningiomas are slow growing, extra axial brain tumors that originating from the meningothelial cells of the arachnoid. Meningiomas are the most common brain tumors, however secretory meningiomas are extremely rare variant and can be confused radiologically and pathologically with malignant tumors. A 48 years old male was presented with a complaint of headache and visual impairment in the left eye. Magnetic resonance imaging revealed an extra axial mass lesion in the left pontocerebellar region, 57x48x30 mm in size, and marked edema around the mass. Microscopically, a tumoral lesion consisting ofuniform meningothelial cells with oval-round nuclei, thin vesiculated chromatin, and moderately eosinophilic cytoplasm was observed. The presence of epithelial differentiation characterized by pseudopsammom structures formed by lumens containing eosinophilic secretion was appointed among the tumor cells. Eosinophilic secretion within the lumens were highlighted with histochemical periodic acid schiff (PAS) stain. Immunoreaction was observed in the pseudopsammom structures and the surrounding tumor cells with carcinoembryonic antigen (CEA), epithelial membrane antigen (EMA) and pan cytokeratin (Pan-CK). Tumor cells were immunopositive with Vimentin and progesterone receptor (PR). Ki67 proliferation index was determined as &amp;lt;1%. No necrosis or mitosis were detected. The tumor was reported as ‘‘secretory meningioma’’. No additional treatment was applied to the case and no recurrence was detected in 32 month follow-up after surgical resection. Although secretory meningiomas are not clinically important due to their benign course, differantial diagnosis is critical, since they can be confused with malign tumors because of marked brain edema radiologically, elevated serum CEA levels and epithelial differentiation.

Antigenicity Preservation Is Related to Tissue Characteristics and the Post-Mortem Interval: Immunohistochemical Study and Literature Review.

The main aim of this study was to investigate the post-mortem proteolytic degradation process of selected tissue antigens and correlate it to the post-mortem interval. During the autopsy of 12 cadavers (time interval ranging 1 day–2 years after death) samples of skin, liver, kidney, and spleen were collected. All samples were formalin-fixed and paraffin-embedded. Four µm paraffin sections were used for hematoxylin–eosin staining and immunohistochemical analysis (Ki67, Vimentin, Pan cytokeratin, and CD20). Data reported here show that immunohistochemical reactivity preservation was related to the characteristics of the tissues. In particular, the most resistant tissue was the skin, where the autolysis phenomena were not appreciable before 5 days. On the contrary, the liver and the spleen underwent early autolysis, while the kidney displayed an early autolysis of the tubules and a late one of the glomeruli. As concerns specific antigens, immunoreactivity was lost earliest for nuclear antigens as compared to cytoplasmic ones. In conclusion, our results demonstrate that immunohistochemical detection of specific antigens may be useful in estimating the post-mortem interval, especially when we need to know whether the post-mortem interval is a few days or more than 7–10 days.

Establishment and characterization of novel autologous pair cell lines from two Indian non‑habitual tongue carcinoma patients.

Oral tongue squamous cell carcinoma (OTSCC) is one of the major causes of fatality in India due to very high percentage of patients with habits of smoking and chewing tobacco and associated products. Being highly heterogeneous in nature, every patient poses a different challenge clinically. To understand disease progression in an improved way, knowledge of cross‑talk between tumor stroma and the tumor cells becomes indispensable. Patient‑derived invitro cell line models are helpful to understand the complexity of diseases. However, they have very low efficiency of establishment from the tumor samples, particularly the cancer‑associated fibroblasts (CAFs). In the present study, two novel autologous pairs were immortalized spontaneously from non‑habitual, HPV‑positive patients, who presented with OTSCC. The epithelial and fibroblast cell lines had typical polygonal and spindle‑shaped morphology, respectively. Positive staining with epithelial specific Pan‑cytokeratin (PanCK) and fibroblast specific protein (FSP‑1) further confirmed their epithelial and fibroblast origin. Unique Short Tandem Repeat (STR) profile of the cultures confirmed their novelty, while the similarity of the STR profiles between the epithelial and fibroblast cells from the same patient, confirmed their autologous nature. DNA analysis revealed aneuploidy of the established cultures. An increase in the tumorigenic potential of the established epithelial cultures upon treatment with CAF‑conditioned medium proved the 'CAF‑ness' of the established fibroblast cells. The established cultures are the first of their kind which would serve as a useful platform in understanding the tumor‑stroma cross‑talk in tongue cancer progression.

Localised cutaneous Amyloidosis Revealed by Immunohistochemistry: an Interesting Diagnostic Tool

A 85-year-old woman had a lightly pruritic firm plaque on the forearm for 28 years. She had no major medical trouble and she didn’t receive any medical treatment before visiting dermatology’s department. Biopsy specimen from the lesion revealed an amorphous a cellular eosinophilic deposits (black star) in the papillary dermis (Figure 1 A and B). The overlying epidermis was of normal thickness without hyperkeratosis (Figure 1 A and B). The deposits were negative for Congo red staining (Figure 1C), without typical green birefrengence on polarizing microscopy (Figure 1D). On immunohistochemistry, they stained diffusely positive with pan cytokeratin AE1/AE3