Association Between Microsatellite Instability and 18F-FDG PET/CT Data in Colorectal Cancer Patients

Aim: Our aim in this study was to evaluate the relationship between microsatellite instability (MSI) status and 18F-FDG PET/CT data in patients with colorectal cancer.Materials and Methods: Our study included 74 patients who underwent PET/CT in preoperative staging with the diagnosis of colorectal cancer and then underwent surgical resection. In the immunohistochemical examination, nuclear staining was considered positive for all antibodies. Normal colon mucosa and lymphocytes in tissue were used as an internal positive control group. The absence of nuclear staining in tumor cells was considered "loss" in "Mismatch Repair Gen" proteins. MSI status of patients was divided into 3 groups according to the occurrence of MLH1, PMS2, MSH2, MSH6 gene proteins, and also the number of MSI genes. It is defined as a high frequency of microsatellite instability (MSI-H) when two or more of the five markers in the tumor DNA were positive. If only one marker was positive, the tumor is termed as low frequency of MSI (MSI-L). And MSS is determined when all of the five markers were negative. Results: While MSI was not detected in 56 of the patients (75.7%), MSI was detected in 18 patients (24.3%). 4 patients (5.4%) had MSH-L, while 14 patients (18.9%) had MSH-H. In the analysis made considering all 3 groups, there was no statistically significant relationship between MSI status and SUVmax (p=0.835). Liver metastases were present in 11 (36.7%) of 30 patients who were metastatic at the time of diagnosis. Distant metastasis incidence was lower in 18F-FDG PET/CT in patients with MSI-H (p=0.010). In addition, a significant correlation was found between the presence of liver metastases and MSI, and liver metastasis was not observed in any of the 14 patients with MSI-H (p=0.041).Conclusion: Although not statistically significant, SUVmax values were found to be higher in patients with MSI-H. In addition, metastasis rates were found to be lower in patients with microsatellite instability in accordance with the literature data.

ANTICANCER ACTIVITY OF NATURAL COMPOUND FROM LEMON GRASS LEAVES EXTRACT ON LUNG CARCINOMA CELL LINE A549: AN IN VITRO STUDY

Lemon grass is a widely cultivated plant, whose extracts are known to possess anti-cancer properties. However, studies related to the effect of different solvent extracts of lemon grass on lung cancer cell lines are scarce. This study was conducted to study the effect of various extracts on the viability of the A549 lung carcinoma cell line. Four solvents (hexane, chloroform, ethyl acetate and ethanol) were used for extraction of lemon grass. The effect of these solvent extracts on A549 cell line was studied. Cell viability was studied by 3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide (MTT) method. The effect of apoptosis on exposure to aforementioned extracts was observed with help of nuclear staining assays and western blotting techniques. Apoptosis was confirmed by nuclear staining using acridine orange ethidium bromide (Ao/EtBr), while pro- and anti-apoptotic cells expressions analysis was conducted using western blotting. All extracts showed cytotoxicity on lung carcinoma cell line, wherein, ethyl acetate and hexane extracts showed up to 48.6±3.8% and 51.7±1.7% viability at 250µg/mL concentration. Up regulation of pro-apoptotic gene expressions like Caspase-3 and inhibition of anti-apoptotic gene expression like Bcl-2 was observed after 24h. An inhibitory concentration (IC50) of 220.01 µg/mL was obtained for the ethyl acetate extract. When observed under fluorescence microscope, stained cells showed an orange red color in the nuclei, indicating the ethyl acetate extract had induced apoptosis potentially for 24 h exposure cells expression, when compared with control cells. The results show that the ethyl acetate extracts were efficient in inhibiting the A549 cell line under in vitro condition, suggesting this extract can be further used as a potential chemotherapy drug.

WT1 and Cyclin D1 Immunohistochemistry: A Useful Adjunct for Diagnosis of Pediatric Small Round Blue Cell Tumors on Small Biopsies

Pediatric small round blue cell tumors (SRBCTs) are a heterogeneous group of neoplasms with overlapping morphological appearance. Accordingly, their diagnosis is one of the most difficult in the field of surgical pathology. The most common tumors include rhabdomyosarcoma, Ewing’s sarcoma, neuroblastoma, lymphoblastic lymphoma and Wilms’ tumor (the blastemal component). Over time their diagnosis has become more difficult due to the increasing use of small biopsies. However, the advent of immunohistochemistry has improved the quality of diagnosis in most cases by the application of an adequate panel of immunomarkers. Recently, WT1 and Cyclin D1 have been shown to be useful in the differential diagnosis of SRBCTs on surgically-resected specimens, showing a diffuse cytoplasmic positivity of the former in all RMSs and a diffuse nuclear staining of the latter in both EWS and NB. The aim of the present study was to investigate the expression of WT1 and Cyclin D1 on small biopsies from a series of 105 pediatric SRBCTs to evaluate their diagnostic utility. Both immunomarkers were differentially expressed, with a diffuse and strong cytoplasmic staining for WT1 limited to all cases of RMS, and a diffuse nuclear staining for cyclin D1 restricted to all cases of EWS and NB. Notably, the expression of WT1 and cyclin D1 was also retained in those cases in which the conventional tumor markers (myogenin, desmin and MyoD1 for RMS; CD99 for EWS; NB84 for NB) were focally expressed or more rarely absent. The present study shows that WT1 and Cyclin D1 are helpful immunomarkers exploitable in the differential diagnosis of pediatric SRBCTs on small biopsies, suggesting their applicability in routine practice.

Hypoxic in vitro culture reduces histone lactylation and impairs pre-implantation embryonic development in mice

Background Dynamic changes of histone posttranslational modifications are important contexts of epigenetic reprograming after fertilization in pre-implantation embryos. Recently, lactylation has been reported as a novel epigenetic modification that regulates various cellular processes, but its role during early embryogenesis has not been elucidated. Results We examined nuclear accumulation of H3K23la, H3K18la and pan histone lactylation in mouse oocytes and pre-implantation embryos by immunofluorescence with specific antibodies. All of the three modifications were abundant in GV stage oocytes, and both H3K23la and pan histone lactylation could be detected on the condensed chromosomes of the MII oocytes, while H3K18la were not detected. After fertilization, the nuclear staining of H3K23la, H3K18la and pan histone lactylation was faint in zygotes but homogeneously stained both of the parental pronuclei. The signal remained weak in the early cleavage stage embryos and increased remarkably in the blastocyst stage embryos. Comparison of the embryos cultured in four different conditions with varying concentrations of oxygen found that H3K23la, H3K18la and pan histone lactylation showed similar and comparable staining pattern in embryos cultured in atmospheric oxygen concentration (20% O2), gradient oxygen concentration (5% O2 to 2% O2) and embryos obtained from in vivo, but the modifications were greatly reduced in embryos cultured in hypoxic condition (2% O2). In contrast, nuclear accumulation of H3K18ac or H3K23ac was not significantly affected under hypoxic condition. Moreover, the developmental rate of in vitro cultured embryo was significantly reduced by low oxygen concentration and small molecule inhibition of LDHA activity led to decreased lactate production, as well as reduced histone lactylation and compromised developmental rate. Conclusions We provided for the first time the dynamic landscape of H3K23la, H3K18la and pan histone lactylation in oocytes and pre-implantation embryos in mice. Our data suggested that histone lactylation is subjected to oxygen concentration in the culture environment and hypoxic in vitro culture reduces histone lactylation, which in turn compromises developmental potential of pre-implantation embryos in mice.

THE ROLE OF EZH2 AND ARID1A IN THE DIAGNOSIS OF FLAT UROTHELIAL LESIONS WITH ATYP

Background. Diagnosis of urothelial carcinoma in situ is of great importance because it has prognostic and therapeutic value.We aim to determine the utility of EZH2 and ARID1A as a new tool in the diagnosis of carcinoma in situ.Material and Methods. This retrospective cross-sectional study included Twenty-four specimens of flat urothelial lesions, twenty specimens of CIS, and 10 of normal adjacent urothelium that was taken by cystoscopic resection biopsy procedure. immunohistochemical expression of EZH2 and ARID1A. were evaluated in all studied cases.Results. All normal urothelium specimens showed high nuclear staining for ARID1A and negative nuclear staining for EZH2. High EZH2 expression was observed in 80 % of CIS specimens compared to 20 % of flat urothelial lesions with atypia (p=0.001 ), while high ARID1A expression was observed in 70.8 % of flat urothelial lesions with atypia compared to 25 % of CIS specimens (p=0.001). EZH2 was more accurate and specific in the diagnosis of carcinoma in situ.Conclusion. EZH2 and ARID1A are promising diagnostic markers for urothelial CIS. EZH2 is more accurate and specific than ARID1A in the diagnosis of carcinoma in situ versus other flat urothelial lesions.

Loss of LKB1-NUAK1 Signalling Enhances NF-κB Activity in a Spheroid Model of High-Grade Serous Ovarian Cancer

High-grade serous ovarian cancer (HGSOC) is an aggressive malignancy often diagnosed at an advanced stage. Although most HGSOC patients respond initially to debulking surgery combined with cytotoxic chemotherapy, many ultimately relapse with platinum-resistant disease. Thus, improving outcomes requires new ways of limiting metastasis and eradicating residual disease. We identified previously that Liver kinase B1 (LKB1) and its substrate NUAK1 are implicated in EOC spheroid cell viability and are required for efficient metastasis in orthotopic mouse models. Here, we sought to identify additional signalling pathways altered in EOC cells due to LKB1 or NUAK1 loss-of-function. Transcriptome analysis revealed that inflammatory signalling mediated by NF-κB transcription factors is hyperactive due to LKB1-NUAK1 loss in HGSOC cells and spheroids. Upregulated NF-κB signalling due to NUAK1 loss suppresses reactive oxygen species (ROS) production and sustains cell survival in spheroids. NF-κB signalling is also activated in HGSOC precursor fallopian tube secretory epithelial cell spheroids, and is further enhanced by NUAK1 loss. Finally, immunohistochemical analysis of OVCAR8 xenograft tumors lacking NUAK1 displayed increased RelB expression and nuclear staining. Our results support the idea that NUAK1 and NF-κB signalling pathways together regulate ROS and inflammatory signalling, supporting cell survival during each step of HGSOC pathogenesis. We propose that their combined inhibition may be efficacious as a novel therapeutic strategy for advanced HGSOC.

Small Bowel Obstruction as a Rare Complication of Progressive Sclerosing Mesenteritis

Introduction/Objective Sclerosing Mesenteritis is an uncommon, idiopathic, localized inflammatory syndrome involving the small intestine and colonic mesentery. It is considered a benign condition that commonly occurs in elderly with a gender predilection for males, and its etiology remains unknown. Small Bowel Obstruction (SBO) is a rare, unexpected, but detrimental complication of progressive Sclerosing Mesenteritis. Herein, we present a case of an enlarging, progressive Sclerosing Mesenteritis with extensive involvement of the small bowel and mesentery requiring two consecutive major surgical interventions. Methods/Case Report A 72-year-old male with Myelodysplastic Syndrome (MDS) and recent history of loop ileostomy due to distal intestinal obstruction secondary to enlarging Sclerosing Mesenteritis, presented to our institution with non-specific symptoms of worsening abdominal pain and multiple episodes of gastrointestinal bleeding. Radiographic investigation revealed SBO and he subsequently underwent exploratory laparotomy resulting in total enterectomy with excision of mesenteric mass, extended right colectomy, Whipple procedure, and gastrostomy. The specimens were sent to pathology for histopathological evaluation and gross examination revealed several bosselated, tan-white, firm and rubbery, fibrotic lesions with associated lobulated fibroadipose tissue. Microscopic examination showed extensive mesenteric fibrosis with dense bundles of collagen fibers, areas of fat necrosis, mucosal ischemia and calcification involving the small bowel and serosal surface of large intestine and peritoneum. The lesional cells showed minimal atypia, mitoses, and lacked the Beta-catenin nuclear staining seen in mesenteric fibromatosis. Given the clinical history and histopathological findings of the lesion, we favored the diagnosis of Sclerosing Mesenteritis. Results (if a Case Study enter NA) N/A Conclusion The etiology of Sclerosing Mesenteritis is not well-understood and there are cases of Sclerosing Mesenteritis reported in the literature in association with trauma, surgery, malignancy, and IgG4-related disease. Our patient’s post-operative history was complicated by short gut syndrome, and he is currently requiring small bowel transplant. We report this case for its unusual and aggressive clinical presentation, and to heighten clinical awareness for detrimental consequences of this seemingly benign condition.

A case of TFE-3-positive non-neoplastic pseudodecidualized endometrium presenting as a cervical mass

Introduction/Objective TFE-3 gene encodes a transcription factor that promotes the expression of genes involved in cell growth and proliferation. Its overactivation can result in oncogenic activity. Although TFE-3 seems to be almost universally expressed in normal tissues, this expression should be at very low levels and strong nuclear expression of TFE-3 is seen almost exclusively in tumors containing or lacking the TFE-3 gene fusion. These include renal cell carcinoma, alveolar soft part sarcomas, epithelioid hemangioendotheliomas, PEComas, granular cell tumour, solid pseudopapillary neoplasm of the pancreas, and ovarian sclerosing stromal tumors. It must be emphasized that only nuclear expression of TFE-3 is of diagnostic value, as non-specific cytoplasmic staining is common. Methods/Case Report A 30-year-old woman with pelvic pain, heavy vaginal bleeding and ureteral stricture on oral contraceptive pill was found to have a cervical mass on exam. Cervical biopsy showed fragments of benign squamous epithelium and polypoid endometrial tissue with atrophic glandular component, stromal pseudodecidualization and abundant mixed inflammation. The stroma was positive for CD10, and negative for P16, desmin, cytokeratin ae1/ae3, CD34, calretinin. There was patchy moderate to strong nuclear staining for TFE-3 (Anti-TFE-3 rabbit monoclonal primary antibody, Cell MarqueTM). No evidence of a neoplastic process was seen, and the overall findings fit with either prolapsed endometrial tissue or endometriosis. TFE-3 by FISH showed no rearrangement of the TFE-3 gene region, ruling out alveolar soft part sarcoma. Results (if a Case Study enter NA) NA Conclusion The Human Protein Atlas, a program mapping all the human proteins in cells and tissues, shows that endometrial stromal and glandular cells can have moderate TFE-3 nuclear expression, using Anti-TFE-3 rabbit polyclonal antibody (Prestige Antibodies ®). In our case, focal strong expression was seen using a monoclonal antibody. In the pathology literature this finding has not been previously reported. Pathologists should be aware of the possibility of strong nuclear expression of TFE-3 in non-neoplastic endometrium to avoid potential misdiagnosis.

Effusion tumor cell (ETC) detection from cytology samples following long-term storage

Introduction/Objective Circulating tumor cells (CTCs) are shed from solid tumors into blood. The RareCyte CTC system involves multiplexed fluorescence whole-slide imaging of blood for antibody-based identification of EpCAM/cytokeratin-positive and CD45-negative CTCs. We have previously established an assay to detect carcinoma effusion tumor cells (ETCs) from remnant pleural fluid samples employing RareCyte, providing a potential avenue for tumor cell detection from any cytology sample. To facilitate this, our aim is to evaluate slide storage conditions. We hypothesize that long-term storage of pleural fluid ThinPrep slides at -20°C prior to RareCyte analysis will yield similar ETC numbers and characteristics to freshly-prepared slides. Methods/Case Report ETCs were identified and counted via RareCyte according to our previously established criteria (EpCAM mean fluorescence intensity (MFI) > 100 arbitrary unit (a.u.), CD45 MFI < 100 a.u., and cellular morphology). We analyzed 7 paired fresh-stored pleural fluid samples. Stored samples were maintained at -20°C for 154 or 385 days. ETCs counts between the fresh and stored conditions were compared using a Wilcoxon signed-rank test. Marker intensities from the 35 EpCAM-brightest cells across one paired fresh-stored sample were compared using a two-tailed independent t-test. The cutoff for significance is p<0.05. Results (if a Case Study enter NA) The median (25%, 75%) number of cells detected in the 7 fresh and stored samples was 36 (14, 129) and 13 (5, 22), respectively, and was not significantly different (p=0.08). Although the MFIs for DAPI nuclear staining (p=0.12) and CD45 (p=0.82) were not significantly different between the fresh and stored 35 EpCAM-brightest cells, cytokeratin (p=0.02) and EpCAM (p<0.00001) MFIs were significantly less in stored samples. Conclusion Detecting ETCs is possible after long-term storage at -20°C despite a significant decrease in EpCAM and cytokeratin MFIs in stored samples. In this small sample, paired ETC counts were not significantly different. These preliminary results provide a foundation for larger studies to define storage conditions.

An Uncommon Case of Colonic Neuroendocrine Carcinoma with Scalp Metastasis

Introduction/Objective Neuroendocrine neoplasms of the colon account for <1% of all colorectal malignancies. While visceral metastasis of neuroendocrine neoplasms is commonly observed, cutaneous distant metastasis has infrequently been reported and correlates with an advance stage and progression of disease. To our knowledge, there have been only 10 cases of neuroendocrine neoplasms with metastasis to the scalp reported in the literature. Herein, we report an unusual case of colonic neuroendocrine carcinoma with scalp metastasis, that can be microscopically indistinguishable from the highly aggressive cutaneous neuroendocrine carcinoma, Merkel Cell Carcinoma. Methods/Case Report A 47-year-old female with a history of ileocecal neuroendocrine carcinoma and status post right hemicolectomy had developed liver metastasis and subsequently had an orthotopic liver transplant. PET scan later revealed multiple areas of increased activity involving the ribs, scalp and cervical lymph node that were concerning for malignancy. The scalp lesion consisted of a 7mm non-tender, mobile, violaceous, erythematous dermal nodule that was clinically concerning for cutaneous metastasis. A skin punch biopsy microscopically revealed a subcutaneous infiltrate of nests composed of neoplastic monotonous blue cells with the classic nuclear “salt and pepper” chromatin and scant eosinophilic cytoplasm. The lesional cells showed positive immunoreactivity for synaptophysin and chromogranin. With the given patient’s clinical history and presentation, the observed histological findings and immunophenotypic expression of the tumor cells supported a diagnosis of metastatic neuroendocrine carcinoma. Results (if a Case Study enter NA) N/A Conclusion Metastatic neuroendocrine carcinoma to the scalp is a rare entity and is infrequently encountered in dermatopathology. Given the location and the gross appearance of the scalp lesion, a wide differential diagnosis would include both benign and malignant tumors. In particular, Merkel Cell Carcinoma can grossly and histologically mimic metastatic colonic neuroendocrine carcinoma. Both entities would show synaptophysin and chromogranin uptake. However, metastatic tumors originating from the colon will demontrate CDX2 and SATB2 nuclear staining. We share this rare case of metastatic colonic neuroendocrine carcinoma as it is an important differential diagnosis for primary cutaneous Merkel Cell Carcinoma.