Sialyltransferase ST6GAL-1 mediates resistance to chemoradiation in rectal cancer.

Locally advanced rectal cancer is typically treated with chemoradiotherapy followed by surgery. Most patients do not display a complete response to chemoradiotherapy, but resistance mechanisms are poorly understood. ST6GAL-1 is a sialyltransferase that adds the negatively charged sugar, sialic acid (Sia), to cell surface proteins in the Golgi, altering their function. We therefore hypothesized that ST6GAL-1 could mediate resistance to chemoradiation in rectal cancer by inhibiting apoptosis. Patient-derived xenograft and organoid models of rectal cancer and rectal cancer cell lines were assessed for ST6GAL-1 protein with and without chemoradiation treatment. ST6GAL-1 mRNA was assessed in untreated human rectal adenocarcinoma by PCR assays. Samples were further assessed by Western blotting, Caspase-Glo apoptosis assays, and colony formation assays. The presence of functional ST6GAL-1 was assessed via flow cytometry using the Sambucus nigra lectin, which specifically binds cell surface α2,6-linked Sia, and via lectin precipitation. In patient-derived xenograft models of rectal cancer, we found that ST6GAL-1 protein was increased after chemoradiation in a subset of samples. Rectal cancer cell lines demonstrated increased ST6GAL-1 protein and cell surface Sia after chemoradiation. ST6GAL-1 was also increased in rectal cancer organoids after treatment. ST6GAL-1 knockdown in rectal cancer cell lines resulted in increased apoptosis and decreased survival after treatment. We concluded that ST6GAL-1 promotes resistance to chemoradiotherapy by inhibiting apoptosis in rectal cancer cell lines. More research will be needed to further elucidate the importance and mechanism of ST6GAL-1-mediated resistance.

Videoconference microscopy is a reliable alternative to conventional microscopy in the evaluation of Barrett's esophagus: Zooming into a new era.

Telepathology, practicing pathology from a distance, allows experts to review cases without the need to transfer glass slides. Due to significant intra- and inter-observer variabilities in the histological evaluation of Barrett's esophagus (BE), current guidelines recommend expert consultation in cases of dysplasia. We aimed to determine whether telepathology using microscope videoconferencing can be reliably used for evaluation of BE. Biopsies from 62 patients with endoscopic findings of salmon colored mucosa extending ≥1 cm proximal to the gastroesophageal junction were randomly selected to represent benign esophagus, non-dysplastic BE, low-grade dysplasia, high-grade dysplasia, and adenocarcinoma. Three gastrointestinal-trained pathologists reviewed the cases via videoconference microscopy followed by conventional microscopy. Intra-observer and pairwise inter-observer agreements between the conventional microscopy and videoconference methodologies were calculated for each of the three pathologists using Fleiss-Cohen weighted kappa (K) analysis. The intra-observer agreement for each pathologist's assessment of videoconference microscopy and glass slide readings showed very good reliability (K = 0.94, 95% confidence interval = 0.89-0.99; 0.88, 95% confidence interval = 0.79-0.98; 0.93, 95% confidence interval = 0.90-0.97). Mean pairwise inter-observer agreement was 0.90 for videoconference and 0.91 for conventional microscopy. Diagnosis and grading of BE using videoconference microscopy show similar reliability as conventional microscopy. Based on our findings, we propose that videoconferencing pathology is a valid instrument for evaluating BE.

Developing 3D Organoid Raft Cultures from Patient-Derived Xenografts as Rapid Models to Screen Efficacy of Experimental Therapeutics.

Reliable preclinical models are needed for screening new cancer drugs. Thus, we developed an improved 3D tumor organoid model termed "organoid raft cultures" (ORCs). Development of ORCs involved culturing tumors ex vivo on collagen beds (boats) with grid supports to maintain their morphological structure. The ORCs were developed from patient-derived xenografts (PDXs) of colon cancers excised from immune-deficient mice (NOD/SCID/IL2Rgammanull). We utilized these new models to evaluate the efficacy of an investigational drug, Navitoclax (ABT-263). We tested the efficacy of ABT-263, an inhibitor of BCL-2 family proteins, in these ORCs derived from a PDX that showed high expression of antiapoptotic BCL2 family proteins (BCL-2, BCL-XL, and BCL-W). Hematoxylin and eosin staining evaluation of PDXs and corresponding ORCs indicated the retention of morphological and other histological integrity of ORCs. ORCs treated with ABT-263 showed decreased expression of antiapoptotic proteins (BCL2, BCL-XL and BCL-W) and increased proapoptotic proteins (BAX and PUMA), with concomitant activation of caspase 3. These studies support the usefulness of the ORCs, developed from PDXs, as an alternative to PDXs and as faster screening models.

CDX-2 Expression in Esophageal Biopsies Without Goblet Cell Intestinal Metaplasia May Be Predictive of Barrett's Esophagus.

BACKGROUND: CDX-2 is a nuclear homeobox transcription factor not normally expressed in esophageal and gastric epithelia, reported to highlight intestinal metaplasia (IM) in the esophagus. Pathological absence of goblet cells at initial screening via hematoxylin and eosin (HE) and alcian blue (AB) staining results in patient exclusion from surveillance programs. AIMS: This study aimed to determine whether non-goblet cell IM, as defined by CDX-2 positivity, can be considered to be a precursor to Barrett's esophagus (BE). METHODS: This study received IRB approval (17,284). Patients with gastroesophageal reflux disease (n = 181) who underwent upper-gastrointestinal endoscopy with biopsies of the distal esophagus to rule out BE using HE/AB staining and CDX-2 immunostaining were followed for 3 years. Initial and follow-up staining results were evaluated for age/sex. RESULTS: Differences between development of goblet cell IM in CDX-2-negative and CDX-2-positive groups were evaluated. A Kaplan-Meier curve showed that, out of the 134 patients initially positive for CDX-2, 25 (18.7%) had developed goblet cell IM after 2 years and 106 (79.1%) after 3 years. Conversely, of the 47 patients initially negative for CDX-2, 8 (17.9%) developed goblet cell IM after 24 months and only 11 (23.8%) after 40 to 45 months (P = .049; age-adjusted Cox proportional hazard regression model). CONCLUSION: In cases that are initially AB negative and CDX-2 positive, CDX-2 was demonstrated to have a potential prognostic utility for early detection of progression to BE. CDX-2 expression is significantly predictive for risk of goblet cell IM development 40 to 45 months after initial biopsy.

NUT Midline Carcinoma: A Rare Malignancy.

Nuclear protein of the testis (NUT) midline carcinoma can present in the head, neck, and mediastinum. In general, it presents in young adult men and has a poor prognosis. We report on a case of NUT midline carcinoma of the mediastinum in a man 27 years of age without any prior malignancy. Due to the location of the tumor, mediastinal lymphoma and germ cell tumor were initially considered; however, immunohistochemistry was performed using NUT antibody that revealed it to be NUT midline carcinoma. Although guidelines exist for squamous cell carcinoma of the head, neck, and mediastinum, no such specific guidelines are available for NUT midline carcinoma, which looks morphologically similar to squamous cell carcinoma but behaves more aggressively and carries a poor prognosis.

Immunohistochemistry in the workup of prostate biopsies: Frequency, variation and appropriateness of use among pathologists practicing at an academic center.

OBJECTIVES: We studied the frequency, inter-pathologist variation, appropriateness and utility of immunohistochemistry (IHC) performed on prostate biopsies (PB) to determine the significance of foci of suspicious glands/atypical small acinar proliferations (ASAP). METHODS: We calculated the rate of IHC use and diagnostic rate of ASAP and adenocarcinoma in PB from 01/01/2008 to 06/30/2015 for individual pathologists working in a tertiary academic institution, and correlated them with the pathologists' experience, subspecialization and PB volume with the aim of determining the interpathologist variation and appropriateness of use of IHC according to recently published recommendations, and the usefulness of IHC to resolve foci of ASAP as either benign or adenocarcinoma. RESULTS: IHC was used in 966/2652 (36.4%, 95% CI 33.4-39.4%) PB cases and 1915 of 16,359 (11.7%, 95% CI 11.2%-12.2%) of PB blocks and allowed definitive diagnosis of either benign or malignant in 75.8% (95% CI 73.9-77.7%) of blocks. By pathologist, IHC use rates varied more than twofold (22.8-50.5%); higher use was found for pathologists with genitourinary pathology specialization, higher PB volume and more experience, and correlated with higher rates of both ASAP and adenocarcinoma diagnoses. The use of IHC stains was considered appropriate in 822/966 (85.1%, 95% CI 82.9-87.4%) cases. CONCLUSIONS: Despite the fact that the use of IHC stains was considered useful and deemed appropriate in the majority of cases, it showed wide variation between pathologists, suggesting monitoring of IHC use rates may be useful to standardize its use.

Combination of dual JAK/HDAC inhibitor with regorafenib synergistically reduces tumor growth, metastasis, and regorafenib-induced toxicity in colorectal cancer.

BACKGROUND: Treatment with regorafenib, a multiple-kinase inhibitor, to manage metastatic colorectal cancers (mCRCs) shows a modest improvement in overall survival but is associated with severe toxicities. Thus, to reduce regorafenib-induced toxicity, we used regorafenib at low concentration along with a dual JAK/HDAC small-molecule inhibitor (JAK/HDACi) to leverage the advantages of both JAK and HDAC inhibition to enhance antitumor activity. The therapeutic efficacy and safety of the combination treatment was evaluated with CRC models. METHODS: The cytotoxicity of JAK/HDACi, regorafenib, and their combination were tested with normal colonic and CRC cells exhibiting various genetic backgrounds. Kinomic, ATAC-seq, RNA-seq, cell cycle, and apoptosis analyses were performed to evaluate the cellular functions/molecular alterations affected by the combination. Efficacy of the combination was assessed using patient-derived xenograft (PDX) and experimental metastasis models of CRC. To evaluate the interplay between tumor, its microenvironment, and modulation of immune response, MC38 syngeneic mice were utilized. RESULTS: The combination therapy decreased cell viability; phosphorylation of JAKs, STAT3, EGFR, and other key kinases; and inhibited deacetylation of histone H3K9, H4K8, and alpha tubulin proteins. It induced cell cycle arrest at G0-G1 phase and apoptosis of CRC cells. Whole transcriptomic analysis showed that combination treatment modulated molecules involved in apoptosis, extracellular matrix-receptor interaction, and focal adhesion pathways. It synergistically reduces PDX tumor growth and experimental metastasis, and, in a syngeneic mouse model, the treatment enhances the antitumor immune response as evidenced by higher infiltration of CD45 and cytotoxic cells. Pharmacokinetic studies showed that combination increased the bioavailability of regorafenib. CONCLUSIONS: The combination treatment was more effective than with regorafenib or JAK/HDACi alone, and had minimal toxicity. A clinical trial to evaluate this combination for treatment of mCRCs is warranted.

Intestinal manifestation of Buerger's disease in a middle-age female with subsequent transverse colon perforation: A case report and review of literature.

Thromboangiitis obliterans, or Buerger's disease, is a relatively rare nonatherosclerotic, segmental inflammatory and obliterative vascular disease that affects the small- and medium-sized arteries, veins, and nerves. In the acute phase, the lesion presents as an inflammatory, nonsuppurative panarteritis or panphlebitis with vascular thrombosis without necrosis. In the late stage of the disease, the thrombus becomes organized leading to varying degrees of recanalization and subsequent gangrene and amputation. There have been rare reports of thromboangiitis obliterans with involvement of the gastrointestinal trace and even more unusual is the occurrence of this manifestation of disease in women. Here, we report a case of a 45-year-old female patient with a history of thromboangiitis obliterans who presented with ischemic colitis.

Spindle Cell Sarcoma of the Uterus Harboring MEIS1::NCOA1 Fusion Gene and Mimicking Endometrial Stromal Sarcoma.

MEIS1::NCOA1/2 sarcomas are a newly recognized group of exceedingly rare low-grade spindle cell sarcomas that often involve the genitourinary and gynecologic tracts. Due to its deceptively low-grade morphology and the non-specific immunoprofile, these neoplasms may pose a diagnostic challenge by histologically mimicking other entities such as endometrial stromal sarcoma, smooth muscle tumor, or uterine perivascular epithelioid cell tumor (PEComa). Histologically, MEIS1::NCOA1/2 sarcomas typically show spindle cell proliferation with hyperchromatic nuclei and a generalized cytologic uniformity, arranged in short fascicles and exhibiting alternating zones of hypo- and hypercellularity. Among the previously reported cases, molecular analysis revealed the MEIS1::NCOA2 fusion as the most commonly detected fusion gene, whereas the MEIS1::NCOA1 fusion gene has been reported in only a single case that involved kidney. Herein we report the first case of uterine sarcoma harboring the MEIS1::NCOA1 fusion gene that was initially misclassified as low-grade endometrial stromal sarcoma, demonstrating its clinicopathologic features, and highlighting the essential role of molecular pathology to arrive at the accurate diagnosis that may alter disease classification and inform therapy.

CALFAN (Low γ-Glutamyl Transpeptidase (GGT) Cholestasis, Acute Liver Failure, and Neurodegeneration) Syndrome: A Case Report with 3-Year Follow-Up after Liver Transplantation in Early Adulthood.

CALFAN syndrome is an extremely rare disease consisting of recurrent pediatric acute liver failure (PALF), neurodegenerative diseases, and skeletal abnormalities associated with SCYL1 gene mutation. To date, three of 18 patients reported underwent liver transplantation in infancy and early childhood (7-23 months). Here, we report a case of CALFAN syndrome with infantile onset, recurrent jaundice/PALF requiring liver transplantation in early adulthood. At the most recent follow-up, 3 years after transplantation, the patient is doing well.

Socs3 expression in myeloid cells modulates the pathogenesis of dextran sulfate sodium (DSS)-induced colitis.

Introduction: Myeloid cells play a critical role in the pathogenesis of Inflammatory Bowel Diseases (IBDs), including Ulcerative Colitis (UC) and Crohn's Disease (CD). Dysregulation of the JAK/STAT pathway is associated with many pathological conditions, including IBD. Suppressors Of Cytokine Signaling (SOCS) are a family of proteins that negatively regulate the JAK/STAT pathway. Our previous studies identified that mice lacking Socs3 in myeloid cells developed a hyper-activated phenotype of macrophages and neutrophils in a pre-clinical model of Multiple Sclerosis. Methods: To better understand the function of myeloid cell Socs3 in the pathogenesis of colitis, mice with Socs3 deletion in myeloid cells (Socs3 ΔLysM) were utilized in a DSS-induced colitis model. Results: Our results indicate that Socs3 deficiency in myeloid cells leads to more severe colitis induced by DSS, which correlates with increased infiltration of monocytes and neutrophils in the colon and increased numbers of monocytes and neutrophils in the spleen. Furthermore, our results demonstrate that the expression of genes related to the pathogenesis and diagnosis of colitis such as Il1ß, Lcn2, S100a8 and S100a9 were specifically enhanced in Socs3-deficient neutrophils localized to the colon and spleen. Conversely, there were no observable differences in gene expression in Ly6C+ monocytes. Depletion of neutrophils using a neutralizing antibody to Ly6G significantly improved the disease severity of DSS-induced colitis in Socs3-deficient mice. Discussion: Thus, our results suggest that deficiency of Socs3 in myeloid cells exacerbates DSS-induced colitis and that Socs3 prevents overt activation of the immune system in IBD. This study may provide novel therapeutic strategies to IBD patients with hyperactivated neutrophils.

Immunophenotypic profiles and prognosis for colorectal mucinous adenocarcinomas are dependent on anatomic location.

BACKGROUND: The prognostic value of mucinous adenocarcinomas (MCAs, exhibiting >50% extracellular mucin) of the colorectum, in relation to their anatomic location is not well studied. MATERIALS AND METHODS: We compared MCAs (n = 175) with non-MCAs (NMCAs, n = 1015) and the cancer-specific survival rates were evaluated, based on their anatomic site, by univariate Kaplan-Meier and multivariate Cox methods. Subsets of these tumors were immunostained for MUC1, MUC2, Bcl-2, and p53. RESULTS: MCAs were more commonly found in the right colon, were of high-grade, and were more prevalent in younger patients (<40 years). They exhibited strong expression of MUC2 and Bcl-2 and showed less p53 nuclear staining. In contrast, most NMCAs were low-grade with high expression of MUC1. MCAs of the rectum were associated with poorer outcomes relative to NMCAs (HR 1.85, CI 95% 1.15-2.97), even though the distributions of advanced-stage tumors were similar. CONCLUSION: Late-stage disease and age were poor independent prognostic indicators of cancer-specific deaths across all tumor locations. In summary, rectal MCAs have a poor prognosis.

RNA-seq analysis identifies transcriptomic profiles associated with anal cancer recurrence among people living with HIV.

BACKGROUND: Chemoradiation therapy (CRT) is the standard of care for squamous cell carcinoma of the anus (SCCA), the most common type of anal cancer. However, approximately one fourth of patients still relapse after CRT. METHODS: We used RNA-sequencing technology to characterize coding and non-coding transcripts in tumor tissues from CRT-treated SCCA patients and compare them between 9 non-recurrent and 3 recurrent cases. RNA was extracted from FFPE tissues. Library preparations for RNA-sequencing were created using SMARTer Stranded Total RNA-Seq Kit. All libraries were pooled and sequenced on a NovaSeq 6000. Function and pathway enrichment analysis was performed with Metascape and enrichment of gene ontology (GO) was performed with Gene Set Enrichment Analysis (GSEA). RESULTS: There were 449 differentially expressed genes (DEGs) observed (390 mRNA, 12 miRNA, 17 lincRNA and 18 snRNA) between the two groups. We identified a core of upregulated genes (IL4, CD40LG, ICAM2, HLA-I (HLA-A, HLA-C) and HLA-II (HLA-DQA1, HLA-DRB5) in the non-recurrent SCCA tissue enriching to the gene ontology term 'allograft rejection', which suggests a CD4+ T cell driven immune response. Conversely, in the recurrent tissues, keratin (KRT1, 10, 12, 20) and hedgehog signaling pathway (PTCH2) genes involved in 'Epidermis Development,', were significantly upregulated. We identified miR-4316, that inhibit tumor proliferation and migration by repressing vascular endothelial growth factors, as being upregulated in non-recurrent SCCA. On the contrary, lncRNA-SOX21-AS1, implicated in the progression of many other cancers, was also found to be more common in our recurrent compared to non-recurrent SCCA.Our study identified key host factors which may drive the recurrence of SCCA and warrants further studies to understand the mechanism and evaluate their potential use in personalized treatment.Key MessageOur study used RNA sequencing (RNA-seq) to identify pivotal factors in coding and non-coding transcripts which differentiate between patients at risk for recurrent anal cancer after treatment. There were 449 differentially expressed genes (390 mRNA, 12 miRNA, 17 lincRNA and 18 snRNA) between 9 non-recurrent and 3 recurrent squamous cell carcinoma of anus (SCCA) tissues. The enrichment of genes related to allograft rejection was observed in the non-recurrent SCCA tissues, while the enrichment of genes related to epidermis development was positively linked with recurrent SCCA tissues.

Carcinosarcoma with chondroid differentiation and neuroendocrine carcinoma: unusual triphasic tumour arising from intracholecystic papillary neoplasm of the gall bladder.

Herein, we report a man in his 70s with gallbladder mass. Microscopically, the tumour demonstrated moderately differentiated adenocarcinoma, sarcoma with focal chondroid differentiation and high-grade neuroendocrine tumour component arising in an intracholecystic papillary neoplasm. The patient did not receive adjuvant chemotherapy in the setting of complete surgical resection. Patient presented with extensive metastasis after 47 months and died 3 months later. Due to the low incidence and poor prognosis of this tumour, it is essential to gather all the individual experience-based information. To our knowledge, this is the first reported case of carcinosarcoma arising from intracholecystic papillary-tubular neoplasm.

Benefit of Gene Expression Profiling in Gastrointestinal Neuroendocrine Tumors of Unknown Primary Origin.

BACKGROUND/AIM: Cancer of unknown primary (CUP), representing 3-5% of all newly diagnosed cancers in the United States, is a presumptive, non-definitive diagnosis rendered when a primary tumor site cannot be identified after exhaustive diagnostic evaluation, including cases of neuroendocrine neoplasms (NENs). CUPs are characterized by findings that are challenging to reconcile, including inconclusive immunohistochemical (IHC) stains, an undifferentiated morphologic phenotype, history of multiple cancers, a clinical presentation that is discordant from histologic findings, an atypical distribution of metastases, or lack of expected response to treatment. For a significant subset of NENs (10%), traditional diagnostic evaluation is unable to determine a primary tumor site using histomorphology and IHC stains. Gene expression profiling (GEP) of either mRNA or microRNA is the technique utilized in the three commercially available platforms that provide a prediction of tumor type in cases of diagnostic uncertainty of CUPs, including those with neuroendocrine differentiation. Case Series Report: Here we present four cases of NENs, where the diagnosis based upon histomorphological and IHC features presented a unique challenge that ultimately benefited from the integration of molecular tumor classification using the validated assay. CancerTYPE ID by Biotheranostics is based on a quantitative RT-PCR assay that uses a computational algorithm to measure the collective expression of 92 genes (87 cancer-related genes and 5 control genes). This case series reports five appropriate clinical scenarios that highlight the utility of a GEP-based assay to effectively provide a molecular tumor classification to identify NEN subtypes and tumor primary site of origin. CONCLUSION: These cases demonstrated that the CancerTYPE ID test was able to resolve challenging diagnoses for primary and metastatic NENs. These cases emphasize the clinical need of utilizing a GEP-based assay for determining the anatomic site of origin and NEN subtyping, both essential for the appropriate clinical management of NENs.

Gastric Xanthomatosis Secondary to Lipoprotein X in Primary Biliary Cholangitis.

Primary biliary cholangitis (PBC) is a rare autoimmune disease characterized by intralobular bile duct destruction. Patients typically present with generalized symptoms including fatigue and pruritis, and less commonly, manifestations of lipid deposition including xanthomas and xanthelasmas. We report a case of a 31-year-old female with PBC-associated cirrhosis who had cutaneous xanthelasmas and diffuse gastric xanthomas secondary to hyperlipidemia and lipoprotein X that completely resolved following liver transplantation. While gastric xanthomas have been reported in patients with PBC previously, to our knowledge, this is the first case report of diffuse gastric xanthomas secondary to PBC reported to resolve following liver transplantation, suggesting that liver transplantation is curative for gastric xanthomatosis in patients with PBC-related cirrhosis.