Definitive diagnosis of canine oral melanocytic neoplasms is often difficult because of variability in pigmentation and cellular pleomorphism. These neoplasms can resemble carcinomas, sarcomas, and round cell neoplasms, which differ in prognosis and treatment. A variety of immunohistochemical antibodies have been used for diagnosis of melanocytic neoplasms in humans and dogs; however, sensitivity and specificity of many markers have not been determined in amelanotic melanocytic neoplasms in dogs. The authors investigated a comprehensive panel of immunohistochemical markers in 49 canine oral amelanotic melanocytic neoplasms-namely, Melan-A, PNL2, HMB-45, microphthalmia transcription factor (MiTF), S-100, tyrosine hydroxylase, tyrosinase, tyrosinase-related proteins 1 and 2 (TRP-1 and TRP-2), and CD34. Ten well-differentiated cutaneous soft tissue spindle cell sarcomas were negative controls. Melan-A, PNL2, TRP-1, and TRP-2 were highly sensitive and 100% specific for the diagnosis of canine oral amelanotic melanocytic neoplasms. S-100 and MiTF showed high sensitivity but were less specific; that is, they also labeled a proportion of the soft tissue spindle cell sarcomas. HMB-45, tyrosinase, and tyrosine hydroxylase were 100% specific but had low sensitivities. CD34 did not label any of the melanocytic neoplasms but did label 80% of the soft tissue spindle cell sarcomas. A cost-effective and efficient immunodiagnostic cocktail containing antibodies against PNL2, Melan-A, TRP-1, and TRP-2 was created that had 100% specificity and 93.9% sensitivity in identifying canine oral amelanotic melanocytic neoplasms. The spindloid variant was the variant with the lowest sensitivity to the cocktail. The likelihood of correctly diagnosing canine oral amelanotic melanocytic neoplasms was dramatically higher when biopsy samples contained ample overlying and adjacent epithelium.

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The initial B cell repertoire contains a considerable proportion of autoreactive specificities. The first major B cell tolerance checkpoint is at the stage of the immature B cell, where receptor editing is the primary mode of eliminating self-reactivity. The cells that emigrate from the bone marrow have a second tolerance checkpoint in the transitional compartment in the spleen. Although it is known that the second checkpoint is defective in lupus, it is not clear whether there is any breakdown in central B cell tolerance in the bone marrow. We demonstrate that receptor editing is less efficient in the lupus-prone strain MRL/lpr. In an in vitro system, when receptor-editing signals are given to bone marrow immature B cells by antiidiotype antibody or after in vivo exposure to membrane-bound self-antigen, MRL/lpr 3-83 transgenic immature B cells undergo less endogenous rearrangement and up-regulate recombination activating gene messenger RNA to a lesser extent than B10 transgenic cells. CD19, along with immunoglobulin M, is down-regulated in the bone marrow upon receptor editing, but the extent of down-regulation is fivefold less in MRL/lpr mice. Less efficient receptor editing could allow some autoreactive cells to escape from the bone marrow in lupus-prone mice, thus predisposing to autoimmunity.

This study was undertaken to determine the frequency, and the clinicopathologic and genetic features, of colon cancers driven by neurotrophic receptor tyrosine kinase (NTRK) gene fusions. Of the 7008 tumors screened for NTRK expression using a pan-Trk antibody, 16 (0.23%) had Trk immunoreactivity. ArcherDx assay detected TPM3-NTRK1 (n=9), LMNA-NTRK1 (n=3), TPR-NTRK1 (n=2) and EML4-NTRK3 (n=1) fusion transcripts in 15 cases with sufficient RNA quality. Patients were predominantly women (median age: 63 y). The tumors involved the right (n=12) and left colon unequally and were either stage T3 (n=12) or T4. Local lymph node and distant metastases were seen at presentation in 6 and 1 patients, respectively. Lymphovascular invasion was present in all cases. Histologically, tumors showed moderate to poor (n=11) differentiation with a partly or entirely solid pattern (n=5) and mucinous component (n=10), including 1 case with sheets of signet ring cells. DNA mismatch repair–deficient phenotype was seen in 13 cases. Tumor-infiltrating CD4/CD8 lymphocytes were prominent in 9 cases. Programmed death-ligand 1 positive tumor-infiltrating immune cells and focal tumor cell positivity were seen in the majority of cases. CDX2 expression and loss of CK20 and MUC2 expression were frequent. CK7 was expressed in 5 cases. No mutations in BRAF, RAS, and PIK3CA were identified. However, other genes of the PI3K-AKT/MTOR pathway were mutated. In several cases, components of Wnt/β-catenin (APC, AMER1, CTNNB1), p53, and TGFβ (ACVR2A, TGFBR2) pathways were mutated. However, no SMAD4 mutations were found. Two tumors harbored FBXW7 tumor suppressor gene mutations. NTRK fusion tumors constitute a distinct but rare subgroup of colorectal carcinomas.

Abstract. Diagnostic laboratories are frequently required to assess the antemortem nutritional condition of deceased animals. The percentage of fat in the bone marrow is used to diagnose starvation because this fat depot is typically the last in the body to be depleted. Diagnosticians rely on measurement of bone marrow adipose content using fat solvent-extraction methods; however, the effects of tissue storage conditions before processing have not been fully assessed. The current study focuses on evaluating the effects of 3 storage conditions (refrigeration [4uC], freezing [220uC], and ambient temperature [9.9-34.4uC]) on the percentage of fat in the bone marrow from 3 species. Equine, bovine, and canine humeri and femurs were removed within 24 hr of death from adult animals in adequate body condition and then stored as described for a minimum of 30-60 days. Bone marrow was harvested from these tissues at the time of necropsy and after 30-60 days. Percentage of fat was measured using an automated solvent extractor. Mean percentage of fat in the bone marrow in initial equine, bovine, and canine samples was 81.75%, 86.33%, and 59.96%, respectively. The results indicate that bovine and equine percentage of fat in bone marrow does not change after 30-60 days, regardless of the storage condition, whereas the fat content from canine tissues varies when stored at ambient temperatures. Results suggest that postmortem interval and environmental conditions of samples must be considered in the postmortem evaluation of bone marrow fat concentration in at least some species.

This study determined the frequency and the clinicopathologic and genetic features of colorectal carcinomas driven by oncogenic fusions of the anaplastic lymphoma kinase gene (ALK). Of the 8150 screened tumors, 12 (0.15%) were immunohistochemically ALK-positive with D5F3 antibody. These cancers harbored CAD-ALK (n=1), DIAPH2-ALK (n=2), EML4-ALK (n=2), LOC101929227-ALK (n=1), SLMAP-ALK (n=1), SPTBN1-ALK (n=4), and STRN-ALK (n=1) fusions, as detected by an RNA-based next-generation sequencing assay. ALK fusion carcinomas were diagnosed mostly in older patients with a 9:3 female predominance (median age: 72 y). All tumors, except a rectal one, occurred in the right colon. Most tumors were stage T3 (n=7) or T4 (n=3). Local lymph node and distant metastases were seen at presentation in 9 and 2 patients. These tumors showed moderate (n=6) or poor (n=3) glandular differentiation, solid medullary growth pattern (n=2), and pure mucinous morphology (n=1). DNA mismatch repair–deficient phenotype was identified in 10 cases. Tumor-infiltrating lymphocytes were prominent in 9 carcinomas. In 4 carcinomas, tumor cells showed strong, focal (n=3), or diffuse programmed death-ligand 1 immunoreactivity. CDX2 expression and loss of CK20 and MUC2 expression were frequent. CK7 was expressed in 5 tumors. Four patients died of disease within 3 years, and 7 were alive with follow-up ranging from 1 to 8 years. No mutations in BRAF, RAS, and in genes encoding components of PI3K-AKT/MTOR pathway were identified. However, 1 tumor had a loss-of-function PTEN mutation. Aberration of p53 signaling, TP53 mutations, and/or nuclear accumulation of p53 protein was seen in 9 cases. ALK fusion colorectal carcinomas are a distinct and rare subtype of colorectal cancers displaying some features of mismatch repair–deficient tumors.

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619 Background: The STARTRK-2 trial is a potentially registration-enabling Phase 2 global basket trial of the tyrosine kinase inhibitor entrectinib in patients with solid tumors harboring NTRK1, NTRK2, NTRK3, ROS1, or ALK gene fusions. Phase 1 studies of entrectinib reported a 79% ORR across multiple histologies in patients with gene fusions of these targets who were naïve to inhibitors of these targets, received an effective dose, and had extracranial disease. Patients harboring these fusions are typically rare in the cancer population ( < 3%); however, they have been seen across a wide variety of GI tumors. In this presentation we report on the occurrence of these fusions in GI tumors, based on Ignyta’s internal and partner testing. We discuss the development of an assay for GI samples and immunohistochemistry (IHC) screening efficiency rates in a clinical cohort of 157 GI samples. Methods: The occurrence of NTRK, ROS1, and ALK gene fusions in GI tumors was studied from Ignyta internal and partner collaborations. We developed a 2-step diagnostic test to identify fusions in FFPE clinical specimens. IHC screening using a pan-receptor tyrosine kinase cocktail of antibodies precedes an RNA-based anchored multiplex PCR next generation sequencing (NGS) assay. Results: IHC positivity rates ranged from 0 to 24 percent depending on histology and averaging 8% positivity rate across 15 tumor locations. The screen is particularly effective in cholangiocarcinoma, colon, esophagus, gastric, peritoneum, rectum and small bowel. It was highest in pancreas (24%). Out of 157 GI samples, no instances were detected where the IHC was screened negative and gene fusions were observed by NGS. The IHC cocktail enriches detection of the patient population for gene fusions over 11-fold and has a 100% negative predictive value. Conclusions: This 2-step assay is an efficient method for detection of gene rearrangements in both high-volume clinical testing and studies of archival formalin-fixed paraffin-embedded specimens. Depending on how it is implemented in clinical trials, the staged approach can substantially decrease the costs and tissue expended for NGS. Screening efficiency may be improved even further as additional diagnostic antibodies become available. Clinical trial information: NCT02568267.