An Undergraduate Course in CRISPR/Cas9-Mediated Gene Editing in Zebrafish.

We have developed a one-credit semester-long research experience for undergraduate students that involves the use of CRISPR/Cas9 to edit genes in zebrafish. The course is available to students at all stages of their undergraduate training and can be taken up to four times. Students select a gene of interest to edit as the basis of their semester-long project. To select a gene, exploration of developmental processes and human disease is encouraged. As part of the course, students use basic bioinformatic tools, design guide RNAs, inject zebrafish embryos, and analyze both the molecular consequences of gene editing and phenotypic outcomes. Over the 10 years we have offered the course, enrollment has grown from less than 10 students to more than 60 students per semester. Each year, we choose a different gene editing strategy to explore based on recent publications of gene editing methodologies. These have included making CRISPants, targeted integrations, and large gene deletions. In this study, we present how we structure the course and our assessment of the course over the past 3 years.

16 year experience with aspiration of noncomplex breast cysts: cytology results with focus on positive cases.

We performed a retrospective review of 5375 aspirations performed during the last 16 years of noncomplex cysts. Cytology results and outcomes following aspirations of simple and complicated cysts performed by the senior author at our institution were reviewed. Complex cysts with associated solid components which were core biopsied are excluded from this review. We present our data as the largest series to date performed by one breast radiologist at a single institution. Our data separate cysts with atypia or malignant cytology into those sampled concurrently with solid neoplasms and those which were isolated lesions. Various technical issues which have not been previously addressed in the literature are discussed. Noncomplex cysts are benign 99% of the time. Cysts with papillary cytology require no further workup. Margin-negative seromas do not require cytologic analysis of fluid. Sixteen malignancies were revealed (0.3%), eight of which were solitary cysts (0.1%). Atypical cytology predicted malignancy in 21%; therefore, atypical cytology requires further workup. Malignant cytology was associated with breast cancer in 90.9% of patients; therefore, all patients with malignant cytology require biopsy.

Unilateral Cysticercosis of the Parotid Gland: Case Report, Diagnosis, and Clinical Management.

Cysticercosis is a systemic parasitic infection caused by the establishment of the larval form of the parasitic cestode, Taenia solium. Cysticercosis is acquired via the fecal-oral route and is prevalent in low- and middle-income countries (LMICs). Patients typically manifest with skeletal muscle, subcutaneous, or central nervous system involvement. Though there are reports of oral mucosa involvement, solitary involvement of the parotid gland is rare. This is a rare case of a 57-year-old man diagnosed with parotid cysticercosis by imaging and FNA. He was successfully treated by anthelminthic therapy and needle aspiration. The patient has been seen back several times. The cyst is not palpable, and he is satisfied. Parotid cysticercosis should be considered in the differential of a parotid mass in patients who have traveled to endemic regions. Though prior reports have indicated the importance of surgical excision, this patient was treated medically.

Molecular subtypes of breast cancer in relation to paclitaxel response and outcomes in women with metastatic disease: results from CALGB 9342.

INTRODUCTION: The response to paclitaxel varies widely in metastatic breast cancer. We analyzed data from CALGB 9342, which tested three doses of paclitaxel in women with advanced disease, to determine whether response and outcomes differed according to HER2, hormone receptor, and p53 status. METHODS: Among 474 women randomly assigned to paclitaxel at a dose of 175, 210, or 250 mg/m2, adequate primary tumor tissue was available from 175. Immunohistochemistry with two antibodies and fluorescence in situ hybridization were performed to evaluate HER2 status; p53 status was determined by immunohistochemistry and sequencing. Hormone receptor status was obtained from pathology reports. RESULTS: Objective response rate was not associated with HER2 or p53 status. There was a trend toward a shorter median time to treatment failure among women with HER2-positive tumors (2.3 versus 4.2 months; P = 0.067). HER2 status was not related to overall survival (OS). Hormone receptor expression was not associated with differences in response but was associated with longer OS (P = 0.003). In contrast, women with p53 over-expression had significantly shorter OS than those without p53 over-expression (11.5 versus 14.4 months; P = 0.002). In addition, triple negative tumors were more frequent in African-American than in Caucasian patients, and were associated with a significant reduction in OS (8.7 versus 12.9 months; P = 0.008). CONCLUSION: None of the biomarkers was predictive of treatment response in women with metastatic breast cancer; however, survival differed according to hormone receptor and p53 status. Triple negative tumors were more frequent in African-American patients and were associated with a shorter survival.

Villous adenoma of the extrahepatic biliary tract: a rare entity.

Patients presenting with large obstructing extrahepatic biliary tumors often are presumed to have cholangiocarcinoma and are labeled with a grim disease with a poor prognosis, given little hope for a cure, and may actually opt for palliative care only. In some instances, however, the diagnosis is that of biliary adenoma (benign until it undergoes malignant degeneration), which can be confirmed via resection and pathologic evaluation of the lesion. Removal of the tumor in its benign stage then provides curative treatment of the obstructing lesion with excellent patient recovery and overall prognosis. We present a rare instance of observation of the presence of high-grade dysplasia in a large villous adenoma arising from the left hepatic duct with relief of biliary obstruction and curative resection.

Primary adnexal adenocarcinoma of the vulva: A diagnosis of exclusion based on location, immunohistochemistry, and pattern of spread.

► Vulvar adenocarcinomas are rare comprising less than 0.1% of primary malignancies of the vulva. ► The diagnosis of primary vulvar adenocarcinomas remains a challenge due to its rarity, variation in histological appearance, and limited literature. ► The basis of the diagnosis includes morphology, immunohistochemistry, clinical history and pattern of spread.

p53 Expression in node-positive breast cancer patients: results from the Cancer and Leukemia Group B 9344 Trial (159905).

PURPOSE: p53 as a prognostic and predictive factor in early-stage breast cancer has had mixed results. We studied p53 protein expression, by immunohistochemistry, in a randomized clinical trial of stage II patients treated with adjuvant doxorubicin and cyclophosphamide with or without paclitaxel [Cancer and Leukemia Group B (CALGB) 9344, INT0148]. PATIENTS AND METHODS: Epithelial p53 expression was evaluated using two immunohistochemical antibodies (DO7 and 1801) in formalin-fixed, paraffin-embedded tissue from patients with node-positive breast cancer who were randomized to four cycles of cyclophosphamide and one of three doses of doxorubicin (60, 75, or 90 mg/m(2); AC) and to receive four subsequent cycles of paclitaxel (T) or not. Prognostic and predictive value of p53 protein expression was assessed, independent of treatment assignment, for escalating doses of doxorubicin or addition of T with endpoints of relapse-free (RFS) and overall survival (OS). RESULTS: Of 3,121 patients, 1,887 patient specimens treated on C9344 were obtained, passed quality control, and evaluated for p53 expression. Expression was 23% and 27% for mAbs 1801 and D07, respectively, with 92% concordance. In univariate analysis, p53 positivity was associated with worse OS with either antibody, but only p53 staining with monoclonal antibody 1801 had significantly worse RFS. In multivariate analysis, p53 was not predictive of RFS or OS from either doxorubicin dose escalation or addition of paclitaxel regardless of the antibody. CONCLUSION: Nuclear staining of p53 by immunohistochemistry is associated with worse prognosis in node-positive patients treated with adjuvant doxorubicin-based chemotherapy but is not a useful predictor of benefit from doxorubicin dose escalation or the addition of paclitaxel.

Cancer and Leukemia Group B Pathology Committee guidelines for tissue microarray construction representing multicenter prospective clinical trial tissues.

Practice-changing evidence requires confirmation, preferably in multi-institutional clinical trials. The collection of tissue within such trials has enabled biomarker studies and evaluation of companion diagnostic tests. Tissue microarrays (TMAs) have become a standard approach in many cooperative oncology groups. A principal goal is to maximize the number of assays with this precious tissue. However, production strategies for these arrays have not been standardized, possibly decreasing the value of the study. In this article, members of the Cancer and Leukemia Group B Pathology Committee relay our experiences as array facility directors and propose guidelines regarding the production of high-quality TMAs for cooperative group studies. We also discuss statistical issues arising from having a proportion of patients available for TMAs and the possibility that patients with TMAs fail to represent the greater study population.

Dendriform pulmonary ossification, a form of diffuse pulmonary ossification: report of a 26-year autopsy experience.

CONTEXT: Dendriform pulmonary ossification (DPO) is an uncommon form of diffuse pulmonary ossification that typically affects the pulmonary interstitium in a setting of interstitial fibrosis. OBJECTIVE: To determine the incidence and characteristics of DPO and correlate the clinical, radiographic, and pathologic features in order to better understand its pathogenesis and behavior and facilitate proper therapy. DESIGN: Adult autopsies performed at a tertiary care center from 1978 to 2004 were reviewed to identify cases of DPO. Clinical, radiographic, and pathologic findings, including ultrastructural studies in select cases, were analyzed and correlated. RESULTS: Eight cases of DPO were identified from 1393 adult autopsies. None of the cases was diagnosed antemortem. The prevalence of DPO was 0.5%, and the incidence was 0.28 cases per year. Most patients had a history of chronic lung disease, and all were 65 years of age or older. CONCLUSIONS: Dendriform pulmonary ossification is an uncommon and unusual entity seen incidentally at autopsy and associated with chronic lung disease. It is well demonstrated in postmortem examination, confirmed by microscopy and ultrastructural study, but rarely diagnosed and virtually never considered clinically. Clinical diagnoses include bronchiectasis and interstitial pneumonitis based on radiographic evidence. With its associated radiographic/pathologic findings, DPO should be considered in the differential diagnosis of chronic lung disease.