Neuropathological Applications of Microscopy with Ultraviolet Surface Excitation (MUSE): A Concordance Study of Human Primary and Metastatic Brain Tumors.

Whereas traditional histology and light microscopy require multiple steps of formalin fixation, paraffin embedding, and sectioning to generate images for pathologic diagnosis, Microscopy using Ultraviolet Surface Excitation (MUSE) operates through UV excitation on the cut surface of tissue, generating images of high resolution without the need to fix or section tissue and allowing for potential use for downstream molecular tests. Here, we present the first study of the use and suitability of MUSE microscopy for neuropathological samples. MUSE images were generated from surgical biopsy samples of primary and metastatic brain tumor biopsy samples (n = 27), and blinded assessments of diagnoses, tumor grades, and cellular features were compared to corresponding hematoxylin and eosin (H&E) images. A set of MUSE-treated samples subsequently underwent exome and targeted sequencing, and quality metrics were compared to those from fresh frozen specimens. Diagnostic accuracy was relatively high, and DNA and RNA integrity appeared to be preserved for this cohort. This suggests that MUSE may be a reliable method of generating high-quality diagnostic-grade histologic images for neuropathology on a rapid and sample-sparing basis and for subsequent molecular analysis of DNA and RNA.

MR imaging findings of a rare pediatric parotid tumor: Mammary analogue secretory carcinoma.

We present magnetic resonance imaging findings of an 11-year-old girl with a mammary analogue secretory carcinoma (MASC) of the parotid gland. MASC is a recently described tumor of the salivary glands that is genetically and histologically similar to secretory breast carcinoma. To date, a few cases have been reported in the pediatric population, with limited information of its imaging characteristics. We suggest that decreased T2 signal of the solid component of the MASC representing cellular components with associated complex cystic parts may be a helpful imaging finding and can make a substantial contribution in differentiating this new entity from other rare pediatric parotid masses. Although there are no characteristic imaging findings at this time, MASC should be considered in the differential of salivary gland tumors in the pediatric population as well.

Digital Whole Slide Imaging Compared With Light Microscopy for Primary Diagnosis in Surgical Pathology.

CONTEXT.­: The adoption of digital capture of pathology slides as whole slide images (WSI) for educational and research applications has proven utility. OBJECTIVE.­: To compare pathologists' primary diagnoses derived from WSI versus the standard microscope. Because WSIs differ in format and method of observation compared with the current standard glass slide microscopy, this study is critical to potential clinical adoption of digital pathology. DESIGN.­: The study enrolled a total of 2045 cases enriched for more difficult diagnostic categories and represented as 5849 slides were curated and provided for diagnosis by a team of 19 reading pathologists separately as WSI or as glass slides viewed by light microscope. Cases were reviewed by each pathologist in both modalities in randomized order with a minimum 31-day washout between modality reads for each case. Each diagnosis was compared with the original clinical reference diagnosis by an independent central adjudication review. RESULTS.­: The overall major discrepancy rates were 3.64% for WSI review and 3.20% for manual slide review diagnosis methods, a difference of 0.44% (95% CI, -0.15 to 1.03). The time to review a case averaged 5.20 minutes for WSI and 4.95 minutes for glass slides. There was no specific subset of diagnostic category that showed higher rates of modality-specific discrepancy, though some categories showed greater discrepancy than others in both modalities. CONCLUSIONS.­: WSIs are noninferior to traditional glass slides for primary diagnosis in anatomic pathology.

Myopericytoma of the Neck Originating From the Middle Scalene: A Case Report.

We report the case of a myopericytoma of the neck. A 23-year-old female noticed a small, nontender mass in her left supraclavicular fossa. The mass grew over a period of 5 months, prompting the patient to seek evaluation. There were no motor or sensory deficits. Imaging suggested a mass originating from the middle scalene muscle. Computed tomography-guided core needle biopsy demonstrated a spindle cell neoplasm with smooth muscle differentiation. Complete surgical excision was performed. Histopathological and immunohistochemical evaluation of the tissue sample suggested myopericytoma. Myopericytoma is an extremely rare tumor of the head and neck. To our knowledge, this is the first reported case of a myopericytoma originating from a scalene muscle.

Intratumor Heterogeneity of the Estrogen Receptor and the Long-term Risk of Fatal Breast Cancer.

Background: Breast cancer patients with estrogen receptor (ER)-positive disease have a continuous long-term risk for fatal breast cancer, but the biological factors influencing this risk are unknown. We aimed to determine whether high intratumor heterogeneity of ER predicts an increased long-term risk (25 years) of fatal breast cancer. Methods: The STO-3 trial enrolled 1780 postmenopausal lymph node-negative breast cancer patients randomly assigned to receive adjuvant tamoxifen vs not. The fraction of cancer cells for each ER intensity level was scored by breast cancer pathologists, and intratumor heterogeneity of ER was calculated using Rao's quadratic entropy and categorized into high and low heterogeneity using a predefined cutoff at the second tertile (67%). Long-term breast cancer-specific survival analyses by intra-tumor heterogeneity of ER were performed using Kaplan-Meier and multivariable Cox proportional hazard modeling adjusting for patient and tumor characteristics. Results: A statistically significant difference in long-term survival by high vs low intratumor heterogeneity of ER was seen for all ER-positive patients (P < .001) and for patients with luminal A subtype tumors (P = .01). In multivariable analyses, patients with high intratumor heterogeneity of ER had a twofold increased long-term risk as compared with patients with low intratumor heterogeneity (ER-positive: hazard ratio [HR] = 1.98, 95% confidence interval [CI] = 1.31 to 3.00; luminal A subtype tumors: HR = 2.43, 95% CI = 1.18 to 4.99). Conclusions: Patients with high intratumor heterogeneity of ER had an increased long-term risk of fatal breast cancer. Interestingly, a similar long-term risk increase was seen in patients with luminal A subtype tumors. Our findings suggest that intratumor heterogeneity of ER is an independent long-term prognosticator with potential to change clinical management, especially for patients with luminal A tumors.

Myopericytoma of the neck originating in the middle scalene muscle: A case report.

We report a case of myopericytoma of the neck. A 23-year-old woman noticed a small, nontender mass in her left supraclavicular fossa. The mass had grown over a period of 5 months, prompting her to seek evaluation. On examination, no motor or sensory deficits were present. Imaging suggested that a mass had originated in the middle scalene muscle. Computed-tomography-guided core needle biopsy demonstrated a spindle-cell neoplasm with smooth-muscle differentiation. Complete surgical excision was performed. Histopathologic and immunohistochemical evaluations of the tissue sample suggested a myopericytoma. Myopericytoma is an extremely rare tumor of the head and neck. To the best of our knowledge, this is the first reported case of a myopericytoma originating in a scalene muscle.

Delayed phlegmon with gallstone fragments masquerading as soft tissue sarcoma.

Complications from lost gallstones after cholecystectomy are rare but varied from simple perihepatic abscess to empyema and expectoration of gallstones. Gallstone complications have been reported in nearly every organ system, although reports of malignant masquerade of retained gallstones are few. We present the case of an 87-year-old woman with a flank soft tissue tumor 4 years after laparoscopic cholecystectomy. The initial clinical, radiographic and biopsy findings were consistent with soft tissue sarcoma (STS), but careful review of her case in multidisciplinary conference raised the suspicion for retained gallstones rather than STS. The patient was treated with incisional biopsy/drainage of the mass, and gallstones were retrieved. The patient recovered completely without an extensive resectional procedure, emphasizing the importance of multidisciplinary sarcoma care to optimize outcomes for potential sarcoma patients.

Comparing Raman and fluorescence lifetime spectroscopy from human atherosclerotic lesions using a bimodal probe.

Fluorescence lifetime imaging (FLIm) and Raman spectroscopy are two promising methods to support morphological intravascular imaging techniques with chemical contrast. Both approaches are complementary and may also be used in combination with OCT/IVUS to add chemical specificity to these morphologic intravascular imaging modalities. In this contribution, both modalities were simultaneously acquired from two human coronary specimens using a bimodal probe. A previously trained SVM model was used to interpret the fluorescence lifetime data; integrated band intensities displayed in RGB false color images were used to interpret the Raman data. Both modalities demonstrate unique strengths and weaknesses and these will be discussed in comparison to histologic analyses from the two coronary arteries imaged.

Small cell mesothelioma: A rare entity and diagnostic pitfall mimicking small cell lung carcinoma on fine-needle aspiration.

Small cell mesothelioma (SCM) is an extremely rare variant of epithelioid mesothelioma that can be mistaken for other forms of small round blue cell tumors, particularly small cell lung carcinoma (SCLC). Here, we describe a fine-needle aspiration (FNA) from a pleural lesion in a 75-year-old man with a history of known asbestos exposure. The FNA revealed cohesive clusters of uniform small round blue cells with high nuclear-to-cytoplasmic ratio, finely powdery chromatin, small inconspicuous nucleoli, and scant amount of cytoplasm. Mitoses were infrequent and nuclear molding was absent. Immunochemical profile supported a mesothelial origin, which was later confirmed by pleurectomy with a diagnosis of SCM. This report demonstrates the difficulties in cytologic evaluation of lung FNAs in differentiating SCM from SCLC or other small round blue cell tumors. As therapy differs for SCM, early recognition of the cytologic features is essential in making the correct diagnosis needed for appropriate clinical management. Diagn. Cytopathol. 2016;44:526-529. © 2016 Wiley Periodicals, Inc.

Preliminary fsLIBS study on bone tumors.

The aim of this study is to evaluate the capability of femtosecond Laser Induced Breakdown Spectroscopy (fsLIBS) to discriminate between normal and cancerous bone, with implications to femtosecond laser surgery procedures. The main advantage of using femtosecond lasers for surgery is that the same laser that is being used to ablate can also be used for a feedback system to prevent ablation of certain tissues. For bone tumor removal, this technique has the potential to reduce the number of repeat surgeries that currently must be performed due to incomplete removal of the tumor mass. In this paper, we performed fsLIBS on primary bone tumor, secondary tumor in bone, and normal bone. These tissues were excised from consenting patients and processed through the UC Davis Cancer Center Biorepository. For comparison, each tumor sample had a matched normal bone sample. fsLIBS was performed to characterize the spectral signatures of each tissue type. A minimum of 20 spectra were acquired for each sample. We did not detect significant differences between the fsLIBS spectra of secondary bone tumors and their matched normal bone samples, likely due to the heterogeneous nature of secondary bone tumors, with normal and cancerous tissue intermingling. However, we did observe an increase in the fsLIBS magnesium peak intensity relative to the calcium peak intensity for the primary bone tumor samples compared to the normal bone samples. These results show the potential of using femtosecond lasers for both ablation and a real-time feedback control system for treatment of primary bone tumors.

"Score the Core" Web-based pathologist training tool improves the accuracy of breast cancer IHC4 scoring.

Hormone receptor status is an integral component of decision-making in breast cancer management. IHC4 score is an algorithm that combines hormone receptor, HER2, and Ki-67 status to provide a semiquantitative prognostic score for breast cancer. High accuracy and low interobserver variance are important to ensure the score is accurately calculated; however, few previous efforts have been made to measure or decrease interobserver variance. We developed a Web-based training tool, called "Score the Core" (STC) using tissue microarrays to train pathologists to visually score estrogen receptor (using the 300-point H score), progesterone receptor (percent positive), and Ki-67 (percent positive). STC used a reference score calculated from a reproducible manual counting method. Pathologists in the Athena Breast Health Network and pathology residents at associated institutions completed the exercise. By using STC, pathologists improved their estrogen receptor H score and progesterone receptor and Ki-67 proportion assessment and demonstrated a good correlation between pathologist and reference scores. In addition, we collected information about pathologist performance that allowed us to compare individual pathologists and measures of agreement. Pathologists' assessment of the proportion of positive cells was closer to the reference than their assessment of the relative intensity of positive cells. Careful training and assessment should be used to ensure the accuracy of breast biomarkers. This is particularly important as breast cancer diagnostics become increasingly quantitative and reproducible. Our training tool is a novel approach for pathologist training that can serve as an important component of ongoing quality assessment and can improve the accuracy of breast cancer prognostic biomarkers.

Diffuse pulmonary meningotheliomatosis: A diagnostically challenging entity on fine-needle aspiration cytology.

Diffuse pulmonary meningotheliomatosis (DPM) is an exceedingly rare entity consisting of multiple minute pulmonary meningothelial-like nodules profusely involving the lungs. To the best of our knowledge, we present the first cytologic description of this uncommon lesion from a 57-year-old nonsmoking woman. Computerized tomographic-guided fine-needle aspiration cytology from a left upper lobe nodule showed whorled/nested clusters of elongated cells with oval nuclei, clear pseudonuclear inclusions, nuclear grooves/indentations, smooth nuclear contours, fine granular chromatin, inconspicuous nucleoli, and abundant fibrillary cytoplasm with indistinct cell borders. The subsequent pulmonary wedge resections confirmed the diagnosis of DPM. As this condition is exceptionally rare, familiarity with these cytologic features is of the essence to accurately establish this challenging diagnosis.

Fluorescence Lifetime Imaging Combined with Conventional Intravascular Ultrasound for Enhanced Assessment of Atherosclerotic Plaques: an Ex Vivo Study in Human Coronary Arteries.

This study evaluates the ability of label-free fluorescence lifetime imaging (FLIm) to complement intravascular ultrasound (IVUS) for concurrent visualization of human coronary vessel composition, structure, and pathology. Co-registered FLIm and IVUS data from 16 coronary segments were correlated to eight distinct pathological features including thin-cap fibroatheroma (TCFA). The sensitivity, specificity, and positive predictive value for combined FLIm-IVUS (89, 99, 89 %) were better than FLIm (70, 98, 88 %) and IVUS (45, 94, 62 %) alone in distinguishing between pathologies. FLIm can assess compositional changes in luminal surface through variations in fluorescence lifetime values (<3.5 ns for lipid-rich areas; >4 ns for collagen-rich areas) enabling detection of macrophages in fibrous caps (sensitivity, 86 %) and distinguishing between relatively stable thick-cap fibroatheromas and rupture-prone TCFA (sensitivity, 80 %) amongst other features. Current results demonstrate the potential of FLIm-IVUS as a new intravascular method for improved evaluation of plaques that may subsequently aid in guiding coronary intervention.

P16 expression predicts necrotic response among patients with osteosarcoma receiving neoadjuvant chemotherapy.

Although pathologic response to neoadjuvant chemotherapy is highly correlated with survival among patients with osteosarcoma, there are currently no established molecular markers to predict response to chemotherapy. The objective of this study was to investigate the relationship of P16 expression in pretreatment osteosarcoma tumors to pathologic necrotic response after neoadjuvant chemotherapy. A tissue microarray was created from paraffin-embedded pretreatment biopsy specimens of 40 patients with osteosarcoma. Immunohistochemistry was performed with commercially available P16 monoclonal mouse antibody. Expression of P16 was defined as nuclear staining in 30% or greater of cells. Percent tumor necrosis was measured in postchemotherapy resection specimens per established protocols, and 90% or greater tumor necrosis was considered "good." Data were abstracted on age, sex, tumor site, and histologic subtype. Univariate and multivariate analyses were performed. The median age was 15 years, 52% were female, and 35% of tumors were located in the femur. P16 expression was present in 62%. Median posttreatment tumor necrosis was 90%, and 55% of patients experienced "good" chemotherapy response (≥90% necrosis). On univariate analysis, P16 expression correlated positively with median percent necrosis and "good" chemotherapy response (P=.004 and .003, respectively). On logistic regression analysis, P16 expression was independently associated with chemotherapy response after controlling for age, subtype, sex, and location (odds ratio, 43.5; 95% confidence interval, 2.64-708.9; P=.008). In summary, immunohistochemical expression of P16 significantly correlates with chemotherapy response in osteosarcoma. P16 expression may be a useful biomarker to guide treatment selection.

Concordance between original screening and final diagnosis using imager vs. manual screen of cervical liquid-based cytology slides.

OBJECTIVE: To compare agreement of pathologists and cytotechnologists and technologist productivity before and after implementation of the Cytyc ThinPrep Imager. STUDY DESIGN: Using the Cytyc ThinPrep Imaging System, a retrospective analysis was conducted, from the first 6 months in 2004 and in 2005. Total cases in January through June were 79,791 in 2004 and 76,887 in 2005. Data on the number and type of changes from one impression to another were collected in a "confusion matrix". The chi2 test with 1 degree of freedom was used to calculate the significance of the difference between the groups. RESULTS: Changes in diagnosis were most frequently seen in negative for intraepithelial lesion, atypical squamous cells of undetermined significance and low-grade squamous intraepithelial lesion. Interobserver agreement before the imager (weighted kappa) was 0.74 and after was 0.73. CONCLUSIONS: The number of high-grade lesions detected increased with the imager (p < 0.01). Technologist productivity increased by an average of 2.2 slides/hour.

Machine scoring of Her2/neu immunohistochemical stains.

OBJECTIVE: To establish the feasibility of a machine scoring method for her2/neu immunohistochemistry in samples of breast carcinoma. STUDY DESIGN: A total of 65 consecutive cases of breast carcinoma with immunohistochemical stainingfor her2/neu by the Herceptest (Dako Corp., Carpinteria, California, U.S.A.) method (DAB chromogen with hematoxylin counterstain) were analyzed using an Extended Slide Wizard (Tripath Imaging, Inc., Burlington, North Carolina, U.S.A.) workstation running prototype software. Representative fields of view from the positive control, negative control and up to 10 fields from the stained tumor sample were captured interactively with a phased alternating line 3 CCD color camera. To determine the amount of specific membrane staining, chromogen separation of nuclear counterstain and membrane-positive stain was performed based on their respective absorption coefficients in the three color channels. The amount of specific membrane staining was scored based on a training set covering the rangefrom 0 to 3 + staining scores according to Dako. Manual scores of 2 + were tested for amplification by fluorescence in situ hybridization. RESULTS: The automated scoring results correlated highly with the manual scores obtained per the Herceptest (Dako) instructions (R2>.92). The results were obtained in real time in the interactive mode. CONCLUSION: Machine scoring of immunohistochemical stains is practical, rapid and inherently reproducible, especially for samples with 1+ and 2+ manual scores.

Cellularity of liquid-based, thin-layer cervical cytology slides.

OBJECTIVE: To study the cellularity of liquid-based preparations (LBPs) for normal, abnormal and false negative cervical cytology cases. STUDY DESIGN: A series of 1,875 LBPs obtained by a split-sample protocol was examined. Slides had been examined by at least one cytotechnologist. All abnormals were reviewed by at least two cytopathologists. The cellular objects were counted using a fully automated microscope. Cellularity was evaluated for the entire population, normal (< LSIL) slides, abnormal (LSIL+) slides and "false negative" slides. False negatives were identified as those with an initial impression of < LSIL and (1) the reference pathologist's impression of the slide was LSIL+, (2) the simultaneous conventional smear was LSIL+, or (3) a cervical biopsy was LSIL+. Descriptive statistics and graphic comparisons were used. RESULTS: There were 192 confirmed abnormal cases and 1,683 normal cases, of which 53 were false negative. The frequency distributions of cellularity for the entire population and each category were skewed-right nonnormal. Median cell counts of the entire series, normals, abnormals and false negatives were 60,510, 59,822, 70,523 and 64,036 respectively. Cell counts at 2.5 percentiles were 8,677, 7,891, 11,864 and 6,009, respectively. The population of abnormal slides tended to have higher cellularity. The population of false negative slides could not be distinguished by their cellularity. CONCLUSION: Cellularity does not provide assurance of adequacy. Any cellularity criterion should be based on measurement of the prevalence of abnormal cells on abnormal slides.