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Background Human epidermal growth factor 2 (HER2) amplification and/or overexpression occurs in 12% to 25% of breast cancers. Accurate detection of HER2 is critical in predicting response to HER2-targeted therapy. Both immunohistochemistry (IHC) and in situ hybridization (ISH) are FDA-approved methods for detecting HER2 status because its protein overexpression is largely attributable to gene amplification. However, variable discordant results between IHC and ISH have been reported. Methods We determined the frequency of HER2 IHC/ISH discordance in these patients and also performed a pooled literature review analysis. Results Of the 1125 consecutive primary or metastatic breast cancers with HER2 IHC and ISH performed simultaneously between 2015 and 2020, 84.6% had an unequivocal HER2 status. Discordance was found in 30 cases from 26 patients, including 13 IHC−/ISH+ and 17 IHC+/ISH−, representing 1.6% and 11.9% of IHC− and IHC+ cases, respectively. Review of the literature between 2001 and 2020 identified 46 relevant studies, with a total of 43,468 cases with IHC and ISH performed. The IHC−/ISH+ and IHC+/ISH− discordances were seen in all antibody clones and ISH methods used. The IHC+/ISH− discordance was significantly higher than IHC−/ISH+ (13.8% vs. 3%, P

The most common sarcomas in the thorax are metastasis from an extrathoracic primary malignancy. Primary intrathoracic sarcomas are rare albeit aggressive malignancies that are diagnosed on histopathology. Although a few imaging characteristics have been described that are common to sarcomas, it is still a diagnosis of exclusion as other tumors are much more common. Like elsewhere, primary thoracic sarcomas are also classified according to their histologic features. They are a rare group of tumors that can arise from the mediastinal structures, lung, pleura, or chest wall. On imaging, differentiating these from more common malignancies like lung cancer is difficult and often requires multimodality workup and tissue sampling. A few sarcomas are very specific to their locations, such as angiosarcoma in the right atrium, leiomyosarcoma in the pulmonary artery, where imaging has high accuracy for the diagnosis. Despite being nonspecific in a majority of cases, imaging plays a pivotal role in determining the organ of origin, tumor extent, invasion of adjacent structures, and thus help to assess the surgical resectability. Although sarcomas arising from chest wall are the most common primary sarcomas in the chest, they are excluded from this review to focus only on primary intrathoracic sarcomas. The article provides a comprehensive imaging and pathology review of the rare primary intrathoracic sarcomas, including but not limited to angiosarcoma, Kaposi sarcoma, fibrosarcoma, malignant transformation of fibrous tumor of pleura, sarcomatoid mesothelioma, leiomyosarcoma, and malignant small round blue cell tumors.Key points:Primary intrathoracic sarcomas are rare but clinically important.Imaging helps to determine local extent, invasion, metastases and appropriate site/mode of biopsy.Role of pathology is paramount in diagnosis and guiding treatment based on immunogenetic/molecular typing.

A case of episodic hypertension related to changes in position with the biochemical diagnosis of pheochromocytoma confirmed on imaging. The positional hypertension due to pheochromocytoma was treated by right open adrenalectomy, but the episodic hypertension recurred after 1 year and remained unresolved.

// Shuko Harada 1, 7 , Rebecca Arend 5, 7 , Qian Dai 1 , Jessica A. Levesque 1 , Thomas S. Winokur 1 , Rongjun Guo 1 , Martin J. Heslin 2, 7 , Lisle Nabell 3, 7 , L. Burt Nabors 4, 7 , Nita A. Limdi 4 , Kevin A. Roth 1 , Edward E. Partridge 7 , Gene P. Siegal 1 and Eddy S. Yang 6, 7 1 Department of Pathology, University of Alabama at Birmingham, Birmingham, AL, USA 2 Department of Surgery, University of Alabama at Birmingham, Birmingham, AL, USA 3 Department of Medicine, University of Alabama at Birmingham, Birmingham, AL, USA 4 Department of Neurology, University of Alabama at Birmingham, Birmingham, AL, USA 5 Department of Obsterics and Gynecology, University of Alabama at Birmingham, Birmingham, AL, USA 6 Department of Radiation Oncology, University of Alabama at Birmingham, Birmingham, AL, USA 7 Department of The Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, AL, USA Correspondence to: Eddy S. Yang, email: shyang@uabmc.edu Keywords: precision medicine, targeted therapy, next generation sequencing, molecular tumor board Received: March 09, 2017 Accepted: May 31, 2017 Published: June 14, 2017 ABSTRACT Background: With rapid advances in genomic medicine, the complexity of delivering precision medicine to oncology patients across a university health system demanded the creation of a Molecular Tumor Board (MTB) for patient selection and assessment of treatment options. The objective of this report is to analyze our progress to date and discuss the importance of the MTB in the implementation of personalized medicine. Materials and Methods: Patients were reviewed in the MTB for appropriateness for comprehensive next generation sequencing (NGS) cancer gene set testing based on set criteria that were in place. Because profiling of stage IV lung cancer, colon cancer, and melanoma cancers were standard of care, these cancer types were excluded from this process. We subsequently analyzed the types of cases referred for testing and approved with regards to their results. Results: 191 cases were discussed at the MTB and 132 cases were approved for testing. Forty-six cases (34.8%) had driver mutations that were associated with an active targeted therapeutic agent, including BRAF, PIK3CA, IDH1, KRAS, and BRCA1 . An additional 56 cases (42.4%) had driver mutations previously reported in some type of cancer. Twenty-two cases (16.7%) did not have any clinically significant mutations. Eight cases did not yield adequate DNA. 15 cases were considered for targeted therapy, 13 of which received targeted therapy. One patient experienced a near complete response. Seven of 13 had stable disease or a partial response. Conclusions: MTB at University of Alabama-Birmingham is unique because it reviews the appropriateness of NGS testing for patients with recurrent cancer and serves as a forum to educate our physicians about the pathways of precision medicine. Our results suggest that our detection of actionable mutations may be higher due to our careful selection. The application of precision medicine and molecular genetic testing for cancer patients remains a continuous educational process for physicians.

OBJECTIVES To compare the effects of two methods of formalin-fixed paraffin-embedded (FFPE) tissue harvesting on DNA quality and next-generation sequencing (NGS) quality metrics. METHODS DNA integrity number (DIN) and NGS quality metrics resulting from DNA extraction and sequencing of 199 sequential samples harvested via the Pinpoint Slide DNA Isolation System and the punch method were compared. RESULTS DNA extracted from FFPE tissue punches had higher DIN than that extracted from Pinpoint samples (mean ± SD, 6.18 ± 0.83 vs 5.09 ± 0.91; P < .0001), indicating less degradation. Lower DIN correlated with lower-quality metrics of NGS, that is lower percentage of unique on-target reads, average depth of coverage, and percentage of positions with coverage depth greater than or equal to 100×, 400×, and 1,000×. CONCLUSIONS Our study demonstrated methods to harvest tissue from FFPE blocks may affect quality of DNA, which in turn has an effect on other NGS quality metrics.

Background With the increased use of molecular testing of thyroid fine-needle biopsies, the frequency and extent of thyroid resection for thyroid nodules has changed. Although the role of frozen-section analysis of the thyroid has been reduced markedly in recent years, many surgeons still routinely use it intraoperatively. We sought to determine the utility of frozen section during thyroidectomy in the era of molecular testing. Study Design We reviewed 236 consecutive patients who had thyroidectomy with intraoperative frozen-section analysis at our institution between November 2015 and October 2017. We re-reviewed the preoperative diagnosis, frozen-section diagnosis, final pathology, and whether operative management changed from the initial plan based on frozen section. Results Mean age of the patients was 55.6 ± 14.1 years, and 83% were female. Of the 236 patients, frozen section did not change the intraoperative management in 225 (95%). Of the 11 patients whose thyroid operation was modified, the operation was either too much or not enough in 6 patients. In only 5 (2.1%) patients, frozen-section analysis correctly changed the extent of thyroidectomy. Conclusions Thyroid frozen-section analysis adds cost and time to thyroid operations without notable benefit. In our cohort, only 2.1% of frozen sections accurately changed intraoperative management. We recommend against its routine use.

Adenomatoid tumor is an uncommon benign neoplasm of mesothelial differentiation that distinctively arises in and around the genital organs. In rare instances, it has been described in extragenital locations. There have been only two reports documenting its occurrence in the anterior mediastinum, and no reports documenting its occurrence in the posterior mediastinum. We report the first case of posterior mediastinal adenomatoid tumor. A 37-year-old Caucasian woman presented with symptoms of bronchitis. Imaging studies identified a 2.0 cm posterior mediastinal mass abutting the T9 vertebral body, clinically and radiologically most consistent with schwannoma. Histologic sections revealed a lesion composed of epithelioid cells arranged in cords and luminal profiles embedded in a fibrotic to loose stroma and surrounded by a fibrous pseudocapsule. Lesional cells showed vacuolated eosinophilic cytoplasm and peripherally displaced nuclei with prominent nucleoli. There was focal cytologic atypia but no mitotic figures or necrosis was identified. The lesional cells expressed cytokeratin, calretinin, and nuclear WT1 but were negative for PAX8, TTF1, p53, chromogranin, CD31, and CD34, and Ki67 showed

Summary Pleomorphic hyalinizing angiectatic tumor of soft parts is a recently recognized, low-grade mesenchymal neoplasm of uncertain lineage. It is characterized by clusters of ectatic, fibrin-lined, thin-walled vessels surrounded by pleomorphic but mitotically inert spindled cells with frequent intranuclear inclusions and a variable inflammatory component. Here, we report a case of recurrent pleomorphic hyalinizing angiectatic tumor in a 37-year-old white woman. Touch imprint cytologic analysis revealed oval and spindled cells with fine chromatin, occasional prominent intranuclear inclusions, and intracytoplasmic pigment. The histologic features of the cells constituting the bulk of tumor were those characteristic of a putative precursor lesion ("early pleomorphic hyalinizing angiectatic tumor") as well as another recently described entity known as hemosiderotic fibrohistiocytic lipomatous lesion. Cytogenetic analysis revealed 2 unbalanced translocations involving chromosomes 1 and 3 and chromosomes 1 and 10, with a karyotype of 45,XX,der(1)t(1;3)(p31;q12),-3,der(10)t(1;10)(p31;q25)[11]/46,XX[4], thus suggesting that this disease is neoplastic. The controversies in classification of these lesions are also discussed.

Objective The objective was to identify whether repeat positron emission tomography scan after neoadjuvant chemoradiotherapy in patients with esophageal cancer predicted a complete response. Methods A retrospective study using a prospective database was performed. Patients had esophageal cancer and underwent neoadjuvant chemoradiotherapy, an initial and repeat positron emission tomography, endoscopic ultrasound with fine-needle aspiration (at the same institution), and Ivor Lewis esophagogastrectomy with lymph node resection. Results There were 221 patients who underwent Ivor Lewis, 86 of whom had their initial and repeat positron emission tomography scans performed at the same center. Of these, 37 patients (43%) were complete responders. The median maximum standardized uptake value of esophageal cancer decreased by 72% in the 37 patients who were complete responders, by 58% in the 31 patients who were partial responders, and by 37% in the 18 patients who had a minimal pathologic response. When the maximum standardized uptake value decreased by more than 64%, the patient was likely to be a complete responder ( P = .003, area under the curve=0.75). Conclusion When initial and repeat positron emission tomography scans are performed at the same center at least 30 days after the completion of preoperative chemoradiotherapy, the percent change in the maximum standardized uptake value is a predictor of the response to chemoradiotherapy by a patient with esophageal cancer. When the maximum standardized uptake value decreases by 64% or more, it is likely that the patient is a complete responder. These data may help guide neoadjuvant therapy and identify patients for a future randomized study that compares observation with surgical resection in patients with esophageal cancer who appear to be complete responders.

Assays that conveniently quantify invasion of carcinoma cells in vitro have generally measured the passage of dissociated cells into a matrix. Although these assays have been helpful in identifying relative differences between different carcinoma cell lines or types, the requirement for dissociation overlooks the possible modulation of invasion by cell-cell interactions among carcinoma cells. Described here is a novel assay that quantifies invasion of a matrix placed above intact, multilayered raft cultures of lung carcinoma cell lines A549 and H520. The assay was performed by placing a porous membrane coated with matrix at the air interface of the raft cultures for varying lengths of time, after which the cells invading the matrix were enumerated. The numbers of cells invading increased in a relatively linear fashion from 24 to 72 h, and the absolute numbers within each cell line were reproducible with multiple sets of raft cultures prepared at different times. It was also found that this assay could quantify differences in invasion caused by changes in matrix composition. It is concluded that this assay can reproducibly quantify carcinoma cell invasion from three dimensional raft cultures.

Background: The molecular diagnostics laboratory faces the challenge of improving test turnaround time (TAT). Low and consistent TATs are of great clinical and regulatory importance, especially for molecular virology tests. Laboratory information systems (LISs) contain all the data elements necessary to do accurate quality assurance (QA) reporting of TAT and other measures, but these reports are in most cases still performed manually: a time-consuming and error-prone task. The aim of this study was to develop a web-based real-time QA platform that would automate QA reporting in the molecular diagnostics laboratory at our institution, and minimize the time expended in preparing these reports. Methods: Using a standard Linux, Nginx, MariaDB, PHP stack virtual machine running atop a Dell Precision 5810, we designed and built a web-based QA platform, code-named Alchemy. Data files pulled periodically from the LIS in comma-separated value format were used to autogenerate QA reports for the human immunodeficiency virus (HIV) quantitation, hepatitis C virus (HCV) quantitation, and BK virus (BKV) quantitation. Alchemy allowed the user to select a specific timeframe to be analyzed and calculated key QA statistics in real-time, including the average TAT in days, tests falling outside the expected TAT ranges, and test result ranges. Results: Before implementing Alchemy, reporting QA for the HIV, HCV, and BKV quantitation assays took 45–60 min of personnel time per test every month. With Alchemy, that time has decreased to 15 min total per month. Alchemy allowed the user to select specific periods of time and analyzed the TAT data in-depth without the need of extensive manual calculations. Conclusions: Alchemy has significantly decreased the time and the human error associated with QA report generation in our molecular diagnostics laboratory. Other tests will be added to this web-based platform in future updates. This effort shows the utility of informatician-supervised resident/fellow programming projects as learning opportunities and workflow improvements in the molecular laboratory.

Pulmonary endarterectomy (PEA) can significantly increase long-term survival in patients with chronic thromboembolic pulmonary hypertension; however, the role of PEA for chronic thromboembolic pulmonary hypertension due to pulmonary valve endocarditis is controversial. A critically ill 61-year-old man with intractable right ventricular heart failure was found to have chronic thromboembolic pulmonary hypertension due to Streptococcus bovis pulmonary valve endocarditis and underwent successful pulmonary valve replacement and PEA. The successful outcome in this case suggests that PEA should be considered in patients with this condition.

During the third trimester of her pregnancy, a 25-year-old carrier of Duchenne's muscular dystrophy developed severe cardiac failure and required mechanical circulatory support and transplantation. Her cardiac function improved during 311 days of circulatory support. However this improvement was not sufficient to allow removal of her left ventricular assist device before transplantation.

Telepathology is a maturing technology that, for a variety of reasons, has not been widely deployed. In addition, clinical validation is relatively modest compared with accepted telemedicine applications such as teleradiology. A prototype telepathology system (Tele-Path(sm)) featuring high-resolution images selected from a remote microscope site has been developed at the University of Alabama at Birmingham (UAB). To validate the diagnostic efficacy of the system, a prospective study was undertaken of parallel diagnoses by conventional microscopy and telepathology with a remotely operated microscope. Slides from 99 intraoperative consultations from 29 tissue/ organ sites in the University of Alabama Hospitals by 9 academic pathologists were used in the study. Each microscopic and telepathology diagnosis was compared with the final diagnosis rendered by a referee pathologist. Diagnoses were classified as correct, false positive, or false negative or classification error. Of the 99 frozen sections evaluated, 3 cases were deferred. Of the remaining 96 cases, 2 received incorrect diagnoses in both the microscopic and telepathology arms of the study. Three errors occurred only in the telepathology arm. There was 1 false-positive diagnosis, 1 false-negative diagnosis, and 1 classification error. Statistical analysis indicated no significant difference between telepathology and conventional microscopy. Qualitative data indicated that the pathologists were generally satisfied with the performance of the system. Telepathology using this system paradigm is sufficiently accurate for real time utilization in a complex surgical environment. Telepathology therefore may be an effective model to support the surgical services of hospitals lacking full-time pathology coverage, resulting in full-time access to anatomic pathology services.

The Nottingham histologic grade (NHG) is a prognostic marker for infiltrating ductal carcinoma. Its usefulness for invasive lobular carcinoma (ILC) has been less clear, given that 2 of the 3 parameters, tubule formation and mitotic activity, show little variation in ILC, placing much of the emphasis on nuclear grade. We have previously reported a trend for improved overall and relapse-free survival in patients with ILC of low nuclear grade, as classified by a 2-tiered nuclear grading system. Given the inherent potential for interobserver variability with any grading system, the goal of this study is to compare interobserver variability in the grading of ILC using a 2-tiered nuclear grade vs the NHG. Thirty-eight cases of ILC were graded independently by 5 pathologists using NHG criteria. Tumors were also categorized by a nuclear grading system as low grade (grade 1 nuclei) or high grade (grades 2-3 nuclei). Pairwise kappa values and interobserver agreement rates were calculated for both NHG and nuclear grade. Results were compared using the paired t test. Mean interobserver agreement rates and kappa values improved with use of the nuclear grading system as compared to NHG (83% vs 70%, 0.4738 vs 0.3228, respectively). The differences between the 2 were statistically significant. Because histologic grade has significant prognostic implications for patients with breast cancer, accurate reporting is paramount. For ILC, where use of the NHG places substantial weight on nuclear pleomorphism, a 2-tiered nuclear grading system may reduce interobserver variability yet still provide useful prognostic information.

The effect of using a 30% cutoff for the proportion of HER2+ cells on the interobserver variability in the interpretation of HER2 immunohistochemical results was evaluated. Immunostained sections from 96 cases of breast carcinoma were reviewed by 10 pathologists and scored as positive (3+) when uniform strong membranous staining was identified in at least 10% of tumor cells; the actual percentage of cells with such staining was also estimated. The agreement rates and the kappa values using a 30% cutoff were compared with those using a 10% cutoff. These proved to be higher in 62% and 66% of measurements, respectively, with average interobserver rates and kappa values of 72% and 0.54 using the 30% cutoff and 70% and 0.49 using the 10% cutoff (P=.001 for all comparisons). Using a 30% cutoff for the percentage of HER2+ cells by immunohistochemical analysis modestly decreased interobserver variability in the interpretation of HER2 immunohistochemical results.

Angiomyolipoma is a benign tumor composed of varying proportions of smooth muscle cells, blood vessels, and adipose tissue that most commonly occurs in the kidney. Sporadic lesions and lesions arising in the setting of the tuberous sclerosis complex have been reported in extrarenal sites. We present the case of an incidentally discovered angiomyolipoma in the anterior mediastinum. Thymoma was suspected clinically, and the lesion was composed mainly of spindled-to-epithelioid cells arranged in a histologic pattern reminiscent of hemangiopericytoma, a pattern that has been described in thymoma. Immunohistochemical stains revealed positivity for smooth muscle actin and HMB-45, revealing the expression of smooth muscle and melanocytic markers characteristic of angiomyolipoma and other lesions in the PEComa family.

This report describes the surgical management of a tumor that filled the left chest of a 58-year-old man. Histopathologic examination showed that this was an angiomyolipoma, a tumor that most commonly occurs in the kidney. The preoperative evaluation and intraoperative management are presented, along with a brief review of this unusual neoplasm.

We examined the effects of transforming growth factor-beta (TGF-beta) on the mRNA expression of the antioxidative enzymes, catalase, manganese superoxide dismutase (MnSOD), and copper-zinc superoxide dismutase (CuZnSOD), as well as the oxidative enzyme, xanthine oxidase (XO), in cultures of cardiomyocytes, cardiac non-myocytes, and fetal bovine heart endothelial cells. TGF-betas alone had little effect on expression of these enzymes, but treatment with a combination of interleukin-1beta, interferon-gamma, and tumor necrosis factor-alpha increased expression of MnSOD, catalase, and XO in some cell types with little effect on CuZnSOD expression. When TGF-betas were added along with these inflammatory cytokines there was a return to control levels of catalase expression, as well as a dramatic reduction in XO expression. In fetal bovine heart endothelial cells, treatment with inflammatory cytokines increased XO mRNA expression 11.5-fold and inclusion of TGF-betas reduced this 4-5-fold: effects on XO enzyme activity paralleled those seen on mRNA expression. Similar changes in XO expression were seen in cardiomyocytes. In contrast, TGF-betas did not change cytokine-induced MnSOD expression. All three mammalian isoforms of TGF-beta showed similar effects. In summary, TGF-betas may be able to decrease superoxide anion production and subsequent tissue damage by decreasing levels of XO.

This case report describes a patient with a known left ventricular mass which was documented by transthoracic echocardiography and which had not changed in size after anticoagulation. The mass was characterized as fatty tumor or hemorrhagic by cardiovascular magnetic resonance imaging study, and surgical removal of the mass was performed. Transesophageal echocardiogram was performed intraoperatively. The tumor appeared homogenous with no echolucencies or echodensities and an overall bright echotexture. In vitro ultrasound studies done using similar instrument gain settings showed the lipoma specimen to have a much brighter appearance than a myxoma. This finding may be helpful in differentiating a lipoma from other left ventricular masses such as myxoma or thrombus.

In this report of three patients with EMF, we report the unusual affection of the pericardium with echocardiographic epicardial thickening and pericardial inflammatory reaction, the presence of schistosomal granuloma in the pericardium, echocardiographic evidence of dense extensive fibrotic endocardial lesions, and microscopic evidences of an ongoing fibrotic process.

Hyperthermia causes changes in expression of TGF-beta mRNA and protein in cultured cardiac cells, as well as in the heart in vivo. 12 h after hyperthermia, primary cultures of neonatal rat cardiomyocytes show a two- to threefold decreased expression of TGF-beta mRNAs which returns to control levels by 48 h after heat shock. In cultures of cardiac fibroblasts, expression of TGF-beta mRNAs increases 5-25-fold, 12-48 h after heat shock, while fetal bovine heart endothelial cells show little change in TGF-beta expression after hyperthermia. In each case, mRNAs for TGF-beta s 1, 2, and 3 are regulated similarly. Hearts isolated from rats exposed to hyperthermia show an initial 20-fold decrease in TGF-beta 1 and 3 mRNA levels which return to control levels by 24 h and subsequently are elevated two- to threefold above normal 48-72 h after heat shock; there is little change in TGF-beta 2 mRNA. Expression of immunoreactive TGF-beta 1 and 3 protein, localized intracellularly in myocytes, follows the same pattern as the mRNA expression. By 72 h, some myocytes show hyperstaining for TGF-beta 1. Staining for extracellular TGF-beta 1/3 exhibits the opposite time course, being most intense 3-6 h after heat shock and returning to control levels by 48 h. The increase in TGF-beta s after hyperthermia could play a role in mediating the reported cardioprotective effects of heat shock.

To examine the role of protein phosphatases in skeletal muscle differentiation, C2C12 myoblasts were treated with okadaic acid, a potent in vitro inhibitor of protein phosphatases 1 and 2A which regulate various cellular events in intact cells. We now show that okadaic acid treatment of the mouse myoblast C2C12 cell line reversibly altered the morphology of the cells and blocked differentiation. At a molecular level, it extinguished expression of the myogenic determination genes, MyoD1 and myogenin, but induced the expression of an inhibitor of differentiation, Id. Analysis of the MyoD1 promoter showed that inhibition of MyoD1 expression by okadaic acid occurs at the transcriptional level. These changes occur 10-20 h after okadaic acid treatment. However, within 1 h of treatment the ability of muscle extracts to support a specific MyoD-dependent gel mobility shift using a MyoD DNA binding site is lost. These data suggest that protein phosphatases play an important role during myogenic differentiation.

Human T-cell lymphotropic virus type I (HTLV-I) has been associated with an adult form of T-cell leukemia as well as tropical spastic paraparesis, a neurodegenerative disease. Adult T-cell leukemia patients express high levels of the type 1 isoform of transforming growth factor-beta (TGF-beta 1), which is mediated by the effects of the HTLV-I Tax transactivator protein on the TGF-beta 1 promoter. To understand further the regulation of TGF-beta 1 expression by Tax, we examined its expression in transgenic mice carrying the HTLV-I tax gene. We show that tumors from these mice and other tissues, such as submaxillary glands and skeletal muscle, which express high levels of tax mRNA selectively express high levels of TGF-beta 1 mRNA and protein. Moreover, TGF-beta 1 significantly stimulated the incorporation of tritiated thymidine into one of three cell lines derived from neurofibromas of tax-transgenic mice, which suggests that the excessive production of TGF-beta 1 may play a role in tumorigenesis and that these mice may serve as a useful model for studying the biological effects of TGF-beta in vivo.

Although most biological activities of transforming growth factor-beta s 1 and 2 (TGF-beta 1 and TGF-beta 2) examined in vitro are similar or identical, recent studies suggest that each of these factors may be independently regulated in vivo. In this study we have used highly sensitive and specific sandwich enzyme-linked immunosorbent assays for TGF-beta 1 and TGF-beta 2 to examine the effects of a variety of treatments on expression of these two TGF-beta isoforms. We show that epidermal growth factor (EGF) induces secretion of TGF-beta 1 and not TGF-beta 2, whereas retinoic acid (RA) induces secretion of TGF-beta 2 and not TGF-beta 1 in NRK-49F normal rat kidney fibroblasts and A549 human lung carcinoma cells. Moreover, treatment with EGF diminishes the levels of TGF-beta 2, while RA decreases the levels of TGF-beta 1 in both cell lines. Dexamethasone (Dex), on the other hand, inhibits the secretion of both TGF-beta 1 and TGF-beta 2 in A549 cells, while selectively inhibiting TGF-beta 1 secretion in NRK-49F cells. The interactive effects of EGF, RA, and Dex on the production of TGF-beta 1 and TGF-beta 2, which were studied on NRK-49F cells, demonstrate that EGF blocks the induction of TGF-beta 2 mRNA and peptide by RA, while Dex inhibits the induction of TGF-beta 1 mRNA and peptide by EGF. These results demonstrate that RA, EGF and Dex are each unique, differential, and interactive regulators of the expression of TGF-beta s 1 and 2.

Transforming growth factor-β1 (TGF-β1) is synthesized and secreted as a biologically latent complex. It has been proposed that one role of the latent complex is to prevent premature interaction of ligand and receptor intracellularly during biosynthesis (Wakefield et al, J. Cell Biol. (1987) 105, 965-9751. To test this hypothesis, the endoplasmic reticulum retention sequence Lys-Asp-Glu-Leu (KDEL) was added to the C-terminus of the wildtype TGF-β1 coding sequence, and to a construct in which mutagenesis of two cysteine residues in the precursor pro region results in the synthesis and secretion of active, as opposed to latent, TGF-β. Addition of either SEKDEL, or the control sequence SEKDVS to the TGF-β1 protein abolished biological activity. Western blot analysis indicated that the extended gene products are synthesized, but that the extension sequence partially interferes with the normal dimerization of the protein product, and totally inhibits the normal proteolytic processing and glycosylation o...

As many as 40% of all primary cutaneous melanomas can have histologic remnants of nevomelanocytic nevi adjacent to the tumor. There is increasing evidence that dysplastic nevi are at least a clinical marker for melanoma risk. Spitz nevi are not known for such an association, but are noteworthy because of their histologic appearances. Spitz and dysplastic nevi were studied by flow cytometry to search for DNA abnormality. The study material consisted of formalin-fixed, paraffin embedded material of 41 dysplastic nevi and 14 Spitz nevi. Four cases of dysplastic nevi were excluded for technical reasons. Of the 37 interpretable histograms from dysplastic nevi, 28 (76%) were diploid and nine (24%) were aneuploid. All the Spitz nevi were diploid. Thus, dysplastic nevi, but not Spitz nevi, share aneuploidy features in some cases with melanoma. Previous authors have demonstrated aneuploidy in melanoma with aggressive behavior and in those in deep vertical growth phase. Aneuploidy may be a feature of early as well as late stages of tumor progression regarding the nevomelanocyte system.

We used double-label immunofluorescence to evaluate the expression of glutathione-S-transferase-P (GST-P) and gamma-glutamyl transferase (gamma GT) in individual liver cells in rats placed on a modified Solt-Farber hepatocarcinogenesis protocol. Four days after treatment with diethylnitrosamine (DEN), single GST-P+ cells were found in the centrilobular and midzonal regions. Most of these were gamma GT-, but the percentage of gamma GT+ cells increased with DEN dose to a high of 32%. At later times (7-8 days) doublets and small clusters of GST-P+ cells predominated; all cells in each pair or cluster displayed the same phenotype (GST-P+ or GST-P+/gamma GT+). Following administration of acetylaminofluorene and partial hepatectomy, lesions were larger and a greater percentage were GST-P+/gamma GT+. However, within these lesions individual cells displayed different phenotypes. These results indicate that the expression of GST-P and gamma GT is discordant in individual cells and small clusters soon after treatment, and suggest that different molecular events may be involved in their expression.

What are we actually speaking of when we use the term “marker”? Practicing pathologists and oncologists have applied the concept for many years without necessarily giving deep thought to what they were doing. Markers are features (of tumors, in the context we are using) that predict future behavior. In fact, the whole exercise of histopathology could arguably be thought of as interpretation of “markers.” Traditionally, the features that proved most useful were tumor stage and grade. As useful as these have been there was always an element of stock market disclaimer to them: “Past performance does not necessarily predict future results.” The effort to identify important features of tumors has moved inexorably to attempts to identify molecular features of tumors or biomarkers. Ideally, biomarkers would impact oncology in five main areas: cancer screening, diagnosis, tumor classification, prognosis, and predicting therapeutic response.

Background: Diagnosis of pancreatic lesions can be accurately performed by endoscopic ultrasound guided fine needle aspiration (EUS-FNA) with onsite cytopathologists to assess specimen adequacy and to determine a preliminary diagnosis. Considerable time is needed to perform on-site assessments. This takes away work time of cytopathologists and prohibits them from serving remote locations. It is therefore logical to ask if real-time telecytopathology could be used to assess specimen adequacy and if telecytopathology diagnosis has the same level of agreement to the final diagnosis as that of onsite evaluation. In this study, we compare agreement between cytodiagnoses rendered using telecytopathology with onsite and final interpretations. Method: 40 Diff-Quik-stained EUS-FNA were re-evaluated retrospectively (patient ages 31-62, 19:21 male:female, 15 non-malignant lesions, 25 malignant lesions as classified by final diagnosis). Each previously assessed by a cytopathologist and finally reviewed by the same or different cytopathologist. Blinded to the final diagnosis, a resident pathologist re-screened all slides for each case, selected a slide and marked the diagnostic cells most representative of the lesion. Blinded to the diagnosis, one cytopathologist assessed the marked cells through a real time remotely operated telecytopathology system (MedMicroscopy). Diagnosis and time spent were recorded. Kappa statistic was used to compare agreements between telecytopathology vs. original onsite vs. final diagnoses. Results: Time spent for prescreening ranged from 1 to 5 minutes (mean 2.6 +/- 1.3 minutes) and time spent for telecytopathology diagnosis ranged from 2-20 minutes (mean 7.5 +/- 4.5 minutes). Kappa statistics, K, was as follows: telecytopathology versus onsite diagnosis K, 95% CI = 0.65, 0.41-0.88, for telecytopathology versus final K, 95% CI = 0.61, 0.37-0.85 and for onsite diagnosis versus final K, 95% CI = 0.79, 0.61-0.98. There is no significant difference in agreement between onsite and telecytopathology diagnoses. Kappa values for telecytopathology were less than onsite evaluation when compared to the final diagnosis; however, the difference was not statistically significant. Conclusion: This retrospective study demonstrates the potential use of telecytopathology as a valid substitute for onsite evaluation of pancreatic carcinoma by EUS-FNA.

cancer associated with pregnancy have been reported in the literature.2 A review of 205 of these cases performed by Bernstein et al3 demonstrated that 85% of these cancers were located below the peritoneal reflection. The symptoms of colorectal cancer include nausea, vomiting, abdominal pain, altered bowel movements, and rectal bleeding. Since these symptoms are commonly found in the pregnant population as a whole, physicians and patients usually attribute them to the usual manifestations of pregnancy without an appropriate evaluation. The delay in initiating the workup for the symptoms related to colorectal cancer is a major contributing factor to the poor prognosis associated with this disease. 4 We present another case of colorectal cancer located below the peritoneal reflection that was diagnosed at an advanced stage.

This preliminary study demonstrates the superiority of live three-dimensional transthoracic echocardiography (3D TTE) over two-dimensional (2D) TTE in the assessment of left atrial (LA) tumors in four patients studied by us (three myxomas, one hemangioma, all subsequently pathologically proven). Because of the unique ability of live 3D TTE to systematically section and view the contents of an intracardiac mass, LA myxomas in the three patients studied could be more confidently diagnosed by noting isolated echolucent areas consistent with hemorrhage/necrosis in the tumor mass. On the other hand, a definite echolucent area was found by 2D TTE in only two of the three patients with myxoma. In the fourth patient with a hemangioma, live 3D TTE showed much more extensive and closely packed echolucencies with little solid tissue as compared to a myxoma consistent with a highly vascularized tumor. In contrast, 2D TTE demonstrated only two isolated echolucencies in the tumor suggesting an erroneous diagnosis of myxoma.

Telepathology is generally defined as the use of telecommunications technologies in the practice of anatomic or surgical pathology. In the usual telepathology scenario, a remotely located pathologist views images of tissues samples in order to render a diagnosis of the biopsy. Some telepathology systems involve interactive remote control of a microscope-based imaging system which delivers diagnostic quality imagery to the remote pathologist. The usefulness of such interactive systems depends on minimizing the end-to-end delays involved in controlling the robotic microscope, manipulating the tissue sample, and acquiring and transmitting the high-resolution image. An approach to minimizing end-to-end delay involves adding "intelligence" to the image acquisition system so that it can gather, classify, rank, and transmit diagnostically useful images in a semiautonomous fashion. In this research, we develop image analysis and ranking techniques which can improve the end-to-end performance of a robotic telepathology imaging system. Our semiautonomous image collection system uses morphological techniques to extract seed points for suspicious regions, a novel region growing algorithm to segment the regions of interest, and heuristically motivated expert system ranking techniques to select diagnostically relevant "next-step" image acquisitions. Diagnostic relevance of our segmentation and ranking algorithms is established via subjective and objective testing of the system. In subjective testing, pathologists Agree or Strongly Agree that all segmented regions are diagnostically relevant with probability greater than 0.75. In objective testing, 84% of "next-step" images acquired by our algorithms coincide with the areas most likely to be chosen by a pathologist.

Repeat positron emission tomography (PET) with 18F-fluorodeoxyglucose (FDG) and chest computed tomography (CT) are used to assess the effectiveness of chemoradiotherapy in patients with non-small cell lung cancer (NSCLC); however, the change in the standardized uptake values (SUV) has not been correlated with the pathologic change of the primary tumor.This is a retrospective cohort study of a prospective database of 56 patients who had NSCLC, FDG-PET, and chest CT scans both before and after neoadjuvant therapy, followed by complete resection of their cancer. Maximum SUVs (maxSUV) and tumor size were measured, and the percentage of change was compared with the percentage of nonviable tumor cells. The primary objective was to measure the degree of correlation between these values.The change in the maxSUV has a near linear relationship to the percent of nonviable tumor cells in the resected tumors. FDG-PET's maxSUV is better correlated to pathology than the change in size on CT scan (r2 = 0.75, r2 = 0.03, p0.001). When the maxSUV decreased by 80% or more, a complete pathologic response could be predicted with a sensitivity of 90%, specificity of 100%, and accuracy of 96%.The change in maxSUV on FDG-PET scan after neoadjuvant therapy holds a near linear relationship with pathologic response. It is a more accurate predictor than the change of size on CT scan. When the maxSUV decreases by 80% or more it is likely that the patient is a complete responder irrespective of cell type, neoadjuvant treatment, or the final absolute maxSUV. These findings may help guide treatment strategies.

The Center for Telecommunications Education and Research at UAB has introduced a new system architecture for telepathology. The prototype TelePath/sup SM/ system combines efficient bandwidth utilization and lossless transfer of "store and forward" digital imaging systems with the real-time control found in video systems. The architecture of the system is constrained by requirements for compatibility with surgical procedures for diagnosis of microscopic tissue biopsies. To date, the prototype system has enabled remotely located UAB pathologists to consult with each other and make accurate diagnoses of "frozen sections" in real-time.

A 12-year-old girl with presumed myocarditis was supported with right and left ventricular assist devices for 68 days before device removal. During this time, the patient underwent echocardiography and right heart catheterization for evaluation of cardiac recovery. This case report serves as the basis for a discussion of criteria for deciding when to terminate mechanical circulatory support in a patient with recovery after acute myocarditis.

Adenocarcinoma (AC), squamous cell carcinoma (SCC) and adenosquamous carcinoma (ASC) of the lung are morphologically distinguished in part by cyto-architectural features. However, little is known about the relative expression and distribution of cyto-architectural proteins among AC, SCC and ASC. Initial microarray analysis revealed significant differences in expression of two cyto-architectural genes in AC, SCC and ASC. Desmoplakin (DP) 1 and 2, which link desmosomes to intermediate filaments, was strongly expressed in SCC relative to AC and ASC. Cytokeratin 18 (CK18), an intermediate filament that is commonly linked to desmoplakin, was strongly expressed in AC and ASC relative to SCC. Western blot analysis demonstrated that AC and ASC had abundant CK18 protein, whereas CK18 was weakly detected in SCC. DP 1 and 2 are strongly expressed in SCC and minimally expressed in AC and ASC. However, the ratio of one to the other is the same in SCC and AC, but DP2 is lost in ASC. Microscopic analysis with fluorescence-labeled antibodies for CK18 and DP 1 and 2 revealed abundant membrane localization of DP and minimal perinuclear localization of CK18 in SCC. In contrast, in both AC and ASC, the CK18 protein was diffusely distributed within the cytoplasm, and DP showed both membranous and cytoplasmic localization. In conclusion, the data here shows that AC, SCC and ASC each have specific patterns of DP 1 and 2 and CK18 gene expression, protein content and biodistribution.

Although transforming growth factor-beta s (TGF-beta s) are expressed widely in both adult and embryonic rat heart, both mRNA and protein expression increase following ischemic injury. Furthermore, exogenous administration of TGF-beta decreases cardiac damage following ischemia-reperfusion in rats. We have found that treatment of primary cultures of neonatal rat cardiomyocytes or cardiac fibroblasts with TGF-beta 1, 2, or 3 results in increased expression of TGF-beta 1, 2, and 3 mRNA. TGF-beta 2 was generally the least effective isoform in inducing TGF-beta expression. In cardiac fibroblasts mRNA expression of all TGF-beta s increased 2-3-fold following 1 h of treatment and decreased to control levels by 8 h which was accompanied by a 2.5- and 2.3-fold increase in TGF-beta 1 and 2 protein secretion, respectively. By 48 h of treatment mRNA levels for TGF-beta s 2 and 3 were less than 10% of control levels. In cardiomyocytes two-five-fold increases in mRNA levels were observed following 1-24 h of TGF-beta 1 treatment, but TGF-beta 1 and 3 mRNA levels returned to control values by 48 h while TGF-beta 2 mRNA expression remained elevated. TGF-beta 1 and 2 protein secreted by the cardiac myocytes was increased 2.9- and 1.7-fold, respectively. Autoinduction of TGF-beta s may play a beneficial role in cardiac wound healing by sustaining transient increases in TGF-beta levels from either endogenous synthesis or exogenous application.

The three isoforms of transforming growth factor-beta (TGF-beta) have previously been implicated in embryonic development of the heart as well as in repair of myocardial damage after ischemia/reperfusion injury. TGF-beta 1 has also been localized intracellularly to both mitochondria and contractile filaments of cardiac myocytes, although its role in these structures has not been defined. We now report that exogenous TGF-beta stabilizes the beating rate of neonatal rat cardiac myocytes cultured on fibroblast matrix, and sustains their spontaneous rhythmic beating in serum-free medium. Moreover, using blocking antibodies to TGF-beta, we show that endogenous TGF-beta secreted by these myocytes acts in an autocrine fashion to maintain their beating rate. In contrast, IL-1 beta, an inflammatory mediator secreted by immune cells during myocardial injury, inhibits the beating of cardiac myocytes, and TGF-beta can overcome this inhibition. The antagonistic effects of TGF-beta and IL-1 were not observed when the myocytes were cultured on gelatin, as compared to native fibroblast matrix. The data indicate that TGF-beta is an important regulator of contractile function of the heart and have significant implications for understanding cardiac physiology in health and disease.

Human T-cell lymphotropic virus type I (HTLV-I) has been associated with an adult form of T-cell leukemia as well as tropical spastic paraparesis, a neurodegenerative disease. Adult T-cell leukemia patients express high levels of the type 1 isoform of transforming growth factor-beta (TGF-beta 1), which is mediated by the effects of the HTLV-I Tax transactivator protein on the TGF-beta 1 promoter. To understand further the regulation of TGF-beta 1 expression by Tax, we examined its expression in transgenic mice carrying the HTLV-I tax gene. We show that tumors from these mice and other tissues, such as submaxillary glands and skeletal muscle, which express high levels of tax mRNA selectively express high levels of TGF-beta 1 mRNA and protein. Moreover, TGF-beta 1 significantly stimulated the incorporation of tritiated thymidine into one of three cell lines derived from neurofibromas of tax-transgenic mice, which suggests that the excessive production of TGF-beta 1 may play a role in tumorigenesis and that these mice may serve as a useful model for studying the biological effects of TGF-beta in vivo.

cDNA probes and antibodies for TGF-beta s 1, 2, 3, and 4 were used to study the expression of these different TGF-beta isoforms in cultured chicken embryo chondrocytes and cardiac myocytes, as well as in developing cartilage and heart tissues. TGF-beta s 2, 3, and 4 mRNAs, but not TGF-beta 1 mRNA, were detected in cultured chondrocytes and myocytes. Expression of TGF-beta s 2 and 4 mRNAs increased with age, while expression of TGF-beta 3 mRNA was independent of age in chondrocytes cultured from 12- to 17-day-old embryos. In contrast, expression of TGF-beta s 2, 3, and 4 mRNAs was constitutive in myocytes cultured from 7- to 9-day-old embryonic hearts; expression of TGF-beta s 3 and 4 mRNAs increased, while expression of TGF-beta 2 mRNA remained unchanged in myocytes from 10-day-old embryos. Immunoprecipitation studies demonstrated expression of TGF-beta in both the conditioned media and the cell lysates of metabolically labeled chondrocyte and myocyte cell cultures. Immunohistochemical staining of cultured chondrocytes and myocytes and of cartilage and heart tissues of developing chicken embryos with antibodies specific for each TGF-beta isoform showed immunoreactive TGF-beta s 1, 2, 3, and 4. Our results demonstrate coordinate expression of these four TGF-beta isoforms in chicken embryo chondrocytes and myocytes, both in vitro and in vivo, with expression of TGF-beta s 2, 3, and 4 mRNA and protein more prominent than that of TGF-beta 1.

Summary Transforming growth factor-βs (TGF-βs) are potent regulators of cell growth and differentiation. Expression of the closely related TGF-β subtypes in vivo is differentially regulated both temporally and spatially. Members of the steroid hormone superfamily may play an important role in this gene- and tissue-specific regulation. We have shown that anti-estrogens induce the production of TGF-β1 in mammary carcinoma cells and fetal fibroblasts, whereas retinoic acid specifically induces TGF-β2 in primary epidermal keratinocytes. The induction of TGF-β2 by retinoids is accompanied by an increase in TGF-β2 mRNAs, but little change in transcription rates, suggesting an effect of retinoids on message stability or processing. In contrast, TGF-β1 mRNA levels are unchanged by anti-estrogen treatment, suggesting these compounds may regulate the translatability of the TGF-β1 message or some post-translational processing event. We have identified a stable stem–loop structure in the 5′ untranslated region (UTR) of the TGF-β1 mRNA that inhibits translation of a heterologous reporter gene, and we are investigating the possibility that anti-estrogens may regulate the activity of this element, and hence the translatability of the TGF-β1 message. A significant fraction (25–90 %) of the TGF-β induced by retinoids and anti-estrogens is in the biologically active rather than the latent form. We have shown that active TGF-β has a much shorter in vivo half-life than latent TGF-β, suggesting that the TGF-β induced by retinoids and steroids may act locally at the site of production. Since many tumor cells retain sensitivity to the growth inhibitory effects of active TGF-β, the use of members of the steroid hormone superfamily for inducing this potent growth inhibitor locally at the tumor site may have therapeutic potential.

We studied the effects of a zinc-inducible metallothionein-ras fusion gene (MTrasT24) in cultured rat liver epithelial (RLE) cells on expression of two genes induced during liver carcinogenesis in vivo: gamma-glutamyltransferase [(5-glutamyl)-peptide:amino acid 5-glutamyltransferase, EC 2.3.2.2] and glutathione S-transferase-P (RX:glutathione R-transferase, EC 2.5.1.18). Expression of MTrasT24 increased steady-state RNA levels of gamma-glutamyltransferase and glutathione transferase-P 6- to 100-fold and 1.6- to 6-fold, respectively; in contrast, levels of alpha-tubulin RNA fell slightly or were unchanged. RNA gel blots verified that gamma-glutamyltransferase and glutathione transferase-P RNAs were of the appropriate size, and results from immunocytochemistry on transfected cells demonstrated that RLE cells carrying MTrasT24 synthesized immunoreactive, appropriately localized gamma-glutamyltransferase and glutathione transferase-P. Zinc induction studies indicated that gamma-glutamyltransferase and glutathione transferase-P RNA levels were directly dependent on MTrasT24 RNA levels. These data suggest that expression of gamma-glutamyltransferase and glutathione transferase-P expression are part of a reorientation of cellular gene expression during carcinogenesis and that activated ras expression, like chemical carcinogens, can bring about this change.