Notable Histologic Findings in a "Normal" Cohort: The National Institutes of Health Genotype-Tissue Expression (GTEx) Project

CONTEXT.­: The National Institutes of Health (NIH) Genotype-Tissue Expression (GTEx) project was designed to evaluate how genetic variation and epigenetic effects influence gene expression in normal tissue. OBJECTIVE.­: To ensure that the grossly normal-appearing tissues collected were free from disease, each specimen underwent histologic evaluation. DESIGN.­: In total, nearly 30 000 tissue aliquots collected from almost 1000 postmortem donors underwent histologic review by project pathologists, and detailed observations of any abnormalities or lesions present were recorded. RESULTS.­: Despite sampling of normal-appearing tissue, in-depth review revealed incidental findings among GTEx samples that included neoplastic, autoimmune, and genetic conditions; the incidence of some of these conditions among GTEx donors differed from those previously reported for other populations. A number of age-related abnormalities observed during histologic review of tissue specimens are also described. CONCLUSIONS.­: Histologic findings from the GTEx project may serve to improve populational awareness of several conditions and present a unique opportunity for others to explore age- and gender-influenced conditions. Resources from the study, including histologic image and sequencing data, are publicly available for research.

The Biospecimen Preanalytical Variables Program: A Multiassay Comparison of Effects of Delay to Fixation and Fixation Duration on Nucleic Acid Quality.

CONTEXT.­: Despite widespread use of formalin-fixed, paraffin-embedded (FFPE) tissue in clinical and research settings, potential effects of variable tissue processing remain largely unknown. OBJECTIVE.­: To elucidate molecular effects associated with clinically relevant preanalytical variability, the National Cancer Institute initiated the Biospecimen Preanalytical Variables (BPV) program. DESIGN.­: The BPV program, a well-controlled series of systematic, blind and randomized studies, investigated whether a delay to fixation (DTF) or time in fixative (TIF) affects the quantity and quality of DNA and RNA isolated from FFPE colon, kidney, and ovarian tumors in comparison to case-matched snap-frozen controls. RESULTS.­: DNA and RNA yields were comparable among FFPE biospecimens subjected to different DTF and TIF time points. DNA and RNA quality metrics revealed assay- and time point-specific effects of DTF and TIF. A quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay was superior when assessing RNA quality, consistently detecting differences between FFPE and snap-frozen biospecimens and among DTF and TIF time points. RNA Integrity Number and DV200 (representing the percentage of RNA fragments longer than 200 nucleotides) displayed more limited sensitivity. Differences in DNA quality (Q-ratio) between FFPE and snap-frozen biospecimens and among DTF and TIF time points were detected with a qPCR-based assay. CONCLUSIONS.­: DNA and RNA quality may be adversely affected in some tumor types by a 12-hour DTF or a TIF of 72 hours. Results presented here as well as those of additional BPV molecular analyses underway will aid in the identification of acceptable delays and optimal fixation times, and quality assays that are suitable predictors of an FFPE biospecimen's fit-for-purpose.

The histopathological classification, diagnosis and differential diagnosis of mucinous appendiceal neoplasms, appendiceal adenocarcinomas and pseudomyxoma peritonei.

The vermiform appendix is the primary site of several distinctive benign and malignant neoplasms. Some can produce the clinical syndrome of pseudomyxoma peritonei (PMP). A consensus on their terminology was reached by an international panel of pathologists and clinicians working under the auspices of the Peritoneal Surface Oncology Group International (PSOGI), and this review discusses the application of the PSOGI classification to routine reporting. We discuss diagnosis and differential diagnosis together with implications for patient management, covering low-grade appendiceal mucinous neoplasms, high-grade appendiceal mucinous neoplasms, serrated polyps, adenomas and adenocarcinomas. We do not cover goblet cell tumours or neuroendocrine neoplasms in this paper.

A Consensus for Classification and Pathologic Reporting of Pseudomyxoma Peritonei and Associated Appendiceal Neoplasia: The Results of the Peritoneal Surface Oncology Group International (PSOGI) Modified Delphi Process.

Pseudomyxoma peritonei (PMP) is a complex disease with unique biological behavior that usually arises from appendiceal mucinous neoplasia. The classification of PMP and its primary appendiceal neoplasia is contentious, and an international modified Delphi consensus process was instigated to address terminology and definitions. A classification of mucinous appendiceal neoplasia was developed, and it was agreed that "mucinous adenocarcinoma" should be reserved for lesions with infiltrative invasion. The term "low-grade appendiceal mucinous neoplasm" was supported and it was agreed that "cystadenoma" should no longer be recommended. A new term of "high-grade appendiceal mucinous neoplasm" was proposed for lesions without infiltrative invasion but with high-grade cytologic atypia. Serrated polyp with or without dysplasia was preferred for tumors with serrated features confined to the mucosa with an intact muscularis mucosae. Consensus was achieved on the pathologic classification of PMP, defined as the intraperitoneal accumulation of mucus due to mucinous neoplasia characterized by the redistribution phenomenon. Three categories of PMP were agreed-low grade, high grade, and high grade with signet ring cells. Acellular mucin should be classified separately. It was agreed that low-grade and high-grade mucinous carcinoma peritonei should be considered synonymous with disseminated peritoneal adenomucinosis and peritoneal mucinous carcinomatosis, respectively. A checklist for the pathologic reporting of PMP and appendiceal mucinous neoplasms was also developed. By adopting the classifications and definitions that were agreed, different centers will be able to use uniform terminology that will allow meaningful comparison of their results.

A Novel Approach to High-Quality Postmortem Tissue Procurement: The GTEx Project.

The Genotype-Tissue Expression (GTEx) project, sponsored by the NIH Common Fund, was established to study the correlation between human genetic variation and tissue-specific gene expression in non-diseased individuals. A significant challenge was the collection of high-quality biospecimens for extensive genomic analyses. Here we describe how a successful infrastructure for biospecimen procurement was developed and implemented by multiple research partners to support the prospective collection, annotation, and distribution of blood, tissues, and cell lines for the GTEx project. Other research projects can follow this model and form beneficial partnerships with rapid autopsy and organ procurement organizations to collect high quality biospecimens and associated clinical data for genomic studies. Biospecimens, clinical and genomic data, and Standard Operating Procedures guiding biospecimen collection for the GTEx project are available to the research community.

Evaluation of colon cancer histomorphology: a comparison between formalin and PAXgene tissue fixation by an international ring trial.

The aim of our study was to evaluate the quality of histo- and cytomorphological features of PAXgene-fixed specimens and their suitability for histomorphological classification in comparison to standard formalin fixation. Fifteen colon cancer tissues were collected, divided into two mirrored samples and either formalin fixed (FFPE) or PAXgene fixed (PFPE) before paraffin embedding. HE- and PAS-stained sections were scanned and evaluated in a blinded, randomised ring trial by 20 pathologists from Europe and the USA using virtual microscopy. The pathologists evaluated histological grading, histological subtype, presence of adenoma, presence of lymphovascular invasion, quality of histomorphology and quality of nuclear features. Statistical analysis revealed that the reproducibility with regard to grading between both fixation methods was rather satisfactory (weighted kappa statistic (k w) = 0.73 (95 % confidence interval (CI), 0.41-0.94)), with a higher agreement between the reference evaluation and the PFPE samples (k w = 0.86 (95 % CI, 0.67-1.00)). Independent from preservation method, inter-observer reproducibility was not completely satisfactory (k w = 0.60). Histomorphological quality parameters were scored equal or better for PFPE than for FFPE samples. For example, overall quality and nuclear features, especially the detection of mitosis, were judged significantly better for PFPE cases. By contrast, significant retraction artefacts were observed more frequently in PFPE samples. In conclusion, our findings suggest that the PAXgene Tissue System leads to excellent preservation of histomorphology and nuclear features of colon cancer tissue and allows routine morphological diagnosis.

Improving the TNM classification: findings from a 10-year continuous literature review.

The Union for International Cancer Control's (UICC) TNM classification is a globally accepted system to describe the anatomic extent of malignant tumors. Since its development seventy years ago, the TNM classification has undergone significant revisions to reflect the current understanding of extent of disease and its role in prognosis. To ensure that revisions are evidence-based, the UICC implemented a process for continuous improvement of the TNM classification that included a formalized system for submitting proposals for revisions directly to the UICC and an annual review of the scientific literature on staging that assessed, criticized or made suggestions for changes. The process involves review of the proposals and literature by a group of international, multidisciplinary Expert Panels. The process has been in place for 10 years and informed the development of the 7th edition of the TNM classification published in 2009. The purpose of this article is to provide a description of the annual literature review process, including the search strategy, article selection process and the roles and requirements of the Expert Panels in the review of the literature. Since 2002, 147 Expert Panel members in 11 cancer sites have reviewed over 770 articles. The results of the annual literature reviews, Expert Panel feedback and documentation and dissemination of results are described.

Non-formalin fixative versus formalin-fixed tissue: a comparison of histology and RNA quality.

Preanalytical handling of tissue samples can influence bioanalyte quality and ultimately outcome of analytical results. The aim of this study was to compare RNA quality, performance in real time RT PCR and histology of formalin-fixed tissue to that of tissue fixed and stabilized with a formalin-free fixative, the PAXgene Tissue System (PAXgene), in an animal model under highly controlled preanalytical conditions. Samples of rat liver, kidney, spleen, intestine, lung, heart muscle, brain, and stomach tissue were either fixed in formalin or fixed in PAXgene or fresh frozen in liquid nitrogen. RNA was extracted from all samples, examined for integrity in microcapillary electrophoresis, and used in a series of quantitative RT PCR assays with increasing amplicon length. Histology of paraffin-embedded samples was determined by staining with hematoxylin and eosin. Histology of all formalin-fixed and PAXgene fixed samples was comparable. RNA with acceptable integrity scores could be isolated from all embedded tissues, 4.0 to 7.2 for formalin and 6.4 to 7.7 for PAXgene, as compared to 8.0 to 9.2 for fresh frozen samples. While RNA with acceptable RINs (RNA integrity number) could be isolated from formalin-fixed samples, in microcapillary electrophoresis this RNA separated with a slower migration rate and displayed diffuse, less focused peaks for ribosomal RNA as compared to RNA from frozen or PAXgene fixed samples. Furthermore, RNA from formalin-fixed tissues exhibited inhibition in quantitative RT PCR assays which increased with increasing amplicon length, while RNA from PAXgene fixed samples did not show such inhibition. In conclusion, our results demonstrate that excluding other preanalytical factors, PAXgene Tissue System preserves histology similarly to formalin, but unlike formalin, does not chemically modify RNA. RNA purified from PAXgene fixed tissues is of high integrity and performs as well as RNA from fresh frozen tissue in RT PCR regardless of amplicon length.

'Evidence-based medicine: the time has come to set standards for staging'. Is a radical overhaul really needed?

This Invited Response addresses concerns and opinions expressed in an Invited Commentary, 'Evidence-based medicine: the time has come to set standards for staging', by Quirke et al., published in this issue of The Journal of Pathology.

A distinctive novel epitheliomesenchymal biphasic tumor of the stomach in young adults ("gastroblastoma"): a series of 3 cases.

This report describes 3 cases of a distinctive, hitherto unreported gastric epitheliomesenchymal biphasic tumor that differs from other biphasic tumors of the stomach and elsewhere: carcinosarcoma, biphasic synovial sarcoma, teratoma, and mixed tumor. The tumors occurred in young adults, 2 males and 1 female, of ages 19, 27, and 30 years. Two tumors were located in the greater curvature in the gastric body and one in the antrum. The tumors measured 5, 6, and 15 cm in maximum diameter, and their mitotic rates were 0, 4, and 30 mitoses per 50HPF. There were 2 components: uniform oval or spindled cells in diffuse sheets, and clusters or cords of epithelial cells occasionally forming glandular structures with small lumens. The epithelial elements were positive for keratin cocktail AE1/AE3, keratin 18, and partly for keratin 7, but were negative for keratins 5/6, 20 and epithelial membrane antigen. The spindle cells were positive for vimentin and CD10. All components were negative for CD34, CD99, estrogen receptor, KIT, smooth muscle actin, desmin S100 protein, p63, calretinin, chromogranin, synaptophysin, CDX2, and thyroid transcription factor 1. In situ hybridization for SS18 rearrangement was negative in all cases separating this tumor from synovial sarcoma. All 3 patients were alive after follow-up of 3.5, 5, and 14 years. Because these tumors have some resemblance to blastomas of other organs, we propose the term "gastroblastoma" for this distinctive, at least low-grade malignant epitheliomesenchymal tumor of the stomach.

Plexiform fibromyxoma: a distinctive benign gastric antral neoplasm not to be confused with a myxoid GIST.

A great majority of gastric mesenchymal tumors are gastrointestinal stromal tumor (GIST). A rare group of non-GISTs include myxoid mesenchymal neoplasms. In this report, we describe 12 cases of a distinctive gastric tumor, named here as plexiform fibromyxoma. These tumors occurred in 5 men and 7 women of ages 7 to 75 years (median, 41 y). All tumors were located in the gastric antrum and 6 of them also extended into extragastric soft tissues or into the duodenal bulb. The tumors measured from 3 to 15 cm (median, 5.5 cm). Histologically typical was a plexiform intramural growth with multiple micronodules containing paucicellular to moderately cellular myxoid to collagenous and fibromyxoid neoplastic elements. A prominent, sometimes plexiform capillary pattern was typically present. Extramural components included subserosal nodules, and sometimes more cellular, solid nonplexiform spindle cell proliferation. The tumor cells varied from oval to spindled and had limited atypia and mitotic activity < 5/50 high-power fields. Frequent ulceration, mucosal invasion, and vascular invasion (4 cases) had no adverse significance in these tumors. Immunohistochemically, the tumor cells were positive for alpha smooth muscle actin, and variably for CD10, and were consistently negative for KIT, DOG1, CD34, desmin, and S100 protein. No KIT or platelet-derived growth factor receptor alpha mutations were present in the 3 examined cases. None of the 4 patients who were followed from 9 to 20 years (median, 19 y) developed recurrences or metastases. Additional 3 patients survived 14 to 25 years with unknown tumor status. Review of large numbers of mesenchymal tumors in the esophagus and intestines did not reveal similar tumors. Plexiform fibromyxoma is a distinctive benign gastric antral neoplasm that should be separated from GIST, nerve sheath tumors, and other fibromyxoid neoplasms.

A uniform residual tumor (R) classification: integration of the R classification and the circumferential margin status.

BACKGROUND: Since the introduction of the TNM residual tumor (R) classification, the involvement of resection margins has been defined either as a microscopic (R1) or a macroscopic (R2) demonstration of tumor directly at the resection margin ("tumor transected"). METHODS: The recognition of the importance of the circumferential resection margin (CRM) in patients with rectal cancer patients raises the need for an alternative definition of resection margin involvement, namely, the importance of delineating tumor with a minimal distance from the CRM of

The IASLC Lung Cancer Staging Project: data elements for the prospective project.

The International Association for the Study of Lung Cancer Retrospective Staging Project culminated in a series of recommendations to the International Union Against Cancer and to the American Joint Committee on Cancer regarding the seventh edition of the tumor, node, metastasis (TNM) classification for lung cancer. The International Staging Committee of the International Association for the Study of Lung Cancer now issues this call for participation in the Prospective Project designed to assess the validity of each component of T, N, and M, and other factors relevant to lung cancer staging and prognosis. In the Retrospective Project, the original data acquisition was typically motivated by interests other than staging. In contrast, the Prospective Project offers online data entry. Alternatively, participants may transfer existing data, provided core objectives are addressed. Cancer Research and Biostatistics will coordinate data management and analysis. The study population is newly diagnosed lung cancer patients. Data elements include patient characteristics, baseline laboratory values, first-line treatment, TNM plus supporting evidence, and survival. Pretreatment TNM will be collected for all cases; postsurgical TNM, if resection is attempted. T descriptors include size and degree of tumor extension, with further description of extent of visceral pleural invasion, venous invasion, carcinomatous lymphangitis, and pleural lavage cytology. M descriptors characterize the newly proposed M1a category and sites of distant metastases. Nodal station involvement is described by means of a newly proposed nodal map, facilitating international participation, and allowing further investigation of nodal zones. Successful collection and analysis of these data can be expected to yield unprecedented improvements in the utility and validity of lung cancer staging.

Gastrointestinal stromal tumors presenting as omental masses--a clinicopathologic analysis of 95 cases.

Gastrointestinal stromal tumors (GISTs), generally KIT-positive and KIT/PDGFRA mutation-driven mesenchymal neoplasms, most commonly originate from the stomach or small intestine, but in rare examples they involve the omentum. In this study, we analyzed 95 GISTs surgically designated as the omental masses. These tumors occurred in 49 males and 46 females with a median age of 60 years (range: 27 to 88 y). They formed single (n=51) or multiple masses (n=39); 5 cases were equivocal in this respect. Of the single tumors, 21 had no evidence of gastrointestinal tract involvement, 25 were attached to stomach, and 3 were attached to small intestine. Clinicopathologic parameters and prognosis of the 2 former groups were similar. Single tumor cases showed a median mitotic count of 2/50 HPFs and median tumor size was 14 cm. Their histologic features were similar to gastric GISTs in 22 cases, and to small intestinal GISTs in 6 cases. These tumors were KIT positive 38/41, CD34 positive 20/33, 8 had PDGFRA mutations, and 6 had KIT exon 11 mutations. The median survival was 129 months (range: 0 to 397 mo) and 14 patients were alive at the end of follow-up. Multiple tumor cases showed median mitotic count of 14/50 HPFs and the main tumor median size was 16 cm. The histologic features were similar to small intestinal GISTs in 21 cases and to gastric GISTs in 7 cases; small intestinal attachment or history of a previous small intestinal GIST were noted in 5 cases, whereas no tumor was attached to stomach. The multiple GISTs were KIT positive 23/24, CD34 positive 7/21, and 5 had KIT exon 11 mutations, 3 had KIT exon 9 mutations, and 2 had PDGFRA mutations. The median survival was for 8 months and all patients died. Omental GISTs are clinicopathologically heterogenous. Patients with solitary tumors usually have gastric GIST-like morphology and a better prognosis than those with multiple tumors, whose tumor usually has small intestinal GIST-like histology. Omental GISTs unattached to gastrointestinal tract often resemble gastric GISTs suggesting that they may be gastric GISTs directly extending or parasitically attached into the omentum, whereas multiple omental GISTs more often resemble small intestinal GISTs suggesting that they may be metastatic or detached from this source. KIT positive Cajal cells were not found in normal omental tissues failing to support the presence of these ancestral cells for GIST in the omentum.

Primary melanomas of the esophagus and anorectum: epidemiologic comparison with melanoma of the skin.

The objective of this study was to use recently available data to describe the epidemiology of melanomas of the esophagus and the anorectum in contrast to melanoma of the skin. The methods used include descriptive epidemiology using cases reported to the Surveillance Epidemiology and End Results registry, 1973-2003. All rates were age adjusted. We found 46 759 cutaneous melanomas, 170 anorectal melanomas and 20 esophageal melanomas, corresponding to age-adjusted rates of 70.1, 0.27, and 0.03 per million population, respectively. Median age of patients with melanoma of the skin was less than those with melanoma of the anorectum or esophagus (55 years vs. 71 years and 69 years, respectively). Rates of melanoma of the skin were 1.5-fold higher for men than for women (87.1 vs. 58.1 per 10); in contrast, rates of anorectal melanoma were 1.6-fold higher for women than for men (0.324 vs. 0.199 per 10). Rates of cutaneous melanoma for whites were 13-fold higher than for blacks (80.6 vs. 6.1 per 10, P<0.001), whereas the rates of anorectal melanoma were 1.7-fold higher among whites than blacks (0.273 vs. 0.173 per 10). Comparing the period 1973-1987 with 1988-2003, the rate of cutaneous melanoma increased 1.4-fold (56.0-80.1 per 10), whereas the rate of anorectal melanoma similarly increased 1.8-fold (0.182 to 0.329 per 10).In conclusion, anorectal melanomas, and especially, esophageal melanomas remain rare malignancies. This is the first report where temporal trends in anorectal melanoma are following the increased incidence of cutaneous melanomas, but it is unclear whether immunohistochemical diagnostic practices have influenced this trend. As gastrointestinal melanomas represent melanocytic transformation in the absence of sunlight, their epidemiology may provide etiologic clues to alternative or systemic transformative factors also operant in cutaneous melanoma.

Gain-of-function PDGFRA mutations, earlier reported in gastrointestinal stromal tumors, are common in small intestinal inflammatory fibroid polyps. A study of 60 cases.

The inflammatory fibroid polyp is a rare benign lesion occurring throughout the digestive tract. It usually forms a solitary mass, characterized by a proliferation of fibrovascular tissue infiltrated by a variable number of inflammatory cells. The etiology of this lesion is unknown and conflicting histogenetic theories have been proposed. Recently, mutations in platelet-derived growth factor receptor (PDGFRA) and PDGFRA expression were reported in gastric inflammatory fibroid polyps. In this study, PDGFRA exons 12, 14, and 18 were screened for activating mutations in 60 small intestinal inflammatory fibroid polyps. In addition, the PDGFRA expression was evaluated immunohistochemically. Mutations in PDGFRA were identified in 33 of 60 (55%) cases, whereas 95% expressed PDGFRA. There were 26 deletions, three deletion-insertions, duplication, and single nucleotide substitution in exon 12, and a single nucleotide substitution and deletion in exon 18. The majority (n=23) of exon 12 deletions were 1837_1851del leading to S566_E571delinsR. However, 1835_1852delinsCGC leading to the same S566_E571delinsR, were found in two tumors. Three inflammatory fibroid polyps had 1836_1850del leading to S566_E571delinsK. A complex deletion-insertion affecting a similar region (1837_1856delinsGATTGATGATC) and leading to S566_I573delinsRIDDL was identified once. In addition, duplication and single nucleotide substitution were found 5' to the common inflammatory fibroid polyp mutational 'hot spot'. These mutations consist of 1808_1828dup leading to I557_E563dup, and 1821T>A resulting in 561V>D substitution. A 2664A>T and 2663_2674del leading to 842D>V and D842_H845del, respectively, were identified in exon 18. Similar gain-of-function PDGFRA mutations reported in gastrointestinal stromal tumors have been considered to be a driving pathogenetic force. This study showed consistent expression and common mutational activation of PDGFRA in small intestinal inflammatory fibroid polyps as in their gastric counterparts, and these lesions should be considered PDGFRA-driven benign neoplasms. We also suggest that these polyps may develop from earlier described PDGFRA-positive mesenchymal cells distributed along the villus membrane after oncogenic PDGFRA activation.

Secondary tumors and tumorlike lesions of the peritoneal cavity: imaging features with pathologic correlation.

Tumors and tumorlike lesions that secondarily involve the mesothelial or submesothelial layers of the peritoneum are a diverse group of disorders that range in biologic behavior from benign to highly malignant. The anatomy of peritoneal ligaments and mesenteries and the normal circulation of peritoneal fluid dictate location and distribution of these diseases within the peritoneal cavity. Peritoneal carcinomatosis is the most common secondary tumor to affect the peritoneal cavity. When it arises from carcinomas of the gastrointestinal tract or ovary, the prognosis is grave. However, when low-grade mucinous adenocarcinoma of the appendix spreads to the peritoneal cavity, the consequence is typically pseudomyxoma peritonei, which is a clinical syndrome, characterized by recurrent and recalcitrant voluminous mucinous ascites due to surface growth on the peritoneum without significant invasion of underlying tissues. Carcinomas from elsewhere in the body, as well as lymphomas and sarcomas, may also produce diffuse peritoneal metastasis. Granulomatous peritonitis is the consequence of disseminated infection such as tuberculosis or histoplasmosis, foreign materials, or rupture of a tumor or hollow viscus. Finally, a group of benign miscellaneous conditions that range from common disorders such as endometriosis and splenosis to very rare conditions such as gliomatosis peritonei and melanosis may also affect the peritoneum diffusely. Secondary tumors and tumorlike lesions of the peritoneum have overlapping imaging features when compared with each other and primary peritoneal tumors. Knowledge of peritoneal anatomy, normal fluid circulation within the peritoneal cavity, and clinical and pathologic features of secondary peritoneal lesions is essential for identification of these lesions.