Spontaneously ruptured endometriomas presenting with symptoms and imaging findings worrisome for carcinomatosis: A case report.

INTRODUCTION AND IMPORTANCE: Endometriomas are the most common presenting subtype of endometriosis. Although most endometriomas are asymptomatic, patients can rarely present acutely with spontaneous rupture causing diffuse peritonitis and severe systemic inflammatory response. CASE PRESENTATION: Here we describe a case of ruptured endometriomas in a 26-year-old nulligravid female with a history of heavy menses, progressive abdominal distension, and a recent urinary tract infection. The patient presented to the emergency department with upper abdominal pain radiating to her back with associated nausea. Computed tomography (CT) scan demonstrated diffuse ascites with a large, multilobulated, and multicystic septated mass arising in the right pelvis and extending into the lower abdomen. Findings were concerning for peritoneal carcinomatosis and the patient was admitted for evaluation. She developed progressive signs of sepsis and was emergently brought to the operating room for surgical exploration on hospital day (HD) number two. She was found to have ruptured pelvic cysts arising from both ovaries with diffuse contamination of the abdomen by cyst contents and bilateral salpingo-oophorectomy (BSO) was performed. Final pathology demonstrated benign bilateral endometriomas. CLINICAL DISCUSSION: Endometrioma rupture is extremely rare and imaging findings may appear to represent disseminated peritoneal malignancy. CT findings demonstrating a pelvic mass with concurrent ascites should raise clinical suspicion for ruptured endometrioma, particularly in younger patients. CONCLUSION: Prompt surgical exploration and complete resection of pathologic tissue may be necessary for diagnosis and treatment in some patients with clinical deterioration related to perforated endometriomas. Combined oral contraceptives are recommended in the postoperative period.

Ruptured granulosa cell tumor of the ovary presenting with catastrophic intra-abdominal hemorrhage: A case report.

INTRODUCTION AND IMPORTANCE: Adult granulosa cell tumor (GCT) is a rare stromal cell neoplasm that most often arises from the ovary. Presenting symptoms are related to external compression of adjacent structures (mass effect) or secretion of hormones such as estrogen. Patients most commonly present with irregular menstruation, postmenopausal bleeding, and abdominal pain. Prolonged estrogen exposure can contribute to endometrial adenocarcinoma development in untreated patients. The highly vascular nature of GCTs can lead to hemorrhagic rupture in rare cases. PRESENTATION OF CASE: We describe a case of adult GCT in a 44-year-old female with a history of irregular menstrual bleeding and anemia. The patient presented with shortness of breath and abdominal pain. Computed tomography (CT) scan demonstrated possible hemorrhagic ascites of unclear etiology and a pelvic mass. The patient was brought to the operating room in hemorrhagic shock for surgical exploration where she was found to have active bleeding of a ruptured ovarian tumor for which she underwent left salpingo-oophorectomy. Postoperative course was unremarkable, and pathology demonstrated ruptured GCT. CLINICAL DISCUSSION: Although rare, ovarian tumors can present with massive bleeding following rupture. Granulosa cell tumors are surreptitious as they grow slowly, and symptoms such as distention, abdominal pain, and irregular vaginal bleeding are nonspecific. CONCLUSION: CT findings demonstrating a pelvic mass in the setting of spontaneous intra-abdominal bleeding should raise clinical suspicion, particularly in patients with histories of menstrual abnormalities. Patients with suspected intra-abdominal hemorrhage due to any cause are best treated by prompt surgical exploration and aggressive resuscitation.

An iOS App to Expedite the Evaluation of Immunohistochemistry Control Tissue.

Control tissues play a vital role in diagnostic immunohistochemistry. They serve to document that the appropriate antibody was used, on the correct control tissue, and run on optimized conditions. As part of the evolving process of standardization in diagnostic immunohistochemistry, specific tissues have been identified based on agreement by experts in this field capable of serving as the benchmark(s) for several antibodies. These tissues are recommended based on known and predictable levels of strong, weak, and no expression of the antigen being queried. These tissues can be used for positive and negative control purposes, respectively, and are regarded as primary positive and/or negative external control tissue. If some of these tissues are not present in sufficient numbers in a laboratory's archive for daily use, they can still be used as the basis to evaluate other tissues that are not as well characterized and chosen to serve as secondary and external positive controls. In this manner, either the former or latter can function as external positive and negative control tissue for the quality assurance of immunohistochemistry done in a laboratory. The use of the selected tissues may be applicable to the detection of several different antigens by a number of separate antibodies, with differences in the staining of specific cells or the localization in staining within those cells. However, the amount of information needed to be familiar with to render a correct interpretation of the control tissue may prove daunting. One means of dealing with this problem would be to create a document capable of serving as a reference guide. Traditional types of references, however, may suffer from issues related to convenience, updating, and mobility. Herein we describe a mobile device application (app) created to serve as a reference for control block tissues. This app can capably house and easily retrieve all the relevant information on all the antibodies and their respective control tissues in a laboratories test menu, thus enabling the use of standardized tissues as control material and spreading the ability to perform immunohistochemical quality control to individual pathologists.

Front-end genomics: using an alternative approach for the recovery of high-quality DNA from core needle biopsies.

AIMS: Determine whether a simple prewash step will provide adequate amounts of high-quality DNA from core needle biopsies for molecular sequencing studies. METHODS: The quantitative and qualitative metrics of DNA recovered from core needle biopsies processed either by 1) formalin fixation and paraffin embedding (FFPE), 2) cells recovered after the core needle biopsy was washed, and 3) frozen sections of the core needle biopsy tissue were evaluated and compared to one another. RESULTS: Fairly equivalent amounts of DNA can be obtained from cells recovered from a prewash step relative to the FFPE and frozen section samples. The number of amplifiable DNA in the wash sample was greater than that from the FFPE samples. The average molecular size of DNA in the wash sample was greater than that of both the FFPE and frozen samples. CONCLUSIONS: Although more starting material in terms of the number of cells was present in both the FFPE and frozen section samples than the wash samples, equivalent to better results were obtained from the latter with regard to quality. This approach may be a means to better aliquot the diminutive amounts of tissue associated with core needle biopsies, allowing dissociated cells to be dedicated for molecular studies while keeping the tissue intact for morphological studies.

Core Needle Biopsy Wash Optimization: Enabling Specimen Integrity for both Cytological and Histological Evaluation.

BACKGROUND: The recovery of cells after washing core needle biopsies represents an under-utilized approach to extend the diagnostic capacity of these diminutive specimens. Recovery of these cells can be dedicated for molecular studies so that the biopsy itself can be used apropos for its intended purpose, diagnosis. Non-enzymatic and enzymatic reagents have the potential to increase the number of cells dissociating from the tissue core, but can also negatively impact the quality of the tissue itself. MATERIALS AND METHODS: Three different means (phosphate-buffered saline, a non-enzymatic and an enzymatic solution) were used to wash core needle biopsies. The washed cells were recovered by traditional preparatory methods and evaluated for cellularity and cytomorphology. The post-washed cores were processed by formalin fixation, paraffin embedding and evaluated for integrity and morphological quality. RESULTS: The enzymatic solution damaged both the cytological and tissue specimens, while the saline and non-enzymatic process allowed for the comparable recovery of cells and tissue for evaluation. CONCLUSION: Adequate numbers of cells are dissociated from the tissue core when needle biopsies are washed. The recovery and preservation of cells and tissue for morphological interpretation was optimal when solutions devoid of enzymes were used for washing.

Maximizing derivable information from cytologic specimens for pathologic and molecular diagnostics.

INTRODUCTION: The advent of precision medicine will increase the demand for molecular testing on patient tumor specimens. Cytology specimens have been shown to be ideal substrates for molecular testing, but their often paucicellular nature can lead to conflicts in prioritizing sample management. A microfluidic platform was investigated to determine whether cytologic and molecular data could be procured from the same cells, obviating the need for partitioning a sample by multiplexing it instead. MATERIALS AND METHODS: Cytology samples were created from a tissue source, stained with a supravital dye, and enriched using immunomagnetic beads. These cells and the attached immunomagnetic beads were then run through a microfluidic channel, temporarily immobilized for cytologic examination, and then recovered. The cytologic characteristics of these cells was compared with cells from the same source prepared by conventional cytologic preparatory means. DNA was extracted from the cells recovered from the microfluidic channel and the nature of their integrity was assessed. RESULTS: Cytologic features between cells run in a microfluidic channel and prepared by conventional means were similar. The DNA recovered from the cells run through the microfluidic channel was of high molecular weight. CONCLUSIONS: Microfluidics enables multiplex testing of cytologic specimens, allowing for cytology-based diagnostic examination and recovery of high-quality DNA. This approach will be of particular benefit for cytology specimens that are paucicellular and will need molecular testing.

Immunohistochemical detection of arginine methylated proteins (MeRP) in archival tissues.

AIMS: To (i) determine whether methylarginine-specific antibodies can be employed for standard immunohistochemical analysis of paraffin-embedded tissues, (ii) analyse methylarginine expression in normal and neoplastic tissues and (iii) correlate methylarginine expression with that of protein arginine methyltransferase (PRMT1), the predominant cellular arginine methyltransferase. METHODS AND RESULTS: Immunohistochemistry of normal and cancer tissues was performed utilizing three commercial polyclonal antibodies: anti-methylarginine-specific antibody (anti-mRG) raised against a methylarginine peptide, Control antibody (anti-RG), a control antiserum raised against a corresponding arginine peptide without any methylated residues and anti-PRMT1. Nuclear and/or cytoplasmic methylarginine expression was detected in all keratinized and non-keratinized epithelia. A preliminary survey of a series of thyroid, pancreatic, colonic and gastric cancers identified a different pattern of methylarginine expression in comparison with normal tissue. A correlation between methylarginine staining and PRMT1 expression was found in all normal and cancer tissues analysed. CONCLUSION: Methylarginine-specific antibodies are capable of recognizing methylarginine proteins (MeRP) in paraffin-embedded tissues. Methylarginine proteins are expressed widely and show differences in subcellular localization in various organs and neoplastic conditions. The efficient detection of methylproteins by standard immunohistochemistry provides a new tool to investigate the role of methylarginine proteins (MeRP) in biological processes including carcinogenesis.

Importance of cell-procurement methods in transforming personalized cancer treatment from concept to reality.

The much-anticipated promise of personalized cancer care is to deliver therapies best suited for a patient based on the knowledge of that individual's genetics and tumor characteristics. This transformative approach will require many changes in the scientific and medical community, one of the most fundamental being the direct study of human tissue biospecimens. Biospecimens will be integral to the elucidation of biomarkers that will help identify and serve as potential diagnostic, prognostic and therapeutic targets in cancer. Despite the vast repositories of fixed-tissue biospecimens that are in existence, a number of flaws exist that hinder their reliable use as instruments from which to enable personalized cancer research and clinical care. A new view of biospecimen worth in the future will mandate that the molecules within its cells are reflective of their in vivo state, and not altered by external variables introduced during the excision and processing of the biospecimen. Research on biospecimen collection is a legitimate field of study that will be necessary for personalized cancer care to become a reality.

A comparative analysis of two tissue procurement approaches for the genomic profiling of clinical colorectal cancer samples.

BACKGROUND: The basis for personalized medicine is the creation of a repository of knowledge about the genetic alterations involved in disease processes. Integral to achieving this goal is the querying of well-preserved, high-quality human tissue samples. Making these findings relevant involves the interrogation of large numbers of samples. The pace with which changes have occurred versus the potential pace with which changes can occur may be indicative of problems associated with traditional approaches on collecting biospecimens. Therefore, transforming personalized medicine from concept to reality may require an alternative approach in the field of tissue specimen procurement. MATERIALS AND METHODS: "Exfoliation and Enrichment" (EE), a recently described rapid and cost-effective approach for procuring cells, was utilized and assessed relative to a more traditional but temporally and economically comparable approach. Material from the same tumor sample, one collected by EE but the other frozen, were procured, the DNA extracted, and the samples analyzed at the global and gene-specific level by array comparative genomic hybridization and quantitative polymerase chain reaction, respectively. RESULTS: Both approaches resulted in the rapid procurement and retrieval of well-preserved cells and nucleic acids. The presence of "contaminating" normal cells in the more traditional approach masked the significance of genetic gains and losses, findings that were more readily apparent from the material derived by the EE method. CONCLUSION: The EE approach represents a cost-effective alternative to traditional cell-procurement methods that results in the generation of superior genomic data.

Universal Reference RNA is not a representative normal sample for oligonucleotide microarray studies.

Translational research has been defined as the scientific study using human material that will ultimately generate patient specific data. A major caveat in human directed study is the availability of high quality and quantities of patient derived homogeneous cells for analysis. Whereas there exist sources for which tumor tissue and blood samples can be made available, the same cannot be said for normal tissue. The absence of normal control tissue has led to the creation of pooled cell lines and tissues for purchase known as "reference RNA". Although initially created for purposes of standardization, the difficulty associated with acquiring normal tissue has led some investigators to use sources of universal pooled RNA for comparative analysis with clinical tissue specimens. In order to study the effects of using Universal Reference RNA on expression profiling experiments we have evaluated the performance of universal RNA compared to RNA obtained from a purified population of colon epithelial cells in defining a set of altered transcripts in colon cancer.

An exfoliation and enrichment strategy results in improved transcriptional profiles when compared to matched formalin fixed samples.

BACKGROUND: Identifying the influence formalin fixation has on RNA integrity and recovery from clinical tissue specimens is integral to determining the utility of using archival tissue blocks in future molecular studies. For clinical material, the current gold standard is unfixed tissue that has been snap frozen. Fixed and frozen tissue however, both require laser capture microdissection to select for a specific cell population to study. The recent development of a sampling method capable of obtaining a viable, enriched cell population represents an alternative option in procuring cells from clinical material for molecular research purposes. The expression profiles of cells obtained by using this procurement approach, in conjunction with the profiles from cells laser capture microdissected from frozen tissue sections, were compared to the expression profiles from formalin fixed cells to determine the influence fixation has on expression profiles in clinical material. METHODS: Triplicate samples of non-neoplastic colonic epithelial cells were recovered from a hemicolectomy specimen using three different procurement methods from the same originating site: 1) an exfoliation and enrichment strategy 2) laser capture microdissection from formalin fixed tissue and 3) laser capture microdissection from frozen tissue. Parameters currently in use to assess RNA integrity were utilized to assess the quality of recovered RNA. Additionally, an expression microarray was performed on each sample to assess the influence each procurement technique had on RNA recovery and degradation. RESULTS: The exfoliation/enrichment strategy was quantitatively and qualitatively superior to tissue that was formalin fixed. Fixation negatively influenced the expression profile of the formalin fixed group compared to both the frozen and exfoliated/enrichment groups. CONCLUSION: The exfoliation/enrichment technique represents a superior alternative in tissue procurement and RNA recovery relative to formalin fixed tissue. None of the deleterious effects associated with formalin fixation are encountered in the exfoliated/enriched samples because of the absence of its use in this protocol. The exfoliation/enrichment technique also represents an economical alternative that will yield comparable results to cells enriched by laser capture microdissection from frozen tissue sections.

Presence of the BRAF V600E point mutation in morphologically benign appearing thyroid inclusions of cervical lymph nodes.

The biologic nature of morphologically bland-appearing thyroid inclusions in cervical lymph nodes continues to be a controversial topic. The diagnosis of benignity entails a much more conservative clinical approach than does malignancy. Arriving at the correct interpretation, however, can be difficult when only morphologic examination is performed. Incorporating the use of the recently identified BRAF V600E point mutation, a highly specific biomarker for papillary carcinoma of the thyroid, may provide a useful adjunct in assessing the biologic nature of morphologically bland-appearing thyroid inclusions in cervical lymph nodes. In this case report, bland thyroid inclusions were noted in addition to a primary papillary carcinoma of the thyroid and morphologically recognizable cervical lymph node metastasis. Cells from these separate entities were procured by lasercapture microdissection, and the DNA was isolated, amplified, and sequenced. Molecular analysis provided integral data indicating these morphologically bland thyroid inclusions were malignant, findings not readily apparent by morphologic examination alone.

Manual exfoliation plus immunomagnetic bead separation as an initial step toward translational research.

CONTEXT: The development of biotechnologic platforms capable of high throughput analysis has ushered in a promising new era of translational medicine. However, most studies to date are based on in vitro cell lines or substitute models for human disease. Although these model systems have proven insightful, it is readily becoming apparent that human clinical tissue must be studied in order to fully understand all the nuances of human disease. Studies that are based on human tissue, however, are limited by qualitative and quantitative issues, factors often precluding their use in high throughput studies. OBJECTIVE: To develop a simple and rapid tissue procurement protocol for use in obtaining a homogeneous epithelial cell population from clinical tissue and the recovery of nucleic acids and proteins of high quality and quantity. Also, to determine if the technique preserves tissue, thereby allowing morphologic correlation with molecular findings. DESIGN: Performance of manual exfoliation to procure cells from clinical resection specimens and use of immunomagnetic beads embedded with the antibody ber-Ep4 for the positive enrichment of a homogeneous epithelial cell population. Nucleic acids and proteins are then separated using a phenol plus guanidine thiocyante solution. Nucleic acids and proteins are quantitated and qualitatively analyzed using standard laboratory techniques. RESULTS: Nucleic acids and proteins of high quality and quantity were recovered following manual exfoliation and immunomagnetic bead separation. Tissue architecture was not destroyed, thus permitting histologic and molecular correlation. CONCLUSIONS: A simple and reproducible protocol is presented that may enable the molecular profiling of clinically resected tissue. Although the technique is currently limited to certain tissue and tumor types, further research will broaden its overall application.

Manual exfoliation of fresh tissue obviates the need for frozen sections for molecular profiling.

BACKGROUND: Simple, rapid tissue processing that preserves macromolecules will enhance translational research capabilities. Traditional fixative-based approaches for specimen preservation are ideal for histologic evaluation but are not conducive to molecular studies of nucleic acids and protein. Tissue cryosections preserve macromolecule integrity, but the process is labor intensive and technically challenging. To the authors' knowledge to date, an alternative method capable of retrieving cells while providing adequate histologic detail yet preserving macromolecule integrity has been lacking. In the current study, the authors evaluated the utility of using manual exfoliation of clinical tissue samples as a means of obtaining cells for molecular analysis. This technique possesses the advantages of fixed and frozen tissue sections without their drawbacks. This simple, rapid, nonfixative based technique is capable of preparing cells from human clinical material for further isolation without compromising the preservation of macromolecules in the tissue. METHODS: Cells from a variety of clinical resection specimens from solid tumors were directly scraped from the tissue samples using the edge of a glass microscope slide and smeared onto another slide for cytologic evaluation. The manually exfoliated cells were evaluated microscopically for cytologic quality and cellular quantity. Pure cell populations were procured by laser capture microdissection (LCM) with subsequent extraction of nucleic acids and proteins. The integrity and suitability of the recovered nucleic acids and proteins for molecular analysis were evaluated using the polymerase chain reaction (PCR), reverse transcriptase-PCR, and reverse-phase protein microarray, respectively. RESULTS: Manual exfoliation permits the selection of homogeneous cell populations by LCM based on well established cytologic characteristics. DNA and mRNA, of comparable quality to frozen sections, can be amplified from the manual exfoliation cells. Proteins of similar quality can be recovered using this technique and quantitated via reverse-phase protein microarray. CONCLUSIONS: Molecular macromolecules of high quality and sufficient quantity can be retrieved from human clinical samples using manual exfoliation and LCM to procure specific cell populations. The manual exfoliation technique does not destroy the original tissue source, thereby allowing subsequent formalin tissue fixation. The technique of manual exfoliation in conjunction with LCM can enable the molecular profiling of a sampled selected cell population. Because it does not destroy the original tissue, histologic correlation can be combined with molecular profiling.