Optimal Method for Reporting Prostate Cancer Grade in MRI-targeted Biopsies.

When multiple cores are biopsied from a single magnetic resonance imaging (MRI)-targeted lesion, Gleason grade may be assigned for each core separately or for all cores of the lesion in aggregate. Because of the potential for disparate grades, an optimal method for pathology reporting MRI lesion grade awaits validation. We examined our institutional experience on the concordance of biopsy grade with subsequent radical prostatectomy (RP) grade of targeted lesions when grade is determined on individual versus aggregate core basis. For 317 patients (with 367 lesions) who underwent MRI-targeted biopsy followed by RP, targeted lesion grade was assigned as (1) global Grade Group (GG), aggregated positive cores; (2) highest GG (highest grade in single biopsy core); and (3) largest volume GG (grade in the core with longest cancer linear length). The 3 biopsy grades were compared (equivalence, upgrade, or downgrade) with the final grade of the lesion in the RP, using κ and weighted κ coefficients. The biopsy global, highest, and largest GGs were the same as the final RP GG in 73%, 68%, 62% cases, respectively (weighted κ: 0.77, 0.79, and 0.71). For cases where the targeted lesion biopsy grade scores differed from each other when assigned by global, highest, and largest GG, the concordance with the targeted lesion RP GG was 69%, 52%, 31% for biopsy global, highest, and largest GGs tumors (weighted κ: 0.65, 0.68, 0.59). Overall, global, highest, and largest GG of the targeted biopsy show substantial agreement with RP-targeted lesion GG, however targeted global GG yields slightly better agreement than either targeted highest or largest GG. This becomes more apparent in nearly one third of cases when each of the 3 targeted lesion level biopsy scores differ. These results support the use of global (aggregate) GG for reporting of MRI lesion-targeted biopsies, while further validations are awaited.

Anal Cancer with Mediastinal Lymph Node Metastasis.

Squamous cell carcinoma of the anal canal remains rare, with metastatic disease even less commonly reported. We present a case of a patient with both a prior history of squamous cell carcinoma of the anal canal as well as breast cancer, who was without evidence of disease for 1 year. She was subsequently found to have FDG-avid mediastinal lymphadenopathy, initially assumed to be related to her more recent breast cancer. However, a biopsy confirmed recurrent anal cancer, with HPV infection. This represents a novel site of spread for anal cancer, one not yet reported in the literature.

Somatic Mutations of TSC2 or MTOR Characterize a Morphologically Distinct Subset of Sporadic Renal Cell Carcinoma With Eosinophilic and Vacuolated Cytoplasm.

The differential diagnosis of renal cell neoplasms with solid or nested architecture and eosinophilic cytoplasm has become increasingly complex. Despite recent advances in classifying a number of entities exhibiting this morphology, some tumors remain in the unclassified category. Here we describe a morphologically distinct group of sporadic renal cell carcinoma (RCC) with predominantly nested architecture, eosinophilic, and remarkably vacuolated cytoplasm retrospectively identified from a cohort of previously unclassified tumors. We examined the clinicopathologic and immunohistochemical features of these tumors and investigated their mutational and copy number alterations using a targeted next-generation sequencing platform. The study included 7 patients with a mean age of 54 years (range: 40 to 68 y) and a male to female ratio of 3:4. All patients presented with a solitary renal mass and had no prior medical or family history raising concern for syndromic conditions. Tumors were well-circumscribed, unencapsulated, and comprised of nests of eosinophilic cells in a hypocellular and often edematous stroma. Tumor cells had round nuclei with prominent nucleoli and granular cytoplasm with striking vacuolization. Thick-walled vessels and calcifications were also frequently present, whereas increased mitotic activity, necrosis, foamy histiocytes or lymphocytic infiltrates were not identified. All cases were positive for PAX8, had retained expression of SDHB and FH, and exhibited a CK7-/CK20- phenotype. While cathepsin-K was positive in 5 cases, none exhibited immunoreactivity to HMB45 or Melan A, or TFE3 immunostaining. Next-generation sequencing identified somatic inactivating mutations of TSC2 (3/5 tumors tested) or activating mutations of MTOR (2/5) as the primary molecular alterations, consistent with hyperactive mTOR complex 1 signaling which was further demonstrated by phospho-S6 and phospho-4E-BP1 immunostaining. Copy number analysis revealed a loss of chromosome 1 in both cases with MTOR mutation. These tumors represent a novel subset of sporadic RCC characterized by alterations in TSC1-TSC2 complex or the mTOR complex 1 pathway. Recognition of their characteristic morphologic and immunophenotypic features will allow them to be readily identified and separated from the unclassified RCC category.

Clues to recognition of fumarate hydratase-deficient renal cell carcinoma: Findings from cytologic and limited biopsy samples.

BACKGROUND: Fumarate hydratase (FH)-deficient renal cell carcinoma (RCC) is rare and highly aggressive and is believed to arise mostly in the setting of hereditary leiomyomatosis-RCC syndrome with a germline mutation of FH. Because of the aggressiveness of these tumors and a frequent lack of ascertainable family history, these tumors may first present as metastases and be sampled by cytology. The cytologic findings of FH-deficient RCC have not previously been reported. METHODS: Cytologic and limited biopsy samples from patients with FH-deficient RCC were reviewed retrospectively. RESULTS: In total, 24 cytologic and limited biopsy samples from 19 patients (6 women and 13 men; age range, 22-69 years) who had FH-deficient RCC and metastasis at presentation were evaluated. These included 21 cytology samples ranging from malignant effusions (n = 7) to metastases (n = 11), to samples of primary kidney tumors (n = 3). The samples exhibited cells, often in clusters and abortive papillae, with voluminous, finely vacuolated cytoplasm and large, pleomorphic nuclei with prominent, viral inclusion-like nucleoli. A distinctive finding of peripheral cytoplasmic clearing frequently was apparent, and intranuclear cytoplasmic pseudoinclusions were less frequent. Of 7 cell block and biopsy samples, several of which represented sampling from the same patient, all demonstrated tissue fragments that had discernable morphologic patterns associated with FH-deficient RCC, including tubulocystic and intracystic papillary growth. CONCLUSIONS: Features characteristic and suggestive of FH-deficient RCC may be identified in cytologic and small biopsy samples. Although the current samples were identified retrospectively in well characterized cases of FH-deficient RCC, the authors argue that, with appropriate clinical correlation, these features are sufficiently distinctive to trigger recognition and confirmatory workup.

A Single Institutional Experience With the Paris System for Reporting Urinary Cytology: Correlation of Cytology and Histology in 194 Cases.

OBJECTIVES: The Paris System for Reporting Urinary Cytology (TPS) is designed to standardize the criteria and terminology used in urinary tract cytology reporting. The aim of this study was to evaluate the impact of implementing TPS and to analyze the correlation with follow-up biopsies in order to assess its reproducibility. METHODS: Urinary tract cytology specimens with follow-up biopsies over a 2-year period were reviewed and reclassified according to TPS criteria. Surgical follow-up diagnoses were correlated with the initial cytology diagnoses and TPS interpretations, and the results were compared. RESULTS: Applying TPS in comparison to our previous reporting system resulted in fewer cases in the atypia category (11.8% vs 24.2%) and higher specificity, accuracy, and predictive value. We observed acceptable interobserver agreement in diagnostic categories of this reporting system. CONCLUSIONS: TPS improves the overall performance of urinary tract cytology by standardizing the criteria and terminology.

Serous Carcinoma Mimicking Primary Urothelial Carcinoma on Clinical Evaluation and Pathology: A Potential Diagnostic Pitfall.

CONTEXT: - Serous carcinoma of the gynecologic tract often involves the external bladder wall and can occasionally mimic primary urothelial carcinoma of the bladder. OBJECTIVE: - To define the spectrum of morphologic and immunohistochemical features that characterize serous carcinoma involving the bladder wall and its distinction from urothelial carcinoma. DESIGN: - We reviewed all cases of serous carcinoma secondarily involving the bladder wall from the University of California San Diego and Polytechnic Institute for histopathologic and immunohistochemical features. RESULTS: - We identified 20 cases of Müllerian high-grade serous carcinoma involving the bladder wall. Five cases were clinical mimics of urothelial carcinoma, including 2 cases that presented as a large, transmural, primary bladder mass without precedent gynecologic history in women younger than 60 years, and 3 cases presumed to be new bladder carcinoma occurring distant to a serous carcinoma diagnosis. A subset of cases were morphologic mimics of urothelial carcinoma, which showed nested growth patterns (2 of 20; 10%), squamouslike foci (2 of 20; 10%), spindled/sarcomatoid growth (2 of 20; 10%), basaloid morphology (3 of 20; 15%), and syncytial growth patterns (1 of 20; 5%). Immunohistochemical stains in 17 cases showed immunoreactivity for CK7 (17 of 17; 100%), WT1 (17 of 17; 100%), uroplakin (UP) II (1 of 17; 6%), p63 (2 of 17; 12%), GATA3 (2 of 17; 12%), and PAX8 (17 of 17; 100%). CONCLUSIONS: - A subset of serous carcinomas involving the bladder wall can mimic urothelial carcinoma. Awareness of this mimicker and use of an immunohistochemical panel that includes CK7, WT1, UPII, PAX8, p63, and GATA3 can be helpful in confirming the diagnosis.

Analysis of T1 Bladder Cancer on Biopsy and Transurethral Resection Specimens: Comparison and Ranking of T1 Quantification Approaches to Predict Progression to Muscularis Propria Invasion.

Urothelial carcinoma of the bladder invasive into lamina propria on biopsy or transurethral resection of bladder tumor, termed "T1" disease, progresses to muscularis propria invasion in a subset of patients. Prior studies have proposed histopathologic metrics to predict progression, although methods vary widely and it is unclear which method is most robust. This poses a challenge since recent World Health Organization and American Joint Commission on Cancer editions encourage some attempt to substratify T1 disease. To address this critical problem, we analyzed T1 specimens to test which T1 quantification method is best to predict progression and to then establish the optimal cut-off. Progression was analyzed for all patients or for patients with definitive muscularis propria only. Multivariate analysis and outcomes modeling controlled for additional histopathologic features. Our results suggest that aggregate linear length of invasive carcinoma (ALLICA) measured by optical micrometer is far superior to other methods (P=3.067×10) and could be applied to 100% of specimens. ALLICA retained significance in multivariate analysis and eliminated contribution of other histopathologic features to progression. The best cut-off for ALLICA using a 30% false-positive threshold was 2.3 mm and using a 10% false-positive threshold at 25 mm, although the latter severely limited patients who could achieve this threshold. After comparison of all proposed methods of T1 quantification, we recommend the adoption of the ALLICA measurement and a cut-off of ≥2.3 mm as the best predictor of progression, acknowledging that additional nonhistopathologic methods may be required to increase broad applicability and further reduce the false-positive threshold.

Nitroxoline induces apoptosis and slows glioma growth in vivo.

BACKGROUND: Nitroxoline is an FDA-approved antibiotic with potential antitumor activity. Here we evaluated whether nitroxoline has antiproliferative properties on glioma cell growth in vitro and in vivo using glioma cell lines and a genetically engineered PTEN/KRAS mouse glioma model. METHODS: The effect of nitroxoline treatment on U87 and/or U251 glioma cell proliferation, cell-cycle arrest, invasion, and ability to induce an apoptotic cascade was determined in vitro. Magnetic resonance imaging was used to measure glioma volumes in genetically engineered PTEN/KRAS mice prior to and after nitroxoline therapy. Induction of apoptosis by nitroxoline was evaluated at the end of treatment using terminal deoxyribonucleotidyl transferase (TDT)-mediated dUTP-digoxigenin nick end labeling (TUNEL). RESULTS: Nitroxoline inhibited the proliferation and invasion of glioblastoma cells in a time- and dose-dependent manner in vitro. Growth inhibition was associated with cell-cycle arrest in G1/G0 phase and induction of apoptosis via caspase 3 and cleaved poly(ADP-ribose) polymerase. In vivo, nitroxoline-treated mice had no increase in tumor volume after 14 days of treatment, whereas tumor volumes doubled in control mice. Histological examination revealed 15%-20% TUNEL-positive cells in nitroxoline-treated mice, compared with ∼5% in the control group. CONCLUSION: Nitroxoline induces apoptosis and inhibits glioma growth in vivo and in vitro. As an already FDA-approved treatment for urinary tract infections with a known safety profile, nitroxoline could move quickly into clinical trials pending confirmatory studies.

The pituitary stalk effect: is it a passing phenomenon?

Most patients with large pituitary tumors do not exhibit hyperprolactinemia as a result of pituitary lactotroph disinhibition (stalk effect). Studies have demonstrated that increased intrasellar pressure is associated with both "stalk effect" hyperprolactinemia and pituitary insufficiency. Our primary hypothesis was that, despite continued disinhibition, lactotroph failure is responsible for normoprolactinemia in patients with large macroadenomas. As a corollary, we proposed that the hyperprolactinemia phase, which presumably would precede the insufficiency/normoprolactinemic state, would more likely be discovered in premenopausal females and go unnoticed in males. Prospective, consecutive surgical series of 98 patients of clinically nonfunctional pituitary adenomas. Lactotroph insufficiency was inferred by the coexistence of insufficiency in another pituitary axis. The existence of pre-operative lactotroph disinhibition was inferred based on comparison of pre- versus post-operative prolactin levels. 87 % of patients with tumor size >20 mm and normoprolactinemia had pituitary insufficiency. Pre-operative prolactin in patients with pituitary insufficiency were lower than those with intact pituitary function. Prolactin levels dropped in nearly all patients, including patients with normoprolactinemia pre-operatively. Premenopausal women had smaller tumors and higher pre-operative prolactin levels compared to males. No premenopausal female exhibited evidence of pituitary insufficiency. Our study provides suggestive evidence that the "stalk effect" pathophysiology is the norm rather than the exception, and that the finding of normoprolactinemia in a patient with a large macroadenoma is likely a consequence of lactotroph insufficiency. In males, the hyperprolactinemia window is more likely to be missed clinically due to an absence of prolactin-related symptoms.

C-terminally truncated form of αB-crystallin is associated with IDH1 R132H mutation in anaplastic astrocytoma.

Malignant gliomas are the most common human primary brain tumors. Point mutation of amino acid arginine 132 to histidine (R132H) in the IDH1 protein leads to an enzymatic gain-of-function and is thought to promote gliomagenesis. Little is known about the downstream effects of the IDH1 mutation on protein expression and how and whether changes in protein expression are involved in tumor formation or propagation. In the current study, we used 2D DIGE (difference gel electrophoresis) and mass spectrometry to analyze differences in protein expression between IDH1(R132H) mutant and wild type anaplastic (grade III) astrocytoma from human brain cancer tissues. We show that expression levels of many proteins are altered in IDH1(R132H) mutant anaplastic astrocytoma. Some of the most over-expressed proteins in the mutants include several forms of αB-crystallin, a small heat-shock and anti-apoptotic protein. αB-crystallin proteins are elevated up to 22-fold in IDH1(R132H) mutant tumors, and αB-crystallin expression appears to be controlled at the post-translational level. We identified the most abundant form of αB-crystallin as a low molecular weight species that is C-terminally truncated. We also found that overexpression of αB-crystallin can be induced by transfecting U251 human glioblastoma cell lines with the IDH1(R132H) mutation. In conclusion, the association of a C-terminally truncated form of αB-crystallin protein with the IDH1(R132H) mutation is a novel finding that could impact apoptosis and stress response in IDH1 mutant glioma.

A review of room temperature storage of biospecimen tissue and nucleic acids for anatomic pathology laboratories and biorepositories.

UNLABELLED: Frozen biospecimens are crucial for translational research and contain well-preserved nucleic acids and protein. However, the risks of freezer failure as well as space, cost, and environmental concerns of frozen biospecimens are substantial. OBJECTIVE: The purpose of the study was to review the current status of room temperature biospecimen storage. METHODS: We searched Pubmed and vendor websites to identify relevant information. RESULTS: Formalin-fixed paraffin embedded (FFPE) tissues have great value but their use is limited by cross-linking and fragmentation of nucleic acids, as well as loss of enzymatic activity. Stabilization solutions can now robustly preserve fresh tissue for up to 7days at room temperature. For longer term storage, commercial vendors of chemical matrices claim real time stability of nucleic acids of over 2 years and their accelerated aging studies to date suggest stability for 12years for RNA and 60years for DNA. However, anatomic pathology biorepositories store mostly frozen tissue rather than nucleic acids. Small quantities of tissue can be directly placed on some chemical matrices to stabilize DNA, however RNA and proteins are not preserved. Current lyophilization approaches can preserve histomorphology, DNA, RNA, and proteins though RNA shows moderate degradation after 1-2years. Formalin-free fixatives show improved but varying abilities to preserve nucleic acids and face validation as well as cost barriers in replacing FFPE specimens. The paraffin embedding process can degrade RNA. CONCLUSION: Development of robust long-term room temperature biospecimen tissue storage technology can potentially reduce costs for the biomedical community in the face of growing targeted therapy needs and decreasing budgets.

Lyophilized brain tumor specimens can be used for histologic, nucleic acid, and protein analyses after 1 year of room temperature storage.

Frozen tissue, a gold standard biospecimen, can yield well preserved nucleic acids and proteins after over a decade but is vulnerable to thawing and has substantial fiscal, spatial, and environmental costs. A long-term room temperature biospecimen storage alternative that preserves broad analytical utility can potentially empower tissue-based research. As there is scant data on the analytical utility of lyophilized brain tumor biospecimens, we evaluated lyophilized (freeze-dried) samples stored for 1 year at room temperature. Lyophilized tumor tissue processed into paraffin sections produced good histology. Yields of extracted DNA, RNA, and protein approximated those of frozen tissue. After 1 year, lyophilized samples yielded high molecular weight DNA that permitted copy number variation analysis, IDH 1 mutation detection, and MGMT promoter methylation PCR. A 27 % decrease in RIN scores over the 1 year suggests that RNA degradation was inhibited though incompletely. Nevertheless, RT-PCR studies on lyophilized tissue performed similarly to frozen tissue. In contrast to FFPE tissues where protein bands were absent or shifted to a lower molecular weight, lyophilized samples showed similar protein bands as frozen tissue on SDS-PAGE analysis. Lyophilized tissue performed similarly to frozen tissue for Western blots and enzyme activity assays. Immunohistochemistry of lyophilized tissue that were processed into FFPE blocks often required longer incubation times for staining than standard FFPE samples but generally provided robust antigen detection. This preliminary study suggests that lyophilization has promise for long-term room temperature storage while permitting varied tests; however, further work is required to better stabilize nucleic acids particularly RNA.

Evidence for orphan nuclear receptor TR4 in the etiology of Cushing disease.

Cushing disease (CD) is a life-threatening disorder attributed to excess pituitary tumor-derived adrenocorticotrophic hormone (ACTH) and adrenal steroid secretion caused by pituitary tumors. Whereas CD was first described in 1932, the underlying genetic basis driving tumor growth and ACTH secretion remains unsolved. Here, we show that testicular orphan nuclear receptor 4 (TR4, nuclear receptor subfamily 2, group C, member 2) is overexpressed in human corticotroph tumors as well as in human and mouse corticotroph tumor cell lines. Forced overexpression of TR4 in both human and murine tumor cells increased proopiomelanocortin transcription, ACTH secretion, cellular proliferation, and tumor invasion rates in vitro. Conversely, knockdown of TR4 expression reversed all phenotypes. Mechanistically, we show that TR4 transcriptionally activates proopiomelanocortin through binding of a direct repeat 1 response element in the promoter, and that this is enhanced by MAPK-mediated TR4 phosphorylation. In vivo, TR4 overexpression promotes murine corticotroph tumor growth as well as enhances ACTH and corticosterone production, whereas TR4 knockdown decreases circulating ACTH and corticosterone levels in mice harboring ACTH-secreting tumors. Our findings directly link TR4 to the etiology of corticotroph tumors, hormone secretion, and cell growth as well as identify it as a potential target in the treatment of CD.

Primary central nervous system histiocytic sarcoma presenting as a postradiation sarcoma: case report and literature review.

Histiocytic sarcoma (HS) is a rare neoplasm that occurs most commonly in the intestinal tract, skin, soft tissue, and lymph node. The incidence of primary central nervous system (CNS) HS is even rarer, with a total of 6 cases reported in the literature. An etiologic link has not been identified for CNS HS, and the current case of primary CNS HS is unique in that an etiologic link to prior radiation therapy is identified, associated with complex cytogenetic abnormalities in the tumor. Although radiation-associated sarcomas can present as any number of different pathologic entities, this is the first reported case of a radiation-associated CNS HS. The pathologic and immunophenotypic characteristics of this case, with a nearly obscuring heavy inflammatory infiltrate and expression of monocytic/histiocytic markers (CD163, CD68, CD4, fascin), are characteristic of CNS HS. A discussion of the differential diagnosis and review of relevant literature are presented.

Detection of 2-hydroxyglutaric acid in vivo by proton magnetic resonance spectroscopy in U87 glioma cells overexpressing isocitrate dehydrogenase-1 mutation.

The arginine 132 (R132) mutation of isocitrate dehydrogenase -1 (IDH1(R132)) results in production of 2-hydroxyglutarate (2-HG) and is associated with a better prognosis compared with wild-type (WT) in glioma patients. The majority of lower-grade gliomas express IDH1(R132), whereas this mutation is rare in grade IV gliomas. The aim of this study was to noninvasively investigate metabolic and physiologic changes associated with the IDH1 mutation in a mouse glioma model. Using a 7T magnet, we compared MRI and proton magnetic resonance spectroscopy (MRS) in U87 glioma cells overexpressing either the mutated IDH1(R132) or IDH1 wild-type (IDH1(WT)) gene in a mouse flank xenograft model. Flank tumors overexpressing IDH1(R132) showed a resonance at 2.25 ppm corresponding to the 2-HG peak described for human IDH1(R132) gliomas. WT tumors lacked this peak in all cases. IDH1 mutant tumors demonstrated significantly reduced glutamate by in vivo MRS. There were no significant differences in T(2), apparent diffusion coefficient (ADC), or perfusion values between the mutant and IDH1(WT) tumors. The IDH1(R132) mutation results in 2-HG resonance at 2.25 ppm and a reduction of glutamate levels as determined by MRS. Our results establish a model system where 2-HG can be monitored noninvasively, which should be helpful in validating 2-HG levels as a prognostic and/or predictive biomarker in glioma.

Bilateral adrenalectomy: lifesaving procedure in severe Cushing syndrome.

OBJECTIVE: To discuss the role of bilateral adrenalectomy in Cushing syndrome, as illustrated in a case of severe hypercortisolism that was unresponsive to combination agent medical therapy. METHODS: We report the clinical, laboratory, imaging, and pathologic findings in a patient with ectopic Cushing syndrome attributable to an adrenocorticotropic hormone (ACTH)-secreting neuroblastoma. In addition, we provide a literature review regarding olfactory neuroblastoma and discuss current and emerging therapeutic options for Cushing syndrome. RESULTS: A 59-year-old man presented with nasal congestion and neck swelling and was noted to have hypokalemia, hypertension, and hyperglycemia. A nasal biopsy demonstrated a poorly differentiated carcinoma with neuroendocrine features. He was subsequently diagnosed as having ACTH-dependent Cushing syndrome, but despite high-dose combination medical therapy, his condition rapidly deteriorated. Urgent bilateral adrenalectomy provided rapid control of the hypercortisolism, and the patient was later able to undergo an uncomplicated total macroscopic resection of his locally metastatic primary tumor. CONCLUSION: This report describes the challenges in the diagnosis and management of ACTH-dependent Cushing syndrome and highlights the important role that bilateral adrenalectomy can still have in patients with severe hypercortisolism causing life-threatening complications.