Lesion-based indicators predict long-term outcomes of pheochromocytoma and paraganglioma- SIZEPASS.

Aim: We seek a simple and reliable tool to predict malignant behavior of pheochromocytoma and paraganglioma (PPGL). Methods: This single-center prospective cohort study assessed size of primary PPGLs on preoperative cross-sectional imaging and prospectively scored specimens using the Pheochromocytoma of the Adrenal Gland Scaled Score (PASS). Multiplication of PASS points with maximum lesion diameter (in mm) yielded the SIZEPASS criterion. Local recurrence, metastasis or death from disease were surrogates defining malignancy. Results: 76 consecutive PPGL patients, whereof 58 with pheochromocytoma and 51 female, were diagnosed at a mean age of 52.0 ± 15.2 years. 11 lesions (14.5%) exhibited malignant features at a median follow-up (FU) of 49 months (range 4-172 mo). Median FU of the remaining cohort was 139 months (range 120-226 mo). SIZEPASS classified malignancy with an area under the curve (AUC) of 0.97 (95%CI 0.93-1.01; p<0.0001). Across PPGL, SIZEPASS >1000 outperformed all known predictors of malignancy, with sensitivity 91%, specificity 94%, and accuracy 93%, and an odds ratio of 72 fold (95%CI 9-571; P<0.001). It retained an accuracy >90% in cohorts defined by location (adrenal, extra-adrenal) or mutation status. Conclusions: The SIZEPASS>1000 criterion is a lesion-based, clinically available, simple and effective tool to predict malignant behavior of PPGLs independently of age, sex, location or mutation status.

Heterogeneous topographic profiles of kinetic and cell cycle regulator microsatellites in atypical (dysplastic) melanocytic nevi.

Atypical (dysplastic) melanocytic nevi are clinically heterogeneous malignant melanoma precursors, for which no topographic analysis of cell kinetic, cell cycle regulators and microsatellite profile is available. We selected low-grade atypical melanocytic nevi (92), high-grade atypical melanocytic nevi (41), melanocytic nevi (18 junctional, 25 compound) and malignant melanomas (16 radial growth phase and 27 vertical growth phase). TP53, CDKN2A, CDKN1A, and CDKN1B microsatellite patterns were topographically studied after microdissection; Ki-67, TP53, CDKN2A, CDKN1A, and CDKN1B expressions and DNA fragmentation by in situ end labeling for apoptosis were topographically scored. Results were statistically analyzed. A decreasing junctional-dermal marker expression gradient was observed, directly correlating with atypical melanocytic nevus grading. High-grade atypical melanocytic nevi revealed coexistent TP53-CDKN2A-CDKN1B microsatellite abnormalities, and significantly higher junctional Ki67-TP53 expression (inversely correlated with CDKN1A-CDKN1B expression and in situ end labeling). Malignant melanomas showed coexistent microsatellite abnormalities (CDKN2A-CDKN1B), no topographic gradient, and significantly decreased expression. Melanocytic nevi and low-grade atypical melanocytic nevi revealed sporadic junctional CDKN2A microsatellite abnormalities and no significant topographic kinetic differences. High-grade atypical melanocytic nevi accumulate junctional TP53-CDKN1A-CDKN1B microsatellite abnormalities, being progression TP53-independent and better assessed in the dermis. Melanocytic nevi and low-grade atypical melanocytic nevi show low incidence of microsatellite abnormalities, and kinetic features that make progression unlikely.

Application of validated severity scores for pneumonia caused by SARS-CoV-2.

OBJECTIVES: Compare the accuracy of PSI, CURB-65, MuLBSTA and COVID-GRAM prognostic scores to predict mortality, the need for invasive mechanical ventilation (IMV) in patients with pneumonia caused by SARS-CoV-2 and assess the coexistence of bacterial respiratory tract infection during admission. METHODS: Retrospective observational study that included hospitalized adults with pneumonia caused by SARS-CoV-2 from 15/03 to 15/05/2020. We excluded immunocompromised patients, nursing home residents and those admitted in the previous 14 days for another reasons. Analysis of ROC curves was performed, calculating the area under the curve for the different scales, as well as sensitivity, specificity and predictive values. RESULTS: 208 patients were enrolled, aged 63 ± 17 years, 577% were men. 38 patients were admitted to ICU (235%), of these patients 33 required IMV (868%), with an overall mortality of 125%. Area under the ROC curves for mortality of the scores were: PSI 082 (95% CI 073-091), CURB-65 082 (073-091), MuLBSTA 072 (062-081) and COVID-GRAM 086 (070-1). Area under the curve for needing IMV was: PSI 073 (95% CI 064-082), CURB-65 066 (055-077), MuLBSTA 078 (069-086) and COVID-GRAM 076 (067-085), respectively. Patients with bacterial co-infections of the respiratory tract were 20 (9,6%), the most frequent strains being Pseudomonas aeruginosa and Klebsiella pneumoniae. CONCLUSIONS: In our study, the COVID-GRAM score was the most accurate to identify patients with higher mortality with pneumonia caused by SARS-CoV-2; however, none of these scores accurately predicts the need for IMV with ICU admission. 10% of patients admitted presented bacterial respiratory co-infection.

OBJETIVOS: Comparar el rendimiento de las escalas pronósticas PSI, CURB-65, MuLBSTA y COVID-GRAM para predecir mortalidad y necesidad de ventilación mecánica invasiva (VMI) en pacientes con neumonía por SARS-CoV-2. Valorar la existencia de coinfección bacteriana respiratoria durante el ingreso. MÉTODO: Estudio observacional retrospectivo que incluyó adultos hospitalizados con neumonía por SARS-CoV-2 del 15/03 al 15/05/2020. Se excluyeron aquellos inmunodeprimidos, institucionalizados e ingresados en los 14 días previos por otro motivo. Se realizó un análisis de curvas ROC, calculando el área bajo la curva para las diferentes escalas, así como sensibilidad, especificidad y valores predictivos. RESULTADOS: Se incluyeron 208 pacientes, con edad de 63 ± 17 años; el 57,7% eran hombres. Ingresaron en UCI 38 (23,5%), precisando de estos VMI 33 (86,8%), con una mortalidad global del 12,5%. Las áreas bajo las curvas ROC para mortalidad de los scores fueron: PSI 0,82 (95% IC 0,73­0,91), CURB-65 0,82 (0,73­0,91), MuLBSTA 0,72 (0,62­0,81) y COVID-GRAM 0,86 (0,70­1). Las áreas para necesidad de VMI fueron: PSI 0,73 (95% IC 0,64­0,82), CURB-65 0,66 (0,55­0,77), MuLBSTA 0,78 (0,69­0,86) y COVID-GRAM 0,76 (0,67­0,85), respectivamente. Los pacientes que presentaron coinfección bacteriana respiratoria fueron 20 (9.6%) siendo los gérmenes más frecuentes Pseudomonas aeruginosa y Klebsiella pneumoniae. CONCLUSIONES: En nuestro estudio el score COVID-GRAM fue el más preciso para identificar los pacientes con mayor mortalidad ingresados con neumonía por SARS-CoV-2, no obstante, ninguno de estos scores predice de forma precisa la necesidad de VMI con ingreso en UCI. El 10% de los pacientes presentó coinfección bacteriana respiratoria.

[Application of validated severity scores for pneumonia caused by SARS-CoV-2]. / Aplicación de escalas pronósticas de gravedad en la neumonía por SARS-CoV-2.

OBJECTIVES: Compare the accuracy of PSI, CURB-65, MuLBSTA and COVID-GRAM prognostic scores to predict mortality, the need for invasive mechanical ventilation in patients with pneumonia caused by SARS-CoV-2 and assess the coexistence of bacterial respiratory tract infection during admission. METHODS: Retrospective observational study that included hospitalized adults with pneumonia caused by SARS-CoV-2 from 15/03 to 15/05/2020. We excluded immunocompromised patients, nursing home residents and those admitted in the previous 14 days for another reasons. Analysis of ROC curves was performed, calculating the area under the curve for the different scales, as well as sensitivity, specificity and predictive values. RESULTS: A total of 208 patients were enrolled, aged 63±17 years, 57,7% were men; 38 patients were admitted to ICU (23,5%), of these patients 33 required invasive mechanical ventilation (86,8%), with an overall mortality of 12,5%. Area under the ROC curves for mortality of the scores were: PSI 0,82 (95% CI: 0,73-0,91), CURB-65 0,82 (0,73-0,91), MuLBSTA 0,72 (0,62-0,81) and COVID-GRAM 0,86 (0,70-1). Area under the curve for needing invasive mechanical ventilation was: PSI 0,73 (95% CI: 0,64-0,82), CURB-65 0,66 (0,55-0,77), MuLBSTA 0,78 (0,69-0,86) and COVID-GRAM 0,76 (0,67-0,85), respectively. Patients with bacterial co-infections of the respiratory tract were 20 (9,6%), the most frequent strains being Pseudomonas aeruginosa and Klebsiella pneumoniae. CONCLUSIONS: In our study, the COVID-GRAM score was the most accurate to identify patients with higher mortality with pneumonia caused by SARS-CoV-2; however, none of these scores accurately predicts the need for invasive mechanical ventilation with ICU admission. The 10% of patients admitted presented bacterial respiratory co-infection.

Histologic criteria for adrenocortical proliferative lesions: value of mitotic figure variability.

This study compared 3 systems and a newly designed stepwise discriminant diagnostic system (SDDS) to assess accuracy, reproducibility, and reliability in adrenocortical nodular hyperplasia (ACNH; n = 82), adenoma (ACA; n = 78), and carcinoma (ACC; n = 32) (diagnoses according to World Health Organization criteria; median follow-up, 135 months). In cross-validations, we studied cortex appearance, growth pattern, cytoplasmic features, nuclear parameters, mitotic figure counting (MFC), necrosis, invasion, and stromal-inflammatory reactions. The SDDS independent predictors were MFC/high-power field SD, anisokaryosis, cortex appearance, and stromal reaction, correctly classifying 100% of ACNH, 91% of ACA, and 88% of ACC cases. The Hough system correctly classified 78% of ACNH, 81% of ACA, and 84% of ACC cases; the Weiss and van Slooten systems correctly classified 100% of ACNH, 0% of ACA, and 92% of ACC cases. MFC variability is the most important adrenocortical malignancy criterion. Accurate malignancy diagnosis requires multiple variable evaluations, provided by SDDS (the most specific system) and the Weiss or van Slooten system (the most sensitive). SDDS is the most useful system for distinguishing tumors from ACNH (myxoid stroma and anisokaryosis).

Topographic molecular profile of pheochromocytomas: role of somatic down-regulation of mismatch repair.

CONTEXT AND OBJECTIVE: Despite extensive molecular investigation of adrenal pheochromocytomas, no information is available on their molecular and mismatch repair (MMR) profiles by topographic compartments. DESIGN AND SETTING: Microdissected samples from the peripheral and internal zones of 143 pheochromocytomas from a referral hospital (95 sporadic and 48 associated with multiple endocrine neoplasia type 2A) were selected for loss of heterozygosity and single nucleotide polymorphism analyses. Five polymorphic DNA regions from TP53, RB1, WT1, and NF1 were systematically studied by PCR-denaturing gradient gel electrophoresis. PATIENTS, OUTCOME MEASURES, AND INTERVENTIONS: Pheochromocytomas were classified as malignant (16 sporadic tumors with distant metastases), locally invasive (30 sporadic tumors showing retroperitoneal infiltration only), and benign (all remaining tumors). Statistical differences were evaluated using Fisher's exact test. MMR was assessed by MLH1/MSH2 sequencing and immunostaining in pheochromocytomas with two or more abnormal microsatellites. No interventions were performed in this study. RESULTS: Loss of heterozygosity/single nucleotide polymorphism involved TP53 in 40 of 134 informative cases (29.9%), RB1 in 22 of 106 informative cases (20.8%), WT1 in 32 of 120 informative cases (26.7%), and NF1 in 32 of 80 informative cases (40.0%). More genetic abnormalities involving the peripheral compartment were revealed in 34 pheochromocytomas (23.8%): 12 of 16 malignant, 10 of 30 locally invasive, and 12 of 97 benign. Multiple and coexistent genetic abnormalities characterized malignant pheochromocytomas (P < 0.001), whereas locally invasive pheochromocytomas showed a significantly higher incidence of NF1 alterations (P < 0.001). No mutations were identified in MLH1/MSH2, but MMR proteins significantly decreased in peripheral compartments. CONCLUSIONS: Multiple microsatellite alterations and topographic intratumor heterogeneity characterize malignant pheochromocytomas, suggesting a multistep tumorigenesis through somatic topographic down-regulation of MMR proteins. Locally invasive pheochromocytomas reveal topographic heterogeneity and single-locus microsatellite alterations, especially involving NF1.

DNA and kinetic heterogeneity during the clonal evolution of adrenocortical proliferative lesions.

Monoclonal adrenocortical lesions show inverse correlation between proliferation and apoptosis, with proliferation being the single most important criterion of malignancy in adrenal lesions. No study yet has evaluated the variability of proliferation regarding the clonal pattern and diagnosis in adrenocortical nodular hyperplasias (ACNHs), adrenocortical adenomas (ACAs), and adrenocortical carcinomas (ACCs). We studied 69 ACNHs, 64 ACAs, and 23 ACCs (World Health Organization criteria) from 156 females. Clonality HUMARA test (from microdissected DNA samples), DNA content and proliferation analysis (slide and flow cytometry), and mitotic figure (MF) counting/50 high-power fields (HPFs) were performed in the same areas. Heterogeneity was assessed by 5cER (percentage of nonoctaploid cells with DNA content exceeding 5c) and standard deviation of MF/HPF. Statistics included analysis of variance/Student t tests regarding the clonal patterns and diagnosis. Polyclonal patterns were observed in 48 of 62 informative ACNHs and 7 of 56 informative ACAs, and monoclonal in 14 of 62 ACNHs, 49 of 56 ACAs, and 21 of 21 ACCs, with all hyperdiploid lesions (14 ACCs and 13 ACAs) being monoclonal. The standard deviation of MF/HPF progressively increased in ACNH-ACA-ACC (0.048 +/- 0.076, 0.110 +/- 0.097, 0.506 +/- 0.291, respectively; P = .0023), but did not differentiate ACNH/ACA. Only tetraploid percentage (P = .0496) and 5cER (P = .0352) distinguished polyclonal (3.64 +/- 2.20 and 0.14 +/- 0.15) from monoclonal (7.25 +/- 7.52 and 1.00 +/- 1.74) benign lesions. Malignancy significantly correlated with a low diploid percentage and high tetraploid percentage. Cell kinetic heterogeneity is the hallmark of adrenocortical neoplasms: tetraploid/hypertetraploid cell accumulation characterizes monoclonal lesions (suggesting nondisjunctional mitoses), whereas heterogeneously distributed mitotic figures and decreased diploid percentage define ACCs.

Complementary analysis of microsatellite tumor profile and mismatch repair defects in colorectal carcinomas.

Microsatellite instability (MSI) is a prognostic factor and a marker of deficient mismatch repair (MMR) in colorectal adenocarcinomas (CRC). However, a proper application of this marker requires understanding the following: (1) The MSI concept: The PCR approach must amplify the correct locus and accurately identify the microsatellite pattern in the patient's normal tissue. MSI is demonstrated when the length of DNA sequences in a tumor differs from that of nontumor tissue. Any anomalous expansion or reduction of tandem repeats results in extra-bands normally located in the expected size range (100 bp, above or below the expected product), differ from the germline pattern by some multiple of the repeating unit, and must show appropriate stutter. (2) MSI mechanisms: MMR gene inactivation (by either mutation or protein down-regulation as frequently present in deep CRC compartments) leads to mutation accumulation in a cell with every cellular division, resulting in malignant transformation. These mechanisms can express tumor progression and result in a decreased prevalence of aneuploid cells and loss of the physiologic cell kinetic correlations in the deep CRC compartments. MSI molecular mechanisms are not necessarily independent from chromosomal instability and may coexist in a given CRC. (3) Because of intratumoural heterogeneity, at least two samples from each CRC should be screened, preferably from the superficial (tumor cells above the muscularis propria) and deep (tumor cells infiltrating the muscularis propria) CRC compartments to cover the topographic tumor heterogeneity. (4) Pathologists play a critical role in identifying microsatellite-unstable CRC, such as occur in young patients with synchronous or metachronous tumors or with tumors showing classic histologic features. In these cases, MSI testing and/or MMR immunohistochemistry are advisable, along with gene sequencing and genetic counseling if appropriate. MSI is an excellent functional and prognostically useful marker, whereas MMR immunohistochemistry can guide gene sequencing.

Kinetic profiles by topographic compartments in muscle-invasive transitional cell carcinomas of the bladder: role of TP53 and NF1 genes.

We evaluated 71 muscle-invasive transitional cell carcinomas (TCCs) of the bladder by tumor compartments. Kinetic parameters included mitotic figure counting, Ki-67 index, proliferation rate (DNA slide cytometry), and apoptotic index (in situ end labeling [ISEL] of fragmented DNA using digoxigenin-labeled deoxyuridine triphosphate and Escherichia coli DNA polymerase [Klenow fragment]). At least 50 high-power fields per compartment were screened from the same tumor areas; results are expressed as percentage of positive neoplastic cells. Mean and SD were compared by tumor compartment. DNA was extracted from microdissected samples (superficial and deep) and used for microsatellite analysis of TP53 and NF1 by polymerase chain reaction-denaturing gradient gel electrophoresis. Significantly higher marker scores were revealed in the superficial compartment than in the deep compartment. An ISEL index of less than 1% was revealed in 63% (45/71) of superficial compartments and 86% (61/71) of deep compartments. Isolated NF1 alterations were observed mainly in superficial compartments, whereas isolated TP53 abnormalities were present in deep compartments. Lower proliferation and down-regulation of apoptosis define kinetically the deep compartment of muscle-invasive TCC of the bladder and correlate with the topographic heterogeneity, NF1-defective in superficial compartments and TP53-defective in deep compartments.

Stromal expression of vascular endothelial growth factor C is relevant to predict sentinel lymph node status in melanomas.

The dissemination of tumour cells to the lymph nodes is a complex process involving the formation of new lymph vessels, or lymphangiogenesis, produced by the tumour itself. The main growth factor involved in lymphangiogenesis is vascular endothelial growth factor C (VEGF-C), which is secreted by several different malignant tumours, including melanoma. Not only has VEGF-C expression been found in tumour cells, it has also been detected in tumour stromal cells like macrophages and fibroblasts. This study aimed to determine whether the expression of VEGF-C in tumour and stromal cells in cutaneous melanoma determines lymphangiogenesis and neoplastic dissemination to lymph nodes. We examined cases from 50 patients with melanoma who underwent selective biopsy of the sentinel lymph node. Immunohistochemical study was done with D2-40 to label lymph vessels, and the expression of VEGF-C was evaluated in tumour and stromal cells. Lymph vessel density was greater in sentinel lymph node-positive than in sentinel lymph node-negative cases, though the difference was not significant (P = 0.075). A significant correlation was seen between lymph vessel density and tumour thickness and the presence of ulceration. The main finding was that the expression of VEGF-C in fibroblasts was highly associated with the presence of metastasis in the sentinel node and with the Clark level. However, VEGF-C expression showed no relation in either tumour cells or macrophages with node status or other prognostic factors, such as the Breslow index or Clark level. Our results highlight the relevance of the stroma in tumour progression in cutaneous melanoma and its role in the spread to lymph nodes.

Coexistent intraurothelial carcinoma and muscle-invasive urothelial carcinoma of the bladder: clonality and somatic down-regulation of DNA mismatch repair.

Muscle-invasive urothelial carcinomas are heterogeneous neoplasms for which the clonal relationship with low-grade urothelial dysplasia and carcinomas in situ remains unknown, and both monoclonal and field change models have been proposed. Low-grade dysplasia (18) and carcinoma in situ (12) associated with muscle-invasive urothelial carcinoma were microdissected and topographically analyzed (intraepithelial and invasive superficial and deep to muscularis mucosa) for methylation pattern of androgen receptor alleles, TP53, RB1, WT1, and NF1 microsatellite analysis to assess clonal identity; MLH1 and MSH2 sequencing/immunostaining. Appropriate controls were run. Carcinoma in situ (100%) and invasive urothelial carcinoma (100%) revealed monoclonal patterns, whereas low-grade dysplasia was preferentially polyclonal (80%). Carcinoma in situ showed aneuploid DNA content and more abnormal microsatellites than the corresponding invasive compartments, opposite to low-grade dysplasia. Absent MLH1 protein expression with no gene mutations were identified in carcinoma in situ and nodular-trabecular urothelial carcinoma with high microsatellite abnormalities. Somatic mismatch repair protein down-regulation and the accumulation of tumor suppressor gene microsatellite abnormalities contribute to a molecular evolution for monoclonal carcinoma in situ divergent from coexistent muscle-invasive urothelial carcinoma. Low-grade dysplasia is however unlikely connected with this molecular progression.