Anal Dysplasia Screening in People Living with HIV: Long-Term Follow-Up in a Large Cohort from Northwest Spain.

Anal squamous cell carcinoma (SCC) is not a common disease in the general population, although its incidence is higher in people living with human immunodeficiency virus (PLWH). Anal SCC is caused by human papillomavirus (HPV) infection and arises from premalignant lesions termed squamous intraepithelial lesions (SILs). SIL surveillance programs are based on the early detection and treatment of SILs, especially those with a higher risk of transforming into cancer. An anal surveillance program has been under development in our institution since 2011. In this context, we performed a retrospective cohort study at the anal dysplasia unit of Álvaro-Cunqueiro Hospital (Spain). Epidemiological and clinical data were gathered from our Infectious Diseases Sample Collection (an open sample cohort including PLWH) from January 2011 to January 2022. A total of 493 PLWH were considered, 122 (24.7%) of whom were diagnosed with anal dysplasia at baseline, including 2 cases of anal SCC. Briefly, most of individuals were young men (median age, 38 years old) born in Spain (76%), whose vaccination rate before their inclusion in the program was scarce (<3%). Throughout the study period, 81 (16.4%) cases were diagnosed with high-grade squamous-intraepithelial lesions (HSILs) and 3 with anal SCC. At the baseline, severe immunosuppression (i.e., nadir CD4+ lymphocyte count below 200 cell/µL), and prior diagnosis of condyloma acuminata were more frequent within the group with SILs. Conversely, the baseline CD4+ lymphocyte count was similar among both groups. HPV-16 was related to a higher risk of HSILs (odds ratio: 2.76). At the end of the follow-up, 385 PLWH had been retained in care; one patient had died of anal cancer. Anal dysplasia was common (25% of cases), especially among patients infected by HPV-16, diagnosed with condyloma acuminata, and who were severely immunosuppressed. HPV-16 was the main risk factor for the presentation of HSILs.

SATB2 as a Marker of the Proximal Nephron: Expression in Nephrogenic Adenoma and Correlation With Other Renal Tubular Markers.

Nephrogenic adenoma (NA) is an infrequent reactive urothelial lesion. The expression of immunohistochemical renal tubular markers has been reported in NA, although a proximal or distal nephron phenotype has not been established. Special AT-rich sequence-binding protein 2 (SATB2) is a marker of a colorectal origin of adenocarcinomas, occasionally reported in renal samples. We have analyzed SATB2 expression in NA, with correlation with other tubular markers, as well as in the normal kidney. Fifty cases of NA were immunostained with PAX8, SATB2, proximal nephron markers [CD10, renal cell carcinoma (RCC) marker, alpha-methylacyl-CoA racemase (AMACR), and CD15], and distal markers (Ksp cadherin, cytokeratin 7, E-cadherin (E-cad), and cytokeratin 19). Ten normal kidney sections were stained with a double method combining SATB2 plus CD10, RCC marker, AMACR, Ksp cadherin, cytokeratin 7, or E-cad. All NA were immunoreactive for PAX8 and 57% for SATB2. Every case was positive for proximal and distal nephron markers: 100% for cytokeratins 7 and 19, 84.1% E-cad +, 81.6% AMACR +, 68.9% Ksp cadherin +, 63% CD15 +, 53.3% CD10 +, and 28.6 % RCC +. In the normal kidney, SATB2 was detected in the straight part of the proximal tubules and the thin descending loops of Henle. NA shows a multiphenotypic pattern with coexpression of both proximal and distal nephron markers, and constant expression of PAX8, cytokeratins 7 and 19. SATB2 is often positive in NA, which should be kept in mind to avoid a possible misdiagnosis of intestinal adenocarcinoma. SATB2 is a marker of the normal proximal nephron.

[Tissue arrays: Two simple and economical methods for manual construction.] / Matrices tisulares: presentación de dos métodos sencillos y económicos de elaboración manual.

OBJECTIVES: Tissue array (TA) technologyis widely used as a method for the in situ investigation oftissue markers in cancer studies. A limitation of this techniqueis the high price of tissue arrayers. We describetwo easy and non-expensive manual methods, that haveproduced small and medium format arrays. MATERIAL AND METHODS: 16 TAs were manuallyconstructed from conventional paraffin blocks using twodifferent techniques. For the first method, a 16G Tru-Cutneedle whose bevel edge had been cut, was used tomake the holes in the donor blocks (80 cases) and thereceptor ones (resulting in 2 TAs each one with 55 casesand two with 25 cases). In the second technique, a 4mm-diameter punch for cutaneous biopsies was appliedto the donor blocks (obtaining 210 cylinders from 108blocks) and to the receptor ones (12 TAs). Hematoxylin-eosin, immunohistochemical and in situ hybridizationstains were performed on sections from these TAs. RESULTS: The tissue loss rate in the sections obtainedfrom the TAs constructed with the first method was26.5%, but as two cylinders were included from eachcase, at least one of them was retained. There was notany loss of tissue in the sections from the TAs constructedwith the second method. The results of all of the stainsperformed were successful. CONCLUSIONS: These two manual methods of elaborationof TAs result rather simple and they are economical.The tissue loss rate is significant in the first methodbut it can be compensated embedding more than onecylinder from each donor block. There was not anyproblem in the sectioning of the TAs constructed with thesecond method.

OBJETIVOS: La tecnología de matricestisulares (MTs) se ha implantado como método de trabajohabitual en la investigación de marcadores tisularesrelacionados con el cáncer. Su inconveniente esla necesidad de contar con un dispositivo especial deprecio elevado. Presentamos dos métodos manuales,económicos, que han sido válidos para construir MTsde pequeño y mediano formato.MATERIAL Y MÉTODOS: Se han elaborado 16 MTs deforma manual a partir de bloques convencionales deparafina, mediante dos técnicas diferentes. En la primerase utilizó una aguja Tru-Cut 16G a la que se cortó el bisel, para realizar los orificios en los bloques donantes(80 casos) y en los receptores (resultando dos MTs de55 casos y dos de 25 casos). Para la segunda técnicase utilizó un dispositivo "punch" para biopsias cutáneas,de 4 mm de diámetro, que se aplicó a los bloques donantes(obteniendo 210 cilindros de 108 bloques) y alos receptores (12 MTs). En las secciones de las MTs obtenidasse realizaron tinciones de hematoxilina-eosina,inmunohistoquímica (IHQ) e hibridación in situ. RESULTADOS: La tasa de pérdida de material en las seccionesobtenidas con el primer método fue del 26,5%,pero al haberse incluído dos cilindros de cada caso, almenos uno de ellos se conservó. En las MTs obtenidascon el método de punch biopsia no hubo pérdidas detejido. Los resultados de todas las tinciones realizadasfueron óptimos. CONCLUSIONES: Estos dos métodos manuales de elaboraciónde MTs resultan relativamente sencillos y soneconómicos. La tasa de pérdida de tejido es sólo significativaen el primero de los métodos pero se puedecompensar incluyendo varios cilindros de cada bloquedonante. En el segundo método no han existido problemasdestacables en la fase de microtomía.

Matrices tisulares: presentación de dos métodos sencillos y económicos de elaboración manual / Tissue arrays: Two simple and economical methods for manual construction

OBJETIVOS: La tecnología de matrices tisulares (MTs) se ha implantado como método de trabajo habitual en la investigación de marcadores tisulares relacionados con el cáncer. Su inconveniente es la necesidad de contar con un dispositivo especial de precio elevado. Presentamos dos métodos manuales, económicos, que han sido válidos para construir MTs de pequeño y mediano formato. MATERIAL Y MÉTODOS: Se han elaborado 16 MTs de forma manual a partir de bloques convencionales de parafina, mediante dos técnicas diferentes. En la primera se utilizó una aguja Tru-Cut 16G a la que se cortó el bisel, para realizar los orificios en los bloques donantes (80 casos) y en los receptores (resultando dos MTs de 55 casos y dos de 25 casos). Para la segunda técnica se utilizó un dispositivo "punch" para biopsias cutáneas, de 4 mm de diámetro, que se aplicó a los bloques donantes (obteniendo 210 cilindros de 108 bloques) y a los receptores (12 MTs). En las secciones de las MTs obtenidas se realizaron tinciones de hematoxilina-eosina, inmunohistoquímica (IHQ) e hibridación in situ. RESULTADOS: La tasa de pérdida de material en las secciones obtenidas con el primer método fue del 26,5%, pero al haberse incluído dos cilindros de cada caso, al menos uno de ellos se conservó. En las MTs obtenidas con el método de punch biopsia no hubo pérdidas de tejido. Los resultados de todas las tinciones realizadas fueron óptimos. CONCLUSIONES: Estos dos métodos manuales de elaboración de MTs resultan relativamente sencillos y son económicos. La tasa de pérdida de tejido es sólo significativa en el primero de los métodos pero se puede compensar incluyendo varios cilindros de cada bloque donante. En el segundo método no han existido problemas destacables en la fase de microtomía

OBJECTIVES: Tissue array (TA) technology is widely used as a method for the in situ investigation of tissue markers in cancer studies. A limitation of this technique is the high price of tissue arrayers. We describe two easy and non-expensive manual methods, that haveproduced small and medium format arrays. MATERIAL AND METHODS: 16 TAs were manually constructed from conventional paraffin blocks using two different techniques. For the first method, a 16G Tru-Cut needle whose bevel edge had been cut, was used to make the holes in the donor blocks (80 cases) and the receptor ones (resulting in 2 TAs each one with 55 cases and two with 25 cases). In the second technique, a 4 mm-diameter punch for cutaneous biopsies was applied to the donor blocks (obtaining 210 cylinders from 108 blocks) and to the receptor ones (12 TAs). Hematoxylin-eosin, immunohistochemical and in situ hybridization stains were performed on sections from these TAs. RESULTS: The tissue loss rate in the sections obtained from the TAs constructed with the first method was 26.5%, but as two cylinders were included from each case, at least one of them was retained. There was not any loss of tissue in the sections from the TAs constructed with the second method. The results of all of the stains performed were successful. CONCLUSIONS: These two manual methods of elaboration of TAs result rather simple and they are economical. The tissue loss rate is significant in the first method but it can be compensated embedding more than one cylinder from each donor block. There was not any problem in the sectioning of the TAs constructed with the second method

Nephrogenic adenoma: an immunohistochemical analysis using biotin-free methods.

Nephrogenic adenoma (NA) has been considered as a metaplastic process of the urothelium. It has been suggested that this lesion is of renal tubular cell origin or differentiation. Immunohistochemical studies of NA emphasize its staining with α-methylacyl-coenzyme A racemase (AMACR), and prostatic adenocarcinoma may be a possible differential diagnosis. This reactivity was recently discussed as an artifact due to endogenous biotin. Kidney-specific cadherin (Ksp-cad) is a marker of distal nephron. CD10 and KIT are also expressed in the kidney. We studied the immunohistochemical expression of AMACR, p63, Ksp-cad, CD10, and KIT in 9 cases of NA (forming a total of 12 lesions). Practically all of the lesions stained for AMACR with 2 different antibodies and 2 high-sensitivity (multimer or polymer based) biotin-free methods (83% and 100%). The staining was similar for both methods in 9 of these 12 lesions. All of the NAs were negative for p63 and KIT, except 1 case, with focal reactivity for KIT. CD10 was expressed very focally in 4 of the 12 lesions (33%). We observed weak staining for Ksp-cad in 6 lesions (50%) and 3 (25%) showed a moderate positivity in 15% to 50% of the cells. In conclusion, positivity of NA for AMACR is not an artifact, as we confirmed using 2 different methods. Besides, p63, a basal cell marker, is usually negative. Immunoreactivity for Ksp-cad seems to support the differentiation of NA to distal nephron cells, at least in some of the cases. Other markers expressed by the nephron, such as CD10 and KIT, are usually negative in NA.

Autoimmune manifestations of Kikuchi disease.

OBJECTIVES: Kikuchi's disease (KD) has been associated with the presence of autoantibodies, systemic lupus erythematosus (SLE), and other autoimmune diseases. The aim of this study was to assess the frequency of autoimmune manifestations in a KD cohort with a long follow-up. METHODS: Twenty patients with histologically confirmed KD since January 1990 until December 2010 were studied; 12 of them were periodically followed up as outpatients. Another 7 patients were contacted by telephone to offer them a specific consultation and a complete autoimmunity study. RESULTS: Thirteen of 20 patients were women (65%) with a mean age of 29 years (range, 15-79). The age at diagnosis was higher in men (44 vs 27 years, P < 0.05). Lymphopenia was present in 75% of the patients (15/20) and was the more frequent hematological abnormality. The mean follow-up of the 17 patients included in the autoimmunity study was 119 months (range, 15-252). Autoimmune diseases were detected in 9 women (53%): SLE was diagnosed in 4 patients (2 SLE before, 1 simultaneous, and 1 after KD), 2 patients developed primary Sjögren's syndrome after KD, 1 thyroiditis before KD, 1 SLE-like, and 1 antiphospholipid antibodies after KD. Leukocytoclastic vasculitis was found in 2 patients; 1 of them eventually developed SLE. Female sex, painful adenopathies, and cytopenias were significantly associated with autoimmune diseases. CONCLUSIONS: Among patients with KD, only women developed autoimmune manifestations. Therefore, long-term follow-up and active surveillance of autoimmune diseases in patients with KD, especially women, are recommended.

A molecular risk score based on 4 functional pathways for advanced classical Hodgkin lymphoma.

Despite improvement in the treatment of advanced classical Hodgkin lymphoma, approximately 30% of patients relapse or die as result of the disease. Current predictive systems, determined by clinical and analytical parameters, fail to identify these high-risk patients accurately. We took a multistep approach to design a quantitative reverse-transcription polymerase chain reaction assay to be applied to routine formalin-fixed paraffin-embedded samples, integrating genes expressed by the tumor cells and their microenvironment. The significance of 30 genes chosen on the basis of previously published data was evaluated in 282 samples (divided into estimation and validation sets) to build a molecular risk score to predict failure. Adequate reverse-transcription polymerase chain reaction profiles were obtained from 262 of 282 cases (92.9%). Best predictor genes were integrated into an 11-gene model, including 4 functional pathways (cell cycle, apoptosis, macrophage activation, and interferon regulatory factor 4) able to identify low- and high-risk patients with different rates of 5-year failure-free survival: 74% versus 44.1% in the estimation set (P < .001) and 67.5% versus 45.0% in the validation set (P = .022). This model can be combined with stage IV into a final predictive model able to identify a group of patients with very bad outcome (5-year failure-free survival probability, 25.2%).

A TaqMan low-density array to predict outcome in advanced Hodgkin's lymphoma using paraffin-embedded samples.

PURPOSE: Despite major advances in the treatment of classic Hodgkin's lymphoma (cHL), approximately 30% of patients in advanced stages may eventually die as result of the disease, and current methods to predict prognosis are rather unreliable. Thus, the application of robust techniques for the identification of biomarkers associated with treatment response is essential if new predictive tools are to be developed. EXPERIMENTAL DESIGN: We used gene expression data from advanced cHL patients to identify transcriptional patterns from the tumoral cells and their nonneoplastic microenvironment, associated with lack of maintained treatment response. Gene-Set Enrichment Analysis was used to identify functional pathways associated with unfavorable outcome that were significantly enriched in either the Hodgkin's and Reed-Sternberg cells (regulation of the G2-M checkpoint, chaperones, histone modification, and signaling pathways) or the reactive cell microenvironment (mainly represented by specific T-cell populations and macrophage activation markers). RESULTS: To explore the pathways identified previously, we used a series of 52 formalin-fixed paraffin-embedded advanced cHL samples and designed a real-time PCR-based low-density array that included the most relevant genes. A large majority of the samples (82.7%) and all selected genes were analyzed successfully with this approach. CONCLUSIONS: The results of this assay can be combined in a single risk score integrating these biological pathways associated with treatment response and eventually used in a larger series to develop a new molecular outcome predictor for advanced cHL.

Intrauterine myocardial infarction: unsuspected diagnosis in the delivery room.

We present a case study of intrauterine myocardial infarction. A female neonate suffered from cardiopulmonary arrest at birth. She presented with refractory cardiogenic shock and died. Echocardiogram showed absence of movement of the posterolateral wall of the left chamber. Autopsy showed severe and extensive myocardial necrosis confined by a band of dystrophic calcification that suggested intrauterine origin. The circumflex artery and anterior descending coronary artery both presented with very narrow calibers. Sympathicomimetics administered to the mother also could have played a role in the development of infarction. Myocardial infarction should be suspected if signs of ischemia are present and there is no clear hypoxic-ischemic insult around delivery. Prognosis is poor especially if the main stem of the left coronary is involved. Cardiac transplantation can be considered.

Variability in the expression of polycomb proteins in different normal and tumoral tissues. A pilot study using tissue microarrays.

In spite of the known function of polycomb group (PcG) genes in stem cell self-renewal, control of cellular proliferation and differentiation, its role in cancer pathogenesis is still poorly understood. We studied the expression by immunohistochemistry of several PcG-maintenance complex proteins (RING1, RNF2, BMI1, MEL18, HPH1 and RYBP) in nontumoral (154 samples) and tumoral (550 samples) human tissues using Tissue Microarrays. For selected genes (BMI1 and RING1) FISH analysis has been also carried out. PcG proteins had a tissue- and cell-type-specific expression pattern. Some of them were highly selectively expressed, such as HPH1, which was detected in germ cells in testis, pituitary and parathyroid glands and Langerhans islets, and RYBP, which was found in placenta, umbilical cord and thyroid gland. By contrast, RING1 was ubiquitously expressed in every normal tissue analyzed. Changes in expression associated with tumoral transformation have been found for BMI1 and RNF2, which exhibited increased expression in a large series of tumors, including gastrointestinal tumors, pituitary and parathyroid adenomas, and lymphomas, compared with their expression in normal-cell counterparts. The high level of expression of BMI1 protein observed in mantle-cell lymphomas and pituitary adenomas is associated in some cases with amplification of BMI1 locus. These findings imply that upregulation of BMI1 may constitute a malignancy marker in different types of cancer, mainly in lymphoid and endocrine tumors. RING1 was lost in a group of renal-cell carcinomas and testicular germ-cell tumors. Lastly, RYBP is anomalously expressed in Hodgkin's lymphomas and oligodendrogliomas, among others tumors. A significant finding of the study is the identification of unique PcG profiles for some tumors, such as testicular germ-cell tumors, which have high levels of HPH1 expression and loss of RING1 and/or BMI1; pituitary adenomas, which expressed every PcG protein analyzed; and clear-cell renal-cell carcinoma, which was the only tumor other than testicular germ-cell tumors that did not express RING1.

p53 and ki67 expression as prognostic factors for cancer-related survival in stage T1 transitional cell bladder carcinoma.

OBJECTIVE: To determine prognostic factors for survival in bladder transitional cell carcinoma (TCC), and the prognostic value of p53 and ki67. MATERIAL AND METHODS: A study was made of patients with stage T1 primary bladder TCC (n = 175). The immunohistochemical study was carried out using DO7 and MIB-1 monoclonal antibodies, for p53 and ki67, respectively. Kaplan-Meier methodology was used for the survival analysis, and the log-rank test was applied in order to determine accumulated probability rates of survival. Moreover, Cox's multivariate regression analysis was also used to establish the variables associated with survival. Receiver operating characteristic (ROC) curves were also drawn, with the aim of determining the prognostic capacity of p53 and ki67. RESULTS: The average follow-up period was 7.3 years. Cancer-related survival rates at 5 and 10 years were 89.51 and 80.68%, respectively. The increase in p53 and ki67 expressions paralleled the histological grade, both markers showing significant inter-group differences (P = 0.0000). The variables which modified cancer-related survival significantly in the univariate analysis were the following: tumour multifocality, solid microscopic morphology, large cell nucleus and a high expression of p53 and ki67. Independent cancer-related survival variables were: age, tumour size of >3 cm, a solid microscopic growth pattern and expression of p53. CONCLUSIONS: The expression of p53, increase in age, tumour size of >3 cm and microscopic growth pattern are independent predictors for cancer-related survival. A positive correlation was observed, indicating that, the higher the expression of p53, the greater the probability of death.

Multivariate analysis of survival, recurrence, progression and development of mestastasis in T1 and T2a transitional cell bladder carcinoma.

BACKGROUND: Determination of prognosis factors associated with survival, recurrence, progression, and development of metastasis in T1 and T2a transitional cell carcinoma (TCC) of the bladder is discussed. METHODS: A study was conducted of a group of 210 patients with primary bladder TCC at classification T1 (n = 175) and T2aN0M0 (n = 35). A total of 177 variables were studied in each patient. The monoclonal antibodies used were the following: DO7 (p53) and MIB-1 (Ki-67). Prognosis was obtained using Kaplan-Meier methodology and Cox proportional hazards model. RESULTS: The average follow-up period was 6.7 years. Cancer-related survival rates at 5 and 10 years were 82.96% and 74.78%, respectively. The independent survival variables were the following: age and expression of p53. Recurrence free survival at 5 and 10 years stood at 51.80% and 42.71%, respectively. The independent recurrence variables were T2a classification, tumor multifocality, tumor size of greater than 3 cm, carcinoma in situ in random biopsy, and expression of Ki-67. Progression free survival rates at 5 and 10 years were 75.31% and 69.16%, respectively. The independent progression variables were age, T2a classification, and expression of p53. Metastasis free survival rates at 5 and 10 years stood at 87.23% and 84.55%, respectively. The expression of p53 was the sole variable to provide an independent prediction of metastasis. CONCLUSIONS: The expression of p53 clearly has an independent effect on the prediction of survival, progression and development of metastasis, showing a dose-response effect. Tumor multifocality and T2a classification are the variables that best predict recurrence.