E2F1 and E2F2 prevent replicative stress and subsequent p53-dependent organ involution.

Tissue homeostasis requires tight regulation of cellular proliferation, differentiation and apoptosis. E2F1 and E2F2 transcription factors share a critical role in tissue homeostasis, since their combined inactivation results in overall organ involution, specially affecting the pancreatic gland, which subsequently triggers diabetes. We have examined the mechanism by which these E2Fs regulate tissue homeostasis. We show that pancreas atrophy in E2F1/E2F2 double-knockout (DKO) mice is associated with mitochondrial apoptosis and activation of the p53 pathway in young animals, before the development of diabetes. A deregulated expression of E2F target genes was detected in pancreatic cells of young DKO animals, along with unscheduled DNA replication and activation of a DNA damage response. Importantly, suppression of DNA replication in vivo with aphidicolin led to a significant inhibition of the p53 pathway in DKO pancreas, implying a causal link between DNA replication stress and p53 activation in this model. We further show that activation of the p53 pathway has a key role in the aberrant phenotype of DKO mice, since targeted inactivation of p53 gene abrogated cellular apoptosis and prevented organ involution and insulin-dependent diabetes in mice lacking E2F1/E2F2. Unexpectedly, p53 inactivation unmasked oncogenic features of E2F1/E2F2-depleted cells, as evidenced by an accelerated tumor development in triple-knockout mice compared with p53(-/-) mice. Collectively, our data reveal a role for E2F1 and E2F2 as suppressors of replicative stress in differentiating cells, and uncover the existence of a robust E2F-p53 regulatory axis to enable tissue homeostasis and prevent tumorigenesis. These findings have implications in the design of approaches targeting E2F for cancer therapy.

Predictors of axillary lymph node metastases in patients with invasive breast carcinoma by a combination of classical and biological prognostic factors.

The presence of axillary lymph node metastases (ALNMs) is the most important prognostic factor in breast carcinoma. If ALNMs were predictable without performing axillary lymph node dissection (ALND), this procedure would not be necessary in selected patients. Using a combination of some of the new biological markers with the classical ones, our objective was I) to identify the best set of predictors of ALNMs, and II) to define predictive models with either high or low probability of ALNMs. We studied 102 patients with invasive breast carcinoma. All patients underwent ALND, and at least 10 axillary lymph nodes per case were obtained. In the primary tumour we evaluated size, histological subtype and grade, lymphatic/vascular invasion and margin. Hormone receptor status, MIB1 index, microvessel density, c-erbB-2 and cathepsin D expression were assessed by immunohistochemistry, and DNA ploidy and S-phase by flow cytometry. Risk factors for ALNMs were estimated by nonlinear logistic regression analysis. The best predictors of ALNMs were: tumour size > 2 cm [OR 6.45, 95% confidence interval (CI) 21.74 to 1.91], presence of lymphatic/vascular invasion [OR 4.95, CI (14.50 to 1.69)], infiltrative margin [OR 9.87 CI (37.44 to 2.60)] and high MIB-1 index [OR 8.39, CI (33.47 to 2.10)]. Two subsets had a very high risk of ALNMs: I) tumour size > 2 cm, with lymphatic/vascular invasion and infiltrative margin; 26 (89.66%) of 29 patients of this subgroup had ALNMs, and (II) tumour size > 2 cm, with lymphatic/vascular and high MIB1 index.; eight of the nine (89%) patients of this subgroup had ALNMs. We could also identify a two-variable model with a very low risk of ALNMs constituted by tumour with circumscribed margin and low MIB-1 index. Of the 19 patients showing these features, only 1 (5.26%) had ALNMs. Therefore, pathological features of the primary tumour can help to assess the risk for ALNM in invasive breast carcinoma. Such risk assessment might avoid regional surgical overtreatment.

Cathepsin D in host stromal cells is associated with more highly vascular and aggressive invasive breast carcinoma.

AIMS: To determinate the relationship between tumoral angiogenesis and cathepsin D (CD) expression in tumour and host stromal cells of invasive breast carcinoma, and to examine its association with classical prognostic factors such as lymph node status, histological grade, tumour size, mitotic rate, peritumoral lymphovascular invasion and oestrogen receptor (ER) status. METHODS AND RESULTS: Sections from 102 invasive breast carcinoma were cut from the archival formalin-fixed, paraffin-embedded tissue blocks and stained using immunohistochemistry for the endothelial cell adhesion molecule (CD31) and CD. Microvessel density was assessed by counting vessels in the three most vascular areas at x400 field. The counts were expressed as the highest counts within any x400 field. The evaluation of immunostaining for CD was performed separately in both the parenchymal and stromal cells. Microvessel density was correlated positively with histological grade and peritumoral lymphovascular invasion, and correlated inversely with ER status. Positive CD staining of tumour cells was more frequent in positive ER tumours and was not significantly associated with the other classical prognostic factors. However, moderate to strong staining of host cells was correlated with higher histological grade, higher mitotic index and lack of ER protein. There was a statistically significant association between CD expression of host stromal cells and higher vessel count. CONCLUSIONS: CD in host stromal cells is associated with more aggressive tumours and with a higher intratumoral microvessel density. Evaluation of CD in combination with angiogenic activity may be of some help in more accurately predicting the biological behaviour of breast carcinoma.

Selective nuclear morphometry as a prognostic factor of survival in renal cell carcinoma.

In the present study, we sought to determine the predictive value of selective nuclear morphometry (SNM) for patient outcome in renal cell carcinoma (RCC). Tumor samples of 140 renal adenocarcinomas diagnosed and treated with radical nephrectomy and hilar lymphadenectomy between 1970 and 1988 with a minimum follow up of 5 years in all the cases were studied by SNM. The morphometric analysis was performed in the most malignant tumor selected zone. Selection was based on cytological criteria including nuclear grade. Nuclear morphometric features analyzed were: area, perimeter, major diameter, major and minor diameter of the equivalent ellipse, volume of the equivalent ellipse and sphere, circumference diameter, and shape factors. The results showed that in the selected zone tumor nuclei were larger than in the zones selected at random. There was an inverse correlation between morphometric parameters and survival and a direct one between tumoral grade and stage. Tumors of the long-term survival group of patients presented nuclei with smaller morphometric measurements than tumors of short term survival group, with significant differences between them (p < 0.05). In the survival analysis carried out by the Kaplan-Meier method significant differences existed between different groups formed from break point for: area, perimeter, major diameter, major and minor diameter of the ellipse, volume of the ellipse and sphere, circumference diameter and perimeter shape factor. In the multivariate analysis carried out by the Cox method, the feature with the most predictable value related to survival, was the tumor stage. Morphometric value with the highest punctuation in the test was major nuclear diameter. The rest of the morphometric values (except elliptic shape factor and elongation factor) were also significant but they did not improve prognostic information of the major nuclear diameter. SNM offers a useful aid in a more objective grading of RCC. Multivariate Cox analysis revealed additional value of karyometry to tumor stage. SNM can be a useful tool for stratification of patients with RCC.

Prognostic factors in supraglottic laryngeal carcinoma.

We carried out a retrospective study of patients with supraglottic carcinomas who were treated surgically at the Marques de Valdecilla Hospital (Santander, Spain) between 1978 and 1987 and who were followed up for at least 5 years. The Kaplan-Meier survival curves were calculated for 24 clinical, histologic, and morphometric parameters. Multivariate analysis was then performed by means of the Cox regression model. In the univariate analysis, survival was related to presence of capsule rupture of the involved lymph nodes (p = 0.00001), number of metastatic lymph nodes (p = 0.0002), postoperative TNM stage (p = 0.004), grade of cell differentiation (p = 0.001), presence of intratumoral necrosis (p = 0.01), and type of invasion (p = 0.04). The nuclear area did not have an influence on survival. Only the presence or absence of capsule rupture of the metastatic lymph nodes and the grade of cell differentiation were included in the final Cox model and proved to be parameters with independent prognostic significance.

[Prognostic significance of DNA content measured by flow cytometry in supraglottic laryngeal carcinoma]. / Valor pronóstico del contenido de ADN por citometría de flujo en el carcinoma supraglótico de laringe.

Eighty-five patients with squamous that were cell carcinoma of the supraglottis treated surgically in the Department of Otolaryngology at the Marqués de Valdecilla Hospital in Santander (Spain) over a 10-year period from January 1, 1978 to December 31, 1987 were studied retrospectively. In 81 cases a flow cytometric study was made from the surgical specimen included in paraffin blocks, to determine the DNA index and the percentage of cells in the S phase of the cellular cycle. An statistical analysis was made to determine the correlation between these parameters and survival was analyzed using the Kaplan-Merer method. Survival curves were compared using the log-rank test. In 67 cases (82.7%) DNA content was diploid and in 14 cases (17.3%) it was aneuploid. The percentage of cells in S phase was 3.8 and 55.4, with a mean value of 15.19. Comparison of with the five these parameters years survival showed no statistical differences between groups, although tumors with more than 19% S phase had worse prognosis (p < 0.1) and tumors in advanced stages were more frequently aneuploid and had higher S phase values (p = 0.1).

Constitutive expression of bcl-2 in B cells causes a lethal form of lupuslike autoimmune disease after induction of neonatal tolerance to H-2b alloantigens.

The bcl-2 protooncogene has been shown to provide a survival signal to self-reactive B cells, but it fails to override their developmental arrest after encounter with antigen. Furthermore, constitutive expression of bcl-2 in B cells does not promote the development of autoimmune disease in most strains of mice, indicating that signals other than those conferred by bcl-2 are required for long-term survival and differentiation of self-reactive B cells in vivo. To further examine the factors that are required for the pathogenesis of autoimmune disease, we have assessed the effect of bcl-2 overexpression on the development of host-versus-graft disease, a self-limited model of systemic autoimmune disease. In this model, injection of spleen cells from (C57BL/6 x BALB/c)F1 hybrid mice into BALB/c newborn parental mice induces immunological tolerance to donor tissues and activation of autoreactive F1 donor B cells through interactions provided by allogeneic host CD4+ T cells. BALB/c newborns injected with spleen cells from (C57BL/6 x BALB/c)F1 mice expressing a bcl-2 transgene in B cells developed high levels of anti-single-stranded DNA and a wide range of pathogenic autoantibodies that were not or barely detectable in mice injected with nontransgenic spleen cells. In mice injected with transgenic B cells, the levels of pathogenic autoantibodies remained high during the course of the study and were associated with long-term persistence of donor B cells, development of a severe autoimmune disease, and accelerated mortality. These results demonstrate that bcl-2 can provide survival signals for the maintenance and differentiation of autoreactive B cells, and suggest that both increased B cell survival and T cell help play critical roles in the development of certain forms of systemic autoimmune disease.

Glandular inclusions in inguinal hernia sacs: a clinicopathological study of six cases.

Glandular inclusions in inguinal hernia sacs are not frequent. We present six cases of inguinal hernia with this finding, which represents an incidence of 2.6% in males and shows a predominance in the prepubertal stage. Five patients showed cryptorchidism and two cases were related to congenital malformations of the single umbilical artery type and 47,XY chromosome disorder with chromosomal marker. The most important differential diagnosis must be made with normal histological structures such as the vas deferens or epididymis. The mean diameter of the inclusions was 0.1988 mm and there was a significant difference in size between the inclusions and the vas deferens, but not the epididymis. Differentiation from the latter is based on the absence of a well-developed muscular coat in the wall of the inclusions. It is important to recognize that these inclusions can occur in hernia sacs because of the clinical and medicolegal implications that arise if they are confused with true epididymis or vas deferens. They may arise from paratesticular embryonal remnants.

Epidermoid carcinoma of the lung in stage I: factors of prognostic interest.

Factors affecting stage I epidermoid cancer of the lung were studied in a series of 29 patients treated only by surgery and followed up for ten years. A set of 13 variables with a possible influence on prognosis were investigated. The application of the Cox Univariate Analysis to the different variables showed the grade of cell differentiation and the mitotic index to be predictors. In the Cox Multivariate Analysis, the proportional regression equation revealed two independently significant variables (p < 0.01), which were the Mitotic Index and Nuclear Area. Grouping patients on the basis of the prognostic variables indicated allows a better prediction for survival to be made for this series of patients.

The value of nuclear area as a prognostic factor in T1 papillary transitional cell carcinoma of the bladder.

In a series of 131 T1 papillary transitional cell carcinomas of the bladder, the nuclear areas of 100 nuclei (50 from the external papillary zone and 50 from the internal papillary zone) were measured. An attempt was made to correlate retrospectively the value of the mean nuclear area with histological grade and with survival. A higher value was obtained for the mean nuclear area of the internal papillary zone than for that of the external papillary zone. A better survival rate was found after 10 years' follow-up for those tumours whose mean nuclear area in the internal papillary zone was < or = 28 microns2, which suggests that it is in this zone that the nuclear area should be measured. A correlation was observed between the increase in the mean nuclear area value and higher histological grade.

Prognostic correlation of morphometric values with survival in invasive transitional cell carcinoma of the bladder.

A morphometric study was made of 95 invasive bladder tumours, differentiating 34 papillary and 61 solid carcinomas. In the invasive papillary tumours, the deeper the zone of the tumour measured, the higher the value of the mean nuclear area. A higher histological grade was also seen to correspond to a higher mean nuclear area value except for Grade IV tumours, whose nuclear area was no larger than that for Grade III tumours. This led us to separate Grade IV tumours from transitional cell carcinomas and classify them as undifferentiated. A better prognosis was found for those tumours whose mean nuclear area was < or = 30 microns2 in category T2. For T3 and T4A tumours no conclusive results were obtained in this respect.

Nuclear morphometry in prognosis of renal adenocarcinoma.

Nuclear morphometry was carried out on 95 parenchymatous adenocarcinomas of the kidney treated by radical nephrectomy and hilar lymphadenectomy and followed up for at least five years. The study assessed nuclear area, nuclear perimeter, major diameter, nucleolar area, nuclear shape factor, and nuclear size. There was a significant statistical correlation between survival and the morphometric parameters and between the parameters themselves except for nuclear shape factor. The multiple regression proved that nuclear area is the factor which shows the greatest statistical significance for prognosis. Taking a mean nuclear area of 35 microns 2 allowed two prognostic groups to be established regardless of stage, with those below the threshold having a good prognosis and those above it having a poor prognosis: 96.7 percent of patients with a good prognosis survived after five years (60 months) compared with 17.2 percent of those with a poor prognosis.

Self-limited autoimmune disease related to transient donor B cell activation in mice neonatally injected with semi-allogeneic F1 cells.

BALB/c mice injected at birth with 10(8) semi-allogeneic (C57BL/6 x BALB.IgHb)F1 spleen cells develop a lupus-like syndrome in which autoantibodies bear exclusively the donor allotype. We have analyzed the evolution of donor B cell chimerism and the autoimmune manifestations during the first year of life in these mice. Anti-DNA, -histone, and -cardiolipin IgG antibodies as well as circulating immune complexes appeared in the second week of life, reached the highest values around the sixth week, and then progressively dropped to normal values after the sixth month in most mice. The kinetics of the evolution of the autoimmune manifestations, as well as the kinetics of serum donor Ig allotype, were parallel to the kinetics of donor B cell chimerism, which was particularly prominent in the spleens in early weeks of life, and progressively decreased after remission of the autoimmune syndrome. Membrane-proliferative glomerulonephritis, which was followed as the more representative histological abnormality in this model, was particularly evident after 10 weeks of life, but disappeared by the end of the follow-up. Interestingly, when mice with a self-limited disease were re-injected with 10(8) F1 spleen cells i.v., a flare in the serological manifestations was observed. In these re-injected mice a predominance of anti-DNA, IgG1 antibodies bearing exclusively the donor allotype was also observed, as in the early weeks of life.(ABSTRACT TRUNCATED AT 250 WORDS)

Kidney cortical necrosis induced by mitomycin-C: a morphologic experimental study.

Experimental studies on mitomycin-C nephrotoxicity are scanty and mention the occurrence of cortical hemorrhage, tubular necrosis, or hydronephrosis secondary to papillomatous hyperplasia of the uroepithelium. To our knowledge, only one experimental study has mentioned morphological lesions similar to the hemolytic uremic syndrome in the human. In the present study 40 female Wistar rats were studied following unilateral renal perfusion of the left kidney with 2 mg/kg of mitomycin-C. Renal lesions corresponded to cortical necrosis with the presence of large bizarre nuclei. The presence of these nuclear atypias supports a direct toxic effect (alkylation) of the mitomycin or its metabolites on cells.

[Prognosis factors in adenocarcinoma of the kidney. I. Staging, clinical and macroscopic factors of the tumor]. / Factores pronósticos en el adenocarcinoma renal. I. Estadiaje, factores clínicos y macroscópicos del tumor.

In 95 cases of renal adenocarcinoma (32 stage I, 18 II, 26 III, and 19 IV) submitted to radical nephrectomy and hilar lymphadenectomy with a follow-up of 5 years, we evaluated the prognostic value of the Robson classification (p less than 0.01), categories T (p less than 0.01), N (p less than 0.01), M (p less than 0.01) and V (p = NS), 3 clinical parameters (sex, age, duration of symptoms and signs) which proved to be of no prognostic value, and 6 macroscopic features of the tumor (solid or cystic nature of the tumor, presence of intrarenal tumor satellite modules, ureteric invasion, tumor size, site and intrarenal location of the tumor). Four of the foregoing proved to influence prognosis.

[Prognosis factors in adenocarcinoma of the kidney. II. Microscopic factors]. / Factores pronósticos en el adenocarcinoma renal. II. Factores microscópicos.

The prognostic value of several microscopic parameters were evaluated in 95 cases of renal adenocarcinoma (32 stage I, 18 II, 26 III and 19 IV) submitted to radical nephrectomy and hilar lymphadenectomy with a 5-year follow-up. No prognostic significance was observed for cell type, architectural pattern, borders, and degree of peritumoral lymphocyte infiltration. A worse prognosis was observed for the high (G 3 + 4) in comparison with the low (G 1 + 2) nuclear grade (p less than 0.01). However, analysis according to stage revealed this parameter only affected prognosis in those cases pertaining to intermediate stages II and III. The mean proliferation rate was 2.97 mitoses/10 fields (500 x). A worse prognosis was observed for a proliferation rate greater than 5 mitoses/10 fields (500 x) in comparison to those with a lower rate (p less than 0.01) but evolves parallel to stage.

[Prognosis factors in adenocarcinoma of the kidney. III. Nuclear area]. / Factores pronósticos en el adenocarcinoma renal. III. Area nuclear.

Nuclear morphometry was performed with the MOP-Videoplan morphometry device in 95 cases of renal adenocarcinoma submitted to radical nephrectomy and hilar lymphadenectomy with a 5-year follow-up. For each case, 100 nuclei were randomly selected and delineated (magnification: 1,000x). A statistically significant correlation (p less than 0.05) was observed for the nuclear area and the overall as well as stage-related survivorship. Using a nuclear size of 35 microns 2, the study population can be divided into two groups: those with a good and those with a poor prognosis regardless of stage. A statistically significant difference was observed for the overall as well as the stage-related survivorship for the group with a nuclear area less than 35 microns 2 (good prognosis) in comparison with the group with a nuclear area greater than 35 microns 2.

Xanthomatous endometritis.

A further case of xanthomatous endometritis, characterized by the presence of histiocytic cells in the endometrium, in the absence of carcinoma is described. This lesion seems to be a rare complication of hematometra with cervical occlusion. The foam cells would appear to be macrophages, components of a nonspecific inflammatory reaction, which have phagocytosed breakdown elements of retained endometrial hemorrhage.

Staging of carcinoma of the colon and rectum.

A retrospective study was carried out upon 393 patients diagnosed from the 1 January 1970 through 1 December 1978 for carcinoma of the colon and rectum, 90 per cent of whom had a complete follow-up study during a five year period or until death. A classification system by stage, which has given us good results since there are no crossovers in the survival evolution of the different subgroups, is described. The system shows great uniformity since a linear equation with a slope of 15.76 per cent and a linear correlation coefficient (r) = -0.999 relates almost perfectly the decrease in survival time as the stages advance.