Mitotically active haemorrhagic cellular (apoplectic) leiomyoma.

Apoplectic leiomyoma is a distinctive smooth muscle tumour usually occurring in women either taking oral contraceptives or who are pregnant or recently postpartum. Most of these tumours show 0-2 mitoses per 10 high power fields, but a mitotic index of up to 8 per 10 high power fields is allowed in such tumours. We describe an apoplectic leiomyoma with a number of atypical features including a high mitotic index (up to 20 per 10 high power fields) in a 47-year-old woman. Follow-up clinically and by computerised tomography (CT) for 3 years demonstrates no recurrence.

High, persistent hepatocellular proliferation and apoptosis precede hepatocarcinogenesis in growth hormone transgenic mice.

AIMS/BACKGROUND: Growth hormone (GH) transgenic mice are known to develop hepatocellular adenomas and carcinomas. In order to understand more about hepatocarcinogenesis in the GH-transgenic mouse model we quantitated the rates of hepatocellular proliferation and apoptosis in these mice. METHODS: Two lines of GH-transgenic mice and non-transgenic control mice were generated and sacrificed at regular intervals between one and nine months. Hepatocellular replication was measured by in vivo incorporation of bromodeoxyuridine (BrdU) and counting BrdU-positive nuclei in histological liver sections. Serial sections taken from these mouse livers were also assessed for rates of hepatocellular apoptosis using the in situ end-labelling of fragmented DNA (TUNEL) method. RESULTS: High levels of hepatocellular replication were sustained life-long in this model. Increased rates of hepatocellular proliferation preceded the onset of hepatic inflammation, a prominent feature in the liver pathology of GH-transgenic mice. In tumour tissue, cellular proliferation was up to 17-fold greater than in surrounding non-tumour tissue. Apoptosis rates were also elevated in non-tumour regions of GH-transgenic mouse livers compared to controls. Interestingly, large dysplastic hepatocytes were common in the fraction of cells undergoing apoptosis, especially in older mice with inflamed livers. The increase in the rate of hepatocellular apoptosis in GH-transgenic animals largely balanced the augmented levels of proliferation seen in these mice. In tumour tissue, however, the profound increase in the number of proliferating tumour cells outstripped the increase in apoptosis. CONCLUSION: Relatively high and enduring levels of hepatocellular replication and apoptosis precede hepatocarcinogenesis in GH-transgenic mice. Increased cellular proliferation and resistance to apoptosis were evident in tumour growth in older animals.

Analysis of bleomycin- and cytosine arabinoside-induced chromosome aberrations involving chromosomes 1 and 4 by painting FISH.

The genomic frequency of chromosomal aberrations obtained by chromosome painting is usually extrapolated from the observed frequency of aberrations by correcting for the DNA content of the labelled chromosomes. This extrapolation is based upon the assumption of random distribution of breakpoints from which aberrations are generated. However, the validity of this assumption has been widely questioned. While extensive investigations have been performed with ionizing radiation as chromosome breaking agent, little efforts have been done with chemical clastogens. In order to investigate interchromosomal differences in chemically-induced chromosome damage, we have used multicolour chromosome painting to analyse bleomycin-induced aberrations involving chromosomes 1 and 4, two chromosomes that differ in gene density. In addition, we have measured the effect of cytosine arabinoside upon the repair of bleomycin-induced DNA damage in chromosomes 1 and 4. Our results show that these chromosomes are equally sensitive to the clastogenic effect of bleomycin with a similar linear dose-effect relationship. However, the high gene density chromosome 1 appeared to be more sensitive to repair inhibition by Ara-C than chromosome 4. This enhanced sensitivity to repair inhibition in chromosome 1 could be mediated by preferential repair of open chromatin and actively transcribed regions.

Mutations in exons 5-8 of the p53 gene, independent of their type and location, are associated with increased apoptosis and mitosis in invasive breast carcinoma.

In breast cancer, mutations located in the zinc-binding functional domains of the p53 gene have been reported to predict a worse prognosis and a worse response to treatment with doxorubicin, compared with mutations in other parts within exons 5-8 of the gene. Similarly, mutations in residues of p53 that directly contact DNA have been associated with a poor prognosis. To investigate whether these specific p53 mutations are associated with differences in the rate of apoptosis and/or mitosis, or expression of the anti-apoptotic Bcl-2 protein, these parameters were evaluated in 89 invasive breast cancers with a confirmed p53 mutation in exons 5-8 and in 99 tumours without a p53 mutation in exons 5-8. Neither mutations located in the zinc-binding functional domains nor mutations in residues that directly contact DNA were associated with alterations in mitotic or apoptotic activity. However, compared with the wild-type p53 tumours, both apoptotic and mitotic indices showed an approximately two-fold increase in the mutant p53 group ( p< 0. 001). The presence of a p53 mutation was also associated with the presence of tumour necrosis ( p< 0.001), high tumour grade ( p< 0. 001) and low expression of Bcl-2 ( p< 0.001). Our data support the concept that in invasive breast carcinoma, loss of p53 function is involved in enhanced proliferation rather than decreased apoptosis and that the resulting acceleration of cell turnover may enhance clonal evolution and tumour progression.

DNA index shift with disease progression in colorectal adenocarcinoma: a morphological and flow cytometric study.

DNA index (DI) values seen in 86 sporadic colorectal adenocarcinomas were related to clinical, morphological, and disease progression features. DI, whose overall distribution was bimodal with peaks in the diploid and from hypotriploid to tetraploid ranges, was related to pathological lymph node staging (pN), staging, lymphoid reaction, and tubular configuration. With increasing severity in pathological features, an irregular shift in DI class prevalence was seen, with no steady increase from diploidy to higher degrees of aneuploidy. All UICC stage I tumors (13% of total) were aneuploid, 50% being hypertriploid; diploidy (35%) and hypertriploidy (22%) prevailed in stage II carcinomas (41% of total), diploidy (35%) and hypotriploidy (30%) in stage III (30% of total), and triploidy (33%) in stage IV (15% of total). Amongst features related to stage (lymphoid reaction, depth of neoplastic embolization, grading, tubular configuration, and polymorphism), few were associated with DI, and none influenced DI shift and class prevalence through the stages. The biological capabilities of colorectal adenocarcinoma in relation to stage are expressed by certain aneuploid DI classes (hypertriploidy: absence of extracolonic spread; hypotriploidy: lymph node metastases; triploidy: distant metastases). Diploidy is unrelated to criteria defining stage above I and predicts 50% of cases with development of metachronous metastases. Irregular DI class shift through the stages may be attributable to different pathways of cancerogenesis and disease progression in diploid versus aneuploid carcinomas. Alternatively, assuming that the diploid fraction in aneuploid tumors contains neoplastic cells, pure diploid carcinomas represent the selection of a vital clone that may give rise to a further mixed population whose aneuploid DI is different and best fitted to express the biological capabilities of that given stage.

Apoptotic glial cell death and kinetics in the spinal cord of the myelin-deficient rat.

This study examined the glial cell kinetics and death in the thoracic spinal cord of normal and myelin-deficient (md) rats between 1 and 21 days of age and determined whether the observed glial cell death primarily affected oligodendrocytes and had the morphologic and molecular features of apoptosis. In the md rat spinal cord there was an increase in cell division and death in a pattern that correlated with the onset of myelination. The dying cells were identified as oligodendrocytes ultrastructurally as many had the characteristic distention of the rough endoplasmic reticulum seen in the md rat glia. Double labeling using PLP in situ hybridization and a modified TUNEL method also suggested that the dying cells, in both mutant rats and control littermates, were oligodendrocytes. These findings were compared with previous studies on the md rat optic nerve and those in other PLP mutants.

Cystic neuroblastoma: emphasis on gene expression, morphology, and pathogenesis.

Cystic neuroblastoma (CN) is an unusual variant of neuroblastoma characterized by a grossly visible cyst(s) and almost always distinctive microcysts on light microscopy. Rarely, CN will appear solid grossly, but microcystification will be present. We examined the clinical, pathologic, and biologic features of 17 cases of CN. The majority of CN had been detected by prenatal ultrasound. The tumors were favorable stage, stroma-poor, but with low or intermediate mitotic-karyorhectic indices and had favorable biologic markers reflected by aneuploidy and by an absence of N-myc amplification and chromosome 1p deletions. However, the high trk expression typically identified in good risk tumors was absent. Although the complete natural history of CN is not fully defined, our experience suggests that some tumors progress in size, whereas others may spontaneously regress or mature. The clinical outcome is excellent, as is expected in localized and stage 4S neuroblastoma in infancy.

Human papillomavirus type influences the extent of chromosomal lag during mitosis in cervical intraepithelial neoplasia grade III.

The level of risk for carcinoma in the uterine cervix depends on the type of human papillomavirus (HPV) present. We examined whether the HPV type influences the proliferation rate and occurrence of mitotic figures with lagging chromosomes in the precursor of cervical carcinoma. The study group comprised 180 women who were referred because of cytologic changes indicating dysplasia and who were subsequently diagnosed with cervical intraepithelial neoplasia grade III. The HPV-16-associated lesions showed a significantly higher number of mitoses per 1,000 nuclei than the lesions without HPV (p < 0.001). The HPV-16-associated lesions also showed a significantly higher proportion of mitotic figures with lagging chromosomes than did the HPV-18- or HPV-31-associated lesions and lesions without HPV (p = 0.01, p = 0.007, and p = 0.002, respectively). Our results indicate that the differences in oncogenic potential among HPVs become apparent in the precursor lesion through the differences in the extent of chromosomal lag during mitosis.

Flow cytometric DNA ploidy and cells phase fractions in recurrent human pituitary adenomas. A correlative study of flow cytometric analysis and the expression of proliferating cell nuclear antigen.

Flow cytometric analysis was applied to embedded tissue to measure the proliferative activity and the DNA ploidy of 16 recurrent and 17 nonrecurrent pituitary adenomas. The results were compared with data from a previous study which demonstrated that proliferating cell nuclear antigen (PCNA) labeling index was higher in recurrent adenomas than in nonrecurrent adenomas. Flow cytometric analysis as a tool for predicting aggressive behavior has been useful in a variety of human tumors; however, its prognostic value in pituitary adenomas is controversial. Therefore, we decided to explore the relationship of the results of flow cytometry and proliferating cell nuclear antigen labeling indices with the prognosis of pituitary adenomas. Three out of 16 recurrent adenomas and five out of 17 nonrecurrent adenomas demonstrated a DNA aneuploid pattern. All the nonfunctional recurrent adenomas had a diploid pattern, while only 40% of the functional recurrent adenomas had a diploid pattern. The GO/G1 phase fraction was higher in the recurrent adenomas, than in the nonrecurrent ones (p = 0.0005). In contrast, the S-phase fraction and the coefficient of variation were higher in the nonrecurrent adenomas (5.9 +/- 1.0%, 7.0 +/- 0.75, respectively) than in the recurrent ones (2.5 +/- 0.6%, 4.0 +/- 0.2%, respectively) (p = 0.003 and p = 0.001, respectively). The proliferating cell nuclear antigen labeling indices were higher in the recurrent adenomas (18.9 +/- 4.5%) than in the nonrecurrent adenomas (2.6 +/- 1.6%) (p = 0.003). The S-phase of flow cytometry correlated weakly with the proliferating cell nuclear antigen labeling indices when the recurrent and the nonrecurrent adenomas were considered as one group. (r = -0.356, p = 0.033). But no significant correlations were observed when the groups of recurrent (r = -0.311, p = 0.195) and nonrecurrent tumors (r = -0.019, p = 0.942) were compared separately. The results of flow cytometric analysis suggest that recurrent adenomas may have a higher proportion of cells in the presynthetic phase than the nonrecurrent adenomas. This study suggests that flow cytometric analysis is of limited value in predicting recurrence of pituitary adenomas.

Effect of K-ras mutation on morphogenesis of colorectal adenomas and early cancers: relationship to distribution of proliferating cells.

The authors evaluated the association of the K-ras mutation with the distribution of proliferating cells and the macroscopic appearance of colorectal tumors. A total of 122 colorectal adenomas and 96 early cancers were classified macroscopically as follows: (1) polypoid, exceeding 3 mm in height; (2) flat or hemispherically elevated 3 mm or less; and (3) depressed. The intramucosal areas of these tumors were examined by Ki-67 immunostaining and nested polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) to detect proliferating cells and the K-ras codon 12 mutation. The distribution pattern of the Ki-67-positive cells was of two types: diffuse (D), being positive throughout the crypts, and superficial (S), being positive mainly in the superficial areas of the crypts. The K-ras mutation and the D type distribution of the Ki-67-positive cells were significantly less common (P < .005) in the nonpolypoid, depressed, or elevated tumors versus the polypoid tumors. The incidence of the K-ras mutation was significantly (P < .0001) associated with the D type distribution. The K-ras mutation may involve a disorder of cell proliferation that leads to the polypoid growth of colorectal tumors.

Standardized mitotic counts in breast cancer. Evaluation of the method.

Twenty-one pathologists and technicians participated in a study evaluating the variation present in mitotic counts for prognostication of breast cancer. The participants counted the mitotic figures in 20 breast cancer samples from ten high power fields (mitotic activity index, MAI, giving the results in mitotic figures per 10 fields) and also made a correction for field size and area fraction of the neoplastic epithelium to get the standardized mitotic index (volume fraction corrected mitotic index, or M/VV index, giving the result in mitotic figures per square mm of neoplastic epithelium). The difference in variation between the two methods was not big, but the standardized mitotic index (SMI) showed consistently smaller variation among all participants and different subgroups. Experienced pathologists had the highest variation in mitotic counts, and specially trained technicians, the lowest. The efficiency of the mitotic counts in grading (the grading efficiency) was used to evaluate the mitotic counts. In groups without special training for mitotic counts the mean grading efficiency was lower (experienced and training pathologists both on average had the potential to grade 88% of the cases correctly) than in the group specially trained for the purpose (trained technicians had the potential to grade 95% of the cases correctly). Among the specially trained technicians, the grading efficiency was of the same magnitude as the grading efficiency achieved in determining the S-Phase fraction of cells from paraffin embedded breast cancers by flow cytometry in different laboratories. The results suggest that special training is helpful in making mitotic counts more reproducible, and that in trained hands, the mitotic counts give results comparable to more sophisticated methods of determining proliferative activity in breast cancer.

Nuclear morphometry, immunohistochemical staining with Ki-67 antibody and mitotic index in the assessment of proliferative activity and prognosis of primary malignant melanomas of the skin.

Nuclear morphometry, immunohistochemical staining with Ki-67 antibody and mitotic index were studied in primary cutaneous malignant melanomas. The number of Ki-67 positive cells/ 200 tumor cells did not correlate with any nuclear morphometrical parameters, and it only approached but did not reach significant correlation with melanoma thickness according to Breslow. The nuclear area, short axis and long axis correlated with melanoma thickness, but the nuclear axis ratio (which reflects the sphericity of nuclei) and melanoma thickness did not show significant correlation. Mitotic index was higher in thick melanomas and in melanomas with high Ki-67 positivity, large nuclear area, long nuclear short axis, and small nuclear axis ratio. In Cox's stepwise proportional hazard model, melanoma thickness and the nuclear axis ratio were significant independent prognostic factors for patient survival, while the nuclear area, short axis and long axis, gender, age, Clark level, mitotic index and Ki-67 positivity lacked significant independent prognostic value. The results suggest that the proliferative activity of tumor cells does not alone explain the great importance of tumor thickness as prognosticator in melanoma. The thickness of melanoma measured according to Breslow and the nuclear axis ratio are more efficient prognosticators in melanoma than parameters associated with proliferation.

Immunohistochemical expression of p53 protein, mitotic index and nuclear morphometry in primary malignant melanoma of the skin.

The immunohistochemical expression of the p53 tumor suppressor protein and the nuclear morphometric parameters were studied in 80 primary skin melanomas. The mitotic index was counted in 64 tumors. In 95% of the tumors p53 positive nuclei were detected, but in only 31% of the cases was the proportion of positive nuclei 3% or higher. The mitotic indices and the mean nuclear areas were not significantly different in melanomas with different p53 expression levels. The p53 positive nuclei had a larger mean nuclear area than the p53 negative nuclei. p53 expression did not increase with melanoma thickness. In Cox's stepwise proportional hazards model, the ratio of mean nuclear long and short axis and melanoma thickness had independent prognostic value, while the level of p53 expression, the mitotic index, and the mean nuclear area were not significantly associated with survival. We conclude that the nuclei expressing p53 protein are larger than p53 negative nuclei. The level of immunohistochemical expression of p53 is low in primary skin melanoma, and it is not valuable as a general prognostic marker for this tumor. p53 expression is not associated with melanoma thickness, indicating that high p53 expression is not a late phenomenon in the progression of this tumor.

Ki-67 and biological behaviour in meningeal haemangiopericytomas.

The biological behaviour of meningeal haemangiopericytomas was retrospectively studied using immunohistochemical staining with MIB1, a monoclonal antibody against the Ki-67 antigen, a nuclear protein related to cell proliferation. Paraffin-embedded material from 62 tumours from 40 patients were investigated. The proliferating compartment of the tumours was estimated by evaluating the MIB1 staining index, i.e. the percentage of MIB1 positive nuclei in at least 1000 counted tumour cells in representative areas. The staining index ranged from 1.24% to 39.01%. Statistical analysis revealed no significant correlation between the staining index and recurrence-free survival (chi 2 = 0.3922, P = 0.5311). Long-term observation (> 100 months), however, revealed a tendency to longer survival in the group with a staining index less than 5%. According to our results, the MIB1 staining index does not contribute to the accuracy of predicting the clinical outcome of meningeal haemangiopericytomas.

Section thickness and mitotic counts in ovarian mucinous carcinoma. Methodological study with scanning confocal microscopy.

To study the effect of section thickness on mitotic counts, paraffin sections of 28 cases of mucinous ovarian carcinomas were analyzed. Mitotic counts, estimated with the number of mitoses per mm2 of neoplastic epithelium (M/Vv index) and per mm3 from optical sections and through the whole section thickness were done. Section thickness was measured with scanning confocal microscopy from each specimen of 3 series of sections with different nominal thicknesses (5, 8, and 10 microns). Section thickness varied considerably within each series of sections. The sections of the 5-microns group tended to be thicker and the sections of the 10-microns group thinner than expected on the basis of their nominal values. As expected, mitotic counts through the section gave higher M/Vv index values than counts done using optical sections. M/Vv values obtained using optical sections increased with increasing nominal section thickness. The study suggests that, when the section thickness cannot be measured, it is advisable to do mitosis counting by using optical sections in lightly stained tissue sections in the thickness range of 8-15 microns.

Peripheral primitive neuroectodermal tumour and extra-osseous Ewing's sarcoma; a histological, immunohistochemical and DNA flow cytometric study.

Although peripheral primitive neuroectodermal tumour (pPNET) and extra-osseous Ewing's sarcoma (EES) are thought to be closely related neoplasms, their clinical behaviour differs considerably. To determine the clinical relevance of the Schmidt classification scheme for differentiating pPNET and EES, 20 tumour specimens of poorly differentiated round cell tumours were evaluated. In addition, the diagnostic value of several neural markers and the prognostic value of quantitative morphological variables (DNA ploidy, S-phase fraction, and the mitotic activity) were assessed. Homer-Wright rosettes were present in 9 tumours. Neuron specific enolase (NSE) was expressed in 11 tumours, 8 of which expressed a second neural marker (CD57, S100, or neurofilament). According to the Schmidt classification, 11 pPNET and 5 EES were distinguished. HBA-71 was exclusively expressed in pPNET and EES. The remaining tumours were classified as sarcoma not otherwise specified (n = 2), rhabdomyosarcoma (n = 1), and desmoplastic tumour with divergent differentiation (n = 1). EES611 patients fared significantly better than the pPNET patients (100% versus 42% 5-year survival). Neither DNA ploidy nor S-phase fraction assessed in 12 evaluative histograms (9 pPNET and 3 EES), nor mitotic activity yielded information of additional prognostic value. On the basis of this study and the Schmidt classification scheme, it can be concluded that if the diagnosis of EES and pPNET is based on light microscopy (Homer-Wright rosettes) and/or immunohistochemistry (at least two neural markers, i.e. NSE, S-100, CD57, and neurofilament), the classification provides important clinical information. Furthermore, positivity for HBA-71 is helpful in differentiating pPNET and EES from all other small round cell tumours.

El enclaustramiento cromósomico, la frecuencia de mitosis y su relación con la proporción Carnoy/KCL / The enclosed chromosome, the frecuency of mitoses and their relationship with the Carnoy/KCL ratio

Se investigó el efecto de diferentes proporciones de Carnoy/KCI sobre la frecuencia de mitosis enclaustradas por los residuos de citoplasma y la relación de éstas con el número total de mitosis. Se demostró que la disminución de la proporción (de 1 a 0.5, 0.4 y 0.3) se asoció con una disminución de la frecuencia de mitosis enclaustradas y un aumento en el número total de mitosis. El resultado de esta divergencia, que denominamos Mitosis Efectiva (ME), con la proporción de 1 fue de 0.32 + - 0.24 por ciento (X + - DE) y con proporciones menores de 1 fue de 3.44 + - 2.25 por ciento (p<0.01). Este aumento se observó exclusivamente cuando el KCI se agregó a 37§C; la Microdosis (MD) de Carnoy 1 22§C se añadió al inicio de la incubación con KCI a 37§C y se incluyó un periodo de reposo de 10 minutos a 22§C entre las dos primeras fijaciones con Carnoy a 22§C. Se suprimió el beneficio de la disminución de la proporción sobre el enclaustramiento y la frecuencia de mitosis, cuando se omitió la MD o el reposo, la MD se agregó al final de la incubación con KCI o a 4§C y el KCI o el Carnoy se añadieron a 22§C o 4§C respectivamente.

Efecto de la "microdosis" de Carnoy y de la proporcion KCI/Carnoy sobre el indice mitótico / Effects of Carnoy priming and the KCI/Carnoy ratio on the mititic index

Se estudiaron, en linfocitos de sangre periférica, los cambios en el Indice Mitótico (IM) producidos por la omisión o la adición de la "microdisis" (MD) de Carnoy (50ul), al inicio o al final de la incubación con KCI 75 mM. El efecto de diferentes proporciones y temperaturas de KGI y Carnoy también fue investigado. La prespuesta óptima, evidenciada por un Im de 9.8 +- 2.4 por ciento, se obtuvo del grupo en el que la MD, a 22oC, se agergó al inicio de la incubación con KGI a 37oC y la proporción KGI/Carnoy fue mayor de 2. El análisis conjunto de todos los resultados demostró que la omisión de la MD; que la MD añadida al final de la incubación; que la omisión del reposo entre las dos primeras fijaciones de Carnoy o éste adicionado a 4oC o el KGI a 22oC, produjeron IM bajos con valores comprendidos entre 0.6 +- 0.1 por ciento y 1.2 +- 0.6 por ciento (p <0.001 vs 9.8+- 2.4 por ciento).