Nuclear DNA content as a prognostic factor in T1-2N0 breast cancer

One hundred fifteen patients with postsurgical stage T1-2N0 breast cancer with the minimum follow-up of 22 years and from a defined population were studied to find reliable prognostic factors predicting long-term survival rate. The 30-year survival rate corrected for intercurrent deaths was 75%, and no deaths of breast cancer occurred after the nineteenth year of follow-up. The 30-year crude survival rate was only 28%, indicating that the great majority of these patients ultimately die of other causes than breast cancer. The nuclear DNA content could be determined by flow cytometry in 95 of the 115 cases, and 52 (55%) of them were nondiploid. DNA ploidy (diploid vs. nondiploid, P = 0.11) did not have a significant influence on long-term survival rate, but cancers with the DNA index less than 1.2 had more favorable prognosis in a univariate analysis than those with the DNA index more than 1.2 (P = 0.02). The most important independent prognostic factor in Cox's multivariate analysis was the presence of lymphatic vessel invasion of cancer cells (P less than 0.001). Several other factors, including histologic grade and type, extent of necrosis, type of tumor margin, age at diagnosis, and primary tumor size could be shown to be of prognostic value in univariate and/or multivariate analyses.     

Prediction of relapse or survival in patients with node-negative breast cancer by DNA flow cytometry

More accurate prediction of the prognosis in women with node-negative breast cancer may improve physicians' ability to identify the patients most likely to benefit from systematic adjuvant therapy. With this in mind, we performed DNA flow-cytometric measurements of ploidy and the fraction of cells in the synthesis phase of the cell cycle (S-phase fraction) on 395 specimens of node-negative breast cancer from our bank of frozen tumors, using the aliquots of pulverized frozen tissue from steroid-receptor assays. The median duration of follow-up in patients still alive at the time of analysis was 59 months. Thirty-two percent of the 345 specimens that could be evaluated were diploid, and 68 percent were aneuploid. The probability of disease-free survival at five years was 88 +/- 3 percent in patients with diploid tumors and 74 +/- 3 percent in those with aneuploid tumors (P = 0.02). The S-phase fraction was not a significant additional predictor of disease-free survival in patients with aneuploid tumors. However, the probability of disease-free survival in patients with diploid tumors and low S-phase fractions was 90 +/- 3 percent at five years, as compared with 70 +/- 13 percent in those with diploid tumors and high S-phase fractions (P = 0.007). Similar differences in overall survival were noted. We conclude that DNA flow-cytometric measurements of ploidy and S-phase fraction can be performed on frozen specimens of tumors and are potentially important predictors of disease-free and overall survival in patients with node-negative breast cancer.     

Tumour heterogeneity of DNA cell cycle variables in breast cancer measured by flow cytometry.

AIM/BACKGROUND: Conflicting results have been reported concerning the prognostic value of DNA flow cytometric variables (DNA ploidy, DNA index, %S phase fraction) in breast cancer. Selection bias and differences in treatment may have contributed to these conflicting prognostic results. Differences in tissue processing, the number of nuclei measured, DNA histogram/cell cycle analysis, and intra-tumour heterogeneity may also have played a role. The aim of the present study was to assess intra-tumour heterogeneity of DNA flow cytometric variables in breast cancer. METHODS: Fresh frozen specimens (n = 274) (0.3 x 0.3 x 0.3 cm) of 17 breast cancers and 167 slices, 50 microns thick, of 58 paraffin wax embedded blocks of 21 breast cancers were studied. All samples were prepared individually for DNA flow cytometry. DNA histograms were interpreted by semi-automated cell cycle analysis (MultiCycle) by two observers to avoid biased interpretation. An artificial averaged DNA histogram of each case was composed to simulate a sample prepared from whole tumour tissue. RESULTS: With regard to DNA ploidy, classified as diploid or aneuploid, the fresh frozen and paraffin wax embedded breast cancers showed intra-tumour heterogeneity in 53% and 38% of cases, respectively. For fresh frozen and paraffin wax embedded material, respectively, six samples had to be measured to detect the highest DNA ploidy class in 71% and 86% of cases. Averaged DNA histograms showed a loss of DNA aneuploidy in 36% and 6% of fresh frozen and paraffin wax embedded samples, respectively. High intra-tumour heterogeneity (wide ranges) was found for the %S phase fraction. Average %S phase fraction and average aneuploid %S phase fraction had the widest ranges at 9.5-31.6% and 0.0-62.7%, respectively. There was no correlation between the number of stemlines and intra-tumour %S phase variability on the one hand and tumour size and grade on the other. CONCLUSIONS: High intra-tumour heterogeneity for breast cancer was found for DNA ploidy, the DNA index and %S phase fraction as measured by flow cytometry, which may explain the conflicting prognostic results reported in the literature. To detect aneuploid cells, six samples may have to be prepared and measured separately. Measurement of these variables may be more reliable in paraffin wax sections because the thick slices provide a more representative sample. Prospective studies are required to determine whether the highest %S phase fraction value or the average value is more useful in the clinical context.     

10q23.3 loss of heterozygosity is higher in lymph node-positive (pT2-3,N+) versus lymph node-negative (pT2-3,N0) prostate cancer

Loss of heterozygosity (LOH) in the region of 10q23.3 has been associated with multiple tumors, including glioblastoma multiforme, melanoma, endometrial carcinoma, and prostate carcinoma. The tumor suppressor gene, PTEN/MMAC1, is also located in this region, and, in addition to other tumor types (eg, glioblastoma multiforme, endometrial, and melanoma), PTEN/MMAC1 mutations have been found in prostate cancer cell lines, xenografts, and hormone refractory prostate cancer tissue specimens. The aim of this study was to evaluate LOH at 10q23.3 as a marker of cancer progression in node-positive prostate cancer. Genetic alterations in the region of 10q23.3 were assessed in 23 node-positive (pT2-3, N+) and 44 node-negative prostate (pT2-3, N0) cancers with D10S532, D10S1687, D10S541, and D10S583 flanking polymorphic genetic markers; PTENCA, a genetic marker within PTEN/MMAC1, was also tested. Using DNA from paired normal and microdissected tumor samples, LOH at microsatellite loci was determined after polymerase chain reaction amplification. LOH in at least 1 marker was identified in 14% (6 of 44) of lymph node-negative and 43% (10 of 23) of lymph node-positive prostate cancers (chi-square test, P = .007). This increase in genetic alterations in node-positive prostate cancer suggests that 10q23.3 is a marker for metastatic progression.     

Long term prognostic value of Nottingham histological grade and its components in early (pT1N0M0) breast carcinoma

AIMS: To determine the prognostic usefulness of the Nottingham histological grade (NHG) and its components in a series of 270 patients with stage pT1N0M0 breast cancer with a median follow up of 12.5 years. METHODS: Microscopic slides were re-examined and the degree of tubule formation, nuclear pleomorphism, and mitotic counts were assessed and scored according to the suggested guidelines. The association with cancer specific survival (CSS) was evaluated by univariate and multivariate analyses. RESULTS: Whereas tumour size, patient age, menopausal status, type of surgery, or adjuvant treatment were not related to prognosis, histological type (p < 0.01) and NHG (p < 0.005) were associated with CSS. When evaluating the components of NHG separately, survival was not related to the score for pleomorphism, but was significantly better in tumours with score 1 or 2 for tubule formation (p < 0.007) and in those with score 1 for mitotic counts (p < 0.006). The two components retained independent significance in multivariate analysis. When the proposed cut off points for mitotic counts were replaced by lower ones based on tertile values, the mitotic index became the strongest prognostic factor (p = 0.0001) and histological type was the only additional factor of independent prognostic significance. CONCLUSIONS: These findings confirm the prognostic value of NHG in pT1N0M0 breast carcinoma, show that the evaluation of tubule formation and mitotic rate provides independent prognostic information, and suggest that the proposed cut off points for mitotic counts may be too high for this particular group of tumours.     

Clinical importance of DNA content in rectal cancer measured by flow cytometry.

The DNA content of 369 rectal cancers was measured by flow cytometry. One hundred and four (28%) were diploid, 252 (68%) were aneuploid, and 13 (3.5%) were tetraploid. Diploid cancers were associated with an improved 5 year survival (p less than 0.001) and were more likely to present at an early stage. DNA content, however, did not confer independent prognostic information in a Cox model based on four discrete pathological variables. Patients were classified by a new system of prognostic grouping and those with a very good or a very poor outlook were removed leaving 137 prognostic group III patients. No further substratification of this group by DNA content or by four additional pathological variables could be achieved. As the new prognostic system is not improved by the addition of ploidy, routine adoption of flow cytometry in the assessment of rectal cancer cannot be recommended.     

Image analysis confirmation of DNA aneuploidy in flow cytometric DNA distributions having a wide coefficient of variation of the G0/G1 peak

With nuclei extracted from paraffin-embedded tissues, resolution of normal diploid DNA and abnormal near-diploid/aneuploid populations by flow cytometry (FCM) is especially difficult. These samples, compared with fresh tissue, tend to show a broader DNA distribution, appearing as a wide (high) coefficient of variation (CV) of the G0/G1 peak. To address the question of whether there may be aneuploid populations hidden in wide CV diploid G0/G1 peaks, the authors measured DNA content in nuclei extracted from paraffin-embedded tumor tissue by morphometric image analysis (IA) in addition to FCM. Of 29 samples showing little evidence of DNA aneuploidy by FCM, in 20 of 20 with G0/G1 CVs greater than or equal to 5.50% there was an aneuploid population when analysis was performed by IA. Of the remaining nine samples with CVs less than or equal to 4.41%, all were diploid in the G0/G1 region by both FCM and IA. The presence of aneuploid populations in FCM distributions with wide CV G0/G1 peaks can be confirmed by IA.     

Poorly differentiated clusters (PDCs) as a novel histological predictor of nodal metastases in pT1 colorectal cancer

The practical use of histological factors such as submucosal (SM) invasion depth, poor differentiation, presence of lymphovascular invasion (LVI) and tumour budding to establish the risk of nodal dissemination in pT1 colorectal cancer (CRC) is limited by their low standardization and high inter-observer variability. It was recently suggested that the presence in CRC histological sections of poorly differentiated clusters (PDCs), defined as ≥5 cancer cells with no gland formation, may predict the metastatic potential of CRC. In addition, PDC assessment was shown to be more reproducible than the evaluation of the other aforementioned histological predictors. Hence, in this study, we investigated and compared the predictive value of PDC and other histological parameters on the risk of nodal involvement in pT1 CRC. The presence of PDC, SM invasion depth ≥1,000 μm and LVI was significantly associated with N+ status in pT1 CRC (P?<?0.0001). Among these parameters, SM invasion depth had the highest sensitivity to identify N+ pT1 CRC but with the lowest specificity. When the analysis was restricted to CRCs with SM invasion depth ≥1,000 μm, the presence of PDC was the only independent risk factor for nodal metastases and allowed the identification of 87.5 % of N+ cancers. In conclusion, in this study, we demonstrate that the presence of PDC is associated with the metastatic potential of pT1 CRC. The combination of this parameter with SM invasion depth may allow identifying most of the pT1 CRC with nodal metastases.     

Marked intratumoral heterogeneity of c-myc and cyclinD1 but not of c-erbB2 amplification in breast cancer

Intratumoral heterogeneity mirrors subclonal diversity and might affect treatment response. To investigate molecular heterogeneity of primary breast cancer specimens, we determined the amplification status of growth regulatory genes (c-erbB2, topoisomerase IIalpha, c-myc, and cyclinD1) in macroscopically and microscopically separate areas of individual tumors (n = 21). Using laser-assisted microdissection and quantitative PCR, we found marked intratumoral heterogeneity with different patterns for each gene. Molecular heterogeneity in amplification pattern could be demonstrated between both macroscopically (0.5 to several centimeters) and microscopically (10 to several hundred micrometers) distant tumor areas. C-erbB2 amplification proved to be the most stable amplification in individual tumors, with heterogeneity occurring in only 36% of amplified cases. By contrast, amplification of c-myc and cyclinD1 revealed varying patterns in the vast majority of amplified cases (100% and 83%). The constancy of c-erbB2 amplification underlines its presumed importance in breast cancer biology. We conclude that the molecular heterogeneity of breast cancer as evidenced in this study requires thorough and representative sampling of different tumor areas when the biologic significance of somatic mutations is considered. Patterns of heterogeneity can be used to trace the clonal evolution within different compartments of an individual tumor.     

Prediction of response to adjuvant chemotherapy in premenopausal lymph node positive breast cancer patients with morphometry, DNA flow cytometry and HER-2/neu oncoprotein expression. Preliminary results.

The value of morphometry, DNA flow cytometry and HER-2/neu oncoprotein expression for prediction of response to adjuvant chemotherapy in premenopausal lymph node positive breast cancer patients was evaluated in a group of CMF treated patients and controls with long-term follow-up. In the treated group, the Morphometric Prognostic Index (cutpoint 1.1) was the best prognosticator (p less than 0.0001, MC = 16.9), followed by the Mitotic Activity Index, the volume percentage epithelium and the number of positive nodes. For the controls, only the % HER-2/neu oncoprotein expression revealed significant differences (p less than 0.0001, MC = 16.3). When directly comparing treated patients and controls stratified for a certain parameter, no significant differences were obtained, although a trend towards improved survival in the treated group was present for some of the subgroups for several parameters. These preliminary results indicate that morphometric features and quantitative HER-2/neu oncoprotein expression may be important factors for identifying cases that will or will not respond to adjuvant chemotherapy.     

流式细胞仪每秒进样细胞数对细胞G0/G1期峰的荧光强度值及变异系数值的影响

在生物学研究中,利用细胞内DNA双螺旋结构碱基对与某些核酸荧光染料有良好的亲和力,荧光染料在光的激发下,产生特定的荧光,运用流式细胞仪检测、分析其荧光强度（Mean值）,及其变异系数（CV值）,了解细胞内DNA含量,已成为科学分析细胞DNA含量及其所处周期状态的一种手段。然而,流式细胞仪对操作者技术要求高,同一样本不同实验室检测的结果不尽相同;即使是同一实验室,不同操作人员检测结果亦不一样。     

Auto-antibody production and glomerulonephritis in congenic Slamf1-/- and Slamf2-/- [B6.129] but not in Slamf1-/- and Slamf2-/- [BALB/c.129] mice

Several genes in an interval of human and mouse chromosome 1 are associated with a predisposition for systemic lupus erythematosus. Congenic mouse strains that contain a 129-derived genomic segment, which is embedded in the B6 genome, develop lupus because of epistatic interactions between the 129-derived and B6 genes, e.g. in B6.129chr1b mice. If a gene that is located on chromosome 1 is altered through homologous recombination in 129-derived embryonic stem cells (ES cells) and if the resultant knockout mouse is backcrossed with B6, interpretation of the phenotype of the mutant mouse may be affected by epistatic interactions between the 129 and B6 genomes. Here, we report that knockout mice of two adjacent chromosome 1 genes, Slamf1(-/-) and Slamf2(-/-), which were generated with the same 129-derived ES cell line, develop features of lupus, if backcrossed on to the B6 genetic background. By contrast, Slamf1(-/-) [BALB/c.129] and Slamf2(-/-) [BALB/c.129] do not develop disease. Surprisingly, Slamf1(-/-) [B6.129] mice develop both auto-antibodies and glomerulonephritis between 3 and 6 months of age, while disease fully develops in Slamf1(-/-) [B6.129] mice after 9-14 months. Functional analyses of CD4(+) T cells reveals that Slamf2(-/-) T cells are resistant to tolerance induction in vivo. We conclude that the Slamf2(-/-) mutation may have a unique influence on T-cell tolerance and lupus.     

Comparative assessment of proliferation and DNA content in breast carcinoma by image analysis and flow cytometry.

Although tumor DNA content and proliferation are usually determined by flow cytometry (FCM), quantitative microscopic image analysis is a viable alternative technique that also provides important histologic correlations. To compare these methods, we measured DNA content and proliferation in 54 consecutive breast cancers and 15 benign breast lesions by FCM and IA. DNA content determination was concordant in 49 of 54 cancers measured by FCM and IA. Four of the discordant cases were aneuploid by IA and diploid by FCM. There was good correlation between the DNA index (DI) measured by FCM and IA (r = 0.89, P less than 0.0001). Proliferation was assessed by IA quantitation of Ki-67 and PCNA/Cyclin antibody staining, as well as by flow cytometric S-phase fraction (SPF). Ki-67 positivity was greater in breast cancer than in benign controls (21.6% +/- 13.1% vs. 7.9% +/- 5.6% [P less than 0.0001]), as was PCNA/Cyclin positivity (10.2 +/- 6.7% vs. 2.7 +/- 2.5% [P less than 0.0001]). S-phase fraction measured by FCM was 7.9% +/- 5.7% for carcinomas and 3.17% +/- 2.1% for benign controls (P less than 0.003). Ki-67 and Cyclin staining, as well as SPF, were significantly increased in aneuploid compared to diploid tumors, and increased staining was associated with worsening nuclear grade. There were significant correlations between SPF and Ki-67 staining (r = 0.48, P less than 0.0001) and SPF and Cyclin staining (r = 0.48, P less than 0.0001). We conclude that FCM and IA provide comparable measurements of DNA content, although occasional discrepancies occur. Image analysis provides a valuable alternative method for assessing tumor cell proliferation and may offer certain advantages over FCM.     

Gli1 induces G2/M arrest and apoptosis in hippocampal but not tumor-derived neural stem cells

Sonic hedgehog (Shh) is necessary for sustaining the proliferation of neural stem cells (NSCs), yet little is known about its mechanisms. Whereas Gli1, Gli2, and Gli3, the primary mediators of Shh signaling, were all expressed in hippocampal neural progenitors, Shh treatment of NSCs induced only Gli1 expression. Acute depletion of Gli1 in postnatal NSCs by short-hairpin RNA decreased proliferation, whereas germline deletion of Gli1 did not affect NSC proliferation, suggesting a difference in mechanisms of Gli1 compensation that may be developmentally dependent. To determine whether Gli1 was sufficient to enhance NSC proliferation, we overexpressed this mitogen and were surprised to find that Gli1 resulted in decreased proliferation, accumulation of NSCs in the G2/M phase of cell cycle, and apoptosis. In contrast, Gli1-expressing lineage-restricted neural precursors demonstrated a 4.5-fold proliferation enhancement. Expression analyses of Gli1-expressing NSCs identified significant induction of Gadd45a and decreased cyclin A2 and Stag1 mRNA, genes involved in the G2-M transition and apoptosis. Furthermore, Gadd45a overexpression was sufficient to partially recapitulate the Gli1-induced G2/M accumulation and cell death of NSCs. In contrast to normal stem cells, tumor-derived stem cells had markedly higher basal Gli1 expression and did not undergo apoptosis with further elevation of Gli1. Our data suggest that Gli1-induced apoptosis may serve as a protective mechanism against premature mitosis and may give insight into mechanisms by which nonmalignant stem cells restrain hyperproliferation in the context of potentially transforming mitogenic signals. Tumor-derived stem cells apparently lack these mechanisms, which may contribute to their unrestrained proliferation and malignant potential.     

Types of tumor lymphoid response and sinus histiocytosis. Relationship to five-year, disease-free survival in patients with breast cancer

Diffuse (D), perivascular (PV), and a combination form (DPV) of lymphoid infiltrates were found in 95% of invasive breast cancers. The DPV and D patterns were associated with tumor necrosis, and the D pattern with cancers of nuclear grade 3 (most anaplastic) and histologic grade 3 (most malignant). An absent cell reaction was significantly related to absent nodal metastases. Life-table analyses disclosed a higher incidence of patients' being disease free for five years when no cell reaction was encountered. There was no significant association between the presence or absence of any particular type of sinus histiocytosis (SH) of regional lymph nodes and five-year, disease-free survival. It is uncertain whether lymphoid reactions and SH represent immunologic host responses, but if they do, they appear to be biologically ineffectual and, in the former instance, perhaps even detrimental.     

Correlation of tissue inhibitor of metalloproteinase-2 with proliferative activity and patients' survival in breast cancer.

Tissue inhibitors of metalloproteinases (TIMPs) are endogenous regulators of matrix metalloproteinases (MMPs). They are believed to possess several distinct cellular functions, particularly the contradictory activities of inhibiting MMPs and promoting tumor cell growth. Immunohistochemistry was performed to detect TIMP-2 protein in 136 infiltrative breast carcinomas. TIMP-2 protein was analyzed in parallel with clinicopathologic features (tumor size, histologic type, nuclear and histologic grade, stage), patients' overall survival and ER, PR, Ki-67, topo IIalpha, c-erbB-2, p53 and bcl-2 proteins. Statistical analysis was performed using univariate and multivariate models analysis. Immunoreactivity for TIMP-2 was observed in cancer cells and stromal fibroblasts in 106 (77.94%) and 104 (76.47%) of 136 cases, respectively. TIMP-2 protein expression in stromal fibroblasts showed a statistically significant inverse correlation with tumor size (P =.014). An inverse correlation was also observed between TIMP-2 epithelial immunoreactivity and nuclear and histologic grade (P =.036 and P =.007, respectively). TIMP-2 protein reactivity showed statistically significant positive associations with topo IIalpha and bcl-2 in stromal and cancer cells, respectively (P =.032 and P =.001, respectively). TIMP-2 protein expression in cancer and stromal cells was associated with better patients' overall survival (P =.002 and P =.038, respectively). When evaluated by the Cox's proportional hazard regression model, this association was further established, but only as far as TIMP-2 expression in tumor epithelium was concerned (P =.019). Our results support the multifunctional potential of TIMP-2 through its correlation on the one hand to a favorable outcome, due to its MMP inhibitory activity and on the other to topo IIalpha contributing to its growth factor activity.     

Phosphatases PP1 and PP2A act after the G0/G1 interface in lymphocyte activation.

An understanding of the progressive events required for lymphocyte activation is a prerequisite to understanding the molecular mechanism of immunosuppressive reagents, and is of central relevance to autoimmunity and immune tolerance. Although reversible phosphorylation is a major control mechanism in T cell activation, few facts are known about phosphatases in T cells. It has recently become possible to block selectively the serine/threonine specific protein phosphatases PP1 and PP2A by okadaic acid, a C38 polyether fatty acid produced by dinoflagellates. Here we have used this new and powerful biochemical probe to identify and quantify the activities of PP1 and PP2A at various times during lymphocyte activation. The selected model was mouse T cell activation by concanavalin A because this gave a reproducible experimental system that allowed large scale experiments in defined, serum-free conditions. Our results show the following: (1) levels of PP1 and PP2A in lymphocytes appear to be unusually low, effects being measured at 10 nM okadaic acid compared to 1 microM reported for other cell types; (2) the okadaic acid effect was readily reversible; (3) okadaic acid stimulated mitogenesis when present in early G1 but inhibited mitogenesis in late G1; (4) levels of PP1 and PP2A remained relatively constant over the first 7 h following stimulation; (5) lymphocytes activated in the presence of okadaic acid became resistant to subsequent treatment with FK506; and (6) combined or consecutive treatment with okadaic acid and FK506 resulted in additive drug effects. We conclude that PP1 and PP2A are not involved in the G0-G1 transition as defined by the FK506-sensitive step.(ABSTRACT TRUNCATED AT 250 WORDS)