Prognostic significance of DNA image cytometry in libyan breast cancer

Background: We evaluated the relation of nuclear DNA content and clinicopathological features and prognosis in primary breast cancer of female Libyan patients with variable stage and grade and different treatment regimes. Patients and Methods: Histological samples from 104 patients of breast carcinoma were retrospectively studied by computerized nuclear DNA cytometry. Isolated nuclei from paraffin sections were stained with Feulgen stain and DNA was measured using a computer-assisted image analysis cytometry system. In each case, 200 nuclei were measured and the DNA histograms, S phase fraction (SPF) and number of cells above 5c and 9c were determined. We applied different approaches in the analysis of DNA to compare the DNA histograms with different clinicopathological features and survival. Results: The mean of DNA ploidy mode for all tumors was 3.43; 82.7% of tumors were aneuploid and 17.3% were diploid. The median SPF was 3.5% for DNA diploid and 13.5% for DNA aneuploid tumors. DNA aneuploid tumors and high SPF were associated with advanced stage, distant metastasis, high histological grade and lymph node involvement. The SPF was also associated with large tumor size and with younger patients (<50 years). In the overall population (median follow-up 51 months), patients with aneuploid DNA histograms and high SPF values had shorter survival times than those with diploid DNA histograms and low SPF values (p = 0.001, p < 0.0001, respectively). Also, short survival was associated with a multiploid DNA histogram and with DNA aneuploid cells ≥5 cells (p < 0.0001, p = 0.001, respectively). In a Cox multivariate analysis, DNA ploidy (p = 0.010), age (p = 0.038) and clinical stage (p = 0.001) were independent predictors of overall survival, and DNA ploidy (p = 0.018) and clinical stage (p = 0.001) also proved to be independent predictors of disease-specific survival. The SPF cutoff point of 11% might be applied to separate patients into good and poor prognosis groups. Conclusions: DNA image cytometry with careful analysis of the histograms may provide valuable prognostic information in Libyan breast cancer, with potential clinical implications in patient management, particularly in predicting the patients at high risk for metastasis and recurrence who should be considered as candidates for combined adjuvant therapy.     

DNA content flow cytometry as a prognostic factor for node-positive breast cancer. The role of multiparameter ploidy analysis and specimen sonication

The DNA content was analyzed in paraffin-embedded material from 167 patients with node-positive breast cancer to learn whether specimen sonication and multiparameter ploidy analysis (MPPA) (using DNA content and light scatter) could improve the strength of ploidy as a prognostic variable. Sonicated specimens were found to have fewer aggregates, a lower percentage of cells in S-phase (%S) and G2M phase than the corresponding nonsonicated specimens. The results using MPPA predicted the prognosis better because they allowed detection of small aneuploid peaks in histograms classified as diploid or tetraploid using DNA content alone. Ploidy was a significant univariate factor, and patients with tetraploid tumors had the best survival. In the multivariate analysis, if other routine factors were examined preferentially, ploidy and %S did not provide additional prognostic information for survival. This study of paraffin-embedded breast cancers suggested that sonication and MPPA may improve the ploidy analysis in certain cases and that tetraploidy may be a favorable ploidy pattern in this group.     

Sequential determination of immunocytochemical estrogen receptor and nuclear DNA content in fine needle biopsies from breast carcinoma

The application of fine needle biopsy as a tool for early detection of breast cancer is becoming extensive, therefore parameters reported to be associated with prognosis should be standardized in this material. We propose the sequential determination of estrogen receptor (ER) status and DNA ploidy on the same smear obtained from a fine needle biopsy of a breast carcinoma, since both parameters seem to reflect properties associated with tumor behaviour and biological aggressiveness. Fifty fine-needle biopsies were investigated for presence of ER by the monoclonal antibody D75 followed by DNA content quantification using Feulgen-DNA cytophotometry. Overall, 66% of the tumors showed immunoreactivity for ER and 66% were classified as aneuploid. Forty-one percent of the aneuploid tumors were negative for ER, while only 7% of the diploid tumors showed no immunoreaction (p<0.05). The significant association between absence of immunocytochemical ER and DNA aneuploidy on the same fine-needle smear is consistent with data obtained through other methods previously reported using much larger tissue samples.     

乳腺癌细针穿刺标本中端粒酶活性的临床意义

目的　研究乳腺细针穿刺细胞标本中的端粒酶活性 ,探讨其在乳腺癌辅助诊断中的临床意义 ;研究端粒酶表达与PR、ER、PCNA、c erbB2及p5 3之间的可能关系。方法　采用PCR TRAP技术检测 99例乳腺细针穿刺标本中的端粒酶活性。采用免疫组化技术检测 35例乳腺癌组织标本中孕激素受体 (PR)、雌激素受体 (ER)、细胞增殖核抗原 (PCNA)、癌基因c erbB2及p5 3蛋白表达水平。结果　 83例乳腺癌标本 ,在 6 9例细胞学阳性标本中 6 1例端粒酶阳性 ,7例细胞学可疑标本中 5例端粒酶阳性 ,7例细胞学阴性标本中 4例端粒酶阳性 ,端粒酶敏感性为 84.3% (70 / 83) ,端粒酶活性检测和细胞学诊断总符合率为 77.1% (6 4/ 83) ;12例乳腺良性病变标本中 ,4例端粒酶阳性 ;4例乳腺炎标本均为阴性。端粒酶表达与乳腺癌组织学类型、淋巴结转移、肿瘤大小、临床分期无相关性 ,与PR、ER状态、PCNA、c erbB2 表达亦无相关性 ,但与p5 3蛋白表达水平呈负相关。结论　乳腺细针穿刺标本端粒酶活性检测在乳腺癌临床诊断中有辅助诊断价值 ;其与p5 3蛋白表达水平之间的关系有待进一步阐明。     

乳腺癌DNA倍体类型与雌激素受体

采用流式细胞计和荧光组织化学法测定了61例新鲜乳腺癌组织细胞DNA倍性及雌激素受体(ER)并分析了DNA倍体类型与ER状况及组织学分级关系。结果表明,61例乳腺癌中,35例为非整倍体癌(57.4％),其余为二倍体癌。ER阳性乳腺癌34例(55.7％)。二倍体乳腺癌多为ER阳性,非整倍体多为ER阴性,二者之间具有显著性关系(P<0.01)。另外,组织分化越差,非整倍体的发生率也越高(P<0.001),乳腺癌细胞集群中ER阳性细胞百分比与DNA非整倍体发生率呈反比关系,随ER阳性细胞比的减少,非整倍体的发生率则显著增加。本组结果提示,DNA倍性可作为估价乳腺癌预后的新指标,将它与ER结合应用,有可能进一步改善乳腺癌的内分泌治疗。     

Flow cytometrically determined DNA content of breast carcinoma and benign lesions: correlations with histopathological parameters

The relative DNA content of cellular samples from 54 patients affected by breast carcinomas and 20 affected by benign breast lesions (including 11 fibroadenomas) was measured by flow cytometry. All normal tissue samples and 17/20 (85%) specimens from benign lesions exhibited a cytometrically diploid DNA distribution, 3/20 (15%) benign lesions an abnormal DNA content, and 35/54 (65%) carcinomas at least one aneuploid cell subpopulation. Furthermore, 9/54 (17%) tumors were characterized by the presence of more than one aneuploid cell subpopulation. The results also indicate that flow cytometry can be used to recognize lymph nodes infiltrated by aneuploid cells. Statistically significant correlations were evidenced between the occurrence of aneuploidy or the ploidy level measured as DNA index and the nodal infiltration status. The percentage of S cells can also be extracted from DNA content distribution histograms. Statistically significant differences (p less than 0.01) were also observed for the percentage of S cells between normal tissues (6.2 +/- 3.2 SD) and benign lesions (11.1 +/- 6.6 SD), normal tissues (6.2 +/- 3.2 SD) and aneuploid tumors (19.7 +/- 10.3 SD), benign lesions (11.1 +/- 6.6 SD) and aneuploid tumors (19.7 +/- 10.3 SD), and diploid (7.9 +/- 4.0 SD) and aneuploid tumors (19.7 +/- 10.3 SD).     

DNA flow cytometry and prognostic factors in 1331 frozen breast cancer specimens

Breast cancer proliferative capacity as determined by the DNA thymidine labeling index, along with estrogen and progesterone receptor status, is highly predictive for risk of relapse and overall survival. Recently, DNA ploidy and proliferative capacity (S-phase fraction [SPF]) as determined by flow cytometry have also shown significant prognostic value. The authors have developed a technique which allows a 50 to 100 mg aliquot of the same frozen breast tumor specimen routinely employed in steroid receptor assays, to be assayed for both DNA ploidy and SPF by flow cytometry. Of the 1331 tumors examined, DNA histograms were evaluable for ploidy in 89% (1184) of specimens examined; 57% of these were aneuploid. Adapting a trapezoidal model to estimate SPF in both diploid and aneuploid tumors, the authors found 81% (1084) to be evaluable for SPF, with a median SPF of 5.8% for the entire population. The median SPF was significantly lower in diploid tumors (2.6%) than in aneuploid tumors (10.3%, P less than 0.0001). Both aneuploidy and high SPF were strongly associated with absence of steroid receptors. Aneuploid tumors showed more striking differences in the frequency of high S-phase values with respect to receptor status and age or menopausal status, whereas diploid but not aneuploid tumors showed lower SPF in node-negative versus node-positive patients. Because it is particularly important to identify the high-risk minority of node-negative patients, the authors examined the node-negative group separately. High SPF subgroups appeared in each category of receptor status and age or menopausal status within the node-negative group, suggesting that SPF will be an independent prognostic factor. With the DNA flow cytometric methods used here, it is now practical to determine ploidy and SPF for nearly every breast cancer patient. These factors, which show associations with established prognostic factors, such as receptor status can now be fully evaluated for their prognostic significance in broad patient populations.     

Prognostic significance of S-phase fraction in good-risk, node-negative breast cancer patients.

PURPOSE: Formalin-fixed, paraffin-embedded tissues from axillary node-negative breast cancer patients were analyzed by flow cytometry to determine the prognostic significance of DNA ploidy and S-phase fraction (SPF). PATIENTS AND METHODS: All patients were registered on a good-risk control arm of an intergroup clinical trial. They had small- to intermediate-sized (less than 3 cm), estrogen receptor (ER)-positive tumors and received no adjuvant therapy after modified radical mastectomy or total mastectomy with low axillary-node sampling. The median follow-up was 4.8 years. RESULTS: Assessable ploidy results were obtained from 92% of the 298 specimens studied (51% diploid, 49% aneuploid), and SPFs were assessable for 83% of the tumors. SPFs for diploid tumors ranged from 0.7% to 11.9% (median, 3.6%), compared with a range of 1.2% to 26.7% (median, 7.6%) for aneuploid tumors (P less than .0001). No significant differences in disease-free or overall survival were observed between patients with diploid and aneuploid tumors. Using different SPF cutoffs by ploidy status (4.4% for diploid, 7.0% for aneuploid), patients with low SPFs had significantly longer disease-free survival rates than patients with high SPFs (P = .0008). The actuarial 5-year relapse rates were 15% and 32% for patients with low (n = 142) and high SPFs (n = 105), respectively. Similar relationships between SPF and clinical outcome were observed for patients with diploid tumors (P = .053) and for patients with aneuploid tumors (P = .0012). CONCLUSION: S-phase fraction provides additional prognostic information for predicting disease-free survival for axillary node-negative breast cancer patients with small, ER-positive tumors.     

Correlation of pretreatment proliferative activity of breast cancer with the response to cytotoxic chemotherapy

That most cytotoxic agents act specifically against actively proliferating cells is well-recognized. In this study, we attempted to correlate pretreatment S-phase fractions (SPF) measured on DNA histograms with regression of the tumor mass after the administration of neoadjuvant chemotherapy. Tumor cells were obtained from 60 previously untreated, premenopausal patients with no metastases and with noninflammatory disease by fine needle sampling without aspiration. We could evaluate DNA ploidy in all patients and SPF in 50 or 83% of them. Tumor responsiveness was significantly related to SPF. The 12 patients who had SPF of 10% or more showed demonstrable regression; six had complete responses. None of the other parameters tested, i.e., DNA ploidy, histopathologic grade, or hormone receptor content, correlated with response. We believe this information may prove valuable for clinicians as they make their decisions regarding patient therapy.     

An immunohistological study of gastric cancer--with special reference to the expression of carbohydrate antigens and nuclear DNA ploidy patterns

The expression of carbohydrate antigens specific to Span-1, CA 19.9, and SLEX in cancer tissues and nuclear DNA ploidy patterns were studied from tissue specimens of lesions excised from 137 patients with gastric cancer. The frequency of detection of each antigen was augmented with advanced invasion depth and progress in regional lymph node metastasis. In the cases which were positive for all three kinds of antigens, lymph node metastasis and lymphatic or venous invasion were detected with significantly higher frequencies than in the negative cases. The DNA histograms showed a DNA ploidy pattern of Type Ia or Ib in the cases negative for these antigens and a non-diploid or aneuploid pattern of Type II or III in many of the positive cases. These findings suggest that an immunohistological study of gastric cancer using monoclonal antibodies, combined with a nuclear DNA ploidy analysis, might be useful for understanding malignancy of the tumour.     

Breast cancer proliferation measured on cytological samples: a study by flow cytometry of S-phase fractions and BrdU incorporation

Cell kinetics have been shown to be an important predictor of clinical evolution of operated breast cancer. We established a method for the estimation of the proliferative activity of tumour cells obtained by fine needle sampling without aspiration (FNS), using simultaneously S-phase fractions (SPF) measured on DNA histograms and 5-bromodeoxyuridine (BrdU) labelling index (BLI) measured by flow cytometry. Biparametric BrdU/DNA flow cytometry could be performed in 122 of 189 (65%) consecutive patients. The mean BLI of the cytologically malignant FNS (118) was of 3.0 and the median of 2.2%. One hundred and forty-eight DNA histograms (78%) were suitable for SPF analysis, of which 141 presented malignant cells, showing a mean of 4.5 and a median of 3.5%, comparable to BLIs. These results were obtained from fluorescence peak area histograms with doublet discrimination and background subtraction allowing the measurements of SPFs as low as 0.4%. An excellent correlation was thus observed between BLIs and SPFs, for the 94 cases for which both results were available (r = 0.85). Infrequent discordances (9%) were noted with SPFs considerably higher than BLIs. Seven patients had three consecutive FNS of their tumour at weekly intervals before treatment. Some variability in the proportions of multiple subpopulations of tumour cells was observed on the DNA histograms. In contrast, proliferation indices (SPF or BLI) were reproducible, suggesting homogeneous growth rates. We conclude that an estimation of the proliferative activity of breast tumours at any stage of the disease is possible routinely by SPF and/or BLI analysis of FNS. At least one quantitative proliferation index could be obtained for 91% of patients.     

A comparison of static cytofluorometry and flow cytometry for the estimation of ploidy and DNA replication in human breast cancer

Summary 332 primary invasive breast carcinomas were analysed by static cytofluorometry and flow cytometry. The ploidy distributions were similar, and 54% of the tumors were judged DNA aneuploid by both methods.The coefficient of variation of the G₀–G₁ peaks ranged from 2.0 to 8% with both techniques, but the mean was somewhat lower with flow cytometry — 4.1%, compared to 4.9% for the static measurements.The proportion of S-phase cells was possible to estimate from 80% of the flow histograms and 70% of the static histograms. S-phase was not estimated from the static histograms if less than 150 tumor cells were measured. With 160 tumors S-phase was measured by both methods. The range was 0 to 27% with the static measurements and 0.7 to 25% with flow cytometry. Corresponding mean values were 7.6% and 8.2%, which are similar to thymidine labeling index results with breast cancers reported in some studies. A close correlation was obtained (r = 0.927) comparing S-phase fractions estimated from aneuploid tumors with flow cytometry and static cytofluorometry if more than 200 cells were measured with the latter. The proportion of S-phase cells was significantly lower for the diploid tumors.We conclude that both techniques can be useful for the estimation of DNA ploidy and replication in human breast cancer.     

Ploidy in invasive colorectal cancer. Implications for metastatic disease

A retrospective study was performed to determine the ploidy of superficial (above muscularis propria) and deep (below muscularis propria) biopsy specimens from the primary colorectal cancer of 88 patients with Dukes Stage C2 and D colorectal tumors. The ploidy of lymph node and liver metastases was compared with that of the superficial and deep specimen from the corresponding primary tumor. Among the tumors studied, 78% exhibited nondiploid stemlines. In 19% of the tumors, the ploidy of the superficial biopsy differed from that of the deep biopsy. Among these discordant tumors, all of the deep biopsy specimens corresponded in ploidy to the liver metastases, whereas most of the superficial specimens were similar in ploidy to the lymph node metastases. Our observations suggest that measurement of a single site may not be sufficient to detect nondiploid stemlines within a tumor. They also suggest that measurement of deeper parts of invasive tumors may be more reflective of the phenotype of distant metastases than measurement of superficial specimens.     

DNA ploidy in intraductal breast carcinomas

Cellular DNA-ploidy in 74 clinically detected intraductal breast carcinomas (IDCs) was analysed by flow cytometry. The histograms were classified as either diploid or aneuploid, and the DNA ploidy pattern compared with that of invasive breast carcinomas and normal breast tissue. All normal breast tissues were diploid while 28 (38%) of the IDCs were aneuploid, the DNA indices ranging from 1.32 to 2.00. The frequency of aneuploidy in invasive ductal carcinomas (73%) was significantly higher (P = 0.003), DNA index ranging from 1.34 to 2.92, compared with that in IDCs. Retrospectively, 14.5% of the patients had invasive breast cancer 16–166 months after the diagnosis of IDC. Neither DNA ploidy nor histopathological classification alone predicted clinical outcome, but patients with DNA diploid non-comedo IDC had a more favourable course.     

乳腺癌组织中bcl-2蛋白表达及其与DNA倍体的关系

目的：探讨乳腺癌中bcl-2蛋白表达及DNA倍性与患者临床病理因素的关系，方法：采用流式细胞光度术(FCM)定量分析63例原发性乳腺癌癌组织、相应癌旁组织和22例乳腺良性肿瘤中bcl-2蛋白表达及DNA倍性结果：乳腺癌组织中bcl-2蛋白表达水平及DNA指数(DI)、S期比例(SPF)和增殖指数(PI)显著高于相应癌旁组织和乳腺艮性肿瘤(P＜0．05)，而后两者在上述指标上未见显著性差异(P＞0．05)。浸润性非特殊类型癌患者DI、SPF和Pl明显高于早期浸润癌患者和浸润性特殊类型癌患者(P＜0．05)；有淋巴结转移组患者DI和SPF显著高于无淋巴结转移组患者(P＜0．05)bcl-2蛋白表达与患者年龄、病理组织学类型、肿瘤大小、淋巴结转移、临床分期均无关(P＞0．05)癌组织bcl-2蛋白表达水平与SPF和PI呈负相关(P＜0．05)结论：bcl-2蛋白表达与乳腺癌发生、发展有关：DI和SPF与淋巴结转移有关；DNA含量可作为判断乳腺癌预后的重要指标     

Pyrimidine nucleoside phosphorylase-activity and biological characteristics of breast-cancer

We investigated whether Pyrimidine nucleoside phosphorylase (PyNPase) activity in breast cancer tissue correlated with biological characteristics of breast cancer. PyNPase activity, ER, PgR, EGFR, DNA ploidy pattern, PCNA positive cells and amplification of the c-erbB-2 gene were determined in specimens from 138 patients. PyNPase activity was significantly higher in ER negative than ER positive carcinomas (p<0.05), in PgR negative than PgR positive carcinomas (p<0.05) and significantly higher in tumors with c-erbB-2 gene amplification compared with tumors with no amplification (p<0.05). The results suggest that PyNPase activity in breast cancer tissue may be a new biological characteristic of breast cancer.     

DNA analysis by flow cytometry in breast carcinoma

Using deparaffinized tumor specimens, a flow cytometric DNA analysis was performed in 63 primary breast cancers from patients who were treated from 1976 through 1982. Fourty-three percent of the cases were found to be aneuploid, and 57% were diploid. No trend to increased aneuploid proportion with a more advanced disease was noted. There was no correlation between the level of ploidy and the clinicopathological factors. Aneuploid tumors seem to have a poorer survival, but no significant difference was found between the two patterns of ploidy. Patients with a low S-phase fraction (SPF) had a significantly longer survival than those with a high SPF. A DNA analysis of the tumor cells in breast cancer is felt to be use for a prognostic evaluation.     

Mutations of the D310 mitochondrial mononucleotide repeat in primary tumors and cytological specimens

A mononucleotide repeat (D310) in mitochondrial DNA has been recently identified as a mutational hot spot in primary tumors. We analyzed 56 tumors for insertion/deletion mutations in the D310 repeat. A total of 13 mutations were detected. The highest frequency of mutations was found for cervical cancer, followed by bladder tumors, breast cancer and endometrial neoplasia. No alterations were observed in four patients suspected of malignancy but without evidence of malignant tumor. We detected identical changes in four of four urine sediments from patients with bladder cancer and in three of three fine needle aspirates of patients with breast cancer. Our results indicate that D310 abnormalities are detectable in cytology specimens from patients with cancer and support the notion that D310 analysis may represent a new molecular tool for cancer detection.     

超声引导针吸活检在术前判断乳腺癌腋窝淋巴结状态中的应用

目的：研究对超声异常的腋窝淋巴结进行针吸活检的临床价值.方法：对47例cT1-2N0M0腋窝超声异常乳腺癌患者的腋窝淋巴结进行超声引导下针吸活检,结果与组织学病理结果进行对照.分析超声引导针吸活检术前判断乳腺癌腋窝淋巴结转移的敏感性、特异性、阳性预测值、阴性预测值和诊断准确率.结果：超声引导针吸活检判断腋窝淋巴结转移的敏感性、特异性、阳性预测值、阴性预测值和诊断准确率分别为82.8%、100.0%、100.0%、78.3%、89.4%.结论：超声引导针吸活检是术前评估腋窝淋巴结状态的有效检查方法,其结果对乳腺癌手术方式的选择具有重要参考价值.     

A comparative study of DNA ploidy in 115 fresh-frozen breast carcinomas by image analysis versus flow cytometry.

BACKGROUND. Qualitative and quantitative analysis of cellular DNA content may be clinically useful in the prognostic evaluation of certain types of malignant tumors, including breast carcinoma. Flow cytometric (FCM) analysis has been the most frequently used procedure for DNA analysis, but it requires a reasonably large tissue sample. Computer-based image analysis (IA) now allows imprint, cytospin, and needle aspiration smear preparations and other small tissue samples to be used. METHODS. To resolve concern about the diagnostic efficacy of small tissue samples in the use of IA, the authors performed a comparative study of FCM analysis and IA using 115 fresh-frozen breast carcinomas. Feulgen-stained imprint preparations for IA and single-cell suspensions from the same fresh-frozen tissue for FCM analysis were used, and the respective histograms were compared. RESULTS. The results were concordant in 90.4% (104 of 115) of the cases, but 11 specimens yielded discordant data. IA provided histograms with a somewhat lower resolution and a relatively high coefficient of variation for the G0/G1 peak, thus rendering occasional tumors, which were near-diploid aneuploid by FCM analysis (four cases), not amenable to diagnosis by aneuploid characterization. In three additional cases, FCM analysis showed aneuploid hyperdiploid (two cases) and multiploid (one case) histograms, but IA only demonstrated a diploid peak. Conversely, in four other cases, aneuploid peaks were recognized only by IA. CONCLUSIONS. Computerized IA has significant advantages over FCM analysis, including lower cost, the ability to analyze very small specimens, the capability of detecting rare high ploidy cells, the capacity to classify cellular populations according to specific morphologic type, and the fact that no destructive enzyme or chemical digestion is required for specimen preparation, thereby preserving the integrity of fragile cells.