Evaluation of prognostic factors following flow-cytometric DNA analysis after cytokeratin labelling: I. Breast cancer.

In gynecologic oncology valid prognostic factors are necessary to estimate the course of disease and to define biologically similar subgroups for analysis of therapeutic efficacy. The presented study is a prospective study concerning prognostic significance of DNA ploidy and S-phase fraction in breast cancer following enrichment of tumor cells by cytokeratin labelling. Epithelial cells were labeled by FITC-conjugated cytokeratin antibody (CK 5, 6, 8, and CK 17) prior to flow cytometric cell cycle analysis in 327 fresh specimens of primary breast cancer. Univariate analysis in breast cancer detected the prognostic significance of DNA-ploidy, S-phase fraction and CV (coefficient of variation) of G(0)G(1)-peak of tumor cells for clinical outcome, especially for nodal-negative patients. Multivariate analysis could not confirm prognostic evidence of DNA-ploidy and S-phase fraction.In conclusion, in breast cancer no clinical significance for determination of DNA-parameters was found.     

Evaluation of prognostic factors following flow-cytometric DNA analysis after cytokeratin labelling: II. Cervical and endometrial cancer.

In gynecologic oncology valid prognostic factors are necessary to define biologically similar subgroups for analysis of therapeutic efficacy. This study is the first published prospective study concerning prognostic significance of DNA ploidy and S-phase fraction in cervical and endometrial cancer following enrichment of tumor cells by cytokeratin labelling. Epithelial cells were labeled by FITC-conjugated cytokeratin antibody (CK 5, 6, 8, and CK 17) prior to flow cytometric cell cycle analysis in 91 specimens of cervical cancer and 73 samples of endometrial cancer. In cervical cancer neither DNA-ploidy nor S-phase fraction were relevant prognostic parameters. But CV of the G(0)G(1)-peak showed prognostic relevance in cervical cancer cells, even in multivariate analysis. This interesting observation, however, seems to have no therapeutic consequence due to the small discrimination capacity of CV. In endometrial carcinoma, gross DNA-aneuploidy (DNA-index > 1.3) and a high percentage of proliferating cells (>75th percentile) were univariate and multivariate highly significant prognostic factors for recurrence-free survival. Especially DNA-aneuploidy (DI>1.3) is one of the most important independent molecular biological prognostic factors. While diagnostic curettage we could identify risk patients even preoperatively by determination of the prognostic factors like histologic tumor type, grading, cervical involvement and DNA-ploidy. Thereby these patients could be treated primarily in an oncologic center. In conclusion, our investigations showed that the determination of DNA-ploidy should be done in endometrial carcinoma. In cervical cancer no clinical significance for determination of DNA-parameters was found.     

Cytokeratin analysis of breast and leukemia tumor cell lines by flow cytometry

Using four human tumor cell lines, MCF-7 and T47-D from breast tumors, MOLT-4 and K-562 from leukemia, flow cytometric DNA analysis of pure and mixed cell population was performed using monoclonal antibodies to cytokeratin to distinguish cytokeratin-containing carcinoma cells from leukemia cells which do not contain cytokeratins. Surprisingly, on pure or mixed K-562 cells, we found positive labeling with KL1, CK8, and CK18 antibodies (results confirmed by immunocytology). This preliminary study has allowed a DNA analysis on epithelial cells of human breast tumors.     

Flow cytometric analysis of DNA content and keratins by using CK7, CK8, CK18, CK19, and KL1 monoclonal antibodies in benign and malignant human breast tumors.

We have used a double-labelling flow cytometry analysis of keratin (CK) and DNA in breast cancer. Five monoclonal anti-keratin antibodies were tested: KL1 recognizing Mr 55,000-57,000 keratins, and "anti-glandular epithelia," LE41, RGE-53, and LP2K specific for CK n. 7, 8, 18, and 19 of Moll's classification, respectively. Flow cytometric (DNA-CK) analysis was performed on 10 benign and 19 malignant human breast tumors. All the benign tumors were diploid and 63% of the malignant tumors were aneuploid. This technique permits the analysis of DNA in the epithelial fraction alone. In aneuploid tumors, gating the DNA-keratin-positive population allowed accurate DNA analysis without interference due to debris background and non-epithelial cells. Moreover, double-labelling using the CK19 antibody gave a better identification of near-diploid tumors. An enhancement of keratin expression in malignant tumors was observed with CK 19 (P less than 0.001), KL1 (P less than 0.01), CK 8 (P less than 0.05), and CK18 (n.s.) compared to benign tumors. The comparison of keratin expression in aneuploid and diploid malignant tumors revealed reduced CK8, CK18, and CK19 in the former.     

Flow cytometric bivariate analysis of DNA and cytokeratin in colorectal cancer.

Different opinions about flow cytometric estimates of DNA aneuploidy and/or S-phase fraction (SPF) as supplementary prognostic markers in colorectal cancer are to some degree associated with methodology. Using univariate DNA analysis, we have previously investigated the DNA ploidy in colorectal cancer, its heterogeneity within and between tumors and its relation to survival. To improve detection of DNA aneuploid subpopulations and particularly estimation of their SPF's we investigated a method for bivariate DNA/cytokeratin analysis on fine-needle aspirates of 728 frozen biopsies from 157 colorectal tumors. Unfixed aspirates were stained with propidium iodide and FITC-conjugated anti-cytokeratin antibody in a saponin-buffer. A significant association between SPF and debris was observed. There were no substantial difference in DNA ploidy patterns between univariate and bivariate measurements (concordance was 92-95%). No new DNA aneuploid subpopulations were detected in cytokeratin-gated compared to ungated or univariate histograms. Debris-adjusted SPF's of cytokeratin-gated histograms were significantly higher than of ungated histograms, also for subpopulations with DI>1.4 (p<0.0001). There was no significant association between SPF and survival.     

Use of a mechanical dissociation device to improve standardization of flow cytometric cytokeratin DNA measurements of colon carcinomas.

In order to standardize dual-fluorescence DNA flow cytometry using cytokeratin (CK) antibodies, normal colonic mucosa and tumor tissue were sampled from 308 colorectal surgical specimens. Fresh colon specimens were processed directly and stored frozen until dissociation. The samples were divided into aliquots for manual dissociation with tweezers and scalpel, and parallel dissociation with an automated disaggregation device (Medimachine, DAKO Diagnostika GmbH, Hamburg, Germany). An indirect immunofluorescence method with anti-cytokeratin antibodies and propidiumiodide was applied and measured on a single-laser flow cytometer (FACScan, Becton Dickinson [BDI], Heidelberg, Germany). Evaluation with CellFit (BDI) or MultiPlus (Phoenix Flow Systems, San Diego, CA) showed that dual-parameter fluorescence propidiumiodide (DNA staining) and fluorescein-isothiocyanate (cytokeratin labeling) provides a reasonable staining method for DNA analysis of epithelial cells. No significant differences in coefficient of variation in CK-gated versus ungated cells could be observed. Normal colon mucosa served as a reliable internal, diploid DNA control. Medimachine dissociation led to a significantly higher gain of cytokeratin-positive cells compared to percentage of cytokeratin-positive cells after manual tissue disaggregation. Cytokeratin gating led to a clear-cut separation of S-phase fractions within the respective ploidy groups, irrespective of manual or automated dissociation. The S-phase fraction increased significantly from normal tissue to diploid and nondiploid tumors. In general, automated tissue preparation with the Medimachine allows simple cell-isolation for dual DNA/CK-flow cytometric measurement, improving the gain of CK-positive cells, and facilitating a standardized DNA analysis.     

Application of antibodies to intermediate filament proteins as tissue-specific probes in the flow cytometric analysis of complex tumors

The flow cytometric (FCM) analysis of carcinomas is often hampered by the presence of stromal and inflammatory cells in the cell suspensions obtained from such neoplasms. Therefore, an FCM method was developed to distinguish epithelial from nonepithelial cells by using polyclonal and monoclonal antibodies to (cyto)keratins, the epithelial type of intermediate filament proteins. Using a model system of cultured bladder carcinoma (T24) and leukemia (MOLT-4) cells, we tested our hypothesis and procedures by labeling cell mixtures with these antibodies. After incubation with an appropriate intermediate filament antibody and propidium iodide staining, the DNA content and distribution of T24 cells could be analyzed separately from MOLT-4 cells. When applied to cell suspensions of endometrial carcinomas, bladder carcinomas and Grawitz tumors, only the epithelial (primarily carcinoma) cells were stained for cytokeratin; these cells could thus be analyzed separately from stromal, inflammatory and other nonepithelial cells. In this way, a more accurate FCM analysis of the malignant fraction within a tumor can be achieved.     

Two-color multiparametric method for flow cytometric DNA analysis of carcinomas using staining for cytokeratin and leukocyte-common antigen

Solid tumors contain heterogenous cell populations, resulting in flow cytometric (FCM) DNA quantitations of a mixture of tumor and host cells. Such mixed populations can result in dilution of the tumor cells by the host cells, in difficulty defining the diploid reference mean and in histogram peak overlap, precluding cell-cycle analysis. In this study, epithelial (tumor) cells and contaminating host cells in 100 consecutively accessioned human mammary and colorectal carcinomas were segregated in a multiparametric two-color FCM DNA analysis of intact, ethanol-fixed cells. These two carcinomas and bladder carcinomas contain a cytoskeleton of simple epithelium that is selectively stained with an FITC-labeled monoclonal antibody (MAb) to cytokeratin (CK: CAM 5.2-FITC). This MAb detects the CK 8, CK 18 and CK 19 consistently present in all layers of normal and neoplastic urothelium, colonic epithelium and mammary epithelium. Gating on CK in these tumors enables the nonstaining leukocytes, stromal fibroblasts and endothelial cells to be excluded from DNA analysis. A separate aliquot of each tumor evaluated was labeled with an MAb to leukocyte-common antigen (LCA-FITC) to serve as a patient-specific intrinsic diploid reference standard. Both the CK-labeled and LCA-labeled cells were then dual labeled for DNA with propidium iodide. This method (1) correctly identified the intrinsic diploid (LCA-positive) channel, allowing an accurate definition of normal cell DNA content for calculation of the DNA index; and (2) resulted in an increased sensitivity in the identification of both diploid and abnormal hyperdiploid tumor cell populations. It also (3) limited DNA cell cycle analysis to urothelial, colonic and mammary epithelial cells, the majority of which were neoplastic in carefully selected tumor samples. In addition, this method (4) clarified near-tetraploid populations that overlap the normal nonepithelial G2M region by diminishing the normal G2M peak and accentuating the aneuploid tetraploid G0G1 peak and (5) deconvoluted overlapping histograms composed of normal host and diploid-range or aneuploid tumor cells by gating on tissue-specific markers. This exclusion of host cells in both classes of tumors resulted in more accurate cell-cycle calculations in the former and allowed calculation of the S-phase fractions in the latter.     

Endometrial aspiration cytology for diagnosis of peritoneal lesions in extrauterine malignancies

OBJECTIVE: To evaluate the usefulness of endometrial aspiration cytology for assessing malignant cells of extrauterine origin. STUDY DESIGN: Endometrial cytology was performed on 224 patients with primary ovarian cancer, 10 with fallopian tube cancer and 45 with peritoneal tumors. RESULTS: Of 224 patients with ovarian cancer, 53 (23.7%) had positive endometrial cytology. Positive rates were: stage I, 4.3%; stage II, 25.0%; stage III, 39.7%; stage IV, 34.5%. Histologic positive rates were: serous, 28.7%; mucinous, 11.4%; clear cell, 23.1%; endometrioid and unclassifiable adenocarcinomas, 28.0%. Of 5 patients with ovarian cancer, 2 were asymptomatic, but aspiration cytology was positive. Of 10 patients with fallopian tube cancer, 9 (90.0%) had positive endometrial cytology. The positive rate on endometrial cytology was 56.7% in stomach cancer, 60.0% in breast cancer and 20.0% in colon cancer. Of 1,209 women with stomach cancer, 30 (2.4%) displayed ovarian metastasis. Of these, 7 (23.3%) had Krukenberg's tumor; endometrial cytology was positive in 1 (14.3%). In 7 of 17 patients with positive endometrial cytology, clinical diagnosis was made before stomach cancer therapy. CONCLUSION: Endometrial aspiration cytology is useful for identifying nongynecologic malignant cells, diagnosing ovarian and fallopian tube cancers, and determining peritoneal dissemination and metastasis originating from gastrointestinal and breast cancers.     

Comparison of fresh and paraffin-embedded tissue as starting material for DNA flow cytometry and evaluation of intratumor heterogeneity

Flow cytometric analysis of DNA ploidy and S-phase fraction (SPF) from paraffin-embedded tumors has become an important diagnostic and prognostic tool in clinical pathology and investigative oncology. The present study aimed at elucidating the reliability of the method. About 90% of the 1,400 paraffin-embedded tumors analyzed were evaluable for DNA index and about 70% for SPF, although considerable differences between various tumor types were detected. The within-assay coefficients of variation for determination of tumor DNA index and SPF were 2% and 6%, respectively. Intratumor variation in DNA index was observed in 24% of breast and in 21% of ovarian carcinomas and variation in SPF in 36% and 29% of the respective tumors. Intratumor variation in SPF was greatest in DNA-diploid tumors, which may indicate that SPF values in these tumors may be less reliable owing to variations in the proportions of tumor and nontumor cells. If the methodological variation and the intratumor variation were taken into account, there was a good correlation between DNA indices (r = 0.980) and between SPF values (r = 0.794) obtained from fresh and paraffin-embedded tumors. It is concluded that accurate determination of DNA index and SPF from paraffin-embedded tumors is possible in the majority of cases. Regardless of the type of starting material used for DNA flow cytometry it is advantageous to study several samples from each tumor to account for the intratumor heterogeneity.     

Effect of sonication on paraffin-embedded tissue preparation for DNA flow cytometry

Since the publication of paraffin block extraction procedures, flow cytometric analysis of DNA ploidy and S-phase of tumor specimens has been widely applied. DNA aneuploidy, DNA tetraploid (elevated G2/M), and elevated S-phase are clinically significant in some tumor systems. True DNA tetraploid cell lines will contain a large 4c population and perhaps an 8c population; samples with cell aggregates will also contain a 6c population. Microscopic examination of samples having a 6c peak revealed nuclei with adhering debris and doublets, triplets, and larger nuclear aggregates. After sonication, a uniform suspension of single nuclei without adherent debris was seen. In addition to reducing the percent of G2/M cells, sonication also reduced S-phase percent such that it was closer to the bromodeoxyuridine labeling index. The DNA ploidy classification of specimens was also compared pre- and post-sonication. Four of 96 breast cancer samples changed classification; all were specimens in which the histogram became cleaner and a small DNA aneuploid peak became apparent after sonication.     

Cytokinetic analysis of lung cancer by bromodeoxyuridine labeling of cytology specimens

Recent flow cytometric (FCM) studies have indicated the prognostic value of S-phase cells (SPF) in lung cancer. More refined cytokinetic analysis can be obtained by dual-parameter FCM, labeling S-phase cells with 5-bromodeoxyuridine (BrdUrd), which can be detected using a monoclonal antibody (MoAb) to BrdUrd. Tumor cells obtained through bronchoscopic brush were incubated for 1 hr in RPMI 1640 medium with 10% fetal calf serum and 10 microM BrdUrd. After fixation in ethanol, pepsin treatment, and DNA denaturation, the nuclei were stained with anti-BrdUrd MoAb and propidium iodide. From 14 of 20 patients, sufficient material was obtained (three adenocarcinoma and seven squamous cell, one giant cell, and three small cell carcinoma). The measured SPF ranged from 5.2% to 26%. The labeling index (LI), calculated as the ratio of the number of BrdUrd-labeled cells to the total number of aneuploid cells, or diploid cells in the case of a diploid tumor, ranged from 1.2% to 16.7%; LI and SPF correlated significantly (r = 0.69). In this study, we have demonstrated the feasibility of determining the actively DNA-synthesizing cells on brush material from lung cancer cells. In addition, some extra information can be obtained about the SPF population, including the fraction of unlabeled SPF, which could be of prognostic significance.     

Dilution effect of nontumor cells on flow cytometric DNA S-phase fraction in breast cancer fine needle aspirates

OBJECTIVE: To analyze the proportion of nontumor cells in fine needle aspirates of breast carcinoma and its influence on flow cytometric S-phase fraction (SPF) estimation. STUDY DESIGN: We analyzed the proportion of nontumor cells in fine needle aspiration biopsy smears, performed flow cytometric analysis of DNA ploidy and SPF on freshly aspirated tumor material and analyzed histograms manually and automatically using Multi-Cycle AV software (Phoenix Flow Systems, San Diego, California, U.S.A.) for cell cycle analysis. We corrected SPF of diploid tumors for the dilution effect using an individually established percentage of nontumor cells (individual correction) and the mean proportion of nontumor cells in diploid tumors (factor correction). RESULTS: The proportion of nontumor cells ranged from 0.5% to 76.6% (mean, 12.6; SD, 15.7) in 55 diploid tumors and from 0.5% to 53% (mean, 8.6; SD, 8.9) in 84 aneuploid tumors (p=0.178). In 14 of 139 (10%) samples, the proportion of nontumor cells exceeded 20%. The mean SPF values of diploid tumors without correction were 4.9% (manually) and 6.5% (automatically) and of aneuploid tumors, 9.5% and 11.0%, respectively. In univariate Cox survival analysis, noncorrected SPF was a significant prognostic factor in overall survival (p < 0.001). Neither individual nor factor correction of SPF significantly changed its prognostic value. CONCLUSION: Fine needle aspirates contain low proportions of nontumor cells, having an insignificant dilution effect on SPF estimation. Most probably, SPF could be reliably estimated usingfreshly aspirated tumor material without any correction or adjustment.     

The reliability of microspectrophotometric and flow cytometric nuclear DNA measurements in adenocarcinomas of the breast

The reliability of microspectrophotometric (MSP) and flow cytometric (FCM) nuclear DNA measurements has been studied in 50 human breast adenocarcinomas. The tumor material was obtained by means of fine-needle aspiration biopsy, and all samples except one were found to be highly representative. The results confirm earlier observations that a good correlation exists between modal value (MV) determined by MSP and DNA index (DI) determined by FCM. However, when tumors were classified into low and high malignant variants according to FCM/DI, FCM/S-phase percentages, and MSP histogram types, the concordance was less pronounced. This was found to be due mainly to the fact that in near-diploid tumors a discrepancy exists between MSP and FCM ploidy, as well as between MSP distribution pattern and the estimated percentages of cells in the S-phase region. Another source of discrepancy was observed in tumors with stemlines in the normal tetraploid region, including cells with highly scattered aneuploid DNA values. These tumors were judged by MSP as aneuploid/high malignant and by FCM as euploid/low malignant. In view of this discrepancy, we conclude that the simple determination of the stemline position by MSP/MV or FCM/DI is not sufficient for adequate cytochemical malignancy grading of breast carcinomas. We suggest that a combination of ploidy and percentage of cells scattered outside the modal peaks is a more sensitive method for optimal cytochemical malignancy grading in breast carcinomas.     

Multiparametric evaluation of flow cytometric synthesis phase fraction determination in dual-labelled breast carcinomas.

Multiparametric, two-color DNA and cell cycle analyses were performed on 112 consecutive mechanically dissociated, ethanol-fixed breast carcinomas using a dual-label method with monoclonal antibodies (CAM 5.2) to cytokeratin (CK) and leukocyte common antigen (LCA) with propidium iodide (PI) staining. There was marked intertumoral variation of CK-positive (range, 3-87%; mean, 40%) and LCA-positive (range, 1-28%; mean, 6.5%) events in DNA histograms. Approximately 70% of DNA aneuploid cells were CK positive. CAM 5.2-stained (avidin-biotin technique) Cytospin preparations correlated with flow cytometric (FCM) detection of CK-positive cells in 15/21 (71%) cases. In each discrepant case, FCM detected greater numbers of CK-positive cells. Cytospin controls of tumor suspensions revealed that cytoplasmic loss was the major cause of decreased CK staining. Synthesis phase fraction (SPF) calculation from CK-gated histograms resulted in kinetic indices (mean ungated, 12.3%, vs. mean CK-gated, 16.8%; P less than .01) with improved statistical correlations with tumor grade and estrogen receptor (ER) status. Differences between ungated vs. CK-gated SPF were greatest in cases having less than 20% CK-positive events (P less than .05). Cases with lower CK staining events generally had higher SPF and were more often high grade (below median CK staining, 61% high grade, vs. above median CK staining, 31% high grade) and ER-negative (below median CK staining, 55% ER negative, vs. above median CK staining, 12% ER negative).(ABSTRACT TRUNCATED AT 250 WORDS)     

Bivariate flow cytometric analysis of murine intestinal epithelial cells for cytokinetic studies

The heterogeneous nature of the small intestine and the lack of methods to obtain pure crypt populations has, in the past, limited the application of standard flow cytometric analysis for cytokinetic studies of the proliferating crypts. We describe a flow cytometric technique to discriminate crypt and villus cells in an epithelial cell suspension on the basis of cell length, and to measure the DNA content of the discriminated subpopulations. Our data indicate that bivariate analysis of a mixed epithelial cell suspension can be used to distinguish mature villus cells, G1 crypt cells, and S-phase crypt cells. In addition, continuous labeling studies suggest that the position of a cell on the cell length axis reflects epithelial cell maturity. We applied this flow cytometric technique to determine the cytokinetic nature of epithelial cells obtained by sequential digestion of the small intestine.     

Flow cytometry DNA analysis on tumor cell subpopulation of human tumor specimens by exclusion of lymphohemopoietic cells

We describe a new flow cytometry procedure in which DNA analyses can be obtained selectively on pure, freshly obtained tumor cell subpopulations of human tumor specimens. This procedure is based on exclusion from analysis of the contaminating lymphohemopoietic cells mixed with tumor cells in tumor specimens. This exclusion is made possible by labeling all lymphohemopoietic cells with an antibody to HLe-1 (HLE), which is present on all lymphohemopoietic cells but on no other cells, and by gating against these labeled cells when analyzing for DNA. For the model system, a 1:1 mixture of normal human peripheral blood leukocytes and either of two human cancer cell lines, HEp-2 and MCF-7, normal leukocyte contamination can be reduced to 3.1% while retaining 94.7% of tumor cells for DNA analysis. Four examples of human tumor samples, two cases each of malignant effusions and lymph node metastases, were analyzed with this procedure. The results clearly indicate that this new method will improve ploidy analysis/aneuploidy detection and will make it possible to obtain more accurate cell-cycle analyses of tumor cells than have previously been possible. This new procedure will contribute to clinical and biological studies involving DNA of human tumors.     

Improved prognostic impact of S-phase values from paraffin-embedded breast and prostate carcinomas after correcting for nuclear slicing

Nuclear debris may significantly interfere with the analysis of S-phase fraction (SPF) from paraffin-embedded tumors. We used a background subtraction algorithm to compensate for the effects of slicing of tumor cell nuclei during preparation of paraffin-embedded specimens. DNA histograms were analyzed from 88 node-negative breast and from 78 prostatic carcinomas. Median SPFs corrected for nuclear slicing were lower than uncorrected ones in both breast cancer (7.6% vs. 5.7%) and prostate cancer (6.7% vs. 4.2%). The median SPF value in each group was used as a cut-off point in survival studies. As compared with the uncorrected SPFs, corrected SPF levels resulted in a more significant survival difference between breast cancer patients with above and below median SPF (p = 0.0014 vs. p = 0.014) and in a higher relative risk (RR) of death (4.5 vs. 3.1). The same was true for prostate cancer survival (p less than 0.0001 vs. p = 0.002) and RR (5.3 vs. 3.1). Compared with the exponential background subtraction method, the sliced nuclei correction was more reproducible and could be applied in all evaluable histograms without the risk of overcompensation. In conclusion, our results support the use of background correction with the sliced nuclei model in DNA flow cytometric studies of archival tissues.     

循环肿瘤细胞在女性实体器官肿瘤中的研究进展

循环肿瘤细胞(circulating tumor cells,CTCs)指的是从实体的肿瘤或转移的病灶进入外周血液循环的恶性肿瘤细胞。自发现以来,随着其检验技术日趋成熟,循环肿瘤细胞(CTCs)日渐成为肿瘤学炙手可热的研究对象。因为它将通过外周血的检验来实现监测肿瘤的发生、发展、转移、复发等情况,相对于肿瘤实体活检,"液体活检"不仅让患者易于接受,更有利于医务工作者监测病情变化。本文综述了循环肿瘤细胞(CTCs)的检测方法并综述了循环肿瘤细胞在女性实体肿瘤--乳腺癌、卵巢癌、宫颈癌、子宫内膜癌中的研究进展。其中着重介绍了其在早期乳腺癌及复发转移性乳腺癌中的重大意义以及在评价治疗效果中的分子学特征。实践表明,循环肿瘤细胞(CTCs)与HE-4、CA125的联合应用在评估卵巢癌化疗敏感性中也具有重要的临床意义。