DNA analysis in the differential diagnosis of osteosarcoma

The DNA content of osteosarcomas, and of giant cell tumors, osteoblastomas, aneurysmal bone cysts, and fibrous dysplasias was determined by cytophotometry. Of 158 tumors, 141 were histologically noncontroversial, whereas 17 posed diagnostic difficulties. In the noncontroversial group, all 41 benign tumors had a diploid (normal) DNA content. Ninety-two of 96 high-grade osteosarcomas were hyperploid (increased DNA content). The four analyzed low-grade parosteal osteosarcomas were diploid. Among 17 diagnostically controversial cases, nine were primarily diagnosed and treated as benign. Three of these patients, nevertheless, exhibited a malignant clinical course and two had local recurrence. All five proved to have hyperploid tumors. The four nonrecurrent lesions were diploid. Of eight patients primarily evaluated as malignant, one died and two developed local recurrence. These three patients had hyperploid tumors. Among the five nonrecurrent lesions, two were hyperploid and three diploid. In the diagnostically controversial group, recurrence or death was consistently related to hyperploidy. The present study shows that the vast majority of high-grade osteosarcomas are hyperploid. Benign bone tumors, which may be mixed up histologically with osteosarcoma, are diploid. Routine DNA analysis of primary bone tumors, as an adjunct to histopathologic assessment, can be employed to obtain diagnostic confirmation. In cases presenting histopathologic difficulties, ploidy determination may provide decisive diagnostic information.     

DNA analysis in the differential diagnosis of osteosarcoma

The DNA content of osteosarcomas, and of giant cell tumors, osteoblastomas, aneurysmal bone cysts, and fibrous dysplasias was determined by cytophotometry. Out of 158 tumors, 141 were histologically noncontroversial, whereas 17 posed diagnostic difficulties. In the noncontroversial group all 41 benign tumors had a diploid (normal) DNA content. Ninety-two of 96 high-grade osteosarcomas were hyperploid (increased DNA content). The four analyzed low-grade parosteal osteosarcomas were diploid. Among 17 diagnostically controversial cases, nine were primarily diagnosed and treated as benign. Three of these patients, nevertheless, exhibited a malignant clinical course and two had local recurrence. All five proved to have hyperploid tumors. The four nonrecurrent lesions were diploid. Of eight cases primarily evaluated as malignant, one died and two developed local recurrence. These three patients had hyperploid tumors. Among the five nonrecurrent lesions, two were hyperploid and three diploid. Hence, in the diagnostically controversial group, recurrence or death was consistently related to hyperploidy. The current study shows that the vast majority of high-grade osteosarcomas are hyperploid. Benign bone tumors, which may be mixed up histologically with osteosarcoma, are diploid. Routine DNA analysis of primary bone tumors, as an adjunct to histopathologic assessment, can be employed to obtain diagnostic confirmation. In cases presenting histopathologic difficulties, ploidy determination may provide decisive diagnostic information.     

DNA aneuploidy and cell proliferation in breast tumors

The cellular DNA content of 30 benign and 180 malignant breast tumors was analyzed by means of flow cytometry (FCM). All benign tumors exhibited a normal DNA content (diploid), whereas 65% of the malignant tumors showed an abnormal DNA content (aneuploid). The ploidy distribution of malignant tumors was bimodal with an increasing frequency near diploid DNA index (DI), and a second group had a DI ranging from triploid to tetraploid. In estimating the degree of malignancy eight independent histomorphologic and cytologic criteria were introduced. A good correlation was observed between DNA content abnormalities and the grade of differentiation of breast carcinomas. The percentage of S-phase cells of DNA aneuploid cell lines was significantly higher than in the diploid ones. The highly differentiated breast carcinomas (Grade 1) indicated lower S-phase values as compared to the undifferentiated (Grade 3) ones. S-phase values estimated by FCM were about two times higher than the 3H-thymidine labeling index (LI) obtained by an in vitro procedure. The data estimated in this study showed that DNA determinations as an adjunct to conventional histopathologic assessment may provide objective clinically relevant information with respect to the degree of malignancy and prognosis of patients with breast carcinoma.     

DNA flow analysis of soft tissue tumors

The cellular DNA content of 81 soft tissue tumors was determined by means of flow cytometry and related to conventional histologic classification of the same tumors. Comparison of histologic and cytometric analysis showed that all 23 benign tumors were diploid (normal DNA content), whereas the malignant group included both diploid and aneuploid (abnormal DNA content) lesions. There appeared to be a relationship between tumor grade and ploidy level in that 92% of Grade II, 28% of Grade III, and 11% of Grade IV lesions were diploid. Cell distribution analysis, feasible in 51 cases, disclosed that diploid lesions had a low proportion of S and G2 + M cells and most aneuploid lesions a high proportion, indicating a relationship between ploidy level and proliferative activity. The current study shows that solid mesenchymal tumors may be analyzed by DNA flow cytometry. Regardless of histogenetic type, it appears that benign and low-grade tumors are diploid and high-grade tumors, in general, are aneuploid. As to exceptions, DNA analysis may prove to give information beyond that obtained by subjective histologic interpretation. Thus, adequate follow-up might show that high-grade lesions with a diploid DNA content are associated with a better prognosis than expected from histologic classification.     

Flow cytometric analysis of DNA and cell proliferation in ovarian tumors

DNA content in tumor cells from 50 patients with ovarian tumors was analysed by flow cytometry (FCM). Solid tissue samples were processed to obtain monodispersed cells. Staining for DNA analysis was achieved with ethidium bromide and mithramycin. Peripheral blood lymphocytes were used as reference diploid cell population. All benign ovarian tumors exhibited only diploid cells. DNA aneuploid cell lines were found 66.6% of serous carcinomas and in 80% of malignant granulosa cell tumors. The S-phase fraction of DNA diploid cells in benign ovarian tumors (S = 2.4 +/- 1.2%) was smaller than those of malignant tumors (S = 8.2 +/- 5.2%). DNA aneuploid cell populations in serous carcinomas display a higher S-phase fraction (S = 19.2 +/- 9.3%) than DNA diploid cells (S = 11.7 +/- 3.2%). No major differences were obtained between primary ovarian tumors and their metastases, as far as degree of aneuploidy and S-phase fraction are concerned. A high degree of correlation was established between the grade of differentiation of ovarian tumors and the DNA ploidy abnormalities.     

Tumor heterogeneity in osteosarcoma as identified by flow cytometry

Measurements of the cellular DNA content by flow cytometry were carried out in 25 untreated osteosarcomas to identify the frequency of DNA aneuploidies and heterogeneous DNA stemlines in relation to histopathology. Analyzing multiple specimens from each single tumor (2-16; median, 4), highly malignant osteosarcomas were found to express DNA aneuploidies in 18 of 21 cases (86%) with multiple aneuploid DNA stemlines in 10 cases (48%). In three paraosteal osteosarcomas, no DNA aneuploidy was detected and a significantly lower proportion of cells in S-phase was observed as compared to the highly malignant osteosarcomas (mean 8.6% vs. 18.8%; P less than 0.05). Like in the paraosteal osteosarcomas, no DNA aneuploidy and a low fraction of cells in S-phase was found in the predominant cell population of one of the very rare sclerosing small cell osteosarcomas, which also revealed a second DNA stemline with a DNA index of 2.0. These results demonstrate a high degree of DNA stemline heterogeneity in highly malignant osteosarcomas. The data emphasize the usefulness of DNA measurements for the characterization of bone tumors and indicate the possibility of discriminating highly malignant from low-grade osteosarcomas.     

卵巢恶性肿瘤DNA倍体及其异质性的临床预后意义

目的 探讨卵巢恶性肿瘤DNA倍体、异倍体和异质性与临床预后的关系。方法 应用FCM流式细胞术，对20例卵巢恶性肿瘤、20例卵巢良性肿瘤、10例正常卵巢组织进行了DNA倍体、异倍体和异质性的检测，并分析其与临床预后的关系。结果 卵巢恶性肿瘤DNA异倍体、异质性的患者术后肿瘤复发率、死亡率明显高于卵巢恶性肿瘤DNA二倍体、同质性患者（P＜0．01）。卵巢恶性肿瘤DNA二倍体、同质性患者术后无瘤生存期明显长于卵巢恶性肿瘤DNA异倍体、异质性的患者（P＜0．01）。结论 卵巢恶性肿瘤DNA倍体、异倍体和异质性的测定是判断患者预后的良好指标。     

DNA cytophotometry and prognosis in ovarian tumors of borderline malignancy. A clinicomorphologic study of 80 cases.

Surgical specimens of 80 ovarian tumors of borderline malignancy (OTBM) were investigated by scanning DNA cytophotometry. Diploid or euploid DNA histograms were found for 21 tumors, whereas 59 OTBM showed noneuploid or aneuploid DNA patterns. All patients were followed-up after surgery for at least 3 years (mean observation period, 6.7 years). Follow-up showed 11 cases of recurrent disease and 6 deaths. DNA findings and several other morphologic and clinical details (including patient age, histologic type and stage of disease, and extent of therapy) were correlated to the postoperative course. Statistical analyses disclosed that, of these parameters, only DNA content significantly affected prognosis. Recurrences and deaths resulting from tumor exclusively were observed among patients with noneuploid or aneuploid OTBM, whereas none of the diploid or euploid tumors recurred (P less than 0.05). DNA cytophotometry thus might be regarded as an effective complementary means to assess the prognosis of individual OTBM cases.     

DNA content and prognosis in renal cell carcinoma. A comparison between primary tumors and metastases

Deoxyribonucleic acid (DNA) content was retrospectively determined by single-cell cytophotometry in primary tumors and corresponding metastases from 32 patients with renal cell carcinoma. In 15 of the primary tumors a diploid/near diploid and in 17 an aneuploid DNA content was found. A diploid/near diploid DNA pattern was revealed in 10 metastases and 22 were aneuploid. By comparing the DNA content in the primary tumors with their metastases, 13 of 32 showed a clear divergency, which might illustrate tumor cell heterogeneity of renal cell carcinoma. The DNA pattern showed a close correlation to morphologic grading. A correlation between DNA content in the metastases and survival time was found. Patients, with diploid/near diploid metastases survived significantly longer than those with aneuploid DNA contents (mean, 31.1 and 11.5 months, respectively; P = 0.004). In contrast to this, no correlation was found between DNA content in the primary tumors and survival time.     

Oncocytic tumors of salivary gland type: A study with emphasis on nuclear DNA ploidy

We studied nine cases of oncocytic tumors of salivary gland type in order to evaluate their clinico-pathologic profiles and nuclear DNA patterns as criteria for the differential diagnosis between benign and malignant forms. The tumors were located at the parotid gland, palate, and orbit. The age of the patients ranged between 35 and 85 years and the sex ratio (F:M) was 1:0.8. Seven tumors were capsulated and typical cytology: they were composed of polyhedrical cells with large, eosinophilic, and granular cytoplasm and dark nucleus. The remaining two tumors exhibited malignancy criteria for the oncocytic tumors: atypia, pleomorphism, and mitosis. The evaluation of the nuclear DNA content was also distinct: benign tumors had a DNA diploid pattern and the malignant neoplasms displayed a DNA aneuploid pattern. These observations point to DNA nuclear assessment as an additional criterion to discriminate neoplasms with divergent clinical behavior and prognosis. © 1993 Wiley-Liss, Inc.     

Flow cytometric DNA ploidy analysis of feline mammary tumors

Flow cytometric DNA analysis was performed on biopsies from 9 nonmalignant and 111 malignant (primary and metastatic) feline mammary lesions. In our series, 46.3% of the primary mammary carcinomas appeared to be aneuploid, whereas all but one benign breast lesion were diploid. The degree of aneuploidy in carcinomas was low, with a relatively high number of primary tumors (12 of 82) displaying hypodiploidy. Aneuploidy was not found to be correlated with any specific histological tumor type, vascular invasion, tumor size, or histological malignancy grade or with the separate components thereof. Comparison of the ploidy in primary and metastatic tumors from the same cases revealed a remarkable stability, both in time and location of appearance of the metastases. It is concluded that with respect to DNA ploidy feline mammary carcinoma has more in common with canine mammary carcinoma than with human mammary carcinoma. Further prospective studies are necessary to clarify the implications of aneuploidy in feline mammary carcinoma for tumor behavior and prognosis.     

Flow cytometric DNA analysis of gastric smooth muscle tumors.

BACKGROUND. To better understand the malignant grade of gastric smooth muscle tumors, the DNA content of these tumors was studied. METHODS. In 43 patients with gastric smooth muscle tumors, the cellular DNA content was determined by flow cytometry and compared with the histologic classification and the prognosis. RESULTS. Flow cytometry indicated that all 21 leiomyomas and 9 of the 10 low-grade leiomyosarcomas were diploid; 10 of the 12 high-grade leiomyosarcomas were aneuploid. All patients with leiomyomas and 8 of the 11 patients with diploid leiomyosarcomas had neither local recurrence nor metastasis. By contrast, 8 of the 10 patients with aneuploid leiomyosarcomas died of their disease (mean survival, 41 months; range, 18-78 months). CONCLUSIONS. These results indicate that the DNA ploidy pattern shown by flow cytometry is related closely to the histologic classification and prognosis of gastric smooth muscle tumors.     

Nuclear dna Measurements on Thyroid Carcinoma in Young Patients

Nuclear DNA measurements were performed on thyroid carcinomas from 36 patients aged 20 years or less. Histologic material from the tumors were stained according to the Feulgen technique and measured with slide cytophotometry. Thirty-two of the 36 tumors were of papillary type, 3 were medullary carcinoma and 1 was a follicular carcinoma. of the 32 papillary carcinomas, 6 tumors (19%) were aneuploid and 26 (81 %) were diploid, including 2 cases with lung metastases at diagnosis. of the 3 medullary carcinomas, 2 were diploid and 1 aneuploid. the only follicular carcinoma was aneuploid. the patients were followed between 10 and 35 years, and 34 were alive at the end of the study. Two patients died, both had medullary carcinomas. One patient, with a diploid tumor, died during surgery. the other patient, with an aneuploid tumor, died 5 years after diagnosis of metastatic disease. Six patients had recurrences, all within 7 years. All the primary tumors and the corresponding recurrences showed a diploid DNA content. the results show that the majority of thyroid carcinomas in young patients exhibit diploid DNA profiles which is in agreement with the overall good prognosis in this patient category. However, since also patients with aneuploid tumors exhibited a similar good prognosis it seems that DNA measurements do not contribute additional prognostic information in young patients.     

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).     

Prognostic value of DNA ploidy response to chemotherapy in human osteosarcomas.

We analyzed the DNA ploidy alterations after preoperative chemotherapy in 30 patients with non-metastatic osteosarcomas of the extremities. All of the patients received intensive chemotherapy with doxorubicin, cisplatin and methotrexate as well as wide tumor resection. DNA ploidy was determined by DNA cytofluorometry using isolated and smeared cells from biopsied and resected tumors after preoperative chemotherapy. The results showed that 12 diploid and nine non-diploid osteosarcomas did not change their ploidy pattern, but nine non-diploid tumors changed to a diploid pattern with the disappearance of the aneuploid cells. The nine patients with altered ploidy tumors had a better histologic response to chemotherapy and a better prognosis than the patients with non-altered tumors especially diploid tumors (P = 0.0138). Therefore, we conclude that a decrease in aneuploid cells after chemotherapy is closely correlated with a good prognosis in half of the cases of aneuploid osteosarcoma. These results also suggest that aneuploid cells are more chemosensitive than diploid cells in human osteosarcomas.     

Flow cytometric analysis of DNA ploidy in canine mammary tumors

DNA ploidy has been determined using flow cytometry in 23 nonmalignant and 34 malignant (primary and metastatic) mammary tumors from 46 dogs. This parameter was compared with clinical stage, histology, and estrogen and progesterone receptor analysis. Twenty-one of 34 cancers (61.8%) from 32 dogs were DNA aneuploid. Aneuploidy was also found in 4 of 23 nonmalignant tumors (17.4%) from 20 dogs. Regional lymph nodes were involved in 6 of 10 diploid and 3 of 9 aneuploid cancers of dogs with operable disease. The aneuploidy incidence was higher in dogs that had distant metastasis at initial diagnosis (8 of 11) than in those presented with local or locoregional disease (9 of 19), although this difference was not statistically significant. DNA aneuploidy incidence was not found to be related to histological tumor type, histological malignancy grade, nuclear grade, or steroid receptor presence. Heterogeneity in DNA content was found in 4 of 32 cancers (30 dogs) in samples from primary or locally recurrent lesions. In 3 of 16 cancers that were analyzed both at the primary and at secondary sites of growth, a significant variation in DNA content was observed. The degree of aneuploidy in the dog cancers was much lower than seen for human breast carcinomas with a relatively high frequency of hypoploid stemlines (7 of 34 cancers, 20.6%). The frequency distribution of DNA indices in dog mammary cancers indicates that aneuploidy evolution probably differs from that of human breast cancer.     

DNA content in renal cell carcinoma with reference to tumor heterogeneity

DNA content was successfully determined by flow cytometry in 196 tissue samples from 25 renal cell carcinomas. Twelve tumors (48%) were homogeneously diploid/near-diploid, whereas 11 tumors were aneuploid and 2 tumors were polyploid. Cell clones with different DNA content were found in 11 tumors demonstrating a considerable heterogeneity in the non-diploid tumors; 9 of these 11 heterogeneous tumors contained both aneuploid and diploid cell clones. Tumor samples morphologically classified as grade 1 and 2 were 98% diploid and grade 4 samples were 78% aneuploid. No correlation between DNA distribution and morphologic grade was found for grade 3 tumor samples. Tumor proliferation rate, as determined by the fraction of cells in S-phase, was significantly higher in aneuploid samples compared to normal kidney tissue samples and diploid tumor samples.     

Correlation of DNA ploidy, histopathology, stage and clinical outcome in gastric carcinoma.

The determination of nuclear DNA ploidy from paraffin-embedded specimens was performed by flow cytophotometry on 277 surgically resected primary gastric carcinomas to assess the relationship of various pathological findings and DNA content with survival. The preparation of samples was performed by a modification of Hedley's technique and the staining method of Vindelov. Eighty-nine (32%) carcinomas were DNA diploid, 69 (25%) were DNA tetraploid, and 119 (43%) were DNA aneuploid. DNA non-diploid patterns were significantly associated with macroscopic ulcerative appearance, location of the tumour in the proximal stomach, histological grade, and advanced stage of tumour. Patients with DNA non-diploid cancers, and specifically DNA aneuploid cancers, exhibited significantly poorer survival than patients with DNA diploid tumours. These data support the prognostic value of tumour DNA content in patients with resected gastric carcinoma.     

Evaluation of DNA ploidy and degree of DNA abnormality in benign and malignant melanocytic lesions of the skin using video imaging

BACKGROUND: Making a morphologic distinction between benign and malignant melanocytic tumors of the skin is frequently difficult, especially because "gray zones" between these lesions often exist. DNA image cytometry as an adjuvant method for the diagnosis and prognostic prediction of premalignant lesions and malignant tumors of many other organs is already well established. The aim of this study was to determine whether DNA image cytometry is helpful in distinguishing benign from malignant melanocytic lesions and whether cytometry would give valid information with which to predict the prognoses associated with malignant melanomas. METHODS: DNA image cytometry was performed on 127 benign and 58 primary maligant melanomas of the skin as well as 11 metastatic melanomas, using an enzymatic single cell solution according to a method described by Heiden et al. in Cytometry (1991;12:614-21). RESULTS: DNA aneuploidy was graded by DNA index (DI) and a 2c deviation index (2cDI). In contrast to benign melanocytic lesions (with 16% DNA aneuploidy), primary and metastatic malignant melanomas had significantly higher frequencies of DNA aneuploidy (86% and 73%, respectively). In the degree of DNA aneuploidy, significant differences between benign and malignant melanocytic tumors could be observed. The mean 2cDI of aneuploid benign lesions was 1.0, whereas the primary malignant melanomas had a mean 2cDI of 2.92 and the metastatic melanomas a mean of 6.9. The frequency of DNA aneuploidy increased with Breslow thickness. Twenty-one patients with primary malignant melanoma developed metastases. All metastasizing primary tumors were aneuploid and showed a significantly higher grade of DNA aneuploidy than nonmetastasizing malignant melanomas. Moreover, none of the diploid malignant melanomas developed metastases. CONCLUSIONS: This study reveals that DNA image cytometry is prognostically and diagnostically relevant to the evaluation of melanocytic lesions of the skin. Nevertheless, it cannot be relied on alone to provide enough information for a diagnosis.     

Comparison of DNA aneuploidy of primary and metastatic spontaneous canine osteosarcomas

Spontaneous canine osteosarcomas were analyzed for DNA aneuploidy and percentage of S phase cells using flow cytometry. Forty-eight dogs were studied in which both a primary tumor and subsequent metastases were available. The DNA index distributions for the primary tumors and the metastases were quite similar. However, when individual primary tumors and metastases derived from them were compared, many of the cases had different ploidy values. The tumor cells were also analyzed for percentage of S phase. The diploid metastases had less than 17% S phase cells, whereas the aneuploid metastases had up to 40% S phase cells. There was a direct correlation between the DNA index and the percentage of S phase in the metastases.