DNA Content Of Tumor Cells Research Articles

Neuroendocrine Tumors of the Gastrointestinal Tract

Neoplastic proliferations of neuroendocrine cells (NE) may occur throughout the entire GI tract but affect particularly appendix and ileum ("midgut carcinoids"), rectum ("hindgut carcinoids"), as well as stomach and the duodenum ("foregut carcinoids"). Only more exceptionally, they arise in the esophagus, jejunum and colon. The NE tumors encompass a heterogeneous gross and microscopic structural spectrum, ranging from inconspicuous microproliferations ("mucous membrane nevi") to bulky tumor masses. Their growth patterns are usually characteristic and easily recognized. In doubtful cases their NE differentiation becomes established by a characteristic silver affinity, by the ultrastructurally observed presence of characteristic "endocrine" secretion granules, and by immunohistochemically detectable occurrence of "pan-NE markers" (neuron-specific enolase, chromogranins, and synaptophysin), biogenic amines (mainly serotonin), and neurohormonal peptides. Foregut carcinoids usually contain serotonin, gastrin, and somatostatin, midgut carcinoids often only serotonin and tachykinins, whereas the hindgut carcinoids as a rule are multihormonal with a wide spectrum of hormonal peptides, including even insulin. Most GI NE tumors are found in the appendix (50%) and the ileum (30%). Practically all (98%) of the appendiceal NE tumors are benign. They have recently been proposed as arising from apparently Schwann-cell-related NE cells in the submucosa, whereas the ileal--and probably also all the other non-appendiceal NE tumors--are derived from the totipotential cells in epithelial crypts of the mucosa. Among the ileal NE neoplasms a large number can metastasize and result in a fatal outcome. The ability to metastasize is related to the size and to the multiplicity of the primary tumors at the time of initial diagnosis and, to some extent, to their histopathologic growth pattern. Now, some relationship between the prognosis and the cytochemically assessed nuclear DNA content of the NE tumor cells has also been established; not less than about 1/4 to 1/3 seem to be aneuploid. Almost 90% of the rectal carcinoids are benign. Exceptionally, a highly malignant NE neoplasms can arise from the colon/rectum--as well as from the esophagus--composed of NE cells of small and intermediate size. The NE tumors of the stomach are often composed of ECL (enterochromaffin-cell-like) cells; such ECL cell carcinoids are related to atrophic gastritis with pernicious anemia; experimentally, they can be induced by hypergastrinemia in rats. Duodenal carcinoids often contain psammoma bodies and can be associated with neurofibromatosis.

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.

Prognostic factors in locally advanced breast cancer (T3, T4) with special reference to tumor cell DNA content.

The prognostic value of DNA analysis was studied retrospectively in 91 patients with locally advanced breast cancer (T3, T4) and a follow-up time of 3-7 years. Tumor cell DNA analysis was performed by static cytometry on aspiration biopsy specimens in 42 cases and on tissue sections in 49 cases. The tumors were classified as euploid or aneuploid. Sixty-four percent of the tumors were aneuploid. DNA pattern correlated significantly to histologic grade and axillary perinodal growth and also to survival. DNA pattern, histologic grade and axillary node metastases correlated significantly to disease-free survival (DFS). In this series of patients with locally advanced breast cancer DNA determinations gave important prognostic information.

Aneuploid DNA content and high S-phase fraction of tumour cells are related to poor prognosis in patients with primary breast cancer

The prognostic impact of DNA content and S-phase fraction (SPF) of tumour cells was studied in 93 patients with primary breast cancer. Aneuploid DNA content and high SPF were clearly associated with poor differentiation state of tumours and absence of steroid, especially progesterone receptors. Aneuploidy and high SPF tended to become more common with increasing primary tumour size, with more extensive nodal involvement and with more advanced stage of the cancer. Patients with diploid tumours had a slightly longer disease-free interval and survival than those with aneuploid tumours, whereas below median SPF as compared to above median SPF was associated with significantly longer ( P < 0.01) relapse-free interval and survival in patients with stage II–III cancer. We conclude that the DNA analysis of tumour cells is a promising method for the estimation of prognosis in breast cancer patients.

The application of microspectrocytofluorometric measurement of Feulgen nuclear DNA content in experimental tumors of rat submandibular gland, 2. The correlation between proliferative ability and nuclear DNA content of tumor tissue in autotransplanted areas

We have reported that there is a difference in the variation of the nuclear DNA content of tumor cells among cases of squamous cell carcinoma induced by 9,10-dimethyl-1,2-benzanthracene (DMBA) in the rat submandibular gland. In the present investigation, the relationship between the nuclear DNA content of tumor cells in autotransplanted sections collected from primary lesions of DMBA-induced tumors and their proliferative ability in the subfascial area of the rat abdomen was examined. As a result of autotransplantation, proliferation in the autotransplanted area was observed in 6 of 14 (42.8%) cases of autotransplantation. Five of these had a keratinizing squamous cell carcinoma while the remaining one had a sarcomatoid tumor. The histological type of the tumor of the proliferative lesion in the transplanted area was very similar to that of the tumor tissues in the primary lesion or the transplanted section collected from the primary lesions. In the proliferative group, marked variation of the nuclear DNA content was observed in the tumor cells of the transplanted section. The proliferative index (PI) was high for these tumor cells in this group. There was no variation in the nuclear DNA content in the tumor cells of the nonproliferative group, and the PI was also low. These results were considered to suggest that there was a correlation between the nuclear DNA content of these experimental tumor cell and their proliferative ability in the autotransplanted area. Therefore, the determination of nuclear DNA content by this method can be used as an objective index of the proliferative ability of tumor tissue.

Cytologic transformation in cutaneous T cell lymphoma: a clinicopathologic entity associated with poor prognosis.

The clinical course of cutaneous T cell lymphoma (mycosis fungoides and Sezary syndrome) is generally indolent, but in occasional patients becomes fulminant. We found that biopsies from patients with accelerating disease can reveal cytologic transformation from previously observed small, convoluted lymphocytes to large cells that are similar to cells seen in large-cell lymphoma. The cerebriform nuclei characteristic of malignant T cells can only rarely be identified. Of 150 cutaneous T cell lymphoma patients we treated from 1976 to 1984, cytologic transformation was identified in 12 after review of peripheral blood smears and biopsies from skin, lymph nodes, and visceral sites. Patients who developed cytologic transformation were initially characterized by advanced stage (11 of 12), with lymph node effacement (seven of 11) and erythroderma (five of 12). The tumor cell DNA content after transformation was aneuploid (four of four), and the ability to form rosettes with sheep erythrocytes was retained in transformed cells (three of three). The median time from diagnosis of cutaneous T cell lymphoma to cytologic transformation was 21.5 months (range, 4 to 64), and the median survival from transformation was only 2 months (range, 0 to 19+). We conclude that cytologic transformation in cutaneous T cell lymphoma represents a distinct clinicopathologic entity, characterized by an aggressive clinical course.

Influence of tumor cell DNA ploidy on the natural history of rectal cancer

We have examined tumor cell DNA content as a possible variable in the behavior of early rectal cancer treated by local excision. Flow cytometry assays of tumor cell DNA content were carried out on specimens of archived, paraffin-embedded tissue specimens from 30 patients (11 male and 19 female) whose early rectal cancers were treated by curative local excision more than 60 months previously. The cancers invaded to the muscularis mucosae in 2 patients (1 with aneuploid disease and 1 with diploid disease), into the submucosa in 15 patients (7 with aneuploid disease and 8 with diploid disease), and the muscularis propria in 13 patients (8 with aneuploid disease and 5 with diploid disease). A total of 16 patients had aneuploid disease and 14 had diploid disease. Local recurrence of cancer developed in 12 patients. Of these 12 patients, 10 (83 percent) had aneuploid disease. By contrast, of the 18 patients who remained free of disease, 12 (67 percent) had diploid disease. Seven of the 12 patients with recurrence died. Six of these seven (86 percent) had aneuploid disease. The aggressive clinical behavior of the tumors with aneuploid DNA content was not otherwise predictable by standard histologic features. Aggressive tumor behavior appears to correlate closely with aneuploidy in locally treated rectal cancers, as opposed to a lack of correlation in our patients treated with major resection. The fact that these cancers are being treated by local excision may allow the prognostic impact of DNA content to reflect the natural history of cancer.

DNA content abnormalities and prognosis in uveal melanoma.

Recent advances in flow cytometry allow for study of DNA content in paraffin-embedded pathology material. Using refinements of published techniques, we retrospectively correlated tumor cell DNA content (ploidy) with histologic findings and clinical outcome in 79 patients with uveal melanoma. Patients were included using these selection criteria: enucleation without adjunctive therapy performed at the University of California at San Francisco between 1956 and 1979, tumor located in ciliary body or choroid, and pathology material and complete follow-up data available. The DNA histograms were classifiable as diploid or hyperploid in 64 cases. The mean coefficient of variation for diploid histograms was 6.6%. Twenty-three patients (36%) had hyperploid tumors. Hyperploidy was correlated with worse outcome. The effect was most marked for DNA indexes of over 1.4. A Cox proportional hazards model showed that the effects of tumor diameter and cell type were insignificant in comparison with the effect of the DNA index.

Spontaneous Neurinoma in an African Lungfish, &lt;italic&gt;Protopterus annectens&lt;/italic&gt;, and DNA Repair Studies on Normal and Neoplastic Tissues&lt;xref ref-type="fn" rid="FN1"&gt;2&lt;/xref&gt;

Neurinomas developed in an African lungfish (Protopterus annectens), living in an aquarium in Western Japan. The 2 tumors, measuring 7.5 X 9.0 X 6.5 and 13 X 4 X 6 cm, were located on the skin. As shown by light microscopy, tumor cells were composed of spindle-shaped cells with huge pleomorphic nuclei, which were arranged in parallel rows or whorls in interlacing connective tissue. Long-term culture of these tumor cells was achieved in vitro at 25 degrees C with use of conditioned medium over a period of more than 4 months. The nuclear DNA contents of erythrocytes (normal diploids, 2C) and tumor cells dispersed from the fixed tumor tissues were measured by 4',6-diamidino-2-phenylindole hydrochloride-DNA microfluorometry by using mouse cerebellar small granule cells (normal diploids, 2C) as a reference. The 2C value of the lungfish was approximately 28-fold greater than that of the mouse. Furthermore, consistent with the nuclear pleomorphism observed by light microscopy, the nuclear DNA contents of tumor cells showed a wide distribution from hypo-2C to hyper-4C. DNA repair synthesis was measured autoradiographically in organ cultures of the tumor, lung, and skin, exposed to chemical carcinogens or UV radiation. Considerable repair was observed in the tumor and skin cells exposed to 1-methyl-1-nitrosourea (CAS: 684-93-5), N-methyl-N'-nitro-N-nitrosoguanidine (CAS: 70-25-7), or 254-nm or sunlamp UV light. Only traces of repair synthesis were detected in lung exposed to 1-methyl-1-nitrosourea or N-methyl-N'-nitro-N-nitrosoguanidine. 4-Hydroxyaminoquinoline 1-oxide (CAS: 4637-56-3) did not induce repair in any of the three tissues. The observed values for repair, relative to the amount of DNA, were similar to those in other fishes.

Hyperdiploidy and chromosomal rearrangements define the anaplastic variant of Wilms' tumor.

Flow cytometric measurement of the DNA content of Wilms' tumor cells revealed a striking correspondence with the histologic subtype and treatment outcome. In the 48 cases studied, a hyperdiploid DNA content ranging from 1.7 to 3.2 times the result for normal diploid cells distinguished all but one of the ten anaplastic tumors. Lower values, from 1.0 to 1.4 times the diploid DNA content, characterized the nonanaplastic specimens. By Kaplan-Meier analysis, the probability of achieving 3 years of relapse-free survival was significantly lower in the group with higher DNA content (0.42 v 0.87, P less than .01). Analysis of banded chromosomes for a subset of 22 patients contributed important information beyond the flow cytometric study. Cases of anaplasia associated with poorer responses to therapy showed numerous complex translocations, whereas all others lacked such changes. By combining flow cytometric techniques and conventional methods of chromosome analysis, it should be possible to identify those patients with Wilms' tumor who are most likely to fail therapy. The biologic implication of these findings is that the development of clinical drug resistance in Wilms' tumor is a result of the genetic instability of the malignant clone.

Prognostic Significance of Nuclear DNA Analysis in Histological Sections in Mammary Carcinoma

The nuclear DNA content in tumor cells from invasive ductal breast carcinomas was analyzed in 26 patients who survived more than 10 years and in 23 patients who died within 2 years after operation. The DNA content of individual tumor cells was measured in sections from the originally paraffin-embedded specimens. In short-term survivors, a large proportion of cells with very high DNA values was found in all tumors except one. Only two patients of the long-term survivors had tumors that exhibited such high DNA values. Prognostic information obtained by DNA analysis compared with histologic malignancy grading showed that the specificity using DNA analysis was distinctly higher. The data thus suggest that analysis of DNA content of tumor cells in breast adenocarcinomas can be a useful supplement to histologic malignancy grading to obtain prognostic guidance in individual patients.

Prognostic implications of tumor cell DNA and RNA content in multiple myeloma

Flow cytometric studies of bone marrow DNA and RNA content were conducted in 71 previously untreated patients with multiple myeloma. There was a progressive increase in response rate with rising plasma cell RNA content. The DNA-derived ploidy level also affected chemotherapy sensitivity: only one of 11 patients with either hypodiploidy or biclonal DNA stemlines responded. DNA-RNA-defined marrow plasmacytosis was the only tumor mass-related variable adversely affecting remission induction. Survival was longer in patients with low tumor burden and favorable DNA features. The availability of objective and quantitative pretreatment variables associated with both initial response and survival should permit a risk-based selection of patients for novel treatment approaches.

Prognostic Implications of Tumor Cell DNA and RNA Content in Multiple Myeloma

Flow cytometric studies of bone marrow DNA and RNA content were conducted in 71 previously untreated patients with multiple myeloma. There was a progressive increase in response rate with rising plasma cell RNA content. The DNA-derived ploidy level also affected chemotherapy sensitivity: only one of 11 patients with either hypodiploidy or biclonal DNA stemlines responded. DNA-RNA-defined marrow plasmacytosis was the only tumor mass-related variable adversely affecting remission induction. Survival was longer in patients with low tumor burden and favorable DNA features. The availability of objective and quantitative pretreatment variables associated with both initial response and survival should permit a risk-based selection of patients for novel treatment approaches.

DNA content of lymphocytes and mononuclear tumor cells in Hodgkin's disease

Thirteen lymph node biopsy specimens with Hodgkin's disease (six cases of the nodular sclerosis type; six cases of the mixed cellularity type; one case of the lymphocytic depletion type) were studied with scanning cytophotometric measurements. The DNA content of small lymphocytes, large lymphocytes, atypical mononuclear cells and Hodgkin cells was measured. The sizes of the nuclei and mitotic indices were also measured. There were no lymphocytes with aneuploid DNA stemlines. Large lymphocytes showed proliferative activity which is possibly reactive, although its neoplastic character cannot be excluded. Hodgkin cells exhibited aneuploid values in seven cases and atypical mononuclear cells in only four cases. The nuclear sizes and the DNA content of the atypical mononuclear cells ranged between the values of lymphocytes and Hodgkin cells. The mixed cellularity type showed higher DNA values, more frequently occurrence of aneuploid stemlines and a higher frequency of mitotic figures than did the nodular sclerosis type. The results confirm the different grades of malignancy of the various histopathological categories of Hodgkin's disease.

口くう病変の臨床的悪性度の指標について 多形性せんしゅの核ＤＮＡ量と核面積を中心にして

This study was performed to obtain indices for clinically determining the degree of malignancy of pleomorphic adenomas. Nuclear DNA content and nuclear area of tumor cells were measured and analyzed in comparison with clinical and pathological observations.Twenty-four cases of pleomorphic adenoma were studied. A control group consisted of seven cases of normal salivary gland, two cases of adenolymphoma, and five cases of malignant salivary gland tumor. Most of the cells were isolated from slices of paraffin embedded tumor masses removed from patients. Some cell samples were obtained from imprints of biopsy specimens. Following Feulgen staining, the nuclear DNA content and nuclear area were simultaneously measured with Vickers M86 scanning microdensitometer. The nuclear DNA content of the epithelial cells was calculated with the nuclear DNA content of a control lymphocyte put at 2.0c.The results were as follows:1) The DNA histogram of normal salivary gland showed one distinct peak at 2.0-2.5c. Cells with nuclear DNA content over 4.0c were not observed. The measurements of the nuclear area were distributed in and around the region of 30μ2.2) In one case of adenolymphoma, the DNA histogram and the DNA-area scattergram showed a similar pattern to those of normal salivary gland cells. In another case, a small population of cells over 4.0c was observed and cells diffused slightly on the scattergram.3) In the cases of malignant salivary tumor, the average values and the variances of the nuclear DNA content increased generally. Hyperploid cells over 4.0c and aneuploid cells were observed. The DNA histogram showed a shift rightward from normal modal C value, bi-peak or poly-peak. The scattergram showed a remarkable diffusion and a high correlation.4) In the cases of pleomorphic adenoma, measurements of the nuclear DNA content and the nuclear area varied greatly, ranging from those close to the values of normal salivary gland to the values of malignant salivary tumor. The average values and the variances of the nuclear DNA content as well as the number of cells over 4.0c were apt to increase in patients who showed rapid progress of tumorgenesis, predominance of a solid pattern of tumor cells histologically and prolifelation like cords. Particularly in the cases where the measurements of the nuclear DNA content and the nuclear area resembled those of malignant salivary gland tumor, malignant potential tended to be high.

Heterogeneity of tumor cell DNA content.

Heterogeneity of tumor cell DNA content.

DNS-Ploidie und Proliferationsverhalten beim metastasierten Neuroblastom*

Nuclear DNA contents of tumor cells were measures by fluorescence cytophotometry in bone marrow and touch preparations of 26 children with neuroblastoma, registered in the NBL79 study of the German Pediatric Oncology society. In all cases tumor cells were found to have a higher DNA-content as compared to the normal diploid (2c) DNA-value of granulocytes in the same preparation. Average ploidy values of the histograms of bone marrow metastases were in the tetra- to hypertetraploid (4c-5c) range and showed unimodal distribution in the majority of cases. In the primary tumors there was also a wide distribution of DNA-values in the aneuploid range. A bimodal distribution with stemlines at 2,5c and 5c was found in a stage I, grade I, differentiated primary tumor. Various ploidy patterns found at different sites in the large tumor mass of a newborn might be interpreted as an expression of different degrees of differentiation within the same tumor. In 2 of 4 patients where primary tumor and metastatic lesions could be examined there was a higher ploidy in the 5c to 6c, or even in the 8c range in the metastatic cell population. It appears, thus, that clonal evolution of tumor cells occurs during growth and spread of neuroblastoma.

Relevance of density, size and DNA content of tumour cells to the lung colony assay.

Mouse fibrosarcoma tumours were dissociated and divided into subpopulations of viable cells by centrifugation in linear density gradients of Renografin. Two of these subpopulations, designated Band 2 and Band 4, differed in their clonogenic ability in lung colony assay. The less dense Band 2 cells were significantly more clonogenic than the Band 4 cells (2.9 percent vs 1.4 percent respectively). Each band was further separated on the basis of cell size by centrifugal elutriation. Each size class of cells comprising Band 2 showed higher clonogenic ability than the corresponding size class in Band 4. Thus cell size differences were not responsible for the clonogenic differences between these bands. To determine whether cell-cycle distribution of the tumour cells was responsible for differences in cloning efficiency, flow microfluorometric and premature chromosome condensation methods were utilized. The unseparated and Band 4 populations showed a higher percentage of cells in S and G2 than did the Band 2 populations, but many of the S and G2 tumour cells showed extensive chromosome damage. From this study we conclude that the increased clonogenic ability of the lighter tumour cells is not due to differences in cell size or cell-cycle parameters.

Malignant granular-cell tumor of the cerebral white matter: morphological, cytophotometrical and neurochemical studies (author's transl)

This is a report of the second known case of a primary malignant granular-cell tumor of the cerebral hemispherical white matter. Two cell types may be distinguished apparently representing different developmental stages of otherwise identical tumor cells. Quantitative histochemical and biochemical studies have shown that tumor cells were containing markedly elevated levels of DNA and RNA. Only few ribosomes and polysomes could be detected, however, by electron microscopy. While cytophotometry disclosed only slightly elevated cytoplasmic proteins, there was a distinct increase in total protein concentration of tumor tissue per unit weight, as measured by conventional techniques. It is suggested that this increase is mainly attributable to structural proteins of cellular and subcellular elements of multiplicating malignant cells. A ratio of RNA to DNA in excess of 1 was found for white matter derived from the central tumor, while the ratios of control tissue were lowered to values far below one due to postmortem changes greatly reducing tissue concentrations of RNA. No detailed characterization of RNA was attempted. The nuclear DNA content of tumor cells reached values equivalent to chromosomal hexadekaploidy. This was in sharp contrast to control data from an Abrikossoff tumor of the oral cavity and from a neurohypophyseal tumorette ("choristoma"), respectively, displaying diploidy only associated with a much lesser increase of cytoplasmic RNA and proteins. Qualitative lipid studies were consistent with a marked active demyelination of the tumor centre as indicated by a severe reduction of cerebrosides and sulfatides and the presence of cholesterol esters. In addition there was a striking loss of phosphatidylethanolamine and a lesser one of sphingomyelin of white matter of both the tumor-stricken and the contralateral unaffected hemispherical regions, possibly suggesting a generally disturbed metabolism of myelin. It is not clear whether these general changes were resulting from the presence of the unilateral tumor or from precocious cerebral involution.

The distribution in the cell cycle of normal cells and of irradiated tumour cells in mice.

Flow microfluorometry (FMF) instrumentation was used to make a rapid, quantitative measurement of the DNA content of individual cells from normal tissues and from KHT tumour cells of mice. These normal tissues are some of the limiting normal tissues in radiation therapy. The DNA content of tumour cells was also measured at different times after X-ray exposure. Preliminary results indicated that the cellular DNA content of all normal tissues was about the same and that a large fraction of cells were in the G0 or G1 stage. In the KHT tumour, nearly 75 per cent of the cells were in the G0 or G1 stage, 20 per cent in the S stage, and the remaining 5 per cent in the G2 + M stage. Ten hours after 300 rads of X rays, the number of tumour cells in the G1 stage decreased to about 40 per cent, cells in the G2 + M stage increased to about 30 per cent, and about 30 per cent of the cells were in the S stage. The cellular DNA content was very nearly the same as unirradiated controls at 24 hours after exposure. When the tumours were exposed to 3,500 rads, at 24 hours after exposure the number of cells in the G1 stage was reduced to 25 per cent, the number of cells in the S stage remained nearly the same (25 per cent), and the number of cells in the G2 + M stage increased to about 50 per cent. These studies indicate that FMF instrumentation can be a powerful tool to obtain quantitative information on DNA distribution of normal and tumour cell kinetics during radiation treatment.