Near tetraploid prostate carcinoma. Methodologic and prognostic aspects.

BACKGROUND: The clinical value of DNA ploidy analysis in prostate carcinoma has been an issue for investigation for more than 2 decades. In general, diploid or pseudodiploid tumors are associated with a favorable prognosis and aneuploid tumors with an unfavorable prognosis, irrespective of type of treatment. Tumors with DNA values in the tetraploid region (around 4c) present a diagnostic problem. Such DNA distributions may clearly represent aneuploid tumors with an unfavorable prognosis. However, a 4c distribution may conversely represent a tetraploid tumor (possibly a polyploid variant of the diploid tumor) with a favorable prognosis. Previous data from our laboratory indicate the existence of such a tetraploid subgroup. The goal of the current study was to investigate the diagnostic problem of 4c tumors in greater detail. METHODS: Ploidy classification of cytologic smears by image cytometry was performed in a retrospective study of 334 patients with hormonally treated prostate carcinoma. Follow-up time was 30 years or until death. RESULTS: Three ploidy types were defined: near-diploid (D type), near-tetraploid (T type), and highly aneuploid (A type). Tumors with a modal value within the tetraploid region were found in 27% (92 cases) of the total material. Of these, 9% were defined as T type and 18% as A type. Overall, 37% of the tumors were classified as D type, 9% as T type, and 54% as A type. Of the A type tumors, one-third had modal DNA values in the tetraploid (4c) region. Multivariate analysis showed a statistically significant difference between A type tumors and D and T type, but not between D type and T type. Both D and T type tumors progressed slowly and killed the patients 5 to 30 years after diagnosis, whereas A type tumors progressed rapidly and killed the patients within 6 years of diagnosis. CONCLUSIONS: By image cytometry, prostate carcinoma can be divided into three ploidy types: D, T, and A type. Biologically, however, the tumors fall into only two groups: low grade malignant, pseudodiploid tumors of D or T type, and high grade malignant, highly aneuploid tumors of A type.

Photographic densitometry for quantitative DNA analysis of cytologic and histologic specimens.

We found that photographic densitometry (PD) is a useful technique for quantitative determinations of nuclear DNA content in clinical tumor material. Optimum conditions for the use of PD in clinical cytology and histopathology were worked out. A quantitative evaluation of the method was performed, particularly with respect to errors that may appear when measuring clinical tumor material. Our study showed that PD offers accurate DNA measurements in cytologic and histologic specimens. Ploidy level determinations in tumor cell populations in clinical material could be as accurately performed with PD as with scanning microspectrophotometry (SMP). Nuclear DNA content of individual cells as determined by PD correlated highly with nuclear DNA content determined by SMP (correlation coefficient, 0.96). Since the PD method is less influenced by background variation than are other image techniques (due to measurement of a photographic image), it is particularly useful in measurement of histopathologic sections, in which the background variation can introduce considerable errors. The method is also valuable with clinical cytologic smears, in which the presence of blood and other material disturbs the background. PD represents a valid complement to scanning microspectrophotometry and TV imaging systems, particularly for DNA analysis of tissue sections. Moreover, it can be applied easily in the clinical routine. Relevant tissue areas are selected and photographed by the pathologist or cytopathologist, and the measurement is performed by a laboratory technician.

The prognostic significance of nuclear DNA content in prostatic carcinoma.

The prognostic significance of ploidy level was studied in prostatic carcinoma and compared with the prognostic significance of morphologic grade and clinical stage. A nonselected group of 145 patients was studied in whom prostatic carcinoma was diagnosed by fine-needle aspiration biopsy at the Karolinska Hospital in 1966. All patients had endocrine therapy and were observed for 23 years or until death. Ploidy level was determined from cytophotometric measurements of Feulgen-stained tumor cell nuclei. The original May-Grünwald-Giemsa-stained cytologic slides, on which the cancer diagnoses were based in 1966, were destained and subsequently Feulgen stained for cytophotometric analysis. From the Feulgen-DNA data, the tumors were classified as near-diploid, near-tetraploid, and highly aneuploid, variants D-type, T-type, and A-type, respectively. The A-type tumors progressed rapidly, and 96% of the patients with this type died of the tumor within 5 years and all patients with this type died within 7 years. D-type and T-type tumors progressed much more slowly. None of the patients with these types of tumors died of the tumor disease within the first 5 years after diagnosis. As many as 12% of the patients (crude survival rate; corresponding to a relative survival rate of 43%) were still alive 15 years after diagnosis. According to multivariate Cox regression analysis, ploidy compared with grade and tumor stage was the strongest predictor of survival.

Ploidy level and tumor progression in prostatic carcinoma.

Methods developed for cytophotometric analysis of archival tumor specimens used in retrospective studies were evaluated quantitatively. Up to 20-year-old May-Grünwald-Giemsa stained cytological slides could be Feulgen stained with excellent results. By expressing DNA data from the tumor cells as relative values (c-values) related to the internal staining control (mean value 2c) detailed ploidy level determinations could be made as accurately from measurements of old, destained slides as from measurements of cells from fresh tumor material, which were directly Feulgen-stained. Ploidy level determinations of prostatic carcinoma in 213 patients selected on the basis of survival time were analyzed. By studying the tumors in two extreme groups (extreme group I: 131 patients who died from prostatic cancer within 3 years of diagnosis, and extreme group II: 82 patients who survived more than 15 years after diagnosis) it was possible to evaluate in detail the optimal limits for defining the diploid 2c-region and tetraploid 4c-region. Using these limits to determine the percentage of aberrant tumor cells, i.e. non 2c and non 4c, and combining this with the modal value (in c units) of the tumor cell population the tumors could unambigously be divided into near-diploid-D-, near-tetraploid-T, T- and highly aneuploid A-types. The prognostic significance of ploidy level was studied in prostatic carcinoma in a non-selected group of patients subjected to endocrine therapy and long-term clinical follow-up (up to 23 years). This patient group consisted of all of the patients who were diagnosed as having prostatic carcinoma by means of fine-needle aspiration biopsy at the Karolinska Hospital during 1966. The A-type tumors progressed rapidly and killed 96% of the patients within 5 years (and all patients within 7 years). D- and T-type tumors progressed much more slowly. None of the patients with these tumors died from the tumor disease within the first 5 years after diagnosis, and 12% of the patients (crude survival) were still alive 15 years after diagnosis. Ploidy level was superior to morphological grade and clinical stage (tumor size) as a prognostic indicator.

A quantitative evaluation of cytophotometric DNA analysis in retrospective studies using archival tumor specimens.

Methods developed for the cytophotometric analysis of archival tumor specimens used in retrospective studies were evaluated quantitatively. May-Grünwald-Giemsa-stained cytologic slides up to 20 years old could be restained by the Feulgen reaction with excellent results if they were destained in methanol and refixed in formaldehyde prior to Feulgen staining. Storage time had only a minor influence on Feulgen stainability. However, a considerable variation in the intensity of the Feulgen stain was observed between different slides stained simultaneously; this variation was not related to storage time. As a consequence of this variation, the use of internal staining controls, such as granulocytes, is an absolute necessity in the quantitative comparison of different slides. By expressing DNA data from the tumor cells in relative values (c values) related to the internal staining control (with a defined mean value of 2c), Feulgen ploidy level determinations could be made as accurately from measurements on old, destained slides as on cells obtained from fresh tumor material. The ploidy level could also be accurately determined in most cases of prostatic carcinoma from measurements on histopathologic sections.

Ploidy level determinations in high-grade and low-grade malignant variants of prostatic carcinoma.

Ploidy level determinations of prostatic carcinoma in 213 patients selected on the basis of survival time were analyzed. Two extreme groups were chosen: extreme group 1, 131 patients who died from prostatic cancer within 3 years of diagnosis; and extreme group 2, 82 patients who survived for more than 15 years after diagnosis. All patients were diagnosed by fine needle aspiration biopsy. The DNA measurements were performed on Feulgen stained, destained May-Grünwald-Giemsa smears. The DNA distribution patterns were studied in three benign prostatic lesions as a base for analyses of prostatic carcinoma. By choosing two extreme groups it was possible to evaluate in detail the optimal limits for defining the diploid 2c region and the tetraploid 4c region. Various limits were tested in order to determine those which most clearly separate extreme group 1 from extreme group 2. We found that the optimal upper diploid limit was 2.5c and the optimal tetraploid limits were 3.5c-4.5c. By using these limits to determine the percentage of aberrant tumor cells, i.e., non-2c and non-4c, and combining this with the modal value (in c units) of the tumor cell population the tumors could unambiguously be divided into D-, T-, and A-types. D/T-type tumors were found only in extreme group 2 and had less than 30% aberrant tumor cells, while A-type tumors (high-grade aneuploid) had greater than 50% non-2c and non-4c tumor cells. All A-type tumors were found in extreme group 1. In order to investigate whether the classification of tumors into D-, T-, and A-type was valid in general and could also be applied to patients with survival time between the two extreme groups (3-15 years), a material of 79 patients with a wide range of survival times was tested. The tumors were classified according to the above-mentioned criteria into A-, D-, and T-type tumors. All patients who died within 5 years of diagnosis had A-type tumors. All patients who lived greater than 5 years from diagnosis had D- or T-type tumors.

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 significance of the DNA content in renal cell carcinoma.

The DNA content in renal cell carcinoma as an indicator of prognosis was studied retrospectively in 55 patients without distant metastases at operation. Two groups of patients were selected, differing with respect to survival. Thirty-three patients survived for at least 10 years and 22 succumbed to the disease within four years. DNA measurements in morphologically identified tumor cells were performed in histological sections by single cell cytophotometry. The tumor cells of the surviving patients had a DNA content comparable to that of normal cells. A diploid/near diploid DNA content was the dominant feature in 32 of these 33 tumors. The remaining patient had a tumor with a tetraploid/near tetraploid DNA value. In contrast, all tumors from the non-surviving patients had abnormally increased DNA content, indicating a high degree of aneuploidy in these tumors. The results suggest that DNA content may be superior to other clinical and microscopical parameters as a prognostic indicator.

Prognostic value of nuclear DNA content in follicular thyroid tumours.

The prognostic value of nuclear DNA content in follicular thyroid tumours was studied in 65 patients. Forty-six (20 with follicular carcinoma and 26 with follicular adenoma) were alive at least 10 years after diagnosis, and 19 had died of follicular thyroid carcinoma 2 months to 13 years after diagnosis. DNA was measured in morphologically identified single tumour cells either on material from fine-needle aspiration biopsy or on histologic sections from the primary tumours. Metastases or locally recurrent tumour were also examined in ten cases. The tumours from the surviving patients had cells with DNA content comparable to that in normal cells, whereas the tumours from the nonsurvivors showed higher DNA values. Primary tumours had essentially the same DNA content as metastases or local recurrence in eight of the ten studied cases. The data suggest that DNA measurement in follicular thyroid tumours offers a valuable addition to standard clinical and microscopic analysis.

Prognostic impact of nuclear DNA content in medullary thyroid carcinoma; a retrospective pilot study.

The prognostic significance of DNA content in medullary thyroid carcinoma was studied retrospectively in 16 patients. Five patients died within 3 years of medullary thyroid carcinoma and 11 patients survived for at least 10 years. Clinical data and tumour morphology were studied. DNA measurements on tumour cells in histologic sections were performed with slide cytophotometric technique. The tumours of the survivors had in all but two cases a DNA content comparable to that of normal cells, whereas the tumours of the non-survivors and two of the survivors had higher DNA content. The results indicate that DNA measurements in medullary thyroid carcinoma might be of use in addition to clinical and morphologic data and that further studies are warranted.

Comparison of nuclear DNA content in primary and metastatic papillary thyroid carcinoma.

Nuclear DNA values were determined in 40 primary papillary thyroid carcinomas, as well as in 52 corresponding local recurrences and metastases were observed either at the time of diagnosis or up to 20 years later. The patient population consisted of 34 survivors and 6 nonsurvivors. In survivors, both the primary tumors and their recurrences and metastases exhibited a majority of cells with DNA values within the normal diploid region, whereas nonsurvivors showed increased and scattered DNA values. In all cases, the primary tumors and the corresponding recurrences and metastases showed similar DNA distribution patterns even if many years had passed between the detection of the primary tumor and the metastases. The results indicate that in papillary thyroid carcinomas, the DNA distribution patterns in the primary tumor and the corresponding recurrences or metastases are generally similar throughout the entire period of disease.

Ploidy levels in nonneoplastic and neoplastic thyroid cells.

DNA levels in nonneoplastic and neoplastic human thyroid cells were studied by slide-cytophotometric and flow-cytophotometric techniques. Normal epithelial thyroid cells and cells from nonneoplastic thyroid disorders, i.e., toxic goiter, colloid goiter and chronic lymphocytic thyroiditis, had euploid (diploid, polyploid) DNA levels. The same was the case for benign tumors. Malignant tumors had either euploid DNA levels, indistinguishable from those in nonneoplastic and benign neoplastic cell populations, or aneuploid DNA levels. Taking into account the histopathologic diagnosis, clinical course and DNA level, the results indicate that DNA measurements in thyroid gland lesions are of limited diagnostic help but may contribute valuable prognostic information. To obtain representative and accurate DNA measurements, combined slide-cytophotometric and flow-cytophotometric techniques should be used.

Combined morphologic and cytochemical grading of serous ovarian tumors.

The potentiality of DNA analysis to complement morphologic evaluation in classifying serous ovarian tumors as adenoma, borderline malignancy, or invasive adenocarcinoma was investigated in a series of 54 tumors. The DNA analyses were performed on histologic tumor sections. The primary diagnoses were borderline tumor in 24 cases and invasive adenocarcinoma in 30 (World Health Organization classification). When the specimens were reviewed, 17 of the 54 tumors were reclassified, after which the series consisted of 9 adenomas, 24 borderline tumors, and 21 invasive adenocarcinomas. Rising histologic malignancy grade was associated with increasing numbers of cells showing high DNA content. The DNA levels in the adenomas thus were within the diploid range of a normal cell population. They were somewhat higher in the borderline tumors and were highest in the invasive adenocarcinomas. Though no clear-cut intergroup demarcation was discernible, there was a subgroup of adenocarcinomas with greatly elevated DNA levels, indicating high biologic malignancy. The observations suggested that DNA analyses can complement histologic malignancy grading and can be useful for the recognition or highly malignant tumors among invasive adenocarcinomas.

Biologic considerations and operative strategy in papillary thyroid carcinoma: arguments against the routine performance of total thyroidectomy.

Reasons cited for the routine performance of total thyroidectomy in patients with papillary thyroid carcinoma include: fear of multicentric neoplastic foci causing local recurrence and death; risk of anaplastic transformation of unresected multifocal microscopic carcinoma; toxicity of high-dose radioactive iodine to ablate normal thyroid remnants; and lack of reliable criteria for grading malignancy and identifying patients at high risk. However, autopsy studies have detected microscopic foci of papillary thyroid cancer as incidental findings in up to 24% of patients dead of other diseases. The prevalence of anaplastic transformation of papillary thyroid carcinoma as determined from reports in the literature is less than 1%. A retrospective investigation of 90 patients with papillary thyroid carcinoma derived from the Swedish National Cancer Registry showed no complications from radioiodine ablation of postoperative thyroid remnants in 45 patients. Retrospective analysis of the DNA content of tumors at the time of the initial operation showed a significant difference between a group of 10 patients who died of recurrent and metastatic papillary thyroid carcinoma and a group of 16 patients alive at least 10 years after operation despite distant metastases or recurrent cancer in the thyroid bed and/or cervical lymph nodes. The risk of permanent hypoparathyroidism is higher in patients after total thyroidectomy without apparent improvement in survival rates when compared with less extensive resections. Therefore it is proposed that the criteria for total thyroidectomy in patients with papillary thyroid carcinoma be limited to: tumors that clinically involve both lobes of the thyroid gland, extracapsular spread of cancer requiring enbloc resection, and reoperations where scarring prevents accurate delineation of the extent of the tumor. By differentiating patients at high risk for death from papillary thyroid carcinoma from patients at low risk, the measurement of DNA content may decrease the need for routine total thyroidectomy.

The significance of DNA distribution pattern in rectal carcinoma. A preliminary study.

The DNA distribution pattern was determined retrospectively in 25 rectal carcinomas and the possible correlation to clinical outcome evaluated. The DNA content in individual cells was measured according to a cytophotometric method based on light transmission measurement of Feulgen-stained nuclei. Tumor cells with DNA content exceeding an upper limit, i.e., the 90 percentile of the control cells, were considered to be nondiploid (aneuploid). Virtually all long-term survivors had less than 50 per cent of the tumor cells exceeding the upper diploid level, whereas those developing only a local recurrence had 50 to 70 per cent. Patients with disseminated disease and short survival time had all of their tumor cells exceeding the upper diploid level. There was a highly significant correlation between Dukes' stage and aneuploidy and probably a significant correlation between histologic grading and aneuploidy. The clinical significance of these results lies in the fact that DNA can be measured in biopsy specimens. It might thus be possible to "tailor" the operation according to the future clinical course to be expected. It could be hypothetically argued that patients with a DNA profile heralding disseminated disease and short life expectancy should have surgery that preserves quality of life, whereas those tending to develop a local recurrence should have more aggressive surgery. It may also be possible to define groups of patients thought to prosper from a more intense postoperative surveillance. The scientific basis for these suggestions is still lacking, and further studies on a prospective basis are currently in progress.

The prognostic significance of DNA measurements in endometrial carcinoma.

DNA analysis was performed in 71 cases of endometrial carcinoma, selected from a retrospective series of 445 cases registered at the Department of Gynecology, Radiumhemmet, Karolinska Hospital, Stockholm, during 1973-1975. The histological material from 37 patients surviving more than eight years was compared with that from those who died from cancer within two years. The prognostic value of the DNA distribution pattern of the tumors in relation to the clinical stage and the histological grade of the tumors was evaluated. Patients with near-diploid or -tetraploid tumors were found to have a significantly lower death rate than those with aneuploid tumors. The DNA distribution pattern was also found to correlate better with the survival rate than the clinical stage or the histological grade of the tumors.

Prognostic significance of nuclear DNA content in serous ovarian tumors.

Specimens from 73 serous ovarian cancers were examined with respect to DNA content of the tumor cells. The prognostic value of DNA analysis, as reflected in patient survival, was retrospectively compared with that of conventional histological assessment of cancer. DNA in individual tumor cells was measured in sections from the original paraffin-embedded specimens. High proportions of cells with very high DNA values were identified in tumors from 16 patients, 10 of whom died of the disease during the follow-up period. The histological classification was invasive adenocarcinoma in 47 tumors and borderline cancer in 26. All of the patients who died had invasive adenocarcinoma. Although both DNA analysis and histological evaluation were sensitive predictors of mortality, the specificity of the DNA method was distinctly higher (0.90 versus 0.42). The study suggests that analysis of the DNA content of tumor cells can be a useful supplement to histological assessment of cancer and accordingly can significantly assist in the planning of treatment.