Early detection of precancerous lesions in dysplasias of the lung by rapid DNA image cytometry

Hematoxylin-and-eosin-stained cytologic smears of sputum from 28 patients with dysplastic and suspicious cell findings were subjected to DNA image cytometry after Feulgen restaining. The nuclear DNA contents were measured with a TV-based image-analysis system, the Leitz TAS plus, combined with an automatic microscope. Computation of DNA data was performed according to an algorithm for the diagnosis and grading of malignancy. Of the 19 cases that were proven to be malignant in the follow-up, either by histologic examination, sputum cytology, fine needle aspiration biopsy or autopsy, the algorithm identified 17 as malignant in a stage (dysplasia) in which cytology was not yet able to present a definite diagnosis of malignancy. Only two cases of bronchial carcinoma were not detected in the state of dysplasia by this procedure. The periods between the DNA diagnosis of malignancy in dysplasia and the morphologic evidence of cancer varied from three days up to six months. Of the 11 cases that had been classified as benign by the algorithm, 9 were confirmed as benign during the clinical follow-up. Rapid DNA image cytometry appears able to separate squamous dysplasias of the lung into precancerous and nonprecancerous lesions.     

Rapid diagnostic DNA cytometry with an automatic microscope and a TV image-analysis system

A TV-based image-analysis system (Leitz TAS plus) combined with a Leitz automatic microscope was used for rapid DNA cytometry for diagnostic purposes. Malignant or suspicious cells found by the cytopathologists in conventionally stained smears were automatically relocated and measured. A program for automatic detection and measurement of nuclear DNA content was developed. DNA data are processed by an algorithm for the diagnosis and grading of malignancy. This diagnostic procedure is performed in real time and yields highly reproducible results. With this equipment, diagnostic DNA cytometry may be introduced in the routine cytology laboratory. Our mode of application does not replace the cytopathologist, but assists him or her in cases of doubtful diagnosis and in the grading of malignant tumors.     

Cytophotomeric DNA-analysis of aspirated cells from prostatic carcinoma.

In the present study material obtained from prostatic lesions by transrectal aspiration biopsy was subjected to a comparative morphologic and cytophotometric DNA analysis. Based on the morphologic pattern, the clinical material was divided into benign lesions (prostatic hyperplasia), suspected prostatic malignancy and highly, moderately and poorly differentiated prostatic carcinoma. The cytochemical analysis, based on quantitative cytophotometric measurements of Feulgen stained nuclei, showed that cell nuclei from benign lesions (prostatic hyperplasia) exhibited the normal diploid amount of DNA. Contrary to this, cell populations from prostatic malignancies, were characterized by various degrees of heteroploidy, i.e. the existence of cells with nuclear DNA quantities increased above the normal diploid level. A general correlation between degree of heteroploidy (frequency of heteroploid cells) and degree of clinical malignancy seemed to exist; high grade malignant prostatic carcinoma (poorly differentiated) exhibited a pronounced degree of heteroploidy with more or less distinct aneuploid stemlines, whereas low-grade prostatic carcinomas (highly differentiated) were more similar to benign cell populations, in showing a large proportion of cells with a diploid DNA quantity suggesting the existence of diploid or near diploid stemlines. Cases morphologically classified as moderately differentiated prostatic carcinoma, which previously have been shown to exhibit individual variations in degree of clinical malignancy, also showed large individual variations in degree of hetyroploidy. Approximately half of these cases had cytochemical DNA characteristics similar to that of highly differentiated prostatic carcinoma in showing a modal DNA value in the diploid region, while the other half showed cytochemical characteristics similar to that of poorly differentiated prostatic carcinoma, i.e. aneuploid DNA distributions. However, no morphologiifferentiated prostatic carcinoma. In conclusion it can be stated that the present investigation suggests the possibility that quantitative cytochemical DNA analysis may be used in combination with, and offer additional information to, morphologic analysis in the malignancy grading of prostatic carcinoma. A future clinical follow-up, now in progress, will hopefully give a more definite answer to that idea.     

Susceptibility Weighted Imaging: A New Tool in the Diagnosis of Prostate Cancer and Detection of Prostatic Calcification

Background

Susceptibility weighted imaging (SWI) is a new MRI technique which has been proved very useful in the diagnosis of brain diseases, but few study was performed on its value in prostatic diseases. The aim of the present study was to investigate the value of SWI in distinguishing prostate cancer from benign prostatic hyperplasia and detecting prostatic calcification.

Methodology/Principal Findings

23 patients with prostate cancer and 53 patients with benign prostatic hyperplasia proved by prostate biopsy were scanned on a 3.0T MR and a 16-row CT scanner. High-resolution SWI, conventional MRI and CT were performed on all patients. The MRI and CT findings, especially SWI, were analyzed and compared. The analyses revealed that 19 out of 23 patients with prostate cancer presented hemorrhage within tumor area on SWI. However, in 53 patients with benign prostatic hyperplasia, hemorrhage was detected only in 1 patient in prostate by SWI. When comparing SWI, conventional MRI and CT in detecting prostate cancer hemorrhage, out of the 19 patients with prostate cancer who had prostatic hemorrhage detected by SWI, the prostatic hemorrhage was detected in only 7 patients by using conventional MRI, and none was detected by CT. In addition, CT demonstrated calcifications in 22 patients which were all detected by SWI whereas only 3 were detected by conventional MRI. Compared to CT, SWI showed 100% in the diagnostic sensitivity, specificity, accuracy, positive predictive value(PPV) and negative predictive value(NPV) in detecting calcifications in prostate but conventional MRI demonstrated 13.6% in sensitivity, 100% in specificity, 75% in accuracy, 100% in PPV and 74% in NPV.

Conclusions

More apparent prostate hemorrhages were detected on SWI than on conventional MRI or CT. SWI may provide valuable information for the differential diagnosis between prostate cancer and prostatic hyperplasia. Filtered phase images can identify prostatic calcifications as well as CT.     

Flow cytometric DNA analysis combined with fine needle aspiration biopsy in the diagnosis of palpable metastases

To determine the diagnostic value of flow cytometric (FCM) DNA analysis in combination with fine needle aspiration (FNA) biopsy, the nuclear DNA content was analyzed in FNA specimens from 155 superficial metastases and 60 benign lymph nodes. The results of FCM DNA analysis were considered to be positive for malignancy if DNA aneuploidy was found or if more than 12% S+G2M cells were present in the aspirate in diploid cases. The diagnostic accuracy of FCM DNA analysis alone in detecting malignancy was 92%, with a sensitivity of 91% and a specificity of 95%. FCM suggested the correct diagnosis in 5 of the 7 cytologically false-negative cases and in 9 of the 12 cytologically indeterminate or suspicious cases.     

Determinación de la certeza diagnóstica de la gammagrafía tiroidea con tecnecio 99 sestamibi en pacientes con nódulo tiroideo y resultado histopatológico definitivo

Background99mTc sestamibi scanning and aspiration biopsy can predict the histopathological result of a thyroid nodule fairly accurately.ObjectiveTo determine the accuracy of 99mTc sestamibi scanning in detecting malignancy in patients with thyroid nodule confirmed by definitive histopathological report after thyroidectomy.Material and methodsA total of 69 patients with a solitary thyroid nodule were studied. In all patients, fine needle aspiration, total thyroidectomy for suspected thyroid cancer, and histological analysis of the surgical specimen were performed. There were 54 patients with a positive 99mTc sestamibi scan; of these, malignancy was confirmed by histological analysis in 25 and excluded in 29. There were 15 patients with a negative 99mTc sestamibi scan; of these, three had a final diagnosis of cancer and 12 were confirmed as cancer-free.ResultsThe diagnostic accuracy of 99mTc sestamibi scanning in detecting malignancy in thyroid nodules was determined through a statistical analysis. 99mTc sestamibi scan for thyroid cancer had a sensitivity of 89.28% and a specificity of 29.25%. The positive predictive value was 46.29% and the negative predictive value was 80%.ConclusionsWe believe that 99mTc sestamibi scan should be routinely used in all patients with a thyroid nodule and an indeterminate result on fine needle aspiration. This procedure is most useful in excluding malignancy in patients with a negative 99mTc sestamibi scan.     

DNA cytometric analysis of surgically treated squamous cell cancer of the uterine cervix, stage pT1b1-pT2b

OBJECTIVE: To determine the utility of DNA content and DNA-related variables of proliferative activity regarding prognosis in cervical cancer. STUDY DESIGN: DNA image (ICM) andflow cytometry (FCM) were performed to determine the DNA index (DI), 5c-exceeding rate (5c-ER), S-phase fraction (SPF) and proliferation index (PI) in 163 patients with surgically staged pT1b1-pT2b squamous cell cancer of the uterine cervix and treated with primary radical hysterectomy. ICM was performed on imprint cytology, obtained from fresh tumor tissue, which was also used for FCM. Results were analyzed using the chi2 test and Cox regression analysis for risk of pelvic lymph node involvement, tumor recurrence and recurrence-free survival (RFS). RESULTS: ICM was performed on all 163 and FCM on 133 samples. One-third of the tumors showed DNA aneuploidy. Analysis demonstrated prognostic significance of a DI > or = 1.70, with a (70:30) 2.3-fold risk of recurrence (P=.024) and reduced RFS of 10 months (P=.003) in cases of DI > or = 1.70. A high 5c-ER > 11% was associated with pelvic lymph node involvement and decreased RFS (P < or = .04). Significantly more relapses were found in tumors with SPF > 12% (70.8% vs. 29.2%, P=.007). RFS was markedly reduced in tumors with high SPF (52.3 vs. 61.1 months, P=.011). Low proliferative tumors (PI<25%) were associated with lower stage (P=.036) and increased RFS (61.2 vs. 47.1 months, P=.028). In multivariate analysis of clinicopathologic variables (pT category, nodal status, lymphovascular space involvement) and DNA related variables, pelvic lymph node involvement was the only significant predictor of RFS. In patients with nodal involvement, tumors with DI >1.70 were associated with lessfavorable outcomes. CONCLUSION: DNA-related variables of cell cycle analysis were valuablefor predicting prognosis in cervical cancer patients. Tumors with DI>1.70, 5c-ER >11% and high proliferative activity (SPF>12%, PI>25%) represent a subgroup with a poor prognosis.     

Computer-assisted grading of adenocarcinoma in prostatic aspirates

Conventional cytologic grading of fine needle aspirates of prostatic adenocarcinoma has been shown neither to be reproducible nor to correlate well with histologic grading. This study developed a tumor grade classification based on computerized cytomorphometric features and compared the results to conventional grading of companion tissue sections. The image analysis system evaluated architectural features of the aspirates (mainly cell cluster features and interrelationships) as well as nuclear features. Thirty-five prostatic adenocarcinomas (8 well, 19 moderately and 8 poorly differentiated) were evaluated. Discriminant functions based on data collected at medium and high resolution distinguished between aspirates from low-grade (well-differentiated) and high-grade (poorly differentiated) adenocarcinomas with 81% accuracy. Moderately differentiated cancers could not be classified as a distinct group. This study suggests that accurate grading of prostatic adenocarcinoma in fine needle aspirate smears requires the evaluation of medium-resolution features related to specimen cellularity and uniformity or crowding of cell clusters as well as of high-resolution features of nuclear area, perimeter and coarseness of chromatin texture. These findings are compared to those of other schemes for the cytologic grading of prostatic aspirates.     

DNA grading of malignancy in breast cancer. Prognostic validity, reproducibility and comparison with other classifications

The prognostic significance of the "DNA malignancy grade" (DNA-MG) was tested in a series of 104 breast cancer patients in comparison with TNM staging, histomorphologic grading according to Bloom and Richardson, mean nuclear area (MNA) and DNA-histogram classification according to Auer. The reproducibility and representativity of the grading systems were investigated, and their results in primary tumors and lymph node metastases were compared. The scalar DNA-MG was assessed on monolayer smears prepared from paraffin-embedded tissues; the smears were automatically Feulgen stained and used for rapid interactive DNA cytometric evaluation by an automated microscope and a TV image-analysis system. TNM staging showed the highest correlation with survival, followed by histomorphologic grading and DNA-MG; MNA and the DNA-histogram classification failed to give statistically significant prognostic information. Both histomorphologic grading and DNA-MG were identified as parameters adding independent prognostic information to the TNM staging. However, only DNA-MG demonstrated an acceptable reliability, with small 95% ranges between repeated measurements within the primary tumor (+/- 0.3 DNA-MG) and a strong correlation between the results in the primary tumor and its lymph node metastases. These findings show that the DNA-MG is a valid and reliable prognostic index that adds significant prognostic information to TNM staging.     

Use of nuclear image cytometry, histopathological grading and DNA cytometry to make breast cancer prognosis more objective.

Feulgen-stained tissue sections of 187 invasive ductal carcinomas (94 with lymph node metastases; mean follow-up: 44 months) were studied using computer assisted image cytometry. Based on survival time, the prognostic significance of nuclear image analysis was compared with the results using conventional histopathological grading according to Bloom and Richardson, as well as with image cytometric DNA measurements. The histopathological grading has the disadvantage of poor interobserver reproducibility (71.1%). Despite statistically significant differences between the actuarial survival curves of grade 1 and grade 3 patients, the prognostic significance of the conventional grading method for individual patients seems to be low and the number of grade 2 cases (42.8%) is large. The quantitative morphological method for analyzing nuclear images gives more reproducible results. Compared to histopathological grading, the predictive values for good or poor prognosis are clearly higher and the number of cases with uncertain prognosis is significantly smaller (20.9%). DNA ploidy measurements also make it possible to distinguish statistically significant differences between favorable and unfavorable prognoses with respect to over-all survival time. However, the classification accuracy based on the best single parameter (DNA-histogram type according to Auer) is 70.2% compared with 78.9% for nuclear image analysis.     

Cytology of prostatic carcinoma. Quantification and validation of diagnostic criteria

The validity of diagnostic criteria for the cytologic differentiation between benign and malignant cases and between normal (hyperplastic) and inflammation-activated (prostatitis) prostatic cells was tested by means of semiautomated image analysis and cytophotometry. The following criteria were found to have significant differences between benign and malignant prostatic cells: nucleoli (number, size and size variability), regularity of nuclear arrangement, anisonucleosis, nuclear size, nuclear polymorphism and dissociation of cells. The following criteria were found to be invalid for the detection of prostatic cancer cells: nuclear-cytoplasmic ratio, hyperchromasia and anisochromasia. the cytologic diagnosis of prostatic cancer was achieved by observing a variable constellation of at least three of six pathognomically altered diagnostic criteria. No fixed pattern of diagnostic criteria was found either for prostatic cancer cells in general or for the different malignancy grades.     

Combination of Autoantibody Signature with PSA Level Enables a Highly Accurate Blood-Based Differentiation of Prostate Cancer Patients from Patients with Benign Prostatic Hyperplasia

Although an increased level of the prostate-specific antigen can be an indication for prostate cancer, other reasons often lead to a high rate of false positive results. Therefore, an additional serological screening of autoantibodies in patients’ sera could improve the detection of prostate cancer. We performed protein macroarray screening with sera from 49 prostate cancer patients, 70 patients with benign prostatic hyperplasia and 28 healthy controls and compared the autoimmune response in those groups. We were able to distinguish prostate cancer patients from normal controls with an accuracy of 83.2%, patients with benign prostatic hyperplasia from normal controls with an accuracy of 86.0% and prostate cancer patients from patients with benign prostatic hyperplasia with an accuracy of 70.3%. Combining seroreactivity pattern with a PSA level of higher than 4.0 ng/ml this classification could be improved to an accuracy of 84.1%. For selected proteins we were able to confirm the differential expression by using luminex on 84 samples. We provide a minimally invasive serological method to reduce false positive results in detection of prostate cancer and according to PSA screening to distinguish men with prostate cancer from men with benign prostatic hyperplasia.     

Prostate cancer vs hyperplasia: relationships with prostatic and adipose tissue fatty acid composition

The objective of the present study was to study whether adipose tissue and prostatic tissue fatty acid composition differentiates between prostate cancer and benign hyperplasia patients. In addition, the present investigation aimed at exploring the extent to which prostatic tissue fatty acid composition differentiates between prostate-confined cancer and extraprostatic disease including possible metastasis. The subjects were 71 male patients from the island of Crete. Half the patients (n=35) had been diagnosed with benign hyperplasia of the prostate, half with prostatic malignancy (n=36). Patients were examined at the outpatient clinic of the urology unit, University Hospital, Medical School, University of Crete. Relative to benign hyperplasia patients, cancer patients had elevated adipose tissue saturated and reduced monounsaturated fatty acid levels. Cancer patients had reduced prostate tissue stearic to oleic acid ratios and stearic acid levels as opposed to hyperplasia patients. The most pronounced difference between cancer patients and hyperplasia patients was a 3-fold elevated prostatic palmitoleic acid in the former group. Relative to benign hyperplasia patients, cancer patients had reduced prostate tissue arachidonic and docosahexaenoic acid levels. Finally, there was a significantly reduced omega-3/omega-6 polyunsaturated fatty acid ratio in the prostate cancer patient as opposed to the benign hyperplasia group. The pronounced elevations in prostatic tissue palmitoleic acid in cancer patients highlight a possible role of this fatty acid in neoplastic processes. The decreased arachidonic acid levels in cancer patients possibly stem from enhanced metabolism of arachidonic acid via lipoxygenase and cyclooxygenase pathways, and the formation of derivatives such as 5-HETE, 15-HETE, 12(S)-HETE and PGE(2).     

Clinical applications of image cytometry to human tumour analysis

Image cytometry (ICM) is widely applied to the automated screening, the detection, the diagnosis, the classification, the prognosis and the therapeutic follow-up of different types of cancers (breast, bladder, cervix,...). This review describes the analysis methods and the applications of nuclear image analysis, the determination of DNA content and the analysis of morphometry and of nuclear texture. DNA content analysis can contribute to a prognostic information in addition to other prognostic factors for breast, renal and prostate cancers. For ovarian cancer, aneuploidy seems to be related to prognosis. Bladder tumours with DNA aneuploidy were frequently of high malignancy while ploidy was significantly correlated to relapse risk. For digestive cancers, patients presenting DNA diploid tumours show a better survival than patients with aneuploid ones. Morphometry seems to be a more important criterion than other conventional prognostic factors of invasive breast and digestive carcinomas. A differential diagnosis between normal and neoplastic thyroids is more precise when based on a quantitative evaluation of texture associated to morphometry. Textural parameters permit the discrimination of two populations of patients having a different prognosis and could thus be an aid for prognosis in prostatic cancers. Morphonuclear parameters contribute to separate low and high grade bladder carcinomas. Although ICM was frequently reported, results from the reported examples were not always obvious. In conclusion, the measurements obtained with ICM could be helpful for a decision in several cancers but could not be a substitute for the classical approach of the pathologist.     

Nuclear DNA in histologic sections from squamous-cell carcinoma and adenocarcinoma of the lung

The nuclear DNA content in morphologically identified tumor cells was analyzed in 4-micron histologic sections from 58 patients with lung carcinoma who survived for at least five years. Thirty-three of the carcinomas were invasive squamous bronchial carcinomas and 25 were pulmonary adenocarcinomas. In all squamous carcinomas, the majority of tumor cells were found to exhibit DNA values exceeding the normal tetraploid and/or diploid region. In contrast, some of the pulmonary adenocarcinomas were found to be composed of a majority of tumor cells with DNA values in the normal diploid region. The results indicate that invasive squamous bronchial carcinomas, in general, are tumors with aneuploid DNA patterns indicative of a high malignant potential and that malignancy grading based on DNA measurements does not add any significant prognostic information to that obtained by morphologic diagnosis.     

Posters. P17 Accuracy of fine needle aspiration cytology in diagnosis of thyroid swellings

Introduction Fine needle aspiration cytology is regarded as the gold standard investigation in diagnosis of thyroid swellings. Published data suggest an overall accuracy rate of 75% 1 in the detection of thyroid malignancy. The aim of this study was to determine the accuracy of FNA cytology in detection of thyroid malignancy in our surgical unit. Methods Between 1989–2002, 144 patients who underwent thyroid resection by single consultant surgeon and who had pre‐operative FNA were enrolled in this retrospective study. The pre‐operative FNA results were compared with definitive histological diagnosis following thyroid resection. Fine needle aspiration cytology was performed using aspirate and non‐aspirate techniques on each thyroid swelling. The cytological sample was assessed by a single cytopathologist and was classified as inadequate, non‐neoplastic, neoplastic, suspicious or indeterminate. The histology was classified as non‐neoplastic (benign) and neoplastic (malignant). Results Fine needle aspiration cytology analysis revealed 94 (13.88%) non‐neoplastic, six (65.27%) neoplastic and 20 (4.16%) suspicious aspirates. Twenty (13.88%) samples were inadequate and four (2.77%) samples were indeterminate. Histological analysis showed 118 (81.94%) benign, 26 (18.05%) malignant specimens. Fine needle aspiration cytology had a sensitivity, specificity and accuracy rate of 52.6%, 86.6% and 79.1%, respectively for diagnosing thyroid malignancy. Conclusion The results are comparable with the current published data and demonstrate that FNA cytology in our hands is accurate investigation for pre‐operative diagnosis for the detection of thyroid malignancy.     

Diagnosis of gastric cancer.

A prospective comparison was made of the accuracy of different diagnostic methods for gastric cancer. The basis of the study was a consecutive series of 113 patients thought to have gastric pathology; cancer was the final diagnosis in 32. Endoscopy and radiology were the most accurate investigations, whereas biopsy, cytology, and clinical examination gave disappointing results. A wide range of clinical features and laboratory investigations were studied in all patients in an attempt to identify criteria suggestive of malignancy. Multifactorial computer analysis of these investigations failed to improve upon the radiological diagnosis. A systemic approach designed to make optimal use of limited endoscopic and histopathological resources in the diagnosis of gastric lesions is presented.     

Host Resistance in Relation to Survival in Breast Cancer

The degree of lymphocytic infiltration in and around breast tumours together with sinus histiocytosis and follicular hyperplasia in regional lymph nodes has been studied in 310 cases of breast cancer treated with standard radical mastectomy. The presence of these features was regarded as evidence of host resistance against the tumour and made possible the division of patients into three classes—no or poor reaction, good reaction, and strong reaction. The grading was shown to have a close correlation with prognosis. The relationship between host defensive factor grading and malignancy, nodal metastases, and survival was also examined. The results support the hypothesis that prognosis in breast cancer is closely related to a histological picture of cell-mediated immunity against the tumour.     

DNA-cytometric diagnosis of prospective malignancy in borderline lesions of the uterine cervix

Interactive DNA cytometry was used for the diagnosis of prospective malignancy in 48 smears with borderline lesions (mild and moderate dysplasias) of the uterine cervix. In addition, 183 smears with benign squamous epithelia, 38 with carcinoma in situ and 7 with invasive squamous carcinoma were also measured. Nuclear Feulgen-DNA measurements were performed using various methods, and the resulting data were analyzed by an algorithm for a DNA-cytophotometric diagnosis of malignancy. The results were compared with the data on follow-up and subsequent histologic studies in these cases. There was no false-positive diagnosis in the 183 benign smears and only 1 false-negative diagnosis in the 76 histologically proven squamous-cell carcinomas, which yields a specificity of 100% and a sensitivity of 98.6%. The sensitivity for the detection of subsequent histologically proven malignancy in cases with cytologically mild or moderate dysplasia amounted to 97%. In 13 borderline cases, there was a mean interval of 21 months between the taking of the cytologic smear on which the DNA diagnosis of malignancy was made and the date on which the histologic confirmation of malignancy was made. In 17% of the cytologically dysplastic cases, the DNA diagnosis of malignancy was not verified by subsequent histologic investigation. These results indicate that interactive DNA cytometry is able to detect prospective malignancy in smears from borderline lesions of the uterine cervix with a high sensitivity.     

Automatic segmentation of cell nuclei in Feulgen-stained histological sections of prostate cancer and quantitative evaluation of segmentation results

Digital image analysis of cell nuclei is useful to obtain quantitative information for the diagnosis and prognosis of cancer. However, the lack of a reliable automatic nuclear segmentation is a limiting factor for high-throughput nuclear image analysis. We have developed a method for automatic segmentation of nuclei in Feulgen-stained histological sections of prostate cancer. A local adaptive thresholding with an object perimeter gradient verification step detected the nuclei and was combined with an active contour model that featured an optimized initialization and worked within a restricted region to improve convergence of the segmentation of each nucleus. The method was tested on 30 randomly selected image frames from three cases, comparing the results from the automatic algorithm to a manual delineation of 924 nuclei. The automatic method segmented a few more nuclei compared to the manual method, and about 73% of the manually segmented nuclei were also segmented by the automatic method. For each nucleus segmented both manually and automatically, the accuracy (i.e., agreement with manual delineation) was estimated. The mean segmentation sensitivity/specificity were 95%/96%. The results from the automatic method were not significantly different from the ground truth provided by manual segmentation. This opens the possibility for large-scale nuclear analysis based on automatic segmentation of nuclei in Feulgen-stained histological sections.