DNA image cytometry predicts disease outcome in stage II colorectal carcinoma.

BACKGROUND: Approximately 30% of all colorectal cancer (CRC) patients are diagnosed with stage II disease. Adjuvant therapy is not widely recommended. However, it is well established that a subgroup of patients with stage II are at high risk for recurrence within their lifetime and should be considered for adjuvant chemotherapy. The present work was designed to study the prognostic value of nuclear DNA content in stage II CRC of patients with long-term followup. PATIENTS AND METHODS: Isolated nuclei from 50 microm-thick paraffin sections of tissue samples from 253 patients with stage II CRC, who had undergone bowel resection at Turku University Central Hospital were cytocentrifuged on slides, stained with Feulgen staining, and DNA was measured using a computer-assisted image analysis cytometry system. Different approaches were applied in analysis of DNA histograms. RESULTS: DNA content did not show any relation with age (p < 0.96), sex (p < 0.35), tumor invasion (p < 0.77), or grade (p < 0.31). Aneuploid DNA content was significantly more frequent in the cancer of the left colon and rectum than the right colon (p = 0.02). S-phase fraction analysis revealed that a higher proportion (62%) of the older patients (>65 years) had high proliferation rates than did the younger patients (p < 0.05). Patients with narrow range histograms had a better disease-free survival (DFS) (narrow range: 70%, wide range: 60% at 10 years). Tumors with >9c nuclei were associated with significantly better DFS and disease-specific survival (DSS) as compared with the patients who did not have >9c nuclei in their tumor samples (p < 0.003 and p < 0.0001, respectively). Multivariate survival (Cox) model showed that only classification of the basic pattern of the histogram [odds ratio OR) = 29.14; 95% confidence interval (CI) 2.350-361.57] (p = 0.009) and recurrence (OR = 165.35; 95% CI 48.42-564.7) (p = 0.0001) proved to be independent predictors of clinical outcome. CONCLUSION: Our results seem to suggest it truly is possible, by using DNA cytometry, to find groups with different prognosis among stage II cases. Those with a high recurrence rate should be considered for adjuvant chemotherapy.

PLA2 (group IIA phospholipase A2) as a prognostic determinant in stage II colorectal carcinoma.

BACKGROUND: Approximately 30% of all colorectal cancer (CRC) patients are diagnosed with stage II disease. Adjuvant therapy is not widely recommended. However, it is well established that a subgroup of patients with stage II are at high risk for recurrence within their lifetime and should be considered for adjuvant chemotherapy. The present work was designed to assess the value of group IIA phospholipase A2 (PLA2) as a predictor of disease outcome in stage II CRC patients with long-term follow-up. PATIENTS AND METHODS: The present study comprises a series of 116 patients who underwent bowel resection for stage II CRC during 1981-1990 at Turku University Hospital. Archival paraffin-embedded CRC tissue samples were used to prepare tissue microarray blocks for immunohistochemical staining with PLA2. RESULTS: Fifty-five percent of all tumors were positive for PLA2. There was no significant correlation between PLA2 expression and age, sex, depth of invasion and lymph node status. In Kaplan-Meier survival analysis, there was a significant (P = 0.010) difference in disease-free survival (DFS) between patients with negative tumors (longer DFS) and those with positive tumors. The same was true with disease-specific survival (DSS), patients with PLA2-negative tumors living significantly longer (P = 0.025). In multivariate (Cox) survival analysis, however, PLA2 was not an independent predictor of DFS or DSS. In subgroup analysis, the right-sided tumors with negative PLA2 staining had remarkably better prognosis (P = 0.010) than PLA2-positive left-sided tumors. CONCLUSIONS: Quantification of PLA2 expression seems to provide valuable prognostic information in stage II CRC, particularly in selecting the patients at high risk for recurrent disease who might benefit from adjuvant therapy.

Expression of the cell-cell adhesion molecule beta-catenin in colorectal carcinomas and their metastases.

To study the dynamic events leading to impaired cell-cell adhesion upon transition to the invasive phenotype of colorectal cancer (CRC), we examined three distinct beta-catenin expression patterns (membranous, cytoplasmic, and nuclear) in the paired samples of the primary tumours (P) and their metastatic lesions (M). beta-catenin expression was detected by immunohistochemistry (IHC) in 33 pairs of the primary CRC and their metastases. In a pair-wise (P-M) comparison, the membranous index (MI) was significantly different between P and M (p=0.036, Wilcoxon Signed-Ranks test), while cytoplasmic index (CI) and nuclear index (NI) values did not significantly deviate between P and M. MI in primary tumours was inversely related to the patient's age (p=0.04) and tumour grade (p=0.03), while patients with low MI in M had a high rate of metastasis at diagnosis (p=0.06). CI in P was lower in patients with LN involvement (p=0.02) and in advanced tumour stage (p=0.002). Tumours of the ascending colon had the highest CI in their M (p=0.04). Interestingly, high MI of the M lesions was a significant predictor of favourable overall survival (OS) in univariate (Kaplan-Meier) survival analysis (p=0.035). In conclusion, significant aberrations in beta-catenin expression probably take place in CRC cells during the development of metastatic phenotype, but a change from membrane expression to cytoplamic and/or nuclear expression is not a prerequisite for metastasis in all cases.

Lymph node status as a guide to selection of available prognostic markers in breast cancer: the clinical practice of the future?

Prognosticators evaluating survival in breast cancer vary in significance in respect to lymph node status. Studies have shown e.g. that HER2/neu immunohistochemistry or HER2/neu gene amplification analysis do perform well as prognosticators in lymph node positive (LN +) patients but are less valuable in lymph node negative (LN -) patients. We collected data from different studies and tried to evaluate the relative significance of different prognosticators in LN+/LN- patient groups. In LN+ patients HER2/neu and E-cadherin immunohistochemistry were the statistically most significant prognosticators followed by proliferation associated features (mitotic counts by SMI (standardised mitotic index) or MAI (mitotic activity index), or S-phase fraction). Bcl-2 immunohistochemistry was also significant but p53 and cystatin A had no significance as prognosticators. In LN- patients proliferation associated prognosticators (SMI, MAI, Ki-67 index, PCNA immunohistochemistry, S-phase fraction) are especially valuable and also Cathepsin D, cystatin A, and p53 are significant, but HER2/neu or bcl-2, or E-cadherin less significant or without significance. We find that in studies evaluating single prognosticators one should distinguish between prognosticators suitable for LN+ and LN- patients. This will allow the choice of best prognosticators in evaluating the prospects of the patient. The distinction between LN+ and LN- patients in this respect may also be of special value in therapeutic decisions.

Comparison of CD44 expression in primary tumours and metastases of colorectal cancer.

A better understanding on the development of a metastatic phenotype in colorectal cancer (CRC) is essential to help identify patients at high risk for metastasis. Therefore, we have studied the role of the CD44 family of trans-membrane glycoprotein in the process of CRC metastasis, by examining the expression of CD44s and CD44v6 in primary tumours and their metastatic lesions in 46 patients using immunohistochemistry. The expression of both CD44s and CD44v6 was significantly higher (moderate/strong) in primary tumours as compared to their metastases (p=0.008, p=0.0001, respectively). CD44s expression in metastases increased with the degree of the histological grade (p=0.009) and invasiveness of the primary tumour (p=0.002). Disease-free survival (DFS) was shorter in patients who had metastases with a strong/moderate expression of CD44s as compared to those with negative/weak expression (8.3 months vs 16.9 months p=0.221, respectively). Our finding that CD44s expression in metastatic lesions may reflect the aggressiveness of the primary tumour from which it has originated implicates an important link between the two lesions. CD44 expression may also provide valuable biological information as suggested by the observation that up-regulated CD44s expression in metastases is associated with a shorter DFS.

Prognostic factors in prostate cancer.

Prognostic factors in organ confined prostate cancer will reflect survival after surgical radical prostatectomy. Gleason score, tumour volume, surgical margins and Ki-67 index have the most significant prognosticators. Also the origins from the transitional zone, p53 status in cancer tissue, stage, and aneuploidy have shown prognostic significance. Progression-associated features include Gleason score, stage, and capsular invasion, but PSA is also highly significant. Progression can also be predicted with biological markers (E-cadherin, microvessel density, and aneuploidy) with high level of significance. Other prognostic features of clinical or PSA-associated progression include age, IGF-1, p27, and Ki-67. In patients who were treated with radiotherapy the survival was potentially predictable with age, race and p53, but available research on other markers is limited. The most significant published survival-associated prognosticators of prostate cancer with extension outside prostate are microvessel density and total blood PSA. However, survival can potentially be predicted by other markers like androgen receptor, and Ki-67-positive cell fraction. In advanced prostate cancer nuclear morphometry and Gleason score are the most highly significant progression-associated prognosticators. In conclusion, Gleason score, capsular invasion, blood PSA, stage, and aneuploidy are the best markers of progression in organ confined disease. Other biological markers are less important. In advanced disease Gleason score and nuclear morphometry can be used as predictors of progression. Compound prognostic factors based on combinations of single prognosticators, or on gene expression profiles (tested by DNA arrays) are promising, but clinically relevant data is still lacking.

Ki67 immunohistochemistry: a valuable marker in prognostication but with a risk of misclassification: proliferation subgroups formed based on Ki67 immunoreactivity and standardized mitotic index.

AIMS: Counting mitotic figures is considered to be a reliable prognosticator, but evaluation of Ki67 immunohistochemistry has become more popular in evaluating proliferation. Our previous studies suggested an occasional discrepancy between mitotic figures and Ki67 fraction. The aim of this study was to investigate this more closely and also to study the associations between bcl-2 and p53 expression and proliferation. METHODS AND RESULTS: Two hundred and sixty-five infiltrating breast carcinomas were immunostained for Ki67, p53 and bcl-2. The standardized mitotic index (SMI) was determined. Four proliferation groups were based on Ki67 positivity fraction and SMI at optimal cut-off points. Cox's multivariate model was used to test the power of the prognosticators. SMI and nodal status were the most powerful individual prognosticators. Ki67 was an independent prognosticator if nodal status, tumour size, age and histological grade were included in the analysis but not if analysed with SMI. The group with low SMI and low Ki67 fraction had the best prognosis. Groups with high SMI had the poorest prognosis. The group with low SMI and high Ki67 fraction had a favourable prognosis. Bcl-2 negativity and p53 positivity correlated with proliferation. CONCLUSIONS: We have found a 'wrong positive' Ki67 group with favourable prognosis. SMI cannot be replaced by Ki67 because of the danger of misclassification of some patients.

DNA image cytometry is a useful adjunct tool in the prediction of disease outcome in patients with stage II and stage III colorectal cancer.

BACKGROUND: We assessed the prognostic value of the nuclear DNA content measured in the primary tumours of 123 patients with stage II or stage III colorectal cancer (CRC). METHODS: Isolated nuclei from paraffin sections were stained with the Feulgen reaction, and DNA was measured using a computer-assisted image analysis cytometry system. We applied 4 different approaches in the analysis of DNA histograms: the ABCDE approach, histogram range, peak evaluation and DNA cut-off values. RESULTS: Using the histogram range, the narrow range was rare (3.7%) in patients who died of disease (n = 28) as compared with 16.4% among those alive (n = 74; p = 0.017). Modal peak evaluation was a significant predictor of disease-free survival (DFS; Kaplan-Meier log-rank p = 0.0235). In the range evaluation, the 1st set (low-start gates) was a significant predictor of DFS (log-rank p = 0.0121), where disease recurrence was closely associated with the widest range (1.8->10c; c = haploid DNA content) gates. Recurrence-free survival was 3 times better in narrow-gate histograms than wide-range histograms (p < 0.03). The 1st set also proved to be a significant predictor of disease-specific survival (DSS; log-rank p = 0.0045), which was markedly better (77.8-90.0%) among the patients with the narrow-gate histograms. Grading of the histogram range into 2 categories (with 6.0c as cut-off), was a powerful predictor of both DSS (log-rank p = 0.0092) and 5-year DFS (p = 0.0106) in the whole series, and separately in stage III (but not stage II) disease, with p = 0.0131 and p = 0.0201, respectively. CONCLUSION: The DNA image cytometry with careful analysis of the histograms may provide valuable prognostic information in CRC, with potential clinical implications in patient management, particularly in predicting the patients at high risk for recurrence who should be considered as candidates for adjuvant therapy.

Value of electron microscopy in kidney biopsy diagnosis.

Kidney biopsy reports given during 2003 were collected from the authors' pathology database. A total of 111 biopsies were performed. Five tumor samples were not studied with electron microscopy (EM). Of the remaining 106 biopsies, 85 were studied with EM. EM was not performed in 10/24 transplant biopsies, or in 11/82 cases of suspected primary kidney disease. The role of EM was evaluated by grouping the samples in 3 categories: (1) EM was essential for diagnosis, (2) EM contributed to the interpretation and cleared uncertainties, and (3) EM had no influence on the diagnostic process. In transplant biopsies EM influenced the final diagnosis in 86% of cases (category 2). In biopsies performed for primary kidney disease EM was essential for diagnosis in 18.3% clearly contributed in 53.5%, and had no influence on the final diagnosis in 28.2% of cases. The study suggests that the importance of EM has not decreased during the last few years. Because only about 25% of the EM reports did not have any influence on the diagnostic process, it is recommended that kidney biopsy protocols should include EM in all biopsy cases, or at least tissue should be reserved for EM studies of all cases. Because of the influence of EM on the diagnostic process the need for EM in pathology training should be emphasized.

E-cadherin, CD44s and CD44v6 correlate with tumour differentiation in colorectal cancer.

Cell adhesion molecules (CAMs) are cell surface glycoproteins that are important in cell-cell and cell-matrix interactions and play an important role in cell growth and differentiation. We examined immunohistochemically CD44s, CD44v6 and E-cadherin expression in 86 formalin-fixed, paraffin-embedded primary tumours and 5 metastases. Lower levels of CD44s, CD44v6 and membranous E-cadherin expression were significantly associated with higher tumour grade (p=0.022, p=0.016 and p= 0.041, respectively). Moreover, CD44v6 and membranous E-cadherin expression were correlated with the depth of primary tumour invasion (p=0.030 and p=0.020, respectively), and increased expression of CD44v6 and decreased membranous E-cadherin expression were associated with increased primary tumour invasion. The results suggest that these CAMs are associated with tumour differentiation and invasion in locally advanced and metastatic colorectal carcinoma.

Response to chemotherapy (irinotecan plus 5-fluorouracil) in colorectal carcinoma can be predicted by tumour DNA content.

OBJECTIVE: The aim of this study was to identify markers that might predict response to chemotherapy. Postoperative chemotherapy improves the outcome in stage III colon cancer and is widely accepted as a standard therapy, but there are currently no reliable predictors to identify and select patients that will benefit. METHODS: Using DNA image cytometry, the DNA content was determined from the isolated nuclei of 56 primary colorectal carcinomas of patients who received chemotherapy (either irinotecan or irinotecan plus 5-fluorouracil and folinic acid) for advanced disease. Response to chemotherapy could be reliably evaluated in 53 patients. RESULTS: The modal DNA content (ploidy status) of the tumour correlated with the observed response to chemotherapy (p = 0.01). An objective response was observed in 56% of patients whose tumour histograms displayed tetraploid, peri-tetraploid or multiploid patterns of peaks, compared with 19% in patients with diploid, peri-diploid or aneuploid peaks. Notably, 86% (6/7) of patients whose tumours displayed a multiploid peak pattern showed an objective response and 1 patient had stable disease. CONCLUSIONS: This study suggests that modal DNA content can be used to predict a patient's response to chemotherapy in advanced colorectal carcinoma. This may help in identifying patients who will benefit most from therapy for advanced colorectal cancer.

Apoptotic index and bcl-2 expression as prognostic factors in colorectal carcinoma.

OBJECTIVES: To examine programmed cell death in 57 colorectal carcinomas (49 primary tumours and 8 metastases) and determine the prognostic significance of apoptosis in colorectal cancer. METHODS: Apoptotic index (AI) was ascertained by counting apoptotic bodies, using terminal deoxynucleotidyl transferase mediated digoxigenin nick end labelling (Tunel assay) and the expression of bcl-2 was examined immunohistochemically. Statistical analysis was used to test the value of clinical variables, histopathological data, AI and bcl-2 expression in predicting the clinical outcome of these patients and the survival function was calculated using the Kaplan-Meier method. RESULTS: AI was found to have a significant independent effect on survival (p = 0.0006), with lower values of AI conveying better survival. CONCLUSION: In summary, these findings reveal that AI is a useful prognostic factor in colorectal carcinoma.

Tubular differentiation in African breast cancer.

In an effort to determine numerical thresholds for histological breast cancer grading, we evaluated the fraction of fields with tubular differentiation (FTD) in Nigerian breast cancers (n=300). Analyses were based on Kaplan-Meier survival curves, and univariate and multivariate analyses by Cox's regression. The mean (SD) value of FTD in Nigeria, 16.7 (19.3)%, was much lower than reported in European breast cancer. Decreasing FTD was associated with increasing histological grades (p < 0.0001) and clinical stage (p = 0.0190). The most significant cut-point for FTD in predicting outcome was 15.0%. In univariate analysis, FTD 15% was a significant prognosticator in the whole material, in larger than 5cm tumours, among postmenopausal and premenopausal patients, and LN+ patients. After multivariate analysis with mitotic count, FTD was an independent prognosticator among tumours larger than 5cm, but not in other groups. We conclude that the rational grading of breast cancers need be optimised according to diagnostic and therapeutic environment. We propose FTD 15% as a cut-point to grade breast cancer in Nigerian material.

Morphometrical malignancy grading is a valuable prognostic factor in invasive ductal breast cancer.

The aim of the present study is to augment the prognostic power of breast cancer grading by elaboration of quantitative histopathological methods. We focus on the recently introduced morphometrical grading system in which the three grading sub-features of the WHO grading system are evaluated with the help of computerised nuclear morphometry, and quantitative methods for assessing mitotic activity and tubular differentiation. The prognostic value of the morphometrical grading system is now confirmed in a material of 159 cases of invasive ductal breast cancer. In the current material the morphometrical grading system very efficiently predicted the prognosis of breast cancer by dividing the patients into favourable (grade I), intermediate (grade II), and unfavourable (grade III) outcome (P<0.0001). The morphometrical grading system was especially efficient in identifying patients with the most unfavourable outcome. In our material the morphometrical grade III was associated with a 5.4-fold risk of breast cancer death. In light of the present results, the morphometrical grading can be applied to clinical use as an aid in treatment decisions of patients with invasive ductal breast cancer.

DNA cytometry in diagnostic cytology of the prostate gland.

BACKGROUND: Differential analysis and cytological grouping of fine needle aspiration biopsy (FNAB) samples of the prostate are important in practice. We used image analytical DNA cytometry to achieve this and also studied the best method of interpretation of the histograms. MATERIALS AND METHODS: Sixty-two FNAB samples of the prostate were stained with Feulgen stain and nuclear DNA histograms were produced by image cytometry. The most atypical cell groups were selected for measurements. Also, free epithelial cells between cell groups were studied. The cells presented in the histograms were grouped according to the nuclear DNA content and by application of different gates of observation for diploid status. RESULTS: Several DNA histogram features (histogram classification categories, benign and malignant histogram patterns, presence of >5c-7c cells) showed significant relationships to differential diagnosis. Highly aneuploid (>5c-7c) cells had the potential for distinguishing a progressive-type of prostate cancer. CONCLUSION: The fraction of tetraploid and aneuploid histograms increased from atypical but benign to definitely malignant samples. DNA histograms have potential in the differential diagnosis and evaluation of the progressive character of prostate cancer.

Proliferation in African breast cancer: biology and prognostication in nigerian breast cancer material.

Three hundred cases of invasive breast carcinoma from the University of Calabar Teaching Hospital, Nigeria were subjected to evaluation of proliferative activity by mitotic counts. The prognostic significance and association with other prognostic factors were evaluated. The mitotic activity was expressed as mitotic activity index (MAI), and standardized mitotic index (SMI). Pearson's correlation and univariate and multivariate Cox's regression were used. The mean follow-up time was 25.9 months. The mean values of SMI and MAI were 42.6 mitotic figures per square millimeter and 30.5 mitotic figures per 10 high-power fields, respectively, and these were much higher than values reported for Europe or other Western countries. The SMI had a positive correlation with tumor size (r = 0.31, P <.0001), histologic grade (r = 0.68, P <.0001), nuclear area (r = 0.45, P <.0001), and negative correlation with fraction of fields with tubular differentiation (FTD; r = -0.56, P = <0.0001). There was no statistically significant difference in the mitotic activity between the postmenopausal and the premenopausal patients. Also, lymph node-positive patients had higher counts than did lymph node-negative patients. Earlier determined grading associated decision thresholds divided the patients into groups of favorable and unfavorable prognosis. However, the statistically optimal thresholds for Nigerian material were different (32 and 92 mitotic figures per square millimeter for SMI). Tumor size of 5 cm, SMI, and MAI were independent prognostic factors. Nigerian breast cancers are high-grade, high-stage, and high-proliferating cancers occurring in a younger population than those of the Western countries. Proliferation is also more active. Evaluation of SMI or MAI can improve the distinction between aggressive and less aggressive variants of breast cancer.

Prognostication of invasive ductal breast cancer by quantification of E-cadherin immunostaining: the methodology and clinical relevance.

AIMS: We tried to improve the evaluation of E-cadherin immunostaining in paraffin sections, to distinguish the less aggressive variants of ductal infiltrating breast cancer from other variants. METHODS AND RESULTS: The method graded the membrane staining and estimated the fraction of area of cancer tissue stained at the respective staining grade, resulting in an immunohistochemical staining index. At the cut-point 0.35 the index divided all 157 patients (P=0.0188), and 57 node-positive patients (P= 0.0006) into two groups of different survival. In multivariate analysis (all patients) E-cadherin immunoscore was inferior to mitotic index (SMI) (P=0.0002), but still significant (P=0.0031). Among node-positive patients E-cadherin was even more powerful and superior (P=0.0001) to the still significant SMI (P=0.0023), and E-cadherin immunostaining and the mitotic activity (SMI) combined did not need the support of other prognosticators in the Cox model. CONCLUSIONS: The study suggests that E-cadherin immunostaining can be used efficiently in finding patients with favourable outcome among node-positive patients.

Unbiased morphological measurements show no neuronal loss in the substantia nigra in Alzheimer's disease.

This is the first study to use the unbiased stereological method, the disector, to estimate the total number of pigmented neurons in the pars compacta of the substantia nigra (SNpc) in Alzheimer's disease (AD) patients as compared to healthy controls. The right half of the SNpc of 11 AD patients and 24 controls was studied. We also used single sections to determine the neuronal number and area in different subregions of the SNpc. The results showed that there was no significant difference in the total number of pigmented neurons in the SNpc (154,415+/-13,593 for AD and 160,163+/-8027 for controls) or in the volume of the SNpc between the patients with AD and controls. Studies on single sections revealed that even subregionally there was no significant difference in the neuronal number or area in the SNpc between AD patients and controls.

Breast cancer in Nigeria and Finland: epidemiological, clinical and histological comparison.

BACKGROUND: We compared the histology and patterns of occurrence of breast cancers in Nigeria (n = 297) and Finland (n = 285). PATIENTS AND METHODS: The histology of invasive ductal carcinoma (IDC) was re-evaluated using similar criteria. The clinical data were extracted from medical records. RESULTS: The mean age at presentation was 42.7 (12.2) years in Nigeria vs. 58.7 (12.5) years in Finland. In both populations there was an association between reproductive factors and the occurrence of breast cancer. In Nigeria, 53.2% of cases belonged to stages 3 and 4 (vs. 6.7% in Finland). In Finland there were higher frequencies of lobular, tubular and mucinous types than in Nigeria. The Nigerian material had more medullary type (2.7% vs. 0.7% in Finland), extensive necrosis, nuclear atypia and pleomorphism, with coexisting pleomorphic ductal carcinoma in situ. At 2 years after treatment, the survival figures for Nigeria and Finland were 72.8% and 96.4%, respectively. CONCLUSION: The histology of Nigerian and Finnish cancers clearly differ. Nigerian cancers appear more advanced with higher grade. The atypical in situ component is clearly more common in Nigeria. Part of the differences can be explained by diagnostic and treatment delays associated with misguided socio-cultural beliefs, poor health care access and impaired immunity.

Dependence of DNA-histograms on the sampling techniques in fine needle aspirates of the breast.

48 fine needle aspiration biopsy (FNAB) samples from 25 breast cancer cases, originally used for cytodiagnosis were subjected to DNA cytometry. There were air dried smears stained with the MGG method, and samples stained with HE or PAP stain after 50% ethanol fixation and cytocentrifugation. Different sampling strategies were applied. Four methods were tested: method 1: cell groups measured, method 2: all cells measured, method 3: free cells measured, and method 4: atypical free cells measured. Method 4 showed most often DNA aneuploid histogram patterns, sampling method 1 had the highest number of DNA diploid histogram patterns. Diagnostic approaches may benefit from a sampling method detecting the hiding aneuploid cell population. Grading of neoplasm could potentially benefit from other approaches.