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Background: The DNA ploidy and the S phase fraction (SPF) are associated with advanced tumors progression. The present study aimed to investigate the clinicopathological behavior of oral squamous cell carcinoma(OSCC) and the prognostication of the DNA ploidy and the SPF on its clinical outcomes. Methods: This a prospective longitudinal study of 93 OSCC patients (186 specimens). Using high resolution flowcytometry, DNA analysis and SPF were performed for the primary tumor, field of cancerization and a control normal field. The patients were followed clinically for the development of recurrence or second primary tumor(SPT). Results: All the 93 patients are associated with wide field of cancerization. Out of the total number of cases, 28 (30%) patients developed recurrence. Of them 17 (18.3%) had regional recurrence, and eight (8.6%) had local recurrence. Second primary tumors were identified in 14 (15.1%) of the cases. Nodal metastasis, overall recurrence and SPTs were significantly associated with DNA aneuploidy and high SPF. High SPF and DNA hyperploidy showed a 28% increased risk of local recurrence (P.value = 0.032). Conclusion: In a series of 93 oral cancers who had wide field of cancerization, 30% of the patients developed recurrence, and 15% developed second primary tumors. The DNA ploidy and the SPF were independent prognosticators for the prediction of OSCC outcomes. [ABSTRACT FROM AUTHOR]

Abstract Background The DNA ploidy and the S phase fraction (SPF) are associated with advanced tumors progression. The present study aimed to investigate the clinicopathological behavior of oral squamous cell carcinoma(OSCC) and the prognostication of the DNA ploidy and the SPF on its clinical outcomes. Methods This a prospective longitudinal study of 93 OSCC patients (186 specimens). Using high resolution flowcytometry, DNA analysis and SPF were performed for the primary tumor, field of cancerization and a control normal field. The patients were followed clinically for the development of recurrence or second primary tumor(SPT). Results All the 93 patients are associated with wide field of cancerization. Out of the total number of cases, 28 (30%) patients developed recurrence. Of them 17 (18.3%) had regional recurrence, and eight (8.6%) had local recurrence. Second primary tumors were identified in 14 (15.1%) of the cases. Nodal metastasis, overall recurrence and SPTs were significantly associated with DNA aneuploidy and high SPF. High SPF and DNA hyperploidy showed a 28% increased risk of local recurrence (P.value = 0.032). Conclusion In a series of 93 oral cancers who had wide field of cancerization, 30% of the patients developed recurrence, and 15% developed second primary tumors. The DNA ploidy and the SPF were independent prognosticators for the prediction of OSCC outcomes.

Introduction: DNA ploidy (P), stroma fraction (S), and nucleotyping (N) collectively known as PSN, have proven prognostic accuracy in stage II colorectal cancer (CRC). However, few studies have reported on the prognostic value of the PSN panel in stage III colon cancer patients receiving capecitabine and oxaliplatin adjuvant chemotherapy. Objectives: This study aimed to validate PSN's prognostic impact on stage III colon cancer, identifying candidates for optimized adjuvant chemotherapy duration. Design: A retrospective analysis was conducted on a cohort of stage III colon cancer patients from April 2008 to June 2020. Methods: Postoperative pathological samples from stage III colon cancer patients who underwent radical surgery and postoperative adjuvant chemotherapy at Sun Yat-sen University Cancer Center were retrospectively collected. Automated digital imaging assessed PSN, categorizing risk groups. Kaplan–Meier, Cox regression, and time-dependent receiver operating characteristic analysis compared model validity. Results: Significant differences in 5-year disease-free survival (DFS) and overall survival (OS) were noted among PSN-based low-, moderate-, and high-risk groups (DFS: 92.10% versus 83.62% versus 79.80%, p = 0.029; OS: 96.69% versus 93.99% versus 90.12%, p = 0.016). PSN emerged as an independent prognostic factor for DFS [hazard ratio (HR) = 1.409, 95% confidence interval (CI): 1.002–1.981, p = 0.049] and OS (HR = 1.720, 95% CI: 1.127–2.624, p = 0.012). The PSN model, incorporating perineural invasion and tumor location, displayed superior area under the curve for 5-year (0.692 versus 0.553, p = 0.020) and 10-year (0.694 versus 0.532, p = 0.006) DFS than TNM stage. In the PSN high-risk group, completing eight cycles of adjuvant chemotherapy significantly improved 5-year DFS and OS compared to four to seven cycles (DFS: 89.43% versus 71.52%, p = 0.026; OS: 96.77% versus 85.46%, p = 0.007). Conclusion: The PSN panel effectively stratifies stage III colon cancer, aiding in optimized adjuvant chemotherapy duration determination. [ABSTRACT FROM AUTHOR]

Purpose: Post prostatectomy PSA kinetics and General Grade Groups (GGG) are the strongest prognostic markers of biochemical recurrence (BCR) and prostate cancer (PCa)-specific mortality after radical prostatectomy. Despite having low-risk PCa, some patients will experience BCR, for some, clinically significant BCR. There is a need for an objective prognostic marker at the time of prostatectomy to improve risk stratification within this population. In this study, we investigated the prognostic potential of DNA ploidy. Materials and Methods: Prostatectomy samples from 97 patients with GGG1 and GGG2 with a low-risk CAPRA-S score were included in this study. PCa tissue with the worst Gleason pattern underwent tissue disaggregation, cell isolation and staining with a DNA stoichiometric stain. Using image cytometry, DNA ploidy was measured and a Ploidy Score (PS) was generated. Results: Among the 97 patients, 79 had no BCR, 18 experienced BCR, of which 14 had a PSA doubling time (PSA-DT) >1 year (low-risk group) and 4 had a PSA-DT of <1 year (high-risk group). Using Logistic regression analysis, only pathological T stage (pT) and PS independently predicted BCR with PS being the most significant (p = 0.001). The number of aneuploid cells was significantly higher in the high-risk group compared to the other groups (p = 1.7x10-11). PS combined with GGG diagnosis further stratified risk groups of biochemical recurrence free survival within CAPRA-S low-risk cohort. Conclusion: DNA ploidy is an independent prognostic marker of BCR in low-risk PCa after radical prostatectomy, which could early on identify potentially aggressive PCa recurrences and introduce a more personalized approach to salvage treatments. [ABSTRACT FROM AUTHOR]

Malignant and potentially malignant epithelial lesions are often associated with various abnormalities such as epithelial dysplasia, abnormal DNA content, loss of heterozygosity, and chromosomal number aberrations. Screening and early detection of such abnormalities facilitates proper care and also helps to prevent further progression of potentially malignant lesions to malignancy. In such way, the presence of DNA aneuploidy in oral potentially malignant disorders (OPMDs) may serve as an indicator for the malignant transforming potential. Various assessment methods have been proposed to find the DNA ploidy status of cells. This current systematic review is mainly designed to assess the importance of ploidy status in OPMD while measuring the feasibility of using this biomarker for evaluating the hazard of malignant transformation. As an upshot of this systematic review, we can conclude that use of DNA ploidy status can serve as an independent bio-marker for predicting the malignant transformation of lesions. Furthermore, as a future scope the use of DNA ploidy analysis in normal mucosa of smokers will help to assess the malignancy risk and this technique might also help to predict the genetic predisposition of patients with malignancy. [ABSTRACT FROM AUTHOR]

Malignant and potentially malignant epithelial lesions are often associated with various abnormalities such as epithelial dysplasia, abnormal DNA content, loss of heterozygosity, and chromosomal number aberrations. Screening and early detection of such abnormalities facilitates proper care and also helps to prevent further progression of potentially malignant lesions to malignancy. In such way, the presence of DNA aneuploidy in oral potentially malignant disorders (OPMDs) may serve as an indicator for the malignant transforming potential. Various assessment methods have been proposed to find the DNA ploidy status of cells. This current systematic review is mainly designed to assess the importance of ploidy status in OPMD while measuring the feasibility of using this biomarker for evaluating the hazard of malignant transformation. As an upshot of this systematic review, we can conclude that use of DNA ploidy status can serve as an independent bio-marker for predicting the malignant transformation of lesions. Furthermore, as a future scope the use of DNA ploidy analysis in normal mucosa of smokers will help to assess the malignancy risk and this technique might also help to predict the genetic predisposition of patients with malignancy.

Simple Summary: In this work, we evaluated the prognostic value of the chromatin structure in patients with early-stage lung cancer. We assessed the associations of DNA ploidy, nucleotyping, and tumor–stroma ratio (TSR) with 5-year disease-free survival rates. Clarifying whether patients with homogeneous and heterogeneous chromatin can benefit from adjuvant chemotherapy can guide the decision-making regarding chemotherapy after lung cancer resection, improve the survival rate of patients, and reduce the incidence and cost of adverse events related to lung treatment. (1) Background: Chromatin structure typing has been used for prognostic risk stratification among cancer survivors. This study aimed to ascertain the prognostic values of ploidy, nucleotyping, and tumor–stroma ratio (TSR) in predicting disease progression for patients with early-stage non-small cell lung cancer (NSCLC), and to explore whether patients with different nucleotyping profiles can benefit from adjuvant chemotherapy. (2) Methods: DNA ploidy, nucleotyping, and TSR were measured by chromatin structure typing analysis (Matrix Analyser, Room4, Kent, UK). Cox proportional hazard regression models were used to assess the relationships of DNA ploidy, nucleotyping, and TSR with a 5-year disease-free survival (DFS). (3) Results: among 154 early-stage NSCLC patients, 102 were non-diploid, 40 had chromatin heterogeneity, and 126 had a low stroma fraction, respectively. Univariable analysis suggested that non-diploidy was associated with a significantly lower 5-year DFS rate. After combining DNA ploidy and nucleotyping for risk stratification and adjusting for potential confounders, the DNA ploidy and nucleotyping (PN) high-risk group and PN medium-risk group had a 4- (95% CI: 1.497–8.754) and 3-fold (95% CI: 1.196–6.380) increase in the risk of disease progression or mortality within 5 years of follow-up, respectively, compared to the PN low-risk group. In PN high-risk patients, adjuvant therapy was associated with a significantly improved 5-year DFS (HR = 0.214, 95% CI: 0.048–0.957, p = 0.027). (4) Conclusions: the non-diploid DNA status and the combination of ploidy and nucleotyping can be useful prognostic indicators to predict long-term outcomes in early-stage NSCLC patients. Additionally, NSCLC patients with non-diploidy and chromatin homogenous status may benefit from adjuvant therapy. [ABSTRACT FROM AUTHOR]

Objective To investigate the application value of liquid-based cytology, DNA ploidy and fluorescence in situ hybridization (FISH) in the diagnosis of upper tract urothelial carcinoma (UTUC). Methods From January 2013 to June 2021, 246 cases with suspected UTUC on CT or MRI images in the Army Medical Center of PLA were recruited and retrospectively analyzed. Among them, 190 cases were pathologically diagnosed as UTUC, 43 cases non-tumor lesions, and 13 cases other types of tumors. All the 246 cases were examined with liquid-based cytology of voided urine, 178 cases were also with DNA ploidy analysis and 167 cases with FISH. The diagnostic accuracy of liquid-based cytology, DNA ploidy and FISH alone or in combination was calculated and compared. Results The sensitivities of liquid-based cytology, DNA ploidy and FISH in the diagnosis of UTUC were 44.7%, 82.2%, and 57.9%, respectively; the specificities were 97.7%, 82.4%, and 92.3%, respectively. The diagnostic sensitivities of the above 3 methods for high-grade UTUC were 48.1%, 87.7%, and 60.7%, respectively, and for low-grade UTUC 25.0%, 52.4%, and 37.5%, respectively. When any one of the 3 methods is positive, the diagnostic sensitivity was increased to 93.8% (compared with the 3 methods being used alone, all P < 0.05). When any 2 of them were positive at the same time as the criteria for diagnosing UTUC (both negative or only 1 positive was regarded as negative), the positive predictive value and specificity of this combined diagnostic method were both 100%. Conclusion Urinary liquid-based cytology, DNA ploidy and FISH all are clinically valuable in the diagnosis of UTUC. The combination of the 3 methods can effectively improve the sensitivity or specificity of diagnosis, thereby helping to reduce the risk of missed diagnosis or misdiagnosis.

Abstract Background About 7 million people die from various types of cancer every year representing nearly 12.5% of deaths worldwide. This fact raises the demand to develop new, effective anticancer, onco-suppressive, and chemoprotective agents for the future fighting of cancers. Genistein exhibits pleiotropic functions in cancer, metabolism, and inflammation. It functions as an antineoplastic agent through its effect on the cell cycle, apoptotic processes, angiogenesis, invasion, and metastasis. Aim of the study The current study aimed to study the genistein onco-suppressive effects during 7,12-dimethylbenz[a]anthracene (DMBA)-induced oral carcinogenesis in hamsters’ buccal pouch utilizing flow cytometry analysis (FMA), as a fast-diagnosing tool, in addition to the histopathology. Material and methods The buccal mucosa of adult male Syrian hamsters was painted with paraffin oil only (group 1), DMBA mixed in mineral oil (group 2), or orally administrated genistein along with painting DMBA (group 2B). The buccal mucosa was utilized for flow cytometric analysis and histopathological examination. Results Grossly, DMBA-induced carcinogenesis started at the 9th week. Progressive signs appeared in the following weeks reaching to large ulcerative oral masses and exophytic nodules at the 21st week. Histologically, invasive well-differentiated oral squamous cell carcinoma (OSCC) appeared in the underlying tissues from the 12th week, showing malignant criteria. Genistein had delayed clinicopathological change, which started 6 weeks later, than the DMBA-painted hamsters, as mild epithelial dysplastic changes. This became moderate during the last 6 weeks, without dysplastic changes. Flow cytometry revealed that DMBA led to considerable variation in DNA proliferation activity, aneuploid DNA pattern, in 47.22% of hamsters and significantly raised the S-phase fragment (SPF) values, which drastically reduced after genistein treatment. Conclusion Taken together, genistein could be employed as an onco-suppressive agent for carcinogenesis. Moreover, FMA could be used as an aiding fast tool for diagnosis of cancer.

Abstract Background Colorectal cancer liver metastases (CRLM) has not been identified as a unified disease entity due to the differences in the severity of metastatic disease and tumor aggressiveness. A screen for specific prognostic risk subgroups is urgently needed. The current study aimed to investigate the prognostic value of DNA ploidy, stroma fraction and nucleotyping of initially resectable liver metastases from patients with CRLM. Methods One hundred thirty-nine consecutive patients with initially resectable CRLM who underwent curative liver resection from 2006 to 2018 at Sun Yat-sen University Cancer Center were selected for analysis. DNA ploidy, stroma fraction and nucleotyping of liver metastases were evaluated using automated digital imaging systems. Recurrence-free survival (RFS) and overall survival (OS) were analyzed using the Kaplan-Meier method and Cox regression models. Results DNA ploidy was identified as an independent prognostic factor for RFS (HR, 2.082; 95% CI 1.053–4.115; P = 0.035) in the multivariate analysis, while stroma-tumor fraction and nucleotyping were not significant prognostic factors. A significant difference in 3-year RFS was observed among the low-, moderate- and high-risk groups stratified by a novel parameter combined with the tumor burden score (TBS) and DNA ploidy (72.5% vs. 63.2% vs. 37.3%, P = 0.007). The high-risk group who received adjuvant chemotherapy had a significantly better 3-year RFS rate than those without adjuvant chemotherapy (46.7% vs. 24.8%; P = 0.034). Conclusions Our study showed that DNA ploidy of liver metastases is an independent prognostic factor for patients with initially resectable CRLM after liver resection. The combination of DNA ploidy and TBS may help to stratify patients into different recurrence risk groups and may guide postoperative treatment among the patients.

Background: FxCycleTM Violet (FCV) based flow cytometric (FCM) DNA ploidy analysis is a rapid and simple tool that can substantiate in characterizing the biological behaviour across the spectrum of haematological malignancies and correlates with cytogenetic studies. Materials and Methods: In this prospective study, we performed simultaneous immunophenotyping with FCV based on ploidy analysis in n=132 consecutive new samples, comprising n=110 samples of haemato-lymphoid neoplasms, including acute leukemias (n=67, 60.9%), CML with myeloid blast crisis (n=1, 0.9%), MDS with excess blasts (n=2, 1.8%), mature B cell/ T cell neoplasms (n=37, 33.7%), multiple myeloma (n=3, 2.7%) along with n=22 normal samples. The FCM DNA data was compared with corresponding conventional karyotyping results, wherever available. Results: In FCM ploidy analysis (n=110), the overall DNA index (DI) ranged from 0.81 to 2.17 and S-Phase fraction (SPF) from 0.1-31.6%. Diploidy was seen in n = 90 (81.8%), low-hyperdiploidy in n = 10 (9.1%), highhyperdiploidy in n = 7 (6.4%) with one case each (0.9% each) having near-tetraploidy, high-hypodiploidy and low-hypodiploidy. The DI of all viable cell populations in normal samples ranged from 0.96-1.05. Conventional karyotyping was performed in n=76/110 cases (70%) with n= 11/76 (15%) culture failures. The modal chromosome number ranged from 45 to 63. A concordance of 95.4% (n=62/65) was noted with corresponding FCM DI. Conclusion: FCV-based ploidy is a sensitive technique that provides complementary information and ascertains a strong correlation with conventional cytogenetics across all haemato-lymphoid neoplasms. It can detect aneuploidy in all B-ALL and myeloma cases, even in hemodiluted samples with cytogenetic culture failure; supplement the diagnoses of erythroleukemia, and provide a useful screen for a higher grade lymph node disease in lymphoma cases with SPF > 3%. [ABSTRACT FROM AUTHOR]

Background: Methods to analyze progression of carcinogenesis and stage of cancer are vital nowadays due to the high prevalence of these lesions. DNA ploidy analysis is one such important method in early diagnosis and improving prognosis. Aims and Objectives: The aim of this study was to correlate histopathological grading and DNA ploidy in oral leukoplakia, oral submucous fibrosis (OSMF), and oral squamous cell carcinoma (OSCC) cases. Materials and Methods: Our study included 80 subjects, grouped into 4 groups of 20 each of OSCC, leukoplakia, OSMF, and healthy individuals. Histopathological grading was carried out in study cases, DNA ploidy was estimated using flow cytometry, and both the findings were correlated. Results: Among the 20 cases of leukoplakia group, 6 cases showed aneuploidy and 14 showed diploidy. In the 20 cases of OSF group, 2 cases showed aneuploidy and 18 showed diploidy, and in the 20 cases of OSCC group, 10 showed aneuploidy and 10 showed diploidy. Most of the aneuploidy cases showed severe dysplasia. Conclusion: Analysis of DNA ploidy status can serve as a diagnostic tool for early detection of malignancies owing to the subjective nature of traditional histopathological grading.

Background: FxCycleTM Violet (FCV) based flow cytometric (FCM) DNA ploidy analysis is a rapid and simple tool that can substantiate in characterizing the biological behaviour across the spectrum of haematological malignancies and correlates with cytogenetic studies. Materials and Methods: In this prospective study, we performed simultaneous immunophenotyping with FCV based on ploidy analysis in n=132 consecutive new samples, comprising n=110 samples of haemato-lymphoid neoplasms, including acute leukemias (n=67, 60.9%), CML with myeloid blast crisis (n=1, 0.9%), MDS with excess blasts (n=2, 1.8%), mature B cell/ T cell neoplasms (n=37, 33.7%), multiple myeloma (n=3, 2.7%) along with n=22 normal samples. The FCM DNA data was compared with corresponding conventional karyotyping results, wherever available. Results: In FCM ploidy analysis (n=110), the overall DNA index (DI) ranged from 0.81 to 2.17 and S-Phase fraction (SPF) from 0.1-31.6%. Diploidy was seen in n = 90 (81.8%), low-hyperdiploidy in n = 10 (9.1%), high-hyperdiploidy in n = 7 (6.4%) with one case each (0.9% each) having near-tetraploidy, high-hypodiploidy and low-hypodiploidy. The DI of all viable cell populations in normal samples ranged from 0.96-1.05. Conventional karyotyping was performed in n=76/110 cases (70%) with n= 11/76 (15%) culture failures. The modal chromosome number ranged from 45 to 63. A concordance of 95.4% (n=62/65) was noted with corresponding FCM DI. Conclusion: FCV-based ploidy is a sensitive technique that provides complementary information and ascertains a strong correlation with conventional cytogenetics across all haemato-lymphoid neoplasms. It can detect aneuploidy in all B-ALL and myeloma cases, even in hemodiluted samples with cytogenetic culture failure; supplement the diagnoses of erythroleukemia, and provide a useful screen for a higher grade lymph node disease in lymphoma cases with SPF > 3%.

Background: Colorectal cancer liver metastases (CRLM) has not been identified as a unified disease entity due to the differences in the severity of metastatic disease and tumor aggressiveness. A screen for specific prognostic risk subgroups is urgently needed. The current study aimed to investigate the prognostic value of DNA ploidy, stroma fraction and nucleotyping of initially resectable liver metastases from patients with CRLM. Methods: One hundred thirty-nine consecutive patients with initially resectable CRLM who underwent curative liver resection from 2006 to 2018 at Sun Yat-sen University Cancer Center were selected for analysis. DNA ploidy, stroma fraction and nucleotyping of liver metastases were evaluated using automated digital imaging systems. Recurrence-free survival (RFS) and overall survival (OS) were analyzed using the Kaplan-Meier method and Cox regression models. Results: DNA ploidy was identified as an independent prognostic factor for RFS (HR, 2.082; 95% CI 1.053–4.115; P = 0.035) in the multivariate analysis, while stroma-tumor fraction and nucleotyping were not significant prognostic factors. A significant difference in 3-year RFS was observed among the low-, moderate- and high-risk groups stratified by a novel parameter combined with the tumor burden score (TBS) and DNA ploidy (72.5% vs. 63.2% vs. 37.3%, P = 0.007). The high-risk group who received adjuvant chemotherapy had a significantly better 3-year RFS rate than those without adjuvant chemotherapy (46.7% vs. 24.8%; P = 0.034). Conclusions: Our study showed that DNA ploidy of liver metastases is an independent prognostic factor for patients with initially resectable CRLM after liver resection. The combination of DNA ploidy and TBS may help to stratify patients into different recurrence risk groups and may guide postoperative treatment among the patients. [ABSTRACT FROM AUTHOR]

Background: Methods to analyze progression of carcinogenesis and stage of cancer are vital nowadays due to the high prevalence of these lesions. DNA ploidy analysis is one such important method in early diagnosis and improving prognosis. Aims and Objectives: The aim of this study was to correlate histopathological grading and DNA ploidy in oral leukoplakia, oral submucous fibrosis (OSMF), and oral squamous cell carcinoma (OSCC) cases. Materials and Methods: Our study included 80 subjects, grouped into 4 groups of 20 each of OSCC, leukoplakia, OSMF, and healthy individuals. Histopathological grading was carried out in study cases, DNA ploidy was estimated using flow cytometry, and both the findings were correlated. Results: Among the 20 cases of leukoplakia group, 6 cases showed aneuploidy and 14 showed diploidy. In the 20 cases of OSF group, 2 cases showed aneuploidy and 18 showed diploidy, and in the 20 cases of OSCC group, 10 showed aneuploidy and 10 showed diploidy. Most of the aneuploidy cases showed severe dysplasia. Conclusion: Analysis of DNA ploidy status can serve as a diagnostic tool for early detection of malignancies owing to the subjective nature of traditional histopathological grading. [ABSTRACT FROM AUTHOR]

Background: Epithelioid hemangioendothelioma (EHE) involving serous effusion is extremely rare, and the diagnosis can be challenging. DNA ploidy quantitation of EHE in effusion fluids has not been previously described in the English-language literature. Methods: Specimens of cytological diagnosed with EHE in effusion fluids between 2002 and 2009 were retrieved from the pathology files at MD Anderson Cancer Center. A total of four cases of EHE involving or arising from effusion fluids were found, and we reviewed cytospin, smears, cell block sections, and immunostained slides. DNA image analysis for ploidy and proliferation evaluation was performed on a destained, papanicolaou-stained slide from each case. Results: The tumor cells were epithelioid with prominent cytoplasmic vacuolization and intracytoplasmic inclusions, which could resemble reactive mesothelial cells, mesothelioma, or adenocarcinoma. The tumor cells were positive for endothelial markers. DNA image analysis in three of the four cases revealed predominantly diploid and tetraploid subpopulations, with few aneuploid cells and fairly low proliferation indices, and these patients had fairly prolonged survival. Conclusions: DNA image analysis is useful for differentiating EHE from reactive mesothelial cells and high-grade carcinoma. For accurate diagnosis of EHE in effusion fluids, cytologic features should be considered together with clinical history and ancillary studies. [ABSTRACT FROM AUTHOR]

The potential prognostic significance of DNA flow cytometric measurements (DNA ploidy and S-phase fraction) in breast cancer remains in dispute. Inconclusive data, primarily due to the lack of consistent standardization and quality control programs, have limited its translation into clinical practice. The aim of the present review, based on the 25-year experience of the Portuguese Institute of Oncology of Lisbon, is to assess the clinical relevance and application of DNA flow cytometry for the prognosis of breast cancer. Overall, data from Portuguese Institute of Oncology of Lisbon indicate that DNA flow cytometry provides significant prognostic information that is biologically relevant and clinically useful for the management of patients with breast cancer. Furthermore, this data has demonstrated the independent value of DNA aneuploidy as a prognostic indicator of poor clinical outcome in various subgroups of patients with early or locally advanced breast cancer at short- and long-term follow-up. Notably, aneuploidy identifies subsets of patients with grade (G)1 or G2 tumours who exhibit a poor clinical outcome. These patients may benefit from adjuvant chemotherapy, particularly those with luminal A and luminal B/human epidermal growth factor-2-negative endocrine-responsive breast cancer. In conclusion, data from Portuguese Institute of Oncology of Lisbon reinforces the clinical importance and utility of DNA flow cytometric analysis, particularly DNA ploidy, in the prognostic assessment and therapeutic planning for patients with breast cancer. [ABSTRACT FROM AUTHOR]

Background: The role of DNA ploidy in genomic instability of cancer cells and prognosis has been described in a number of studies. The role of the centrosome in cell cycle has also been reported. Aim: In this study, we aimed to investigate the correlation between the centrosome and DNA ploidy in breast cancer in a search for a cytologic predictive and prognostic marker. Materials and Methods: Cell prints were prepared from cell culture of mesothelial cells, fibroblast cell line MRC5 and breast cancer cell lines MCF7 and T47D. Indirect immunofluorescence was used with anti-γ-tubulin and centrosomes were quantified using a fluorescence microscope. DNA ploidy was scored with the DNA index analyzed by flow cytometry. Results: The normal mesothelial cells (94% of the cells with one detected centrosome) and MRC5 diploid cells (68% with two centrosomes) were used as quality controls. A correlation between the number of centrosomes and DNA ploidy was found in MCF7 cell lines (64% of the cells with a number of centrosomes ≥ 3). It was not observed in invasive breast cancer samples; however, the frequency of cells with centrosomes ≥ 3 was found to be slightly higher in DNA aneuploid samples than in DNA diploid samples (15% vs 13.3%). Conclusion: Quantification of centrosome appears to be correlated to DNA ploidy in breast cancer cell lines and slightly associated to DNA aneuploidy in invasive breast cancer. Studies analyzing a larger number of samples as well as morphological abnormalities of the centrosome are needed.

Objectives: To evaluate DNA ploidy and S-phase fraction (SPF) in non-Lynch colonic adenocarcinoma, ulcerative colitis (UC), Crohn disease (CD) which are known as risk factors, and colitis. We correlated ploidy and SPF with tumor grading, staging and BRAF expression., Methods: All studied adenocarcinomas have intact mismatch repair genes as proved by immunohistochemistry. All were assessed for ploidy by automated image-based DNA cytometry and histograms were drawn. Immunostaining by anti-BRAF V600E was performed. Diagnostic laparoscopy (DL) was done as a preliminary step for staging GI cancers., Results: there is significant difference in DNA ploidy between groups; 77.5% and 17.5% of aneuploid cases are adenocarcinoma and UC. Groups are compared in terms of 2C, 4C, above 4C DNA content and SPF and significant difference is principally found between adenocarcinoma group and others. In adenocarcinomas, DNA ploidy is significantly correlated with tumor staging and grading. Regarding BRAF expression, there is significant difference between groups; all adenocarcinomas, 83.33% of UC were positive, while all cases of colitis, bilharzial colitis, CD were negative. There is significant relation between BRAF and SPF among all diploid cases including adenocarcinoma, and among non-neoplastic diploid cases. There is direct significant relation between BRAF intensity and adenocarcinoma staging. There is no significant difference between BRAF and ploidy among UC cases, although 75% of aneuploid UC are positive. DL helps in GI cancer staging. Routine laparoscopy before laparotomy, especially in cancers which have equivocal operability helps to avoid unnecessary laparotomies., Conclusion: Based on significant difference in ploidy between adenocarcinoma and UC and between SPF and ploidy, assessment of ploidy by DNA cytometry for UC and other colitis could therefore predict impending malignant transformation before development of colonic dysplasia. Also measuring SPF in adenocarcinoma helps to select patients who could greatly benefit from chemotherapy. DL has vital role in staging GI cancers.

Nos últimos dez anos, grandes mudanças ocorreram no tratamento do MM com a utilização de novas drogas. Frente a estas novas opções de tratamento é essencial reconhecermos parâmetros clínicos ou biológicos que orientem a melhor escolha terapêutica. Mais recentemente foi validado um novo e simples sistema de estadiamento, International Staging System (ISS), baseado nos valores dabeta2 microglobulina e albumina sérica. Os pacientes são classificados em três grupos de risco: Estádio I: beta2M 3,5 g/dl. Mediana de sobrevida de 62 meses; Estádio II: beta2 M 3,5 - < 5,5 mg/l. Mediana de sobrevida 49 meses; Estádio III: beta2 > 5,5 mg/l. Mediana de sobrevida de 29 meses. Atualmente, a citogenética e achados moleculares estão sendo amplamente reconhecidos como fatores de prognóstico. A deleção do cromossomo 13/13q-, translocação t(4;14), deleção p53 e, mais recentemente, a amplificação da banda cromossômica 1q21 estão associadas a prognóstico reservado.Over the last 10 years, great changes have occurred in the treatment of multiple myeloma (MM) due to the use of new drugs. Considering the new options, it is essential to recognize clinical and biological parameters to arrive at the best therapeutic choice. More recently the new International Staging System (ISS) for multiple myeloma was validated which utilizes two straight forward laboratory parameters: the beta2 microglobulin (beta2M) and albumin levels. Stage I: beta2M < 3.5 mg/L and albumin level > 3.5 g/dL with a median survival of 62 months; stage II: beta2M < 3.5 and albumin < 3.5 g/dL or beta2M > 3.5 to < 5.5 g/dL with a median survival of 49 months; stage III: > 5.5 g/dL with a median survival of 29 months. The importance of cytogenetics and molecular features as prognostic factors is being recognized. Deletion of chromosome 13 or 13q, the t(4:14) translocation, p53 deletion and amplification of chromosome band 1q21 are all associated with poor prognosis.

OBJECTIVE: To study the importance of NB84, synaptophysin and AgNOR and explore the quantitative association of these factors with diagnosis and outcome as well as the association between NB84 and AgNOR and other tumor and stromal factors in twenty-eight peripheral neuroblastic tumors. METHODS: We assessed AgNORs, NB84, synaptophysin and several other markers in tumor tissues from 28 patients with primary neuroblastic tumors. The treatment included: surgery for stage 1, chemotherapy and bone marrow transplantation for most of stages 3 and 4. Histochemistry, immunohistochemistry and morphometry were used to evaluate the amount of tumor staining for AgNOR, NB84 and synaptophysin; the outcome for our study was survival time until death due to recurrent neuroblastic tumors. RESULTS: Only stage (pOBJETIVO: Estudar a importância dos marcadores NB84 e AgNOR e explorar as relações quantitativas entre esses marcadores com o diagnóstico e prognóstico assim como as relações entre NB84 e AgNOR e outros marcadores tumorais e estromais em 28 tumores neuroblásticos periféricos. MÉTODOS: Examinamos AgNOR, NB84 e sinaptofisina e vários outros marcadores em tecidos tumorais de vinte e oito pacientes com tumors neuroblásticos primários. Tratamento dos pacientes incluiu: cirurgia para o estágio 1, quimioterapia e transplante de medula óssea para a maioria dos pacientes nos estágios 3 e 4. Utilizamos histoquímica, imunohistoquímica e morfometria para avaliar a intensidade e extensão de expressão do AgNOR, NB84 e sinaptofisina, tendo o prognóstico dos pacientes incluído o tempo de sobrevida até a morte por recurrência dos tumores neuroblásticos. RESULTADOS: Estadiamento (p

Amifostine is a cytoprotective drug that was initially used to control and treat nuclear radiation injury and is currently used to provide organ protection in cancer patients receiving chemotherapy. Clinical studies have also found that amifostine has some efficacy in the treatment of cytopenia caused by conditions such as myelodysplastic syndrome and immune thrombocytopenia, both of which involve megakaryocyte maturation defects. We hypothesized that amifostine induced the differentiation of megakaryocytes and investigated this by exposing the human Dami megakaryocyte leukemia cell line to amifostine (1 mmol/L). After 12 days of amifostine exposure, optical microscopy showed that the proportion of Dami cells with diameters >20 μm had increased to 24.63%. Transmission electron microscopy identified the development of a platelet demarcation membrane system, while flow cytometry detected increased CD41a expression and decreased CD33 expression on the Dami cell surface. Ploidy analysis found that the number of polyploid cells with >4N DNA content increased to 27.96%. We did not detect any elevation in the mRNA or protein levels of megakaryocytic differentiation-associated transcription factors GATA-binding factor 1 ( GATA-1) and nuclear factor, erythroid 2 ( NF-E2), but nuclear import assay revealed an increased nuclear translocation of these proteins. These findings indicate that amifostine induced the differentiation of Dami cells into mature megakaryocytes via a mechanism involving increased nuclear translocation of the transcription factors, NF-E2 and GATA-1. [ABSTRACT FROM AUTHOR]

Aim: Atypical lymphocytes usually described as lymphocytes with altered shape, increased DNA amount, and larger size. For analysis of cause of genesis and source of atypical lymphocytes during African swine fever virus (ASFV) infection, bone marrow, peripheral blood, and in vitro model were investigated. Materials and Methods: Atypical lymphocytes under the influence of ASFV were studied for morphologic, cytophotometric, and membrane surface marker characteristics and were used in vivo and in vitro models. Results: This study indicated the increased size, high metabolic activity, and the presence of additional DNA amount in atypical lymphocytes caused by ASFV infection. Furthermore, in atypical lymphocytes, nuclear-cytoplasmic ratio usually decreased, compared to normal lymphocytes. In morphology, they looking like lymphocytes transformed into blasts by exposure to mitogens or antigens in vitro. They vary in morphologic detail, but most of them are CD2 positive. Conclusions: Our data suggest that atypical lymphocytes may represent an unusual and specific cellular response to ASFV infection. [ABSTRACT FROM AUTHOR]

The present study investigated the predictive and prognostic impact of DNA ploidy together with other well-known prognostic factors in a series of non-endometrioid, high-risk endometrial carcinomas. From a complete consecutive series of 4,543 endometrial carcinomas of International Federation of Gynecology and Obstetrics (FIGO) stages I-IV, 94 serous carcinomas, 48 clear cell carcinomas and 231 carcinosarcomas were selected as a non-endometrioid, high-risk group for further studies regarding prognosis. The impact of DNA ploidy, as assessed by flow cytometry, was of particular focus. The age of the patients, FIGO stage, depth of myometrial infiltration and tumor expression of p53 were also included in the analyses (univariate and multivariate). In the complete series of cases, the recurrence rate was 37%, and the 5-year overall survival rate was 39% with no difference between the three histological subtypes. The primary cure rate (78%) was also similar for all tumor types studied. DNA ploidy was a significant predictive factor (on univariate analysis) for primary tumor cure rate, and a prognostic factor for survival rate (on univariate and multivariate analyses). The predictive and prognostic impact of DNA ploidy was higher in carcinosarcomas than in serous and clear cell carcinomas. In the majority of multivariate analyses, FIGO stage and depth of myometrial infiltration were the most important predictive (tumor recurrence) and prognostic (survival rate) factors. DNA ploidy status is a less important predictive and prognostic factor in non-endometrioid, high-risk endometrial carcinomas than in the common endometrioid carcinomas, in which FIGO and nuclear grade also are highly significant and important factors. [ABSTRACT FROM AUTHOR]

Festulolium is a hybrid between Festuca and Lolium species that has valuable agronomic traits from both grass species. The purpose of our breeding program is to produce hexaploid festulolium that introduces tolerance to summer depression into Italian ryegrass (Lolium multiflorum) by crossing it with tall fescue (Festuca arundinacea). However, we found the DNA ploidy of hexaploids was not stable and was reduced in successive generations. We aimed to find out how to obtain stable high-ploidy festulolium. F1 hybrids of L. multiflorum and F. arundinacea were produced. The F3 generation was produced from putative hexaploid F2 individuals by open pollination. The F4 to F6 generations were obtained by polycrossing. The DNA ploidy levels of F2 to F6 individuals were estimated by flow cytometry. Cytological characteristics of the F5 and F6 individuals were investigated by FISH and GISH. The DNA ploidy level of hexaploid festulolium was reduced and stabilized at almost the same level as a tetraploid. Seed fertility was inversely correlated with an increase in ploidy level. GISH revealed no preferential Lolium transmission. FISH with a telomere probe revealed that counting the exact number of chromosomes in festulolium was difficult. DNA ploidy level was strongly correlated with the number of chromosomes. [ABSTRACT FROM AUTHOR]

Phenotypic evaluation of epigenetic mutants is mainly based on the analysis of plant growth and morphological features. However, there are cellular level changes that are not visible to the naked eye and require analysis with higher resolution techniques. In this study, we carried out a phenotypic characterisation of several Arabidopsis thaliana hypomethylation mutants by quantitative image analysis combined with flow cytometry. This phenotyping approach permitted identification of abnormalities at the cellular level in mutants with wild-type morphology at the organ level. Morphometry of adaxial leaf epidermis revealed variations in the size and number of pavement cells, and the density and distribution of stomata in the analysed second rosette leaves from the mutants studied. A direct correlation between DNA ploidy status and leaf pavement cell size in wild type and mutant leaves was observed. Recognition of hidden phenotypic variations could facilitate the identification of key genetic loci underlying the phenotypes caused by modifications of DNA methylation. Thus, this study outlines an easy and fast phenotyping strategy that can be used as a reliable tool for characterisation of epigenetic mutants at the cellular level. [ABSTRACT FROM AUTHOR]

Malignant tumors have telomerase activity, which is thought to play a critical role in tumor growth. However, the relation between telomerase activity and genomic DNA status in tumor cells is poorly understood. In the present study, we examined telomerase activity in 13 clear cell type renal cell carcinomas (CRCCs) with similar clinicopathologic features by telomeric repeat amplification protocol assay (TRAP). Based on TRAP assay results, we divided the CRCCs into two groups: a high telomerase activity group and a low/no telomerase activity group. We then analyzed genomic aberration, DNA ploidy, and telomere status in these two groups by comparative genomic hybridization (CGH), laser scanning cytometry (LSC), and telomere-specific fluorescence in situ hybridization (T-FISH), respectively. CGH showed the high telomerase activity group to have fewer genomic changes than the low/no telomerase activity group, which had many genomic aberrations. Moreover, with LSC, DNA diploid cells were found more frequently in the high telomerase activity group than in the low/no telomerase activity group. In addition, T-FISH revealed strong telomere signal intensity in the high telomerase activity group compared with that of the low/no telomerase activity group. These results suggest that telomerase activity is linked to genomic DNA status and that high telomerase activity is associated with genomic stability, DNA ploidy, and telomere length in CRCC.

Two-color fluorescence in situ hybridization (FISH) with chromosome enumeration DNA probes specific to chromosomes 7, 11, 17, and 18 was applied to CAL-51 breast cancer cells to examine whether the fluorescence intensity of FISH spots was associated with cell cycle progression. The fluorescence intensity of each FISH spot was quantitatively analyzed based on the cell cycle stage determined by image cytometry at the single-cell level. The spot intensity of cells in the G2 phase was larger than that in the G0/1 phase. This increased intensity was not seen during the early and mid S phases, whereas the cells in the late S phase showed significant increases in spot intensity, reaching the same level as that observed in the G2 phase, indicating that alpha satellite DNA in the centromeric region was replicated in the late S phase. Thus, image cytometry can successfully detect small differences in the fluorescence intensities of centromeric spots of homologous chromosomes. This combinational image analysis of FISH spots and the cell cycle with cell image cytometry provides insights into new aspects of the cell cycle. This is the first report demonstrating that image cytometry can be used to analyze the fluorescence intensity of FISH signals during the cell cycle. [ABSTRACT FROM PUBLISHER]

Oral leukoplakia and other potentially malignant disorders (PMD) may progress to oral squamous cell carcinoma (OSCC). The gold standard for assessing the potential for malignant transformation remains histologic examination with the aim of grading the dysplastic changes. However, not all lesions with dysplasia will progress to OSCC. DNA ploidy has been suggested as a method to predict the clinical behaviour of PMD. This study reports on the use of high-resolution flow cytometry to determine the ploidy status of formalin-fixed, paraffin-embedded material from PMD compared to their dysplasia grade on histology. Aneuploidy was found in 13 % of mild, 31 % of moderate, and 54 % of severe dysplasia cases. This difference was statistically significant ( p = 0.011). The differences in ploidy status were more significant when grouping the dysplasia into low-risk and high-risk categories ( p = 0.008). These findings indicate that the ploidy status of PMD as determined by high-resolution flow cytometry may be of value in predicting biological behaviour in PMD such as leukoplakia. [ABSTRACT FROM AUTHOR]

Background: We evaluated the prognostic importance of DNA ploidy in stage I and II endometrioid adenocarcinoma (EAC) of the endometrium with a focus on DNA index. Patients and methods: High-resolution DNA ploidy analysis was carried out in tumor material from 937 consecutive patients with International Federation of Gynecology and Obstetrics (FIGO) stage I and II EAC of the endometrium. Results: Patients with diploid (N = 728), aneuploid tumor with DNA index ≤1.20 (N = 118), aneuploid tumors with DNA index >1.20 (N = 39) and tetraploid tumor (N = 52) had 5-year recurrence rates 8%, 14%, 20% and 12%, respectively. Patients with aneuploid tumor with DNA index >1.20 had a poorer 5-year progression-free survival (67%) and overall survival (72%) compared with the patients with aneuploid tumor with DNA index ≤1.20 (81% and 89%, respectively). Aneuploid tumors with DNA index ≤1.20 relapsed mainly in the vagina and pelvis, whereas aneuploid tumors with DNA index >1.20 relapsed predominantly outside pelvis. Conclusions: The recurrence risk for the patients with aneuploid tumor is higher than the patients with diploid tumor in EAC of the endometrium. Based on DNA index with cut-off 1.20, aneuploid tumors can be separated into two subgroups with different recurrence pattern and survival. [ABSTRACT FROM PUBLISHER]

Background: The role of DNA ploidy in genomic instability of cancer cells and prognosis has been described in a number of studies. The role of the centrosome in cell cycle has also been reported. Aim: In this study, we aimed to investigate the correlation between the centrosome and DNA ploidy in breast cancer in a search for a cytologic predictive and prognostic marker. Materials and Methods: Cell prints were prepared from cell culture of mesothelial cells, fibroblast cell line MRC5 and breast cancer cell lines MCF7 and T47D. Indirect immunofluorescence was used with anti-γ-tubulin and centrosomes were quantified using a fluorescence microscope. DNA ploidy was scored with the DNA index analyzed by flow cytometry. Results: The normal mesothelial cells (94% of the cells with one detected centrosome) and MRC5 diploid cells (68% with two centrosomes) were used as quality controls. A correlation between the number of centrosomes and DNA ploidy was found in MCF7 cell lines (64% of the cells with a number of centrosomes ≥ 3). It was not observed in invasive breast cancer samples; however, the frequency of cells with centrosomes ≥ 3 was found to be slightly higher in DNA aneuploid samples than in DNA diploid samples (15% vs 13.3%). Conclusion: Quantification of centrosome appears to be correlated to DNA ploidy in breast cancer cell lines and slightly associated to DNA aneuploidy in invasive breast cancer. Studies analyzing a larger number of samples as well as morphological abnormalities of the centrosome are needed. [ABSTRACT FROM AUTHOR]

Abstract: DNA ploidy analysis is useful for prognostication in cancer patients, but the genomic details underlying ploidy changes are not fully understood. To improve this understanding, we compared DNA ploidy status with karyotypic and comparative genomic hybridization data on 51 endometrial adenocarcinomas. Out of 34 DNA diploid tumors evaluated by CGH, 16 (47%) showed imbalances, though only two had more than four copy number changes. Ten (29%) had aberrations involving chromosome 1, seven (21%) involving chromosome 10, while one tumor had a chromosome 8 aberration. Four of the seven DNA tetraploid tumors (57%) had imbalances detected by CGH with two (29%) having more than four. Six out of eight DNA aneuploid tumors showed imbalances by CGH, with five (63%) having more than four. The aberrations were observed on chromosomes 1 and 8 in five/eight (63%) cases while four imbalances (50%) involved chromosomes 5, 7 and X. Not surprisingly, we observed a significant correlation between increasing DNA ploidy complexity and increasing number of copy alterations. Gains of material from chromosomes 8 and 7 might be specifically correlated to DNA aneuploidy in endometrial adenocarcinomas since 63% and 50% of the aneuploid compared to 3% of the diploid tumors showed imbalances involving these chromosomes. [Copyright &y& Elsevier]

Background& objectives: Fas receptor and Fas Ligand (FasL) system has been implicated in the resistance to apoptosis, insensitivity to chemotherapy and in providing immune privileged status to most of the tumours. However, no reports are available on Fas and FasL expression in patients with tobacco-related oral carcinoma. Therefore, the present study was undertaken to observe Fas and FasL expression and their correlation with clinicopathoiogical features as well as cell cycle parameters. Methods: Immunohistochemistry for Fas, FasL and DNA flow cytometry for cell cycle parameters was successfully done on 41 paraffin embedded tumour and 10 normal samples. The results were evaluated for possible association of Fas and FasL with clinicopathological features and cell cycle parameters. Results: Weak Fas expression was observed on the cell membrane only in 2 of 41 (5%) oral tumours while FasL immunoreactivity was seen in 26 of 41 (63.4%) tumours. In contrast, all ten normal oral tissues exhibited strong cytoplasmic and membrane Fas receptor immunoreactivity but absence of FasL staining. Older patients, greater tumour size and lymph node positivity were found to be associated with high expression of FasL. Significantly higher (P

Purpose: The quality of life in postmenopause is seriously affected by the symptoms related to vaginal atrophy. To evaluate in a 3-month, prospective, randomized, double blind, study whether vaginal suppositories containing genistein might improve genital symptoms, colposcopical and cytologic findings or modify DNA cytometric features in postmenopausal women affected by vaginal atrophy, in comparison with vaginal suppositories containing hyaluronic acid (HA). Methods: A total of 62 postmenopausal women were randomly assigned to receive intravaginally 97 μg of genistein (group A, n = 31) or 5 mg of HA (group B, n = 31) daily for 15 days continuously/month for 3 months. Vaginal and cervical smear, colposcopy, vaginal biopsy were performed before and at the end of the study. Maturation value (MV) was calculated. Flow cytometric analysis of DNA ploidy (DI) and S-phase fraction (SPF) were performed. Results: After 90 days of study, a significant improvement was obtained in genital symptoms, colposcopy scores and MV ( p < 0.001) in both groups; the improvement obtained by genistein was more effective especially regarding genital score ( p value between groups 0.001). No significant change was found in SPF value and DI. Conclusion: Both treatments improved genital symptoms, colposcopical features and MV, although genistein was more effective on genital score. Both treatments did not significantly influence flow cytometry parameters, although genistein showed slight decrease in DI, with a normalization of the aneuploid content present in some cases that could represent an additional application of intravaginal phytoestrogen therapy, providing an alternative therapy of vaginal atrophy in postmenopausal patients. The results of this investigation should be considered preliminary and need to be verified in larger, prospective studies. [ABSTRACT FROM AUTHOR]