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It is believed that the majority of oral cancers develop from oral potentially malignant lesions (OPML). Though they can be easily detected during screening, risk stratification is difficult. During screening clinicians often find it difficult to distinguish OPMLs from benign lesions, and predicting OPML at risk of malignant transformation is particularly challenging. DNA aneuploidy has been known to be a marker of malignancy in a number of sites including the oral cavity. We performed a systematic review to evaluate the effectiveness of DNA-ICM using brushings in differentiating OPMLs from benign/inflammatory lesions during screening and in predicting malignant transformation. MEDLINE, Pubmed, EMBASE electronic databases were systematically searched using a combination of keywords and subject headings. A total of 11 articles satisfied our inclusion criteria. These studies reported a wide range of sensitivity (16-96.4%) and specificity (90-100%) due to the differences in study design, definitions of high risk or low risk lesions and DNA-ICM protocol used. No long-term longitudinal studies were identified to assess the role of DNA-ICM using brushings in predicting malignant transformation. No studies evaluated the role of DNA-ICM in community screening settings. A number of studies combined DNA-ICM with other techniques like cytology or argyrophilic nucleolar organizer region counts leading to improved test results. In spite of DNA aneuploidy being accepted as a marker of malignancy, there is limited evidence of DNA-ICM using brushings being successful as an adjunct oral cancer screening tool. Longitudinal studies and large community screening studies need to be undertaken to draw stronger conclusion. [ABSTRACT FROM AUTHOR]

Assessing the nuclear C–DNA content and estimating DNA ploidy in succulent species, such as dragon fruit ( Hylocereus spp., Cactaceae), is a challenging task. Dragon fruit tissues are rich in polysaccharides that are released during the nuclei isolation procedure, inducing the formation of clusters of nuclei and hence leading to inaccurate results. Our flow cytometry protocol for dragon fruit species is simple, rapid and reproducible. Our innovations include: modifications to the nuclear isolation buffer to ‘match’ the cytosol composition of the cells; filtration of the samples instead of centrifugation, thereby preventing co-precipitation of nuclei and polysaccharides; and staining of the nuclei with propidium iodide. DNA ploidy of dragon fruit samples were estimated using a reference standard − either external or internal − with a known ploidy level and a known nuclear DNA content. The best results were obtained using new emerging and actively growing stem tips, which have lower concentrations of polysaccharides and a lower frequency of endopolyploid nuclei, thereby simplifying the nuclei isolation procedure. [ABSTRACT FROM AUTHOR]

Objective Histological grade is a well-established prognostic/predictive factor in breast cancer. However, mainly within intermediate categories, patients may have unpredictable outcome. We hypothesised whether ploidy status can distinguish different prognostic groups among breast cancer patients with similar tumour grade. Material and methods The study involved 684 patients with invasive breast carcinoma, and median follow-up of 134.5 months. Pathological staging was evaluated according to WHO classification. Tumour differentiation was assessed using the Nottingham grading system. Ploidy was determined prospectively by DNA flow cytometry. Disease-free survival (DFS) and overall survival (OS) were estimated by the Kaplan–Meier method. Results There were 179 (26.2%) deaths and 239 (33.3%) disease recurrences. For grading, tumours were classified as follows: 163 (23.8%) G1, 356 (52.1%) G2 and 165 (24.1%) G3, while 389 (56.9%) tumours presented aneuploidy. Ploidy and grading are strongly associated (P < 0.001). Patients with aneuploid G2 tumours showed worse DFS (P = 0.001) and OS (P < 0.001), as well as those with aneuploid G1 tumours in relation to OS (P = 0.013). When a subset analysis was performed in early breast cancer patients (n = 451) with Stage I/IIA of disease, it remained the same significant associations of aneuploid G1 (to OS) and G2 tumours (to DFS and OS) with unfavourable prognosis. Conclusions Aneuploidy identifies subsets of breast cancer patients with G1 and G2 tumours who showed poor clinical outcome. The finding has therapeutic implications, as these patients are potential candidates to risk-adapted adjuvant therapy. [ABSTRACT FROM AUTHOR]

Abstract: The centrosome plays an essential role in chromosomal segregation during cell division. Centrosome dysfunction might lead to aneuploidy and chromosomal instability. Invasive breast tumors with centrosome amplification often show aneuploidy. Flow cytometry (FCM) was used to examine the aneuploidy rate in 30 cases of each of the following seven tissue types: normal breast tissue, usual ductal hyperplasia, atypical ductal hyperplasia, low-grade ductal carcinoma in situ, high-grade ductal carcinoma in situ, low-grade invasive ductal carcinoma, and high-grade invasive ductal carcinoma. Centrosomal α, γ-tubulin expression was examined by FCM immunofluorescence and compared between diploid and aneuploid cells. The aneuploidy rate was 0, 6.7%, 26.7%, 30.0%, 46.7%, 56.7%, and 86.7%, respectively, in the seven tissue types. The percentage of cells expressing α- and γ-tubulins was significantly different between the seven groups, and the positive rate of α- and γ-tubulin expression in ADH, DCIS and IDC was higher than that in NBT and UDH. The percentage of cells expressing α- and γ-tubulins in the diploid state was significantly lower than that in the aneuploid state. Expression of centrosomal α- and γ-tubulins seems to be associated with DNA ploidy in breast premalignant lesions and carcinoma during the progression of breast cancer. [Copyright &y& Elsevier]

Summary: The estimated prevalence of oral leukoplakia is worldwide approximately 2%, with an annual malignant transformation rate of approximately 1%. The aim of the present study was to evaluate the possible contribution of ploidy measurement to the prediction of the clinical course, in a well defined cohort of patients with oral leukoplakia. Ploidy was measured by both flow cytometry (FCM-DNA) and image cytometry (ICM-DNA) and we focussed on the comparison of the two different techniques to determine ploidy. A total of 41 patients have been included, with a mean age of 59years (range 36–78years). With FCM-DNA, three lesions were aneuploid, with ICM-DNA, 19 lesions were aneuploid. DNA ploidy was compared with clinicopathological and patients parameters. There were no statistically significant differences between DNA ploidy and any patient factor with both FCM-DNA and ICM-DNA. Using FCM-DNA, DNA aneuploid lesions showed statistically significant more dysplasia (p =0.04) than diploid lesions. Furthermore, DNA aneuploid lesions were more frequently encountered at high-risk locations (p =0.03) as being determined with FCM-DNA. These relations were not found when DNA ploidy was determined with ICM-DNA. [Copyright &y& Elsevier]

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]

Abstract: Lung cancer and head and neck cancer have similar genotoxic risk factors. While the vast majority of lung cancers are caused by cigarette smoking alone, smoking together with heavy drinking are the major etiological agents of head and neck cancer. In addition, human papilloma virus (HPV) has been identified as an important causative factor of tonsillar carcinomas. In contrast, chromosomal instability and aneuploidy identifiable by DNA measurement are predominately associated with cancer progression. This selective review summarizes our studies that aimed to gain a better understanding of the biology and pathology of lung cancer and head and neck cancer. In particular, it was attempted (a) to develop a microscopy-based tumor classification system that provides insight into the genetics of cancer cells and in particular their DNA ploidy, (b) to apply this classification system to lung cancer and head and neck cancer and to correlate it with clinicopathological parameters and molecular biomarkers, and (c) to analyze molecular characteristics and in particular the presence of human papilloma virus in lung cancer and head and neck cancer. Therefore, we developed a core classification based on the semi-quantitative assessment of the size and type of tumor cell nuclei and mitoses. It was found that (1) nuclear and mitosis size correlated with the DNA content of the tumor cells, (2) tripolar mitoses were indicative of cancer with near-triploid DNA content, (3) morphological and DNA parameters indicating variability of the cancer genome were associated with poor prognosis of lung cancer patients, (4) HPV-positive head and neck squamous cell carcinomas were characterized by smaller tumor nuclei and reduced DNA amount compared to HPV-negative carcinomas, and (5) HPV is associated with lung cancer in certain geographical regions of the world. [Copyright &y& Elsevier]

Abstract: The aim of this study was to investigate the potential clinical utility of DNA flow cytometry biomarkers, ploidy, and S-phase fraction (SPF) in predicting overall survival in cervical cancer. This prospective study involved 159 patients with cervical carcinoma (median follow-up, 48 months). Pretreatment clinical staging was done according to the FIGO 2009 update classification. Biopsy tumor samples were used for flow cytometry analysis and histological examination. A prognostic study was performed using both Cox and Bayesian Weibull regression models. Eighty (50.3%) tumors presented DNA aneuploidy, mostly observed in adenosquamous (AS) cell carcinoma (8 of 9 cases) and adenocarcinoma (AC) (12 of 17 cases). The median SPF value (8.6%) was used for discriminating low vs. high tumor cell proliferation. High SPF significantly correlated with aneuploidy (p <0.001). All AS carcinomas had SPF>15%, while all ACs presented SPF<10% (p <0.001). Forty-three (27%) patients died of the disease during follow-up. Log-rank tests revealed significant differences between survival curves for older patients (≥44 years) (p =0.029), advanced clinical staging (p <0.001), and DNA diploidy in stage IIB of disease (p =0.039). Both regression analyses showed that advanced clinical staging and low SPF independently predict worse overall survival of patients. The results suggest that DNA flow cytometry parameters can provide additional predictive information in cervical cancer management. [Copyright &y& Elsevier]

Abstract: Mitochondria are the bioenergetic and metabolic centers of cells and play an important role in the regulation of cell death. The mitochondrial apoptosis pathway is controlled by the bcl-2 protein family. Overexpression of mitochondrial uncoupling protein 4 (UCP4) can promote proliferation and inhibit apoptosis and differentiation. Imprint smears obtained from 124 tumors were studied immunocytochemically, and results were correlated with prognostic markers. There were 112 ductal and 12 lobular carcinomas. The positivity of UCP4 was correlated with lymph node metastases (p =0.005), positive ER and PR expression (p <0.0001 for both), as well as positivity for p53 (p <0.0001) and Ki-67 (p <0.0001). Decreased expression of bcl-2 correlated with increased expression of UCP4 (p =0.001). Regarding DNA ploidy, UCP4 positivity was correlated with aneuploid tumors (p =0.002). Negative expression of bcl-2 was correlated with poorly differentiated carcinomas (p <0.0001), as well as with positive expression of p53 (p <0.0001) and Ki-67 (p <0.0001). Logistic regression revealed that ploidy and p53 expression had an impact on UCP4. These findings encourage future investigations regarding the potential role of UCPs not only into mechanisms underlying breast cancer, but also as a novel candidate to the design and development of more effective therapeutic strategies. [Copyright &y& Elsevier]

Summary: To date there are still no reliable biomarkers for oral potentially malignant disorders (PMDs) to predict the risk of progression to squamous cell carcinoma (SCC). Within a prospective clinical trial of patients with PMDs, DNA content flow cytometry (DNA FCM) was evaluated for 60 PMDs using fresh samples obtained by a dermatological curette. There were 6/42 PMDs without dysplasia, but with DNA aneuploidy, versus 8/18 with both dysplasia and aneuploidy (p =0.02). When the tongue and the buccal mucosa, the two most common sites in the present series of cases were compared, dysplastic PMDs were mainly located on the tongue (p =0.01). Tobacco smokers, who preferentially developed PMDs in the buccal mucosa at a younger age than non-smokers (p =0.002), had fewer dysplastic PMDs than did non-smokers (p =0.01). Dysplasia was significantly linked to DNA aneuploidy (p =0.03) in smokers. The present data suggest that aneuploidy is an early event in oral carcinogenesis and that the influence of tobacco varies according to subsite and patient age. When DNA FCM of PMD samples are obtained by curette scraping, extensive areas can be covered with a minimally invasive, rapid, inexpensive procedure. Moreover DNA FCM of these samples appears easy amenable to routine analysis. Further research on larger numbers of PMDs should be carried out to determine whether DNA FCM plays a role in the prediction of risk of PMD transformation. [Copyright &y& Elsevier]

Abstract: We attempted to establish a microscopy based tumour characterization providing insight into the genetics of cancer cells and in particular their DNA ploidy. The core classification defined semi-quantitative criteria for scoring the nuclear size ranging from small (core score 1) to giant nuclei (core score 4). By listing all nuclear sizes according to their relative frequencies it provided a measure for core size variability as a correlate of nuclear pleomorphism. Additionally, the mitosis size, their variability and the presence/abundance of tripolar and tetrapolar mitoses were determined. This classification was applied to 155 lung cancer samples from all major histologic types and the results were correlated with the analysis by DNA image cytometry and patient survival. The morphological assessments correlated highly significantly with the DNA ploidy parameters, e.g. small cell lung carcinomas showed the smallest values for nuclear size (mean core score of 1.18) and DNA content (DNA index mean of 2.08c) being highly significantly different from adenocarcinomas (1.95/3.10c), large cell lung carcinoma (2.00/3.26c) and squamous cell carcinoma (2.20/3.42c). In non-small cell lung carcinoma (NSCLC) in general and adenocarcinoma in particular, the core size variability correlated significantly with grading and survival. Furthermore, parameters indicative for chromosomal variability, i.e. 2c deviation index and 5c exceeding rate, were predictors of poor survival in NSCLC patients. As a complement to histologic tumour diagnosis the core classification should help to better stratify cancer subtypes. [Copyright &y& Elsevier]

Summary: Oral dysplasia is a potentially precancerous lesion diagnosed histologically. While the risk of progression is associated with histological grade, it is currently impossible to predict accurately which lesions will progress. More accurate markers predicting progression to cancer would enable the targeting of these lesions for more aggressive treatment and closer follow-up. We have performed a systematic review with pooling of data to assess the evidence for the use of biomarkers in predicting transformation of oral dysplasia into cancer. We systematically searched the Cochrane library, MEDLINE, EMBASE, AMED, Cinahl and the Kings Fund electronic databases using the terms: oral dysplasia, leukoplakia, erythroplakia, biomarkers and genetic markers. The following a priori selection criteria were used: longitudinal cohort or case-controlled studies of oral dysplasia that progressed to cancer. Cross-sectional studies and studies reporting only on leukoplakia were excluded. Data were extracted by two reviewers. Quality assessment was carried out using validated tools. We assessed the relative risk of progression form oral dysplasia to cancer and pooled data where possible. 2550 studies were identified, from which 288 were scrutinised in greater detail. Of these, 247 were excluded, mainly due to cross-sectional design. Of the 41 studies containing follow-up data, 28 were excluded, most commonly due to data only being available for lesions once they had progressed to cancer. A lack of clear histological definition of oral lesions was also a common finding. Data were extracted from 13 longitudinal studies. The evidence consists mainly of small, single centre, retrospective studies. In oral dysplasia, loss of heterozygosity (LOH), particularly at the 3p±9p loci, increases the risk of progression to cancer (RR 17.60 (2.77, 108.37) p <0.001), as does survivin (RR 30 (4.25, 197.73), p ⩽0.001), matrix metalloproteinase (MMP 9), (RR 19.00 (1.56, 209.38) p =0.02) and DNA content (RR 12.00 (1.17, 82.10) p = 0.03). Other markers identified by this review including p53, p73, MMP 1 and 2 and cathepsin L mRNA, did not predict progression. LOH, survivin, MMP 9 and DNA content are potential markers for increased risk of progression from oral dysplasia to cancer. Many methodological limitations have been identified by this review, however, and we recommend these results are interpreted with caution. Research into this field should concentrate on longitudinal design, with pooling of data from multiple centres to achieve larger cohorts. We recommend standardisation of definitions to allow appropriate comparisons to be made. [Copyright &y& Elsevier]

Background: The diagnosis of uterine sarcoma is associated with poor outcome for the patient and there is a need for reliable prognostic markers. Most previous studies on the prognostic value of DNA ploidy include few uterine sarcomas and report conflicting results. [ABSTRACT FROM PUBLISHER]

Abstract: Numerous data demonstrate a lower aggressiveness of tumors in aged as compared to young patients. The mechanisms underlying this phenomenon have not yet been completely elucidated. Several mechanisms have been shown, such as reduced tumor cell proliferation, increased apoptosis, immune response modifications and reduced angiogenesis in aged organism tumors. In the present study we report an incidentally found, not yet described mechanism, of the age-related reduced tumor progression, namely a decreased ploidy in B16 melanoma growing in old (near diploidy) as compared to young mice (tetraploidy). We surprisingly observed that tumor cells from aged mice were of smaller cell and nuclear size than those of young animals. Flow cytometry forward scatter data also showed a smaller cell size of melanoma cells from old mice. DNA flow cytometry profile comparison demonstrated that while B16 melanoma cells from young animals contained a high percentage of tetraploid cells, those derived from old animals were mostly close to diploid. A high importance has recently been attributed to aneuploidy as being at the origin of the genetic instability of neoplasia. Our results may support this notion. The transit from tetraploidy to near euploidy in melanoma cells growing in aged mice might avoid the genetic instability inherent to tumor progression. [Copyright &y& Elsevier]

Abstract: Objective: To investigate the correlation of DNA ploidy, S-phase fraction (SPF) and the expression of cyclin A to the clinical prognostic factors in patients with epithelial ovarian cancer. Study design: A prospective analysis was performed on 46 ovarian tumor patients. The DNA ploidy and SPF were determined by flow cytometry and the expression of cyclin A was measured by immunohistochemical staining. Results: Compared with benign and borderline tumors, the malignant tumors expressed higher levels of cyclin A (P =0.007), more aneuploid cells (P =0.018) and higher SPF (P =0.015). With regard to DNA ploidy and the clinical prognostic factors, aneuploid cells increased with tumor grade (P =0.011), the disease stage (P =0.009) and residual volume (P =0.001). When residual tumor was more than 2cm, the expression of cyclin A was increased (P =0.043). Three-year survival was low for patients with tumors expressing cyclin A in over 10% of cells (P =0.003). Conclusions: The data suggest that the assessment of the DNA ploidy, SPF and the expression of cyclin A may provide important information for predicting the prognosis of epithelial ovarian cancer. [Copyright &y& Elsevier]

Summary: Despite new diagnostic and therapeutic strategies (combined radiochemotherapy, EGFR antibody Cetuximab), the prognosis of head and neck squamous cell carcinoma (HNSCC) is still poor and more information regarding prognosis is essential to establish earlier and better treatment options. To elucidate the role of DNA ploidy and cellular proliferation, resected tumors of 48 patients with primary or recurrent HNSCC were analyzed by flow cytometry and in vitro-5-bromodeoxyuridine incorporation (BrdU). The results were compared with histopathological findings such as tumor size, lymph node involvement and tumor differentiation. To assess the influence of intratumoral heterogeneity of these biological parameters, multiple biopsies (>3) were analyzed by flow cytometry and BrdU-incorporation in 12 larger (>4cm diameter) tumors. BrdU-labeling index (LI%) was significantly higher in aneuploid HNSCC and correlated significantly with poor histologic differentiation of the analyzed tumor tissues (P <0.001). Furthermore, a trend for higher LI% in nodal positive tumors was observed. Aneuploid HNSCC showed significantly more often tissue dedifferentiation (P =0.049) and in most cases an advanced tumor stage, especially in tumors with biclonal cell lines. Lymph node involvement was also seen more often in aneuploid and undifferentiated tumors. As in aneuploid tumors recurrent HNSCC showed in most cases a higher LI% and poor tissue differentiation, but as a result of the small collection of samples there was no correlation between aneuploidy and tumor recurrence. To proof the robustness of the acquired data and to estimate the influence of intratumoral heterogeneity to ploidy and LI% multiple biopsies were analyzed in larger tumors. Using a specific statistical algorithm a secure estimation of ploidy and LI% was possible by a single biopsy in these tumors. These findings indicate aneuploidy and proliferative activity as important findings for malignant progression in HNSCC. An estimation of these biological parameters may be useful for identification of patients with high risk for lymph node involvement or tumor recurrence and pre-treatment can be performed by a single biopsy. As a conclusion, these patients may benefit from more aggressive treatment. [Copyright &y& Elsevier]

Summary: We attempted to identify the molecular mechanisms involved in Head and Neck Squamous Cell Carcinoma (HNSCC) pathogenesis by measuring the nuclear DNA content (ploidy) in premalignant (potentially malignant) and malignant patients as compared to normal controls, and to determine whether DNA ploidy could be used to predict the clinical outcome. From March 2001 to December 2003, the analysis was carried out in a set of 41 patients with premalignant lesions and 79 suffering from squamous cell carcinoma of laryngeal, oesophageal, nasopharyngeal, nasal and oral lesions and 50 controls. Representative samples were taken by punch biopsy and processed using standard formol-paraffin technique for histopathological examination. Fifty micrometer thick sections of paraffin-embedded tissues were analyzed to detect the DNA content by image cytometry. Of the potentially malignant patients, 46% had diploid lesions, 37% had tetraploid lesions and 17% had aneuploid lesions. While of the patients with cancer, 90% had aneuploid lesions, 10% had diploid lesions and none had tetraploid lesions. DNA diploidy tended to occur earlier in the progression from premalignant to malignant lesions and this helps us early detection of HNSCC by DNA from lesions in high risk groups and examination of its ploidy. Knowledge of tumor cell ploidy by DNA image cytometry may facilitate the evaluation of malignant and premalignant lesions in HNSCC. The present findings are promising to supplement clinical and histopathological parameters in evaluating prognosis and to demonstrate methods that are readily applicable for routine diagnostic work. [Copyright &y& Elsevier]

Abstract: Background: The aim of the present study was to evaluate the prognostic significance of DNA ploidy and Ki67 expression in non-small cell lung carcinoma (NSCLC). Methods: This prospective study included 96 patients with stages I–IIIA NSCLC who underwent surgical excision. DNA image analysis cytometry was applied on imprints. Calculation of the DNA index (DI) and the 5c exceeding rate (5cER) was performed and the histograms were classified as peridiploid, peritetraploid, and x-ploid–multiploid. The Ki67 immunoreactivity was determined according to the avidin–biotin complex immunoperoxidase method. Results: DNA histogram classification disclosed 30 peridiploid cases, 15 peritetraploid and 51 x-ploid–multiploid. Forty-eight cases (50%) had 5cER>5%. The Ki67 immunoreactivity was below 25% in 53 tumors (62.4%) and above 25% in 32 (32.6%). Our results revealed the existence of a statistically significant relationship of DNA ploidy with nodal status (p =0.042) and grade (p =0.005). Adenocarcinomas and large cell carcinomas were more frequently encountered in x-ploid–multiploid tumors as compared to squamous cell carcinomas, which were more frequently peridiploid (p =0.003). 5cER showed statistically significant association with nodal status (p =0.037). Univariate analysis with respect to survival revealed significant association with stage (p <0.001), nodal status (p <0.001), tumor status (p <0.001), DNA ploidy (p =0.008) and 5cER (p =0.0124). Multivariate analysis revealed stage and ploidy status as independent factors: peridiploid tumors were associated with better survival as compared to x-ploid–multiploid tumors (p =0.022). Conclusion: Our results suggest that DNA ploidy, as determined by image analysis, provides an independent prognostic parameter for patients with NSCLC and thus, could be used to identify a subset of patients with more aggressive tumors. [Copyright &y& Elsevier]

Abstract: Purpose: The study was designed to determine independent prognostic variables in suboptimally debulked advanced ovarian cancer patients entered in the randomised phase III study EORTC 55865. Experimental design: Retrospectively collected paraffin blocks from 169 patients with stages IIb–IV epithelial ovarian cancer, taken at primary debulking surgery, were analysed. All patients were treated with cyclophosphamide and cisplatin (CP), and followed up for a median of 10 years. Expression of p53, bcl-2, P21, Ki-67 and HER-2 status was assessed by immunohistochemistry (IHC). Results: Expression of p21, a downstream effector of the p53 gene, was found to be a favourable prognostic factor for survival (HR 0.58, CI 0.36–0.94, p =0.025) in addition to FIGO stage (HR 1.54, CI 1.08–2.21, p =<0.02). For progression free survival (PFS), both p21 (HR 0.52) and Ki-67 (HR 0.6) were significant factors. Conclusion: P21 overexpression is a positive prognostic factor for survival and PFS in advanced ovarian carcinoma with residual lesions of more than 1cm. [Copyright &y& Elsevier]

Abstract: Aims: To determine the prognostic value of proliferative potential and DNA ploidy in 72 brain tumours (36 grade III and 36 grade IV astrocytomas) using bromodeoxyuridine (BrdUrd) incorporation and flow cytometry. Material and methods: All 72 patients underwent excision, mostly incomplete of the tumour. After surgery, eight patients received conventionally fractionated radiotherapy, 11 patients received accelerated radiotherapy, and 53 patients received hypofractionated radiotherapy. Tumour samples taken during surgery from each patient were incubated in vitro for 1h at 37°C with BrdUrd using the high pressure oxygen method. The percentage of BrdUrd-labelled cells (BrdUrd labelling index [BrdUrd LI]), and the total DNA content were evaluated. Results: The tumours showed variability in the BrdUrd LI values, which ranged from 0.3 to 19.1%. No difference was observed in mean BrdUrd LI between grade III and grade IV sub-groups. A significantly higher percentage of DNA aneuploidy was observed in grade III gliomas (69.4%) than in grade IV gliomas (52.8%). Univariate analysis showed that younger patients (≤51 years) (P =0.021) with grade III gliomas (P =0.030) and low tumour proliferation rate (BrdUrd LI≤2.7%, P =0.028) had significantly higher 5-year survival rates. Tumour ploidy had no influence on patients'' survival (P =0.591). However, Cox multi-variate analysis showed that only age over 51 years, and high tumour proliferation rate (BrdUrd LI>2.7%), were significant unfavourable prognostic factors in patient survival. Conclusion: In this study, independent prognostic factors for patients with high-grade gliomas treated with surgery and post-operative radiotherapy are age and tumour proliferation rate assessed according to the BrdUrd LI. [Copyright &y& Elsevier]

Abstract: Background: The purpose of this study was to explore the potential use of image analysis on tissue sections preparation as a predictive marker of early malignant changes during squamous cell (SC) carcinogenesis in the esophagus. Results of DNA ploidy quantification on formalin-fixed, paraffin-embedded tissue using two different techniques were compared: imprint-cytospin and 6μm thick tissue sections preparation. Methods: This retrospective study included 26 surgical specimens of squamous cell carcinoma (SCC) from patients who underwent surgery alone at the Department of Surgery in CHUV Hospital in Lausanne between January 1993 and December 2000. We analyzed 53 samples of healthy tissue, 43 tumors and 7 lymph node metastases. Results: Diploid DNA histogram patterns were observed in all histologically healthy tissues, either distant or proximal to the lesion. Aneuploidy was observed in 34 (79%) of 43 carcinomas, namely 24 (75%) of 32 early squamous cell carcinomas and 10 (91%) of 11 advanced carcinomas. DNA content was similar in the different tumor stages, whether patients presented with single or multiple synchronous tumors. All lymph node metastases had similar DNA content as their primary tumor. Conclusions: Early malignant changes in the esophagus are associated with alteration in DNA content, and aneuploidy tends to correlate with progression of invasive SCC. A very good correlation between imprint-cytospin and tissue section analysis was observed. Although each method used here showed advantages and disadvantages; tissue sections preparation provided useful information on aberrant cell-cycle regulation and helped select the optimal treatment for the individual patient along with consideration of other clinical parameters. [Copyright &y& Elsevier]

Summary: This study was designed to show the relation between DNA-ploidy in patients with resectable lung cancers and their 5-year survival rate. The results are compared with those from our 2-year follow-up study of the same group of patients published in 2000. The group of 80 patients with SCC who underwent lung resection between 1995 and 96 were re-analyzed. For the statistical analysis the hazard Cox model and an expotential multiple regression model were used. The survival curves were drawn using the Kaplan–Meier method. DNA-aneuploidy was found in 45% of cancer tumors. There was no statistically significant correlation between aneuploidy and gender, age, cancer staging or grading. In the 3-year follow-up the survival rate in patients with aneuploid type tumors was significantly lower than in those with the diploid type. However, this difference was not found after 5 years of follow-up. Tumor ploidy was an independent prognostic factor only in patients between 55 and 60 years of age. The mortality rate in patients with aneuploid tumors was mainly the result of distant metastases while, in patients with diploid tumors, local recurrence was the main reason for death. In the first three years after surgical resection patients with aneuploid tumors are at higher risk of distant metastases than patients with the diploid type. Tumor ploidy can be recognized as an independent prognostic factor in younger (55–60) patients. Aneuploidy promoted the occurrence of early distant metastases while the diploid type was associated with late (after 3 years) local tumor recurrence. [Copyright &y& Elsevier]

Abstract: Purpose. – To assess the significance of S-phase fraction (SPF) and DNA ploidy evaluated by DNA flow cytometry as prognostic markers in stage I or II breast cancer. Patients and methods. – A series of 271 patients, treated by surgery, radiotherapy±systemic therapy was analysed (median follow up: 64 months). Standardized flow cytometry cell preparation from frozen samples and consensus rules for data interpretation were followed. Three SPF classes were defined on the basis of tertiles after adjustment for ploidy. Four groups were defined based on combinations of DNA ploidy (DIP: diploid; ANEUP: aneuploid) and SPF: DIP and low SPF (DL, N =37), DIP and medium or high SPF (DMH, N =76), ANEUP and low SPF (AL, N =24), ANEUP and medium or high SPF (AMH, N =68). Local control rate (LCR), disease-free survival (DFS), metastasis-free survival (MFS), and overall survival (OS) were correlated with DNA ploidy, SPF, DL to AMH groups, T and N stages, SBR grading, age, and hormonal status on univariate and multivariate analysis (Cox model). Results. – On univariate analysis, DFS and LCR were higher for DIP tumours. High SPF values were associated with shorter DFS. LCR, MFS, DFS, and OS rates were significantly different with an increasingly poorer prognosis from DL to AMH. On multivariate analysis, groups DL to AMH, histological node involvement and T stage were independently associated with MFS, and DFS. In N– patients, DL to AMH remained independent for MFS and DFS. For SBR III tumours, MFS and OS were significantly different in DL to AMH groups. These results strongly support the use of combined evaluation of DNA ploidy and SPF as independent parameters in clinical trials for N– stage I and II breast cancer. [Copyright &y& Elsevier]

Summary: The aim of this study on a series of biopsies diagnosed as normal, metaplastic, low-grade squamous intraepithelial lesions (LSILs), and high-grade squamous intraepithelial lesions (HSILs) was dual: to determine the chronology of cell cycle and proliferation abnormalities after human papillomavirus infection during the development of squamous intraepithelial lesions and to determine the best diagnostic indicator(s) linked to the appearance of an HSIL. Ninety-nine cervical biopsies, 18 normal, 9 with metaplastic changes, 29 LSIL, and 43 HSIL (23 cervical intraepithelial neoplasia 2 and 20 cervical intraepithelial neoplasia 3), were analyzed by image cytometry for DNA ploidy and p16INK4A determination, AgNOR counting, MIB-1, and ICBP90 immunostaining quantification. The human papillomavirus status had been previously determined on corresponding cytological smears with the Hybrid Capture II test. Suspect DNA profile and p16INK4A staining were the first significant events that preceded the increase of cell proliferation. Indeed, these markers were the best tests for the detection of a lesion, whatever its grade (positive predictive values of 90% and 100%, respectively). The presence of MIB-1– or ICBP90-positive cells in the upper two thirds of the epithelium was a very accurate feature to select HSIL (sensitivity, 100% for MIB-1) but with a low specificity. The sensitivity of a suspect DNA profile associated with a positive MIB-1 or ICPB90 immunostaining for the detection of an HSIL was, respectively, 92.8% and 92.7%; their specificities were 54.2% and 44%; their positive predictive values were 78% and 73%; their negative predictive values were 81.2% and 78.6%; and the global values were 78.8% and 74.3%. Thus, the most accurate test to distinguish an LSIL from an HSIL was the association of a suspect DNA profile and the presence of MIB-1– or ICBP90-positive cells in the upper two thirds of the epithelium. [Copyright &y& Elsevier]

Summary: Morphologic examination still forms the main diagnostic tool in the differential diagnosis of molar pregnancies. However, the criteria are subjective and show considerable interobserver variability among pathologists. Once a diagnosis of molar pregnancy is made, DNA ploidy studies help to differentiate a triploid partial mole from diploid complete mole (CM). However, with earlier diagnosis and therapeutic evacuation of molar pregnancies, the differentiation of molar pregnancies from early nonmolar placentation is becoming increasingly difficult. The p57KIP2 gene (CDKN1C) is strongly paternally imprinted and expressed from the maternal allele. Because CM lacks a maternal genome, p57KIP2 immunostaining is correspondingly absent, whereas hydropic abortuses and partial mole show positive staining. We compared the use of p57KIP2 staining in the differential diagnosis of 68 morphologically challenging cases of early first-trimester hydropic placentas. Diagnosis based on p57KIP2 staining was compared with the original diagnosis based on morphology and DNA ploidy analysis. Concordant results were obtained in 65 of 68 cases studied. In 2 of 3 cases with a discordant diagnosis, microsatellite DNA genotyping analysis agreed with the results of p57KIP2 staining, confirming that positive p57KIP2 staining is a highly sensitive and specific marker for excluding CM in this setting. In addition, p57KIP2 staining has the advantage of differentiating hydropic abortuses from CMs, a distinction not made by ploidy analysis. p57KIP2 staining can be used in concert with ploidy studies to refine the diagnosis of early molar pregnancies. [ABSTRACT FROM AUTHOR]

DNA ploidy pattern and S-phase fraction (SPF) measured by flow cytometry and expression of the retinoblastoma gene product (pRB) estimated by immunohistochemistry were correlated with outcome in 114 patients who received a curative operation for primary non-small cell lung cancer (NSCLC). One hundred ten tumors yielded an adequate DNA histogram, and all tumors exhibited an assessable immunohistochemical stain. DNA diploidy was detected in 31 tumors and DNA aneuploidy in 79 tumors. The mean SPF was 14.1±6.4%. Eighty tumors were positively stained, and 34 tumors were negative for pRB. Multivariate analysis clarified that both TNM staging and DNA ploidy were prognostic factors after surgery. In 39 recurrent cases, the SPF value was inversely correlated with disease-free interval. With only supportive care after recurrence, high SPF tumors and pRB-negative tumors progressed rapidly, whereas active treatment yielded an equivalent effect on recurrent tumors regardless of the SPF or pRB expression. DNA ploidy is related to the risk of recurrence, while SPF is related to tumor growth rate and the impact of active treatment on recurrence. The utility of pRB expression was limited. The combination of DNA ploidy and SPF allows practical stratification of the biologic aggressiveness of NSCLC. [Copyright &y& Elsevier]

The objective of this study was to determine the clinical and biopsy features associated with outcomes at radical retropubic prostatectomy (RRP) in patients with clinically organ-confined prostate cancers and biopsy Gleason scores (GS) of 6 or less. We reviewed 274 biopsies with GS 6 or less cancers from patients with clinically organ-confined disease between 1995 and 1998 to determine statistically significant predictors for the following outcomes at RRP: tumor volume, small (<0.5 cc), confined (pT2) tumors with RRP GS of 6 or less (potentially “insignificant” tumors), and extraprostatic extension (EPE). Clinical and pathologic features evaluated included age, serum prostate specific antigen (PSA), clinical stage, percent biopsy cores and surface area positive for cancer (tumor extent), perineural invasion, MIB-I proliferation, and DNA ploidy by digital image analysis (DIA). Multivariate analyses showed that biopsy tumor extent (median percent surface area positive 3.3%; P < 0.001 and median biopsy cores positive 28.6%; P = 0.001) and PSA (median 5.5 ng/mL; P = 0.009) predicted tumor volume (median 1.4 cc). Biopsy tumor extent (P = 0.002), PSA (P = 0.002), and percent S-phase nuclei (P = 0.050) predicted potentially “insignificant” tumors at RRP (n = 76, 28%). Percent surface area positive for cancer (P = 0.003) predicted EPE (n = 22, 8%). DNA ploidy (n = 211, 79% diploid) and MIB-I proliferation (median 1.4%) did not add information to predict these RRP outcomes. Biopsy tumor extent and serum PSA were significantly associated with tumor volume. Biopsy tumor extent, serum PSA, and percent S-phase nuclei by DIA were predictive of potentially insignificant tumors. Patients with clinically confined disease, <5% biopsy surface area positive for cancer, <20% biopsy cores positive for cancer, and GS 6 or less, had a 48% chance of having a potentially insignificant tumor at diagnosis if the serum PSA was <10 ng/mL. Percent surface area predicted EPE at RRP. DNA ploidy and MIB-I proliferation by DIA did not provide additional information. [Copyright &y& Elsevier]

Introduction: The clinical value of established prognostic factors seems to be limited since they fail to predict reliably survival of patients after resection of cholangiocarcinoma. DNA ploidy reflecting irregularities of chromosome number and content might be an alternative predictor. In this study, we evaluated the DNA ploidy as a prognostic factor for survival of patients after resection of cholangiocarcinoma. Methods: This prospective study included 34 patients with cholangiocarcinoma which were surgically resected and followed up to death or more than 3 years. Tissue specimens were taken from the liver tissue immediately after resection and DNA ploidy determined. Survival was related to the type of DNA ploidy as well as to five established prognostic factors. Results: Multivariate analysis revealed that in this study only DNA ploidy (P=0.012) was significantly associated with prediction of survival. In contrast, neither tumor stage pT (P=0.073) nor tumor grade pG (P=0.154), resection margins R (P=0.322), metastasis M (P=0.060), lymph node stage pN (P=0.209), age (P=0.13) nor sex (P=0.849) could significantly predict survival. Three-year survival was best for patients with diploid tumors (n=6) of whom 75% survived more than 3 years. Poor prognostic signs associated with short term survival of less than 18 months were tumors classified as aneuploid (n=17), large tumors pT4 (n=8), metastasis pM1 (n=11), undifferentiated tumors pG3 (n=9) and non-tumor-free resection margins R2 (n=14). The best predictor for poor prognosis was aneuploidy since it could identify more patients with a fatal outcome than other prognostic factors. DNA ploidy turned out to discriminate highly significant between diploid, polyploid and aneuploid tumors. Discussion: The most accurate prognostic factor for survival of patients after resection of cholangiocarcinoma was DNA ploidy. Most patients suffering from a diploid tumor turned out to be long term survivors whereas aneuploid tumors indicated a poor prognosis with a rather short survival time of less than 18 months. We conclude that DNA ploidy is a valuable diagnostic tool for identifying subgroups of patients that are at higher risk for tumor progression. [ABSTRACT FROM AUTHOR]

Purpose: The correlation between the DNA content, determined using fresh tumor tissues, and the macroscopic presentation of the lesion was studied prospectively in patients with T1 glottic carcinoma.Methods and Materials: DNA flow cytometry and fiber-optic endoscopic examination were performed for 30 previously untreated patients with T1 glottic carcinoma. The patients received radical radiotherapy at Aichi Cancer Center Hospital.Results: In regard to the type of lesion, 4 (80%) were aneuploid, and 1 (20%) was diploid for the invasive type. There was a tendency to show an invasive appearance in aneuploid tumors. With respect to clinical outcome, there were 3 (43%) local recurrences among the aneuploid tumors that invaded the entire length of one vocal cord, 0 (0%) for medium-sized lesions, and 1 (17%) for small lesions. Aneuploid tumors showed a high correlation between lesion size and local control.Conclusions: The correlation was not strong enough to conclude that DNA content can replace the macroscopic presentation of the lesion. However, the combination of DNA content and tumor size may help predict radiation sensitivity. [Copyright &y& Elsevier]