Manohar Pradhan

Image cytometry DNA ploidy correlates with histological subtypes in endometrial carcinomas

Image cytometric DNA ploidy analysis of endometrial carcinomas was performed to determine whether ploidy status and ploidy-related parameters like DNA index, percentage of cells exceeding 5c and 9c, correlate with histologic subtype. This is a prospective study of 391 patients with stage I endometrial carcinoma which included 331 (85%) endometrioid adenocarcinoma, 22 (6%) serous adenocarcinoma, 7 (2%) clear cell adenocarcinoma, 2 (0.5%) small cell carcinoma, 1 (0.3%) undifferentiated carcinoma, and 28 (7%) unclassifiable adenocarcinoma. Twenty-five percent of endometrioid adenocarcinomas were non-diploid. In contrast, all clear cell adenocarcinomas and 21/22 (95%) of serous adenocarcinomas were non-diploid. Hyperdiploidy (25 cases) was found only in endometrioid adenocarcinomas. Mean DNA index of the stemline in serous adenocarcinoma (1.72) and clear cell adenocarcinoma (1.81) was higher than in endometrioid adenocarcinoma (1.1). The difference in ploidy-related parameters between endometrioid adenocarcinoma and serous adenocarcinoma was highly significant (P<0.001). In addition, Grade 3 endometrioid adenocarcinoma showed significant difference in all ploidy-related parameters compared with grade 1 and grade 2 tumors (P<0.001). Our results show that DNA ploidy-related parameters may be valuable in subtyping histologically difficult cases of endometrial carcinomas.

Prognostic importance of DNA ploidy and DNA index in stage I and II endometrioid adenocarcinoma of the endometrium

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.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.

β-Catenin expression in uterine sarcomas and its relation to clinicopathological parameters

Aberrations in the Wnt/beta-catenin signalling pathway are suggested as mediators of chromosomal instability and carcinogenesis. beta-catenin acts both as a component of the membranous adhesion system, and as a transcription activator in the nucleus. beta-Catenin immunoreactivity was evaluated in 353 uterine sarcomas (US) including 231 leiomyosarcomas (LMS), 82 endometrial stromal sarcomas (ESS), 22 adenosarcomas (AS) and 18 undifferentiated uterine sarcomas (UUS). Up-regulated membranous beta-catenin was observed in 25% of the LMS (p=0.039), 21% of the ESS (p=0.072) and 39% of the UUS (p=0.025). Cytoplasmic beta-catenin was up-regulated in 36% of the LMS (p=0.008) and 33% of the UUS (p=0.028). Nuclear beta-catenin expression was observed in 23% of the LMS (p=0.051), 61% of ESS (p=0.628) and in the sarcoma component of 68% of the AS. In patients with LMS, membranous beta-catenin was associated with poor crude survival in univariate (p=0.045), but not in multivariate analyses. In patients with ESS, nuclear beta-catenin expression was related to spread of tumour (p=0.033), but not to survival. The observation of up-regulated beta-catenin expression in US might suggest a so far undocumented role for the Wnt/beta-catenin pathway in these malignancies.

Tumour heterogeneity poses a significant challenge to cancer biomarker research

Background:The high degree of genomic diversity in cancer represents a challenge for identifying objective prognostic markers. We aimed to examine the extent of tumour heterogeneity and its effect on the evaluation of a selected prognostic marker using prostate cancer as a model.Methods:We assessed Gleason Score (GS), DNA ploidy status and phosphatase and tensin homologue (PTEN) expression in radical prostatectomy specimens (RP) from 304 patients followed for a median of 10 years (interquartile range 6–12). GS was assessed for every tumour-containing block and DNA ploidy for a median of four samples for each RP. In a subgroup of 40 patients we assessed DNA ploidy and PTEN status in every tumour-containing block. In 102 patients assigned to active surveillance (AS), GS and DNA ploidy were studied in needle biopsies.Results:Extensive heterogeneity was observed for GS (89% of the patients) and DNA ploidy (40% of the patients) in the cohort, and DNA ploidy (60% of the patients) and PTEN expression (75% of the patients) in the subgroup. DNA ploidy was a significant prognostic marker when heterogeneity was taken into consideration. In the AS cohort we found heterogeneity in GS (24%) and in DNA ploidy (25%) specimens.Conclusions:Multi-sample analysis should be performed to support clinical treatment decisions.

DNA ploidy heterogeneity in endometrial carcinoma: Comparison between curettage and hysterectomy specimens

DNA ploidy has been reported to be a prognostic marker for patients with endometrial carcinoma. In this study, DNA ploidy and histologic heterogeneity were evaluated by comparing curettage and hysterectomy specimens in 99 consecutive patients diagnosed with endometrial carcinoma. High-resolution DNA ploidy image analysis and review of histologic specimens were performed. The histologic subtypes were identical in 77 (78%) and differed in 22 (22%) cases. The DNA ploidy results were concordant in the curettage and hysterectomy specimens in 72 (72.7%) and discordant in 27 (27.3%) cases. Histologic heterogeneity was significantly associated with DNA ploidy heterogeneity (P=0.03). On the basis of histologic heterogeneity, DNA ploidy-discordant cases were divided into 2 groups. One group (16.2% of cases) consisted of specimens with similar histology in curettage and hysterectomy, all belonging to the endometrioid subtype. This group showed DNA ploidy discordance because of a DNA diploid peak in 1 specimen and an aneuploid peak (DI=1.05–1.2) in the other. The other group (11.1% of cases) consisted of cases with different histologic subtype or grade and showed a more pronounced DNA ploidy difference (diploid vs. aneuploid with DI>1.2). Our results suggest that the DNA ploidy results of the hysterectomy and curettage specimens are not identical. The difference observed, which we believe reflects the intratumoral heterogeneity, should be taken into account when applying DNA ploidy to endometrial carcinoma specimens.

DNA ploidy in curettage specimens identifies high-risk patients and lymph node metastasis in endometrial cancer

Background:Preoperative risk stratification is essential in tailoring endometrial cancer treatment, and biomarkers predicting lymph node metastasis and aggressive disease are aspired in clinical practice. DNA ploidy assessment in hysterectomy specimens is a well-established prognostic marker. DNA ploidy assessment in preoperative curettage specimens is less studied, and in particular in relation to the occurrence of lymph node metastasis.Methods:Curettage image cytometry DNA ploidy in relation to established clinicopathological variables and outcome was investigated in 785 endometrial carcinoma patients prospectively included in the MoMaTEC multicentre trial.Results:Diploid curettage status was found in 72.0%, whereas 28.0% were non-diploid. Non-diploid status significantly correlated with traditional aggressive postoperative clinicopathological features, and was an independent predictor of lymph node metastasis among FIGO stage I–III patients in multivariate analysis (OR 1.94, P=0.033). Non-diploid status was related to shorter disease-specific survival (5-year DSS of 74.4% vs 88.8% for diploid curettage, P<0.001). When stratifying by FIGO stage and lymph node status, the prognostic effect remained. However, in multivariate regression analysis, preoperative histological risk classification was a stronger predictor of DSS than DNA ploidy.Conclusions:Non-diploid curettage is significantly associated with aggressive clinicopathological phenotype, lymph node metastasis, and poor survival in endometrial cancer. The prognostic effect was also observed among subgroups with (presumably) less aggressive traits, such as low FIGO stage and negative lymph node status. Our results indicate curettage DNA ploidy as a possible supplement to existing parameters used to tailor surgical treatment.

Gross genomic alterations and gene expression profiles of high- grade serous carcinoma of the ovary with and without BRCA1 inactivation

BackgroundBRCA1 gene inactivation causes chromosomal instability, leading to rapid accumulation of chromosomal rearrangements and mutations. The loss of BRCA1 function due to either germline/somatic mutation or epigenetic silencing is observed in most high-grade serous carcinomas of the ovary.MethodsDNA ploidy and gene expression profile were used in order to compare gross genomic alteration and gene expression pattern between cases with BRCA1 loss through mutation, BRCA1 epigenetic loss, and no BRCA1 loss in cases of high-grade serous carcinoma with known BRCA1 and BRCA 2 status.ResultsUsing image cytometry and oligonucleotide microarrays, we analyzed DNA ploidy, S-phase fraction and gene expression profile of 28 consecutive cases of ovarian high-grade serous adenocarcinomas, which included 8 tumor samples with BRCA1 somatic or germline mutation, 9 samples with promoter hypermethylation of BRCA1, and 11 samples with no BRCA1 loss. None had BRCA2 mutations. The prevalence of aneuploidy and tetraploidy was not statistically different in the three groups with different BRCA1 status. The gene expression profiles were also very similar between the groups, with only two genes showing significant differential expression when comparison was made between the group with BRCA1 mutation and the group with no demonstrable BRCA1 loss. There were no genes showing significant differences in expression when the group with BRCA1 loss through epigenetic silencing was compared to either of the other two groups.ConclusionsIn this series of 28 high-grade serous carcinomas, gross genomic alteration characterized by aneuploidy did not correlate with BRCA1 status. In addition, the gene expression profiles of the tumors showed negligible differences between the three defined groups based on BRCA1 status. This suggests that all ovarian high-grade serous carcinomas arise through oncogenic mechanisms that result in chromosomal instability, irrespective of BRCA status; the molecular abnormalities underlying this in the BRCA intact tumors remains unknown.

Gross genomic alterations differ between serous borderline tumors and serous adenocarcinomas—an image cytometric DNA ploidy analysis of 307 cases with histogenetic implications

Our objective was to study the gross genomic alterations in serous borderline tumors and serous adenocarcinomas of the ovary. A retrospective analysis of 245 serous borderline tumors and 62 serous adenocarcinomas from 249 patients was performed using high-resolution image cytometric DNA ploidy analysis. DNA ploidy status, S-phase fraction, and DNA index were evaluated. The majority of serous borderline tumors were diploid (225/245 cases, 92%). The remaining 8% showed an aneuploid peak predominantly with DNA index of less than 1.4. Grades 2 and 3 serous adenocarcinomas were more often (80%) nondiploid, mostly with DNA index exceeding 1.4. Grade 1 serous adenocarcinomas were an intermediate group, more similar to serous borderline tumors. The S-phase fraction increased from serous borderline tumors (mean = 0.6%) through grade 1 serous adenocarcinomas (mean = 2.8%), being highest in grades 2 and 3 adenocarcinomas (mean = 6.8%). Our findings support the hypothesis that serous borderline tumors and grades 2 and 3 serous adenocarcinomas are genomically different lesions, with grade 1 serous adenocarcinomas being an intermediate group more close to borderline tumors.

Prostate Cancer Prognosis Defined by the Combined Analysis of 8q, PTEN and ERG

Overtreatment is a major concern in men diagnosed with prostate cancer. The aim of this study was to evaluate the combined prognostic role of three frequent molecular alterations in prostate cancer, namely relative 8q gain, ERG overexpression, and loss of PTEN expression, in a series of 136 patients with prostate cancer treated with prostatectomy and with a long follow-up. Fluorescent in situ hybridization was used to detect the relative copy number of 8q and immunohistochemistry was used for quantitative assessment of ERG and PTEN expression. During a median follow-up period of 117.8 months, 66 (49%) patients had disease recurrence. Relative 8q gain, ERG overexpression, and loss of PTEN expression were observed in 18%, 56%, and 33% of the cases, respectively. No association with patient recurrence-free survival was found for relative 8q gain or ERG overexpression on their own, whereas loss of PTEN expression was associated with worse recurrence-free survival (P = .006). Interestingly, in the subgroup of patients with normal PTEN expression, we found that the combined relative 8q gain/ERG overexpression is associated with high risk of recurrence (P = .008), suggesting that alternative mechanisms exist for progression into clinically aggressive disease. Additionally, in intermediate-risk patients with normal PTEN expression in their tumors, the combination of 8q gain/ERG overexpression was associated with a poor recurrence-free survival (P < .001), thus indicating independent prognostic value. This study shows that the combined analysis of 8q, ERG and PTEN contributes to an improved clinical outcome stratification of prostate cancer patients treated with radical prostatectomy.

Ovarian carcinoma cells in effusions show increased S-phase fraction compared to corresponding primary tumors

The objective of this study was to analyze large‐scale genomic patterns during disease progression from primary tumor to effusion in ovarian carcinoma, and to study the association between DNA ploidy parameters in effusions, proliferation/survival markers, and clinicopathologic characteristics. DNA ploidy status, DNA index (DI), and S‐phase fraction (SPF) were compared in 22 matched primary carcinomas (all prechemotherapy specimens) and effusions (14 prechemotherapy and 8 postchemotherapy specimens) using image analysis. The association between these parameters and previously studied cell survival/proliferation biomarkers, previous administration of chemotherapy, chemotherapy response and survival was analyzed in a larger series of 54 effusions. The majority of specimens were aneuploid irrespective of anatomic site, with no significant differences in DI. SPF was significantly higher in effusions compared to matched primary tumors (P = 0.007 for all 22 pairs, P = 0.011 for 14 matched prechemotherapy specimens). Higher SPF was related to higher Ki‐67 score (P = 0.045), and both SPF and DI were directly associated with higher level of Survivin (P < 0.001 for both). DI and SPF in effusions showed no association with histological grade, FIGO stage, residual disease volume, previous chemotherapy, response to chemotherapy at primary disease, recurrence or survival. Ovarian carcinoma cells in effusions have increased proliferation compared to corresponding primary tumors, as evidence of disease progression. DNA ploidy parameters in cancer cells in effusions are unaltered by chemotherapy and appear to be unrelated to chemotherapy response and to survival, suggesting that large‐scale genomic patterns at this anatomic site are not useful in segregating patients into prognostic groups. Diagn. Cytopathol. 2008;36:637–644.