Aneuploid Ones Research Articles

Could Flow Cytometry Provide New Prognostic Markers in Colorectal Cancer?

Background/Objectives: Colorectal cancer (CRC) is still accompanied by significant mortality, which poses the necessity of novel markers to predict treatment success and patient survival. This study aims to evaluate the prognostic and survival impact of flowytometry (FC) in CRC patients. Methods: In this prospective study, 106 surgically resectable CRC patients were included. Tissue specimens from tumor and normal mucosa were collected and analyzed by FC. DNA and tumor index were calculated. In a subgroup of 46 patients, the CD26 expression on tumor cells was estimated. These parameters were compared with patients' tumor characteristics as stage, histology data, responsiveness to treatment, metastasis/recurrence, and, finally, patients' survival to identify possible new biomarkers. Results: The overall survival and the disease-specific survival in our study group was 76% and 72%, respectively, during the 7-year follow up period. Diploid tumors had better median survival than the aneuploid ones. The DNA index had significant correlation to the tumor index and response to neoadjuvant treatment. Similarly, the tumor index was also significantly related to the response to neoadjuvant treatment. Patients with a higher tumor index had worst survival rates. Surprisingly, CD26 levels were not associated with any of the parameters examined and were negatively related to tumor stage and differentiation. Conclusions: FC is a rapid and reliable method of cell analysis. In CRC, it has been used for prognostic and diagnostic purposes. In this study, we have shown that DNA and tumor index could become predictive biomarkers of tumor response to neoadjuvant treatment and survival of resectable CRC patients.

EMBRYOLOGISTS ARE MORE LIKELY TO CHOOSE EUPLOID EMBRYOS FOR TRANSFER: A PROSPECTIVE BLINDED NON-SELECTION TRIAL

A number of morphologic parameters assessed using conventional microscopy or time-lapse imaging have been correlated with embryo euploidy. However, these parameters have not been validated in prospective studies. Similarly, it has not been established whether morphologic grading systems or other more subjective strategies utilized by embryologists help select euploid embryos over aneuploid ones. In this study, we tested the hypothesis that morphologic grading systems used in the embryology laboratory preferentially select euploid embryos. To test this hypothesis we conducted a prospective non-selection study where embryos were biopsied and underwent PGT-A, but embryologists, physicians, and patients were blinded to the PGT-A results until after the embryo transfer. This study design provided a unique opportunity to determine whether embryologists are more likely to choose euploid embryos for transfer. Women ages 35-42 undergoing their first IVF cycle with PGT-A were included. All embryos were cultured to the blastocyst stage. Embryo grading was performed using the modified Gardner blastocyst grading system, and embryos were selected for transfer based on morphology alone. Genetic testing results were withheld until after frozen embryo transfer was completed. The odds of selecting a euploid versus aneuploid embryo for transfer was calculated. Results were adjusted for patient age. 1,111 blastocysts became available for transfer from 156 patients. The median (IQR) number of embryos per patient was 6 (5-9). A total of 156 single embryo transfers were performed where embryos were selected by embryologists based on morphology. Of the transferred embryos, 129 were later found to be euploid and 27 were aneuploid. Euploid embryos were 1.9 times more likely to be selected for transfer compared to aneuploid embryos (OR 1.99 CI [1.27-3.12]; p=0.0026). When the data was analyzed based on age, euploid embryos were 2.79 times more likely to be chosen for transfer in the <35-year-old group (p=0.012) and 1.7 times more likely in the >35-year-old group (p=0.065), although we were not powered to detect the true association in the latter group. Embryo assessment utilizing morphologic grading preferentially selects euploid embryos for transfer.

P–564 Analysis of efficiency and outcomes of assisted reproductive technology (ART) programs after embryo transfer with a low-level of mosaicism

Abstract Study question How does embryo transfer with a low-level of mosaicism affect the success of ART programs, pregnancy, and live birth in comparison with euploid embryo transfer? Summary answer The transfer of mosaic embryos results in the delivery of a healthy baby however significantly decreases the outcome of ART programs and live birth rate. What is known already Present methods of preimplantation genetic testing of aneuploidy (PGT-A) allow detecting a mixture of euploid and aneuploid cells at the blastocyst stage with high accuracy. Such embryos are classified as mosaics with varying levels according to the guidelines of the International Society for Preimplantation Genetic Diagnosis (PGDIS). Numerous sources describe that number of mosaic embryos can vary from 4 to 22%. Several publications report that mosaic embryos can lead to successful pregnancies and healthy childbirth, but with a lower frequency and higher rates of pregnancy loss compared to euploid embryos. Nevertheless, the effect of mosaicism on ART outcomes remains controversial. Study design, size, duration It has been analyzed 2506 embryos from 648 patients undergoing the ART program with PGT-A at the Institute of Reproductive Medicine for 2018 - 2019. Embryos after PGT-A were classified as euploid, aneuploid, and having mosaicism of less than 40% as low level and more than 40% as high level following PGDIS guidelines. Patients of (group A) were transferred 467 single euploid embryos, and 43 patients (group B) underwent single low-level mosaic embryo transfer. Participants/materials, setting, methods The embryos on day 5 or 6 were graded by Gardner Scoring System. Approximately 5–10 TE cells were biopsied from good quality blastocysts and subsequently vitrified. PGT-A was performed utilizing an array comparative genomic hybridization (aCGH) (Agilent). The transfer of mosaic embryos was performed in the absence of an alternative, only after medical genetic counseling with a risk explanation and the subsequent signing of an informed agreement. Statistical tests processed by Pearson’s chi-squared test. Main results and the role of chance Of all analyzed embryos, the proportion of euploid embryos was 48.6% (n = 1002), the total number of mosaics was 18.6% (n = 384) and aneuploid ones were 32.8% (n = 676). Depending on the level of mosaicism, the ratio between embryos with low-level mosaicism (≤40%) / high-level (≥40%) was 38.3% / 61.7%, respectively. According to the study, there was a significant decrease in the indicator of clinical pregnancy rate after embryo transfer with a low-level of mosaicism of 44.1% versus 63.2% transferred euploid embryo (р&amp;lt;0,01), however, despite an increase losses pregnancy in the group B (26.3%) there was no significant difference (p = 0.16) in comparison with the control group (15.4%). The live birth rate (LBR) significantly decreased (p &amp;lt; 0.001) after the transfer of the mosaic embryo by 32.5%, while in the control group the indicator was 53.9%. In all cases, after the transfer of the mosaic embryo, healthy babies were born. There were 2 cases of high-level mosaic embryo transfer as a result of which pregnancy did not occur. According to the survey, about 70% of patients agree to replant mosaic embryos, 20% are ready to go to the new program, and 10% cannot make a decision. Limitations, reasons for caution The number of patients in group B was significantly lower than in group A. Not enough cases of embryo transfer with a high-level of mosaicism. Wider implications of the findings: The current study might help to develop and to select a more appropriate strategy for transfer mosaic embryos. The next series of studies should focus on obstetric and neonatal outcome data from mosaic embryo transfer to gain a better understanding of the chromosomal and physiological health of children. Trial registration number Not applicable

Comparative DNA cytometry of primary and recurrent soft tissue sarcomas.

e22516 Background: Soft tissue sarcomas (STS) are aggressive tumors with a high degree of recurrence. Radical resection within healthy tissues allows to reduce the recurrence percentage to 25-30% without subsequent therapy. The literary analysis has shown that the study of various biological properties of primary and recurrentsoft tissue tumorsis being conducted. However, currently there is a lack of information to understand the reasons for STS recurrence. The goal of investigation was to reveal the distinctive features of the DNA content and cell distribution in the phases of the cell cycle of recurrent STS. Methods: DNA cytometry in the tumor tissue of 30 primary soft tissue sarcomas and 30 STS recurrences was carried out using the method of flow cytofluorometry. The tumor ploidy and cell distribution in the cell cycle phases were analyzed. Results: A number of differences in the DNA cytometric parameters of primary and recurrent STS have been revealed, they include: an increase in the proportion of aneuploid tumors in case of recurrence, the number of tumors with DNA index within the mitotic cycle, an increase in the proportion of cells in G2+M- phase of diploid and aneuploid tumors and a decrease in S- phase of aneuploid ones. It has been shown that with a G2 differentiation degree, the proportion of cells in G2+M, S- and proliferation index of recurrent STS is significantly lower than the primary parameters. An increase in the proportion of cells in G2+M- phase and a decrease in the rate of proliferation of recurrent STS, depending on the stage, are shown only in case of stage III. Conclusions: The revealed features of DNA content and cell cycle of tumor cells of soft tissue sarcomas will allow to approach to understanding of biological bases of recurrence of this malignant disease.

Deoxyribonucleic acid detection in blastocoelic fluid: a new predictor of embryo ploidy and viable pregnancy

To investigate blastocysts, defined as euploid and aneuploid by trophectoderm (TE) cell analysis, for the presence of DNA in the blastocoelic fluid (BF) detected by whole-genomic amplification (WGA); and to correlate the presence of DNA in BF with the clinical outcome after the transfer of TE-euploid blastocysts. Retrospective study. Invitro fertilization unit. This study included 91 patients performing preimplantation genetic testing for aneuploidy on TEcells from January 2015 to December 2017. In the case of ET, only single blastocyst transfers were performed. Blastocoelic fluids and TEcells were retrieved from 256 blastocysts before vitrification. All blastocysts were diagnosed by array-comparative genomic hybridization (a-CGH) on TEcells. Amplification and a-CGH of DNA from BFs was performed at a later time after TE biopsy and ET. Whole-genomic amplification of BFs, evaluation of the chromosome condition in BFs and TEcells, and correlation of BF results with the clinical outcome of TE-euploid transferred blastocysts. The incidence of amplification after WGA was significantly lower in BFs from TE-euploid blastocysts (n = 32, 45%) when compared with the aneuploid ones (n = 150, 81%), resulting in 182 BFs with successful DNA amplification. When submitted to a-CGH, informative results were obtained from 172 BFs. Comparison of these results with those from the corresponding TEcells gave a ploidy concordance of 93.6% and a mean number of aneuploid events per sample that was higher in BFs than in TEcells (2.0 vs. 1.4, respectively). After the transfer of 53 TE-euploid blastocysts, the clinical pregnancy rate was 77% in the group with BF-failed amplification, and 37% after BF-successful amplification. The same trend was found for the ongoing pregnancy rate (68% vs. 31.5%, respectively). The presence of DNA in BFs detected by WGA is correlated with the blastocyst ploidy condition defined by TEcell biopsy and with the implantation potential of TE-euploid blastocysts. These findings could have a clinical implication for the selection of the most viable embryo for transfer because, after submitting BFs to WGA, priority would be given to TE-euploid blastocysts with BF-failed amplification. Similarly, BF-failed amplification could be an additional selection criterion to prioritize embryos for transfer even in conventional IVF cycles with blastocysts that were vitrified after BF aspiration.

The increase in the aneuploidy rate in embryos is mostly due to an absolute decrease in the number of euploid embryos

This increase on the aneuploidy rate could with the advance of the maternal age could be cause by either an increase of aneuploid embryos per cycle or by a reduction on the absolute number of euploid embryos per cycle. The objective of this study is to ascertain if the source of increase in the rate of aneuploid embryos with advancing maternal age is due to one or the other Retrospective study Retrospective study of 55,403 embryos from 10,497 cycles from 2013 to 2017, from 126 IVF centers analyzed by Next Generation Sequencing (NGS). Embryos were classified by their diagnosis into: euploid, mosaic, complex mosaic, aneuploid, complex aneuploid, “mosaic and aneuploid”, and mixed complex. Cycles were distributed in age groups (<35, 35-37, 38-40, 41-42, >42) plus an egg donor group. For each age group, the average and standard deviation of each type of embryo per cycle were obtained.ANOVA analysis was applied to compare the difference “between” age groups and “inside” age groups for the different types of embryos. Pearson C values were obtained to study the degree of correlation of average number of the different types of embryos with age. The results are summarized in table 1. Euploid and “mosaics with a euploid” line showed strong negative correlations with maternal age but aneuploid and “aneuploid and mosaic” only showed a mild increase with the bulk of the increase in aneuploidy rates being caused by a decrease in euploid embryos, not an increase in aneuploid ones. The dramatic decrease in aneuploidy rates is mostly cause by a decrease in euploid embryos, not an increase in aneuploid ones. That is, there are no more aneuploid eggs made or recruited with age but fewer euploid ones, with euploid ones almost exhausted after age 42. The observation that aneuploid embryos are almost constant with age suggest a different causative mechanism than one in which aneuploid embryos would increase with age and euploid ones remain constant or decrease. Mosaics, being post-meiotic are just a fraction of euploid or aneuploid ones, and thus increase or decrease with them.

DNA aneuploidy relationship with patient age and tobacco smoke in OPMDs/OSCCs.

The aim of this study was to investigate the relationship between tobacco smoke habit, patient age, DNA aneuploidy and genomic DNA copy number aberrations (CNAs) in oral potentially malignant disorder (OPMD) and oral squamous cell carcinoma (OSCC) patients. DNA aneuploidy was detected by high-resolution DNA flow cytometry (hr DNA-FCM) on DAPI stained nuclei obtained from multiple tissue samples from OPMDs/OSCCs in 220 consecutive patients. Nuclear genomic aberrations were determined in a subset of 65 patients by genome-wide array comparative genomic hybridization (aCGH) using DNA extracted from either diploid or aneuploid nuclei suspension sorted by FCM. DNA aneuploidy and mean nuclear genomic aberrations were associated with patients’ age. In particular, DNA aneuploidy strongly associated with age in non-smoker OPMDs/OSCCs patients. OSCCs from smokers showed a lower prevalence of DNA aneuploidy compared to OSCCs from non-smokers. A higher occurrence of DNA aneuploidy (particularly in smokers’ OPMDs) was observed in patients characterized by involvement of a single oral subsite. Our study suggests that: 1) DNA aneuploidy in non-smokers is mainly related to aging; 2) OPMDs/OSCCs involving multiple oral subsites in smokers are less likely to develop DNA aneuploidy compared to non-smokers; 3) OSCC development is characterized by both CIN and CIN-independent mechanisms and that the latter are more relevant in smokers. This study provides evidence that DNA diploid OPMDs may be considered at lower risk of cancerization than DNA aneuploid ones in non-smokers but not in smokers.

Correlation between aneuploidy, standard morphology evaluation and morphokinetic development in 1730 biopsied blastocysts: a consecutive case series study.

Are there correlations among human blastocyst ploidy status, standard morphology evaluation and time-lapse kinetics? Correlations were observed, in that euploid human blastocysts showed a higher percentage with top quality inner cell mass (ICM) and trophectoderm (TE), higher expansion grades and shorter time to start of blastulation, expansion and hatching, compared to aneuploid ones. Embryo quality has always been considered an important predictor of successful implantation and pregnancy. Nevertheless, knowledge of the relative impact of each morphological parameter at the blastocyst stage needs to be increased. Recently, with the introduction of time-lapse technology, morphokinetic parameters can also be evaluated. However, a large number of studies has reported conflicting outcomes. This was a consecutive case series study. The morphology of 1730 blastocysts obtained in 530 PGS cycles performed from September 2012 to April 2014 that underwent TE biopsy and array comparative genomic hybridization was analyzed retrospectively. A total of 928 blastocysts were cultured in a time-lapse incubator allowing morphokinetic parameters to be analyzed. Mean female age was 36.8±4.24 years. Four hunderd fifty-four couples were enrolled in the study: 384, 64 and 6 of them performed single, double or triple PGS cycles, respectively. In standard morphology evaluation, the expansion grade, and quality of theICM and TE were analyzed. The morphokinetic parameters observed were second polar body extrusion, appearance of two pronuclei, pronuclear fading, onset of two- to eight-cell divisions, time between the two- and three-cell (cc2) and three- and four-cell (s2) stages, morulae formation time, starting blastulation, full blastocyst stage, expansion and hatching timing. Of the 1730 biopsied blastocysts, 603 were euploid and 1127 aneuploid. We observed that 47.2% of euploid and 32.8% of aneuploid blastocysts showedtop quality ICM (P <0.001), and 17.1% of euploid and 28.5% of aneuploid blastocysts showed poor quality ICM (P <0.001). Top quality TE was present in 46.5% of euploid and 31.1% of aneuploid blastocysts (P <0.001), while 26.6% of euploid and 38.1% of aneuploid blastocysts showedpoor quality TE (P <0.001). Regarding expansion grade, 81.1% of euploid and 72.4% of aneuploid blastocysts were fully expanded (Grade 5-6; P <0.001). The timing of cleavage from the three- to four-cell stage, of reaching four-cell stage, of starting blastulation, reaching full blastocyst stage, blastocyst expansion and hatching were 2.6 (95% confidence interval (CI): 1.7-3.5), 40.0 (95% CI: 39.3-40.6), 103.4 (95% CI: 102.2-104.6), 110.2 (95% CI: 108.8-111.5), 118.7 (95% CI: 117.0-120.5) and 133.2 (95% CI: 131.2-135.2) hours in euploid blastocysts, and 4.2 (95% CI: 3.6-4.8), 41.1 (95% CI: 40.6-41.6), 105.0 (95% CI: 104.0-106.0), 112.8 (95% CI: 111.7-113.9), 122.1 (95% CI: 120.7-123.4) and 137.4 (95% CI: 135.7-139.1) hours in aneuploid blastocysts (P <0.05 for early and P <0.0001 for later stages of development), respectively. No statistically significant differences were found between euploid and aneuploid blastocysts for the remaining morphokinetic parameters.A total of 407 embryo transfers were performed (155 fresh, 252 frozen-thawed blastocysts). Higher clinical pregnancy, implantation and live birth rates were obtained in frozen-thawed compared to fresh embryo transfers (P =0.0104,0.0091 and0.0148, respectively). The miscarriage rate was 16.1% and 19.6% in cryopreserved and fresh embryo transfer, respectively. The mean female age was lower in the euploid compared to aneuploid groups (35.0±3.78 versus 36.7±4.13 years, respectively), We found an increasing probability for aneuploidy with female age of 10% per year (odds ratio (OR) =1.1, 95% CI: 1.1-1.2, P<0.001). The main limitation of morphology assessment is that it is a static system and can be operator-dependent. In this study, eight embryologists performedmorphology assessments. The main limitation of the time-lapse technology is that it is impossible to rotate the embryos making it very difficult to observe them in case of blastomere overlapping or increased cytoplasmic fragmentation. Although there seems to be a relationship between the ploidy status and blastocyst morphology/development dynamics, the evaluation of morphological and morphokinetic parameters cannot currently be improved upon, and therefore replace, PGS. Our results on ongoing pregnancy and miscarriage rates suggest that embryo evaluation by PGS or time-lapse imaging may not improve IVF outcome. However, time-lapse monitoring could be used in conjunction with PGS to choose, within a cohort, the blastocysts to analyze or, when more than one euploid blastocyst is available, to select which one should be transferred. No specific funding was obtained for this study. None of the authors have any competing interests to declare.

Polyploidy: The Link Between Senescence and Cancer

Polyploidy, a state of increased number of chromosomes, occurs often in plants and less frequently in animals. Polyploidization is recognized as a part of a developmental program and can be achieved via different mechanisms bypassing certain stages of the cell cycle. However, polyploidization also accompanies some pathological conditions as well as ageing. It is believed that tetraploid cells precede aneuploid ones in the early phases of tumor development. Division of tetraploid cells is restricted by the active tetraploid (4N G1) checkpoint. Tetraploid cells that are able to overcome this checkpoint give rise to increased genomic instability and tumor progression. Recently a cellular senescence program activated by oncogene expression was shown to act as a natural barrier against cancer development. Senescent cells were detected in many benign, but not malignant, human and animal tumors. Senescence can actually block cell transformation provided the 4N G1 checkpoint is active. Cancer cells that escaped the 4N G1 block are still able to undergo senescence upon anticancer treatment. Induction of cancer cell senescence is often correlated with high ploidy formation. Some polyploid cells can escape senescence and give progeny with numerical changes of chromosomes. Divisions of polyploid cancer cells on the road to senescence can be responsible for the ineffectiveness of anticancer therapy. Altogether, this implies polyploidy as a link between cellular senescence, cancer development and possible cancer renewal after treatment.

Induction of senescence with doxorubicin leads to increased genomic instability of HCT116 cells

Induction of senescence has been proposed as a possible in vivo tumor response to anticancer treatment. Senescent cancer cells are often polyploid, however, their route to polyploidy is poorly recognized (endoreduplication versus aberrant mitoses). We showed that after treatment of HCT116 cells with a low dose of doxorubicin most of them stopped proliferation as documented by SA-β-galactosidase activity and the lack of Ki67 expression. Increased expression of other common senescence markers, p53, p21 and cyclin D1, was also observed. The cells became giant, polyploid and polymorphic, with multinucleated cells comprising a substantial fraction. The vast majority of the doxorubicin-treated cells did not enter mitoses, as evidenced by mitotic index analysis, as well as by the predominantly cytoplasmic localization of cyclin B1 and a lack of separation of multiplied centrosomes. This allowed us to conclude that doxorubicin-treated HCT116 cells underwent endoreduplication. However, the rare events of aberrant mitoses of polyploid cells observed by us led to aneuploid progeny as was documented by cytogenetic analysis of survivors. Thus, a senescence-inducing treatment of HCT116 cancer cells had a dual effect—it stopped the proliferation of the majority of the cells, but also led to the appearance of proliferating aneuploid ones.

Clinicopathological and Biological Features of DNA Tetraploid Colorectal Cancers

Most studies of colorectal cancers focus on a comparison of DNA diploid to non-diploid tumors consisting of tetraploid and aneuploid tumors. Tetraploid tumors alone have not been well studied. In the present study, clinicopathological and biological features of tetraploid colorectal cancers in contrast to those of diploid and aneuploid ones were investigated. DNA ploidy in 278 primary colorectal adenocarcinomas was determined by flow cytometry. Among 278 cases, 8% of the cases were tetraploidy, 44% were aneuploidy, and 48% were diploidy. Compared to diploid tumors, tetraploid tumors were more frequent in advanced stage, high index of S-phase fraction and apoptosis, higher expression of Cox-2, c-erbB-2 and heat shock protein, but had decreased inflammatory infiltration (P<0.05). Compared to aneuploid tumors, tetraploid tumors had a high frequency of microsatellite instability, high expression of Cox-2 and heat shock protein (P<0.05). Unlike tetraploid tumors, aneuploid tumors had increased p53 expression but did not have microsatellite instability (P<0.05). Tetraploidy and aneploidy predicted a worse prognosis in the subgroups of stages A-C, proximal colon, p53 negative expression and higher S-phase fraction (P<0.05). DNA tetraploid tumors seem, to some extent, to exhibit distinct characteristics of clinicopathology and biology compared with aneuploid or diploid colorectal cancers.

Comparison of Development Rates for PGD/FISH Embryos Based on Gender

Research in bovine and murine embryos suggests an increased development rate in male vs. female embryos. There has been some speculation that blastocyst ET weights gender due to this phenomenon but it has not been proved. With the introduction of IVF/PGD/FISH for aneuploidy screening, the ability to monitor human embryo development against gender can be realized. Because recent advances in PGD technologies have allowed fertility labs to look at the genetics, as well as the morphology, of embryos, this study also evaluates if embryo development in genetically abnormal embryos occurs at an increased rate to genetically normal embryos. A retrospective analysis of all PGD/FISH-Aneuploidy treatment embryos and their gender outcome from 2004, regardless of patient diagnosis. 599 embryos were evaluated based on their euploidy: Group A-euploid embryos; Group B- monsomic embryos; Group C- trisomic embryos; and Group D- complex abnormal embryos. All embryos were cultured in sequential media and isolated into individual culture drops for assessment of development. Only viable embryos were included in this study. The table below shows no significant difference in gender outcome within all study groups. Female embryos were XX and male XY, only. Embryos with abnormal sex chromosomes were excluded. Tabled 1 Tabled 1 118 blastocysts developed by day 5 of culture (20%). A blastocyst included early, full, and hatching blastocysts. Blastocyst development within the 4 study groups is shown in Table 2. There was no significant difference in development of blastocysts from male or female embryos or euploid (47%) vs. aneuploid embryos (53%; 63/118). Human male embryos do not appear to have a developmental advantage over female embryos in vitro, both having the same rate of blastocyst formation. However, euploid embryos develop to the blastocyst stage at an equal rate (467%) as aneuploid ones (53%; 63/118). This data corroborates previous published data that indicate blastocyst development does not eliminate abnormalities and suggests no gender skewing in either blastocyst culture or aneuploidy studies.

Flow cytometry in primary breast carcinomas: prognostic impact of multiploidy and hypoploidy.

The aims of the present work were to study the prognostic impact of multiploidy and/or hypoploidy in breast cancers and their relation to other classic clinicopathologic prognostic factors (T, grade, receptors, and lymph node status). From 3 January 1990 to 7 January 1999, 1984 previously untreated, invasive breast carcinoma samples were snap frozen for flow-cytometry. Multiploid tumors had the same prognosis as the aneuploid ones, and those with one hypoploid peak had a better prognosis than did the other aneuploid tumors. However, the presence of both multiploid and hypoploid peaks was correlated with a poor outcome, even after multivariate analysis. In this series after quality control, 93.4% of the histograms could be evaluated concerning ploidy; of these 81.6% could be assessed concerning S-phase fraction (SPF) in the entire population and 77.1% in the multiploid population. In the entire population, we performed a multivariate analysis including all relevant prognostic factors remaining after monovariate analysis by using a compound factor (proliferative activity) regrouping SPF and mitotic activity. This analysis showed that lymph node status and proliferative activity correlates with every type of survival, whereas receptor status correlates with all types of survival except recurrence free survival size, correlated with non-metastasis and overall survival. Grade and age correlated only with overall survival and vascular permeations only with disease-free survival. SPF is a valuable predictor of survival, can be confidently assessed in multiploid histograms, and thus improves the yield of flow cytometry. When combined with mitotic activity, the prognostic impact of SPF is the same as that of lymph node status. Tumors that are hypoploid and multiploid have a significantly worse prognosis.

Clinical applications of image cytometry to human tumour analysis.

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

744 - Biological factors predicting the outcome of regional chemotherapy in colorectal carcinoma metastases to liver

Purpose: Regional intrahepatic chemotherapy may be beneficial for se• lected subgroup of patients with liver metastases of colorectal carcinoma. However. biological criteria precislng Indications to this demanding type of treatment have not yet been established. Methods: Twenty two cases of coloreclal carcinoma metastases to liver treated by regional chemotherapy were analysed for DNA ploidy and PCNA, Ki67, p53, p21 (WAF1), mdm2, o-erb2, CEA, CA19-9 and P·glycoprotein (MDR) expression and results related to survival. Results: Mean survival of patients with DNA diploid tumors was signifi• cantly longer (20 versus 11 months, P = 0.04) comparing to DNA aneuplOId ones. Only the trend for lower PCNA positiVIty and p53 expression could be observed among DNA diploid tumors. All p53 positive cases were negatIve lor p21 (WAF1) and mdm2. Other parameters were unrelated to outcome 01 treatment. Conclusion: DNA plOidy, perhaps together with p53 overexpression and PCNA positivity, may be predictive of effectiveness of regional Intrahepatic chemotherapy performed for colorectal cancer liver metastases. However, a larger study is needad to confirm these preliminary findings. (Supported by IGA MZ CR grant No.2923-3)

Differential histochemical peanut agglutinin stain in benign and malignant human prostate tumors: Relationship with prostatic specific antigen immunostain and nuclear DNA content

The histochemical binding pattern of the peanut ( Arachis hypogaea) lectin (PNA) was quantitatively described by means of computer-assisted microscope analysis in 28 benign prostatic hyperplasias (BPH), 15 prostatic intraepithelial neoplasias (PIN), and 119 prostatic adenocarcinomas. PNA exhibits noninunune but selective binding to glycoproteins with β-D-galactosyl(1,3)-N-acetyl-D-galactosamine residues. We also investigated whether a relationship existed between the number of histochemical-related PNA acceptors and the histochemical prostate-specific antigen (PSA) stain intensity, and between the number of PNA receptors and DNA ploidy level. The results show that neoplastic prostate tissues and high-grade intraepithelial prostatic neoplasias (PIN2_3) exhibit a significantly higher number of PNA acceptors than benign prostatic hyperplasias and low (PIN1) grade prostatic intraepithelial neoplasias. A statistically significant correlation was observed between the number of histochemically related PNA acceptors and PSA immunostain intensity. Lastly, diploid prostatic tumors, whether benign or malignant, exhibited a significantly higher number of PNA acceptors than aneuploid ones. These results suggest that PNA acceptors play an important role in the biology of prostate tumors.

692 Relationship between DNA ploidy and disease-free survival in node-negative breast cancer patients

Aim of the study is to evaluate prognostic value of DNA ploidy in the group of 106 pts with intraductal invasive adenocarcinoma in stage pT1–2 NO MO and with the Bloom grade II. All pts have been treated by surgery alone. Follow-up was at least 10 years (10–24). Clinical material was subdivided into 2 groups: A-without, B-with dissemination. DNA content was measured by image cytometry. There were 4 types of ploidy: diploidy and low, medium and high grade of aneuploidy. There were significantly higher rate of diploid tumours in the group A (83% vs 17%) and aneuploid tumours in the group B (67% vs 33%). The risk of dissemination was 17% for diploid and 54%, 74%, 100% respectively for aneuploid ones. Among patients with diploidy 83% had 10-years DFS, while nobody from the group of high grade of aneuploidy survive 10 years. The results suggest that high grade aneuploidy is a significant risk factor for dissemination in node-negative breast cancer patients.

Relationship between DNA ploidy level and tumor sociology behavior in 12 nervous cell lines

Cell population sociology was studied in two medulloblastomas and 10 astrocytic human tumor cell lines by means of the characterization of the structure of neoplastic cell colonies growing on histological slides. This was carried out via digital cell image analysis of Feulgen-stained nuclei, to which the Delaunay triangulation and Voronoi paving mathematical techniques were applied. Such assessments were compared to the DNA polidy level (assessed by means of DNA histogram typing). The results show that the cell colony architecture characteristics differed markedly according to whether the cell lines were euploid (diploid or tetraploid) or aneuploid (hyperdiploid, triploid, hypertriploid, or polymorphic). In fact, the cell colonies from the euploid cell nuclei populations were larger and more dense than those from the aneuploid ones. Furthermore, for an identical period of culture, the cell lines from high-grade malignant astrocytic tumors (glioblastomas) exhibited cell colonies that were larger and more dense than those in cell lines from low-grade astrocytic tumors (astrocytomas). In each of these two groups, the diploid cell nuclei populations exhibited cell colonies larger and more dense than the nondiploid colonies. The present methodology is now being applied in vivo to histological sections of surgically removed human brain tumors in order to distinguish between high-risk clinical subgroups and medium-risk subgroups in clearly circumscribed histopathological groups.

DNA image cytometric analysis of paraffin‐embedded sections of small renal cortical neoplasms

DNA ploidy analysis of neoplasms is most frequently performed by flow cytometry of single nuclear suspensions or image analysis of Feulgen-stained cytologic specimens. To assess the prognostic significance of ploidy in renal adenomas (less than 3 cm in diameter), nuclear DNA content of 21 renal cortical neoplasms was analyzed by image cytometry (CAS 200, Cell Analysis Systems, Inc., Elmhurst, IL) on Feulgen-stained 5 microns fixed embedded tissue sections. Five (36%) of 14 small neoplasms (so-called renal adenomas; (mean diameter, 7.2 mm; range, 2-24 mm) were in the aneuploid range. Four (57%) of seven carcinomas (mean diameter, 6.3 cm; range, 3.5-10 cm) were aneuploid, a frequency not significantly different from that in the adenomas (P = 0.4). Mean DNA index (1.38) for aneuploid adenomas and carcinomas was identical. Diploid renal adenomas (mean diameter, 5.9 mm) were smaller, although not significantly so (P > 0.05), than the aneuploid ones (mean, 9.7 mm). Size of diploid (mean, 6.7 cm) and aneuploid (mean, 6.1 cm) renal carcinomas was similar. Clinical follow-up studies to evaluate utility of DNA ploidy and proliferative activity in prognosis and management of small renal cortical neoplasms are indicated.

Mucoepidermoid Carcinoma of Major and Minor Salivary Glands

Forty-three consecutive cases of mucoepidermoid carcinoma of the major and minor salivary glands were studied to evaluate the influence of the following parameters on prognosis: age, sex, location, local extension, mitotic counts, vascular invasion, grade of differentiation, and nuclear DNA ploidy assessed by image cytometry. The age of the patients ranged between 7 and 84 years. Twenty-eight patients were male and 15 female. Thirty-five tumors were in the major salivary glands and 8 in the minor glands of the oral cavity. According to Healey's criteria, 14 tumors were classified as grade 1, 17 as grade 2, and 12 as grade 3 and, according to the 1990 World Health Organization proposal, 17 tumors were classified as low-grade and 26 as high-grade neoplasms. Image cytophotometric DNA analysis revealed 15 euploid and 6 aneuploid tumors. The range of the follow-up study was 3-156 months (mean, 40.7 months). The 5- year actuarial survival for each of the above-cited parameters was: 100% and 59.1 % for patients aged less than 20 and over 55 years, respectively; 49.6% for male and 85.7% for female patients; 61.3% for major gland and 73.3% for minor gland tumors; 100% and 57.5% for intraglandular and extraglandular major salivary gland neo plasms, respectively; 46.2% for cases with high mitotic rate and 83.1% for those with low mitotic rate; 38.7% and 81.6% for tumors respectively with and without vascular invasion; 100% for grade 1 and 64.7% for grade 2; none of the patients with grade 3 neoplasms were alive at 5 years. Survival was of 100% for low-grade and 40.7% for high-grade tumors. Survival was 92.8% for euploid tumors and 61.3% for the aneuploid ones. In conclusion, survival of patients with mucoepidermoid carcinoma is significantly related to the histological grade of differentiation, the mitotic count, the presence of vascular invasion and the DNA ploidy of the tumor cells. Int J Surg Pathol 1(1):3-12, 1993