Roland Sennerstam

Improved Grading of Breast Adenocarcinomas Based on Genomic Instability

Numerous investigations have shown that in primary breast adenocarcinomas DNA aneuploidy in contrast to DNA diploidy indicates high malignancy potential. On the basis of the study of 104 breast carcinomas, we describe a subtype of aneuploidy, which demonstrates a low degree of malignancy. In image cytometric DNA histograms, this subtype possessed a low percentage (≤8.8%) of nonmodal DNA values as measured by the stemline scatter index (SSI), which is defined as sum of the percentage of cells in the S-phase region, the G2 exceeding rate and the coefficient of variation of the tumor stemline. The cut point of SSI = 8.8% (P = 0.03) enabled us to also subdivide diploid and tetraploid tumors into clinically low and high malignant variants. One possible reason for aneuploidy is impaired distribution of chromosomes at mitosis caused by numerical or structural centrosome aberrations. Cyclins A and E seem to be involved in centrosome duplication. Real-time quantitative PCR measurements of cyclin A and E transcript levels and immunohistochemical determination of cyclin A protein expression showed statistically significantly increased values in the tumors with a high SSI (>8.8%), compared with those with a low SSI. A pilot study demonstrated centrosomal aberrations in an average of 9.6% of the measured cells in four aneuploid carcinomas with high SSI values and in an average of 2.5% of the cells in three aneuploid and three diploid tumors with low SSI. Our data indicate that the SSI, most likely reflecting the degree of genomic instability, allows additional classifying of the known aneuploid, diploid, and tetraploid categories of primary breast adenocarcinomas into low and high malignant subtypes.

Cell Cycle Progression in Serum‐Free Cultures of Sf9 Insect Cells: Modulation by Conditioned Medium Factors and Implications for Proliferation and Productivity

Cell cycle progression was studied in serum‐free batch cultures of Spodoptera frugiperda (Sf9) insect cells, and the implications for proliferation and productivity were investigated. Cell cycle dynamics in KBM10 serum‐free medium was characterized by an accumulation of 50−70% of the cells in the G2/M phase of the cell cycle during the first 24 h after inoculation. Following the cell cycle arrest, the cell population was redistributed into G1 and in particular into the S phase. Maximum rate of proliferation (μN,max) was reached 24−48 h after the release from cell cycle arrest, coinciding with a minimum distribution of cells in the G2/M phase. The following declining μN could be explained by a slow increase in the G2/M cell population. However, at approximately 100 h, an abrupt increase in the amount of G2/M cells occurred. This switch occurred at about the same time point and cell density, irrespective of medium composition and maximum cell density. An octaploid population evolved from G2/M arrested cells, showing the occurrence of endoreplication in this cell line. In addition, conditioned medium factor(s) were found to increase μN,max, decrease the time to reach μN,max, and decrease the synchronization of cells in G2/M during the lag and growth phase. A conditioned medium factor appears to be a small peptide. On basis of these results we suggest that the observed cell cycle dynamics is the result of autoregulatory events occurring at key points during the course of a culture, and that entry into mitosis is the target for regulation. Infecting the Sf9 cells with recombinant baculovirus resulted in a linear increase in volumetric productivity of β‐galactosidase up to 68−75 h of culture. Beyond this point almost no product was formed. Medium renewal at the time of infection could only partly restore the lost hypertrophy and product yield of cultures infected after the transition point. The critical time of infection correlated to the time when the mean population cell volume had attained a minimum, and this occurred 24 h before the switch into the G2/M phase. We suggest that the cell density dependent decrease in productivity ultimately depends on the autoregulatory events leading to G2/M cell cycle arrest.

MORPHOLOGY OF LIVE SEMINAL AND POSTCOITAL CERVICAL SPERMATOZOA AND ITS BEARING ON HUMAN FERTILITY

Abstract. Various seminal variables were studied in two groups of infertile couples and in one fertile control group. Sperm morphology was emphasized. Patients with normal postcoital tests and sperm counts exceeding 5 mill/ml were selected and followed prospectively. The two clinical groups could then be identified

Evidence for an intraclonal random shift between two types of cell cycle times in an embryonal carcinoma cell line

In a recent paper we reported the discovery of an intraclonal bimodal-like cell cycle time variation within the multipotent embryonal carcinoma (EC) PCC3 N/1 line growing in the exponential phase in the undifferentiated state. The variability was found to be localized in the G1 period. Furthermore, an inverse relation between cell size and cell generation time was found in the cell system analysed. It was suggested that the bimodal-like intraclonal time variability previously reported was attributable to an intraclonal shift between two types of cell-growth-rate cycles and that the cell-growth cycle has a supramitotic character, being dissociated from the DNA-division cycle. The growth rate heterogeneity in the cell population was found to need three cell cycles to reach full dispersion in time. This was assumed to be due to a decreased inheritance from sister cell pairs to second cousin cell pairs. Thus, the interesting feature is that in one and the same multipotent cell line there was evidence for an intraclonal instability with a random shift between two types of cell cycle differing in the duration of their G1 period.

Anticoagulant medication at time of needle biopsy for breast cancer in relation to risk of lymph node metastasis

Anticoagulant treatment might enhance the natural defense against tumor cell dissemination caused by diagnostic needle biopsy by counteracting thrombocyte coating of such cells. To clarify whether women using anticoagulant treatment at the time of biopsy have a lower occurrence of lymph node metastasis, we conducted a nationwide Swedish cohort study of 26,528 female incident breast cancer patients in 2006–2011. Point risk ratio (RR) of risk of lymph node metastasis among users of anticoagulant treatment adjusted for age, T‐stage, socioeconomic factors, and concomitant medication was RR = 0.94, (95% CI: 0.87–1.03), and lower in younger women (RR = 0.80, 95% CI 0.50–1.29). Although nonsignificant, these associations may underestimate a true negative association since women using anticoagulant treatment are likely to have more concomitant diseases, lead an unhealthier lifestyle, and have lower participation in mammography screening. These findings provide some support for the hypothesis that anticoagulant medications might counteract breast cancer spread caused by needle biopsy.

Hyperdiploidy Tetraploidization and Genomic Instability in Breast Cancer-A Case Report Study

Introduction: Several reports have indicated that tetraploidization is an intermediate step of tumor progression between diploid cells and genomically reorganized aneuploid tumor cells. Tetraploidization is a conserved phenomenon in both plants and animals, which occurs in the human body in reaction to various stress factors.Methods: A breast cancer population was divided into groups according to DNA Index (DI) interval, and three ploidy entities were defined as three different tumor groups: diploid (D-type), tetraploid (T-type) and aneuploid (A-type) tumors. Using a parameter reflecting genomic instability and proliferative activity (Stemline Scatter Index, SSI), we stepwise simulated the ploidy alterations following increase in SSI values. The percentage of each tumor type at each level of accumulated SSI value was estimated and the slopes of the generated curves were compared in linear regression analysis.Results: At diagnosis, 32% of the patients had T-type tumors some of which were established in a pre-diagnostic period. During simulation guided by increasing SSI values a second step of tetraploidization was found during a tumor size interval of 10–20 mm caused by a presumed reaction to anoxic stress. The generated tetraploid cell populations were recruited from diploid cancer cells, indicating that already transformed cells loaded tetraploidgenerated cells with genomic instability. These genomically unstable and altered tetraploid cells were postulated to generate aneuploid cells within a hypotetraploid DI region. A narrow DI interval among D-type tumors was suggested to be responsible for the recruitment of T-type tumors.Conclusion: We present a two-step model in which the first tetraploidization occurs early in breast cancer tumor progression and might represent a reaction to stress factors in benign epithelial tumors and epithelial hyperplasia and a later process of tumor size dependent origin for tetraploidization.

Axillary lymph node metastasis and survival in breast cancer patients with concurrent cardio-cerebral-vascular disease

Objectives Dissemination of tumour cells occurring both spontaneously or caused by diagnostic biopsy procedures is the most serious complication of solid malignancies. In the present work we focus on local tumour spread and how this complication of cancer disease can be counteracted. Design From a cohort of 864 breast cancer patients we selected those who died of their primary cancer and those who died because of a simultaneously existing cardio-cerebral-vascular disease (CCVD) and were exposed to anticoagulants. Setting The study was based on breast cancer patients diagnosed at Karolinska University Hospital during 1991 (n = 519) and 1997–1998 (n = 345). Main outcome measures Axillary lymph node metastasis (ALNM) and survival of breast cancer patients with concurrent CCVD. Results Breast cancer patients belonging to the group that died of CCVD showed ALNM at the time of tumour diagnosis in 27% of the cases compared with 68% diagnosed in the group that died of their breast cancer (p < 0.0001). Likewise we observed a highly significant (p < 0.0001) difference in mean survival time with an average of 102 months in the group of breast cancer patients who died of CCVD and an average of 61 months in the group who died of breast cancer. Conclusion The data presented herein indicate that breast cancer patients regularly involved in treatment with anticoagulants because of simultaneously existing CCVD develop ALNM significantly less frequently and have an increased average survival time compared with breast cancer patients not suffering from CCVD.

Embryonic cell commitment by a simultaneous alteration in nucleo/cytoplasmic ratio in sibling cells between subsequent cell cycles

Studies on murine embryonal carcinoma (EC) cell lines have revealed a mechanism for commitment of early embryonic cells. By means of a particular intraclonal cell surface glycoprotein and intermitotic time heterogeneity found in the EC lines used, we have devised a cell cycle model and written a computer program for cell cycle stimulation. In the present investigation experimental tissue culture data obtained from the EC lines were inserted into the computer program and the simulations represent a good fit to experimental data. It is shown that the dynamics of the driving forces in the ‘cell growth cycle’ and the ‘DNA‐division cycle’, when assumed to be loosely coupled and analyzed in subsequent cell cycles, reveal a mechanism that can commit the mother cell to impel her daughter cells into the next stage in embryonic development by altering the relationship between those two uncoupled subcycles. Thereby the nucleo/cytoplasmic ratio (DNA/mass) is altered in a similar way in the two daughter cells. The simulations increase the reliability of the model and open up possibilities to test other embryonic cell systems.