Tumour-related factors and prognosis in breast cancer detected by screening.

BACKGROUND: Breast cancer detected by screening has an unexplained prognostic advantage beyond stage shift compared with cancers detected clinically. The aim was to investigate biological factors in invasive breast cancer, with reference to mode of detection and rate of death from breast cancer. METHODS: Histology, oestrogen receptor α and ß, progesterone receptor, human epidermal growth factor receptor (HER) 2, cyclin D1, p27, Ki-67 and perinodal growth were analysed in 466 tumours from a prospective cohort, the Malmö Diet and Cancer Study. Using logistic regression, odds ratios were calculated to investigate the relationship between tumour characteristics and mode of detection. The same tumour factors were analysed in relation to standard prognostic features. Death from breast cancer was analysed using Cox regression with adjustments for standard tumour factors; differences following adjustment were analysed by means of Freedman statistics. RESULTS: None of the biological tumour characteristics varied with mode of detection of breast cancer. After adjustment for age, tumour size, axillary lymph node involvement (ALNI) and grade, women with cancer detected clinically had an increased risk of death from breast cancer (hazard ratio 2·48, 95 per cent confidence interval 1·34 to 4·59), corresponding to a 37·2 per cent difference compared with the unadjusted model. Additional adjustment for biological tumour factors studied caused only minor changes. CONCLUSION: None of the biological tumour markers investigated explained the improved prognosis in breast cancer detected by screening. None of the factors was related to ALNI, suggesting that other mechanisms may be responsible for tumour spread.

Risk of recurrence following delayed large flap reconstruction after mastectomy for breast cancer.

BACKGROUND: The aim of this retrospective matched cohort study was to evaluate the rate of recurrence among women with delayed large flap breast reconstruction after mastectomy for breast cancer. The recurrence rate among women treated at a single hospital was compared with that in an individually matched control group of women with breast cancer who did not have reconstruction after mastectomy. METHODS: Between 1982 and 2001, 125 women with previous invasive breast carcinoma underwent delayed large flap breast reconstruction with pedicled musculocutaneous or microvascular flaps (a median of 32 months after mastectomy). They were matched individually with 182 women with breast cancer who had a mastectomy but did not undergo breast reconstruction. Matching criteria were year of diagnosis, age at diagnosis and treating hospital. Medical records were evaluated until October 2007. Histopathological specimens for all included women were re-evaluated. The endpoint was locoregional or distant breast cancer recurrence. The risk of recurrent disease was calculated using a Cox proportional hazards analysis, adjusted for established prognostic factors. RESULTS: Median follow-up for the entire cohort was 146 months. The reconstruction group had a 2·08 (95 per cent confidence interval 1·07 to 4·06) times higher risk of recurrent disease than the mastectomy only group. CONCLUSION: Women with breast cancer who had delayed reconstruction with a large flap in this study had a higher risk of recurrent disease than those with mastectomy alone.

Phosphorylation of the oestrogen receptor alpha at serine 305 and prediction of tamoxifen resistance in breast cancer.

Phosphorylation of oestrogen receptor alpha at serine 305 (ERalphaS305-P) induces tamoxifen resistance in experimental studies, but does not influence response to other endocrine agents, such as fulvestrant. We evaluated ERalphaS305-P using immunohistochemistry in 377 breast carcinomas from premenopausal participants of a randomized trial (n=248) and patients with advanced disease (n=129). Among the premenopausal patients, adjuvant tamoxifen improved recurrence-free survival (RFS) for ERalphaS305-P-negative tumours (multivariate HR=0.53, 95% CI 0.32-0.86, p=0.010), but not for ERalphaS305-P-positive tumours (multivariate HR=1.01, 95% CI 0.33-3.05, p=0.99) (interaction p=0.131). Notably, ERalphaS305-P was not significantly associated with RFS in patients not treated with tamoxifen (multivariate HR=0.64, 95% CI 0.30-1.37, p=0.248), indicating that ERalphaS305-P is a marker for treatment outcome rather than tumour progression. Given the direct experimental link between ERalphaS305-P and tamoxifen resistance and these first clinical data suggesting that premenopausal patients with ERalphaS305-P-positive breast cancer are resistant to adjuvant tamoxifen, further research is encouraged to study whether alternative endocrine treatment should be considered for this subgroup.

Hypoxia, Snail and incomplete epithelial-mesenchymal transition in breast cancer.

BACKGROUND: Hypoxia is an element of the tumour microenvironment that impacts upon numerous cellular factors linked to clinical aggressiveness in cancer. One such factor, Snail, a master regulator of the epithelial-mesenchymal transition (EMT), has been implicated in key tumour biological processes such as invasion and metastasis. In this study we set out to investigate regulation of EMT in hypoxia, and the importance of Snail in cell migration and clinical outcome in breast cancer. METHODS: Four breast cancer cell lines were exposed to 0.1% oxygen and expression of EMT markers was monitored. The migratory ability was analysed following Snail overexpression and silencing. Snail expression was assessed in 500 tumour samples from premenopausal breast cancer patients, randomised to either 2 years of tamoxifen or no adjuvant treatment. RESULTS: Exposure to 0.1% oxygen resulted in elevated levels of Snail protein, along with changes in vimentin and E-cadherin expression, and in addition increased migration of MDA-MB-468 cells. Overexpression of Snail increased the motility of MCF-7, T-47D and MDA-MB-231 cells, whereas silencing of the protein resulted in decreased migratory propensity of MCF-7, MDA-MB-468 and MDA-MB-231 cells. Moreover, nuclear Snail expression was associated with tumours of higher grade and proliferation rate, but not with disease recurrence. Interestingly, Snail negativity was associated with impaired tamoxifen response (P=0.048). CONCLUSIONS: Our results demonstrate that hypoxia induces Snail expression but generally not a migratory phenotype, suggesting that hypoxic cells are only partially pushed towards EMT. Furthermore, our study supports the link between Snail and clinically relevant features and treatment response.

Robotic Mammosphere Assay for High-Throughput Screening in Triple-Negative Breast Cancer.

In order to identify novel treatment principles specifically affecting cancer stem cells in triple-negative breast cancer, we have developed a high-throughput screening method based on the mammosphere and anoikis resistance assays allowing us to screen compounds using a functional readout. The assay was validated against manual protocols and through the use of positive controls, such as the response to hypoxia and treatment with the known cancer stem cell-targeting compound salinomycin. Manual and robotic procedures were compared and produced similar results in cell handling, cell cultures, and counting techniques, with no statistically significant difference produced from either method. The variance between samples processed manually versus robotically was no greater than 0.012, while Levene's test of significance was 0.2, indicating no significant difference between mammosphere data produced manually or robotically. Through the screening of 989 FDA-approved drugs and a follow-up screen assessing the antineoplastic subgroup, we have identified three therapeutic compounds with the ability to modulate the breast cancer stem cell fraction in the triple-negative breast cancer cell line MDA-MB-231, highlighting their potential usage as stem cell-specific adjuvant treatments.

Antibodies to proliferating cell nuclear antigen as S-phase probes in flow cytometric cell cycle analysis.

The usefulness of different anti-proliferating cell nuclear antigen monoclonal antibodies as S-phase probes in flow cytometric analysis was evaluated after various fixation procedures on human cell lines. With a newly developed detergent extraction/fixation method the monoclonal antibody PC10 acted as a selective S-phase marker. A total of 27 human hematopoietic tumors were analyzed using PC10, and all exhibited the characteristic S-phase recognition pattern. The percentage of PC10-positive cells was easily calculated and showed strong correlation to the fraction of S-phase cells determined from DNA histograms. Using alternative fixation procedures PC10, on the other hand, could recognize all actively cycling cells, a feature also observed for the monoclonal antibodies, 19A2 and TOB7.

Telomerase activity is associated with cell cycle deregulation in human breast cancer.

Deregulation of the cell cycle by abnormal expression of one or several cell cycle regulatory proteins is a common finding in malignant tumors and might be a prerequisite for cancer development. Telomerase activity is an immortalization marker that is found in most cancers and for which an association with an active cell cycle has been implicated. In the tissue of 106 human breast carcinomas, we analyzed the relationship between telomerase activity levels and defects in the cell cycle machinery with a focus on the retinoblastoma protein (pRB) pathway(s). The fraction of telomerase-positive tumors was 85%, and large differences in telomerase activity were found. Overexpression of cyclin D1 and/or cyclin E, in combination with a normal pRB, was a typical feature of tumors with high telomerase activity levels. Down-regulation of p16INK4 was not related per se to telomerase activity, but tumors with low p16INK4 in combination with cyclin D1 or E overexpression demonstrated high activity. Tumor cell proliferation, determined by Ki-67 expression, correlated significantly to telomerase activity levels. There was, however, not a strict association between proliferation rate and telomerase activity, because tumors with inactivated pRB had the highest Ki-67 fractions but intermediate telomerase activity. Also, cyclin D1 overexpression was associated with high telomerase levels without an increase in tumor cell proliferation. The present study indicates that telomerase activation occurs preferentially in breast cancers with certain cell cycle regulatory defects and that telomerase activity levels may depend on the specific defect(s).

PO-21 - Stromal fibroblasts in preinvasive breast cancer (ductal carcinoma in situ, DCIS) demonstrate a cancer-like procoagulant phenotypic switch that may facilitate invasion.

INTRODUCTION: Ductal carcinoma in-situ (DCIS) is a preinvasive breast cancer where cancer cells remain confined within the ductal basement membrane. However, genotypic changes have been identified in stroma surrounding DCIS, outside the basement membrane. Stromal fibroblasts undergo phenotypic change in cancer to promote tumour angiogenesis, proliferation, immunosuppression and metastasis and in vivo can induce invasion of DCIS. Phenotypic changes in DCIS stromal fibroblasts may potentially act as a precursor for invasion. AIM: To determine if stromal fibroblasts in DCIS have procoagulant changes similar to those seen in cancer-associated fibroblasts in invasive breast cancer. MATERIALS AND METHODS: As part of the prospective cohort study CHAMPion (Cancer induced Hypercoagulabulity as a Marker of Prognosis), patients with DCIS (n=72) and invasive breast cancer (n=292) were recruited. Stromal fibroblasts in tumour and corresponding normal breast tissue (distant from the cancer) were quantified (percentage IHC stained) for tissue factor (TF), thrombin, PAR1 and PAR2. Fibroblasts were identified morphologically, at a minimum distance of 0.2mm from ductal tissue, to avoid myoepithelial scoring. Scoring was performed in duplicate by two independent pathologists. RESULTS: Fibroblast TF expression was present in normal breast tissue (mean 43% ([SD 27%]) but markedly increased in DCIS (mean 62% [SD 27%], p=0.002). Fibroblast TF expression was further increased in invasive breast cancer (mean 74% [SD 23%], normal vs invasion, p<0.001; DCIS vs invasion, p=0.03). Fibroblast thrombin and PAR2, but not PAR1, expression was increased in DCIS compared to normal (thrombin: 60% vs 42%, p<0.001; PAR2: 58% vs 41%, p=0.002), however no further significant increase was seen in invasive cancer (thrombin 63%, PAR2 61%). Fibroblast tissue factor correlated with fibroblast thrombin expression (p<0.001, r=0.4) and fibroblast PAR2 expression (p<0.001, r=0.5), with thrombin and PAR2 expression also correlating (p<0.001, r=0.4). CONCLUSIONS: Procoagulant phenotypic changes, in terms of increased TF, thrombin and PAR2 expression, occur in stromal fibroblasts at the preinvasive stage. It needs to be determined if this change is functional and therefore a potential therapeutic target for preventing transition to invasion.

The C/EBPδ protein is stabilized by estrogen receptor α activity, inhibits SNAI2 expression and associates with good prognosis in breast cancer.

Hypoxia and inflammatory cytokines like interleukin-6 (IL-6, IL6) are strongly linked to cancer progression, and signal in part through the transcription factor Ccaat/enhancer-binding protein δ (C/EBPδ, CEBPD), which has been shown to promote mesenchymal features and malignant progression of glioblastoma. Here we report a different role for C/EBPδ in breast cancer. We found that the C/EBPδ protein is expressed in normal breast epithelial cells and in low-grade cancers. C/EBPδ protein (but not mRNA) expression correlates with estrogen receptor (ER+) and progesterone receptor (PGR) expression and longer progression-free survival of breast cancer patients. Specifically in ER+ breast cancers, CEBPD-but not the related CEBPB-mRNA in combination with IL6 correlated with lower risk of progression. Functional studies in cell lines showed that ERα promotes C/EBPδ expression at the level of protein stability by inhibition of the FBXW7 pathway. Furthermore, we found that C/EBPδ attenuates cell growth, motility and invasiveness by inhibiting expression of the SNAI2 (Slug) transcriptional repressor, which leads to expression of the cyclin-dependent kinase inhibitor CDKN1A (p21CIP1/WAF1). These findings identify a molecular mechanism by which ERα signaling reduces the aggressiveness of cancer cells, and demonstrate that C/EBPδ can have different functions in different types of cancer. Furthermore, our results support a potentially beneficial role for the IL-6 pathway specifically in ER+ breast cancer and call for further evaluation of the role of intra-tumoral IL-6 expression and of which cancers might benefit from current attempts to target the IL-6 pathway as a therapeutic strategy.

Deregulation of cyclin E and D1 in breast cancer is associated with inactivation of the retinoblastoma protein.

Inactivation of the retinoblastoma protein (pRB) by mutations or abnormal phosphorylation is a mechanism by which tumour cells can subdue normal growth control. Among molecules involved in control of pRB phosphorylation, cyclin D1 and E have been found to be deregulated and overexpressed in various types of cancers. In order to study the cell cycle regulatory mechanisms in breast cancer, we have analysed the protein expression of cyclin D1 and E in 114 tumour specimens from patients with primary breast cancer using Western blotting. Twenty-five out of 34 tumours with overexpression of cyclin E showed uniform low cyclin D1 expression, and by immunohistochemical analysis of pRB we present evidence for the existence of pRB defects in approximately 40% of these tumours in contrast to no pRB defects in the other group of tumours. This result was supported by a high protein expression of the cyclin-dependent kinase inhibitor p16 in 44% of the tumours with high cyclin E and low D1 expression, and all immunohistochemical pRB defect tumours showed a high p16 protein level. Additionally, an abnormal low pRB phosphorylation in relation to a high proliferative activity and loss of heterozygosity of the retinoblastoma susceptibility gene locus were found in all but one tumour with immunohistochemical defect pRB. Interestingly, tumours with high cyclin E and low D1 expression were generally oestrogen receptor negative suggesting a role for cell cycle regulators in the mechanisms leading to oestrogen independent tumour growth. Furthermore, the prognosis differed markedly for the patients in the various groups of tumours, indicating that the heterogeneous nature of breast cancer pathogenesis and the clinical course in part could be explained by different and distinctive sets of cell cycle defects.

Cyclin E dependent kinase activity in human breast cancer in relation to cyclin E, p27 and p21 expression and retinoblastoma protein phosphorylation.

The cell cycle machinery is regulated by cyclin dependent kinases and sets of activating and inhibitory proteins. The G1-S control mechanism is often deregulated in tumours supposedly leading to increased kinase activity, phosphorylation of substrates and subsequent S phase entrance. Increased kinase activity has been proposed to be essential in cell cycle aberrations, but few studies have actually shown enhanced kinase activity related to specific cell cycle defects in primary tumours. In the present study we have determined the cyclin E dependent kinase activity (cyclin E(kinase)) in 59 primary breast cancers, using an H1-kinase assay, and related the activity to the expression of cyclin E, p27 and p21. In a subgroup of 48 tumours, we further characterized the association between cyclin E(kinase), in vivo phosphorylation of the retinoblastoma protein (pRb) and proliferation. The cyclin E(kinase) correlated significantly with cyclin E content and inversely with p27 and p21 expression. P27, but not p21, was associated with low cyclin E(kinase) in specimens with normal/low levels of cyclin E. At elevated cyclin E levels, suppression of cyclin E(kinase) seemed to require high levels of both p21 and p27. The cyclin E(kinase) correlated with the phosphorylation status of pRb as well as with proliferation. Surprisingly, pRb phosphorylation did not correlate with proliferation. Our results support that pRb is a substrate for cyclin E(kinase) in primary breast cancer and that deregulation of cyclin E and p27 act through increased CDK-kinase activity, but cyclin E associated events beside pRb phosphorylation might be rate-limiting for entrance into S phase.

Downregulation of the potential suppressor gene IGFBP-rP1 in human breast cancer is associated with inactivation of the retinoblastoma protein, cyclin E overexpression and increased proliferation in estrogen receptor negative tumors.

The complex insulin-like growth factor network of ligands, receptors and binding proteins has been shown to be disturbed in breast cancer. In addition to defects in proteins controlling cell cycle checkpoints, this type of aberrations could affect tumor growth and survival thereby influencing both tumor aggressiveness and potential response to treatments. We have previously identified the T1A12/mac25 protein, which is identical to the IGFBP-rP1, as a differentially expressed gene product in breast cancer cells compared with normal cells. Here we compare the expression of IGFBP-rP1 in 106 tumor samples with known status of cell cycle aberrations and other clinicopathological data. This was done using a tumor tissue section array system that allows for simultaneous immunohistochemical staining of all samples in parallel. Cytoplasmic staining of variable intensity was observed in most tumors, 15% lacked IGFBP-rP1 staining completely, 20% had weak staining, 32% intermediate and 33% showed strong staining. Low IGFBP-rP1 was associated with high cyclin E protein content, retinoblastoma protein (pRb) inactivation, low bcl-2 protein, poorly differentiated tumors and higher stage. There was a significantly impaired prognosis for patients with low IGFBP-rP1 protein tumors. Interestingly, IGFBP-rP1 showed an inverse association with proliferation (Ki-67%) in estrogen receptor negative tumors as well as in cyclin E high tumors suggesting a separate cell cycle regulatory function for IGFBP-rP1 independent of interaction with the estrogen receptor or the pRb pathway.

Pathology parameters and adjuvant tamoxifen response in a randomised premenopausal breast cancer trial.

BACKGROUND: Subgroups of breast cancer that have an impaired response to endocrine treatment, despite hormone receptor positivity, are still poorly defined. Breast cancer can be subdivided according to standard pathological parameters including histological type, grade, and assessment of proliferation. These parameters are the net result of combinations of genetic alterations effecting tumour behaviour and could potentially reflect subtypes that respond differently to endocrine treatment. AIMS: To investigate the usefulness of these parameters as predictors of the response to tamoxifen in premenopausal women with breast cancer. MATERIALS/METHODS: Clinically established pathological parameters were assessed and related to the tamoxifen response in 500 available tumour specimens from 564 premenopausal patients with breast cancer randomised to either two years of tamoxifen or no treatment with 14 years of follow up. Proliferation was further evaluated by immunohistochemical Ki-67 expression. RESULTS: Oestrogen receptor positive ductal carcinomas responded as expected to tamoxifen, whereas the difference in recurrence free survival between control and tamoxifen treated patients was less apparent in the relatively few lobular carcinomas. For histological grade, there was no obvious difference in treatment response between the groups. The relation between proliferation and tamoxifen response seemed to be more complex, with a clear response in tumours with high and low proliferation, whereas tumours with intermediate proliferation defined by Ki-67 responded more poorly. CONCLUSIONS: Clinically established pathology parameters seem to mirror the endocrine treatment response and could potentially be valuable in future treatment decisions for patients with breast cancer.

Expression of oncoprotein 18 in human leukemias and lymphomas.

Oncoprotein 18 (Op18) is an 18 kDa intracellular phosphoprotein that appears to be up-regulated in certain neoplastic cells. In the present report we have analysed the expression of Op18 in samples of human hematopoietic disorders, mainly leukemias and lymphomas. For this purpose we have developed reagents allowing quantitative Western-blot analysis, and quantification of Op18 on the single cell level by flow cytometric analysis. The data demonstrates that a significant fraction of all lymphoma and leukemia cases express Op18 at levels that are several-fold higher than the Op18 levels ever found in benign proliferating tissue, such as bone marrows in remission and reactive lymph nodes. Thus, the results establish on the single cell level that Op18 is frequently expressed at abnormal levels in lymphoid and myeloid malignancies. Flow cytometric analysis revealed a striking qualitative and quantitative heterogeneity in Op18 expression between patients with lymphoma or leukemia. Moreover, our analysis demonstrates that the abnormal cellular levels of Op18 expression frequently found in high grade lymphoma and acute leukemia samples do not correlate with an increased fraction of cells in S phase. Finally, we also present an example of dramatic changes in Op18 expression pattern in bone marrow cells during the progression of a chemo-resistant acute leukemia.

Quantitative analysis of the expression and regulation of an activation-regulated phosphoprotein (oncoprotein 18) in normal and neoplastic cells.

Activation of protein kinase C results in phosphorylation of a 19-kDa protein termed 19K. Isolation and sequence analysis of a cDNA encoding the 19K protein revealed that this protein has been studied in other systems under different names. The name oncoprotein 18 (Op18) has been proposed on the basis of a postulated up-regulation in neoplastic cells. In the present report we adopt the designation Op18 for the 19K protein, and quantify this phosphoprotein in a series of leukemia/lymphoma cell lines, a panel of non-transformed cells and some terminally differentiated cell types. For this purpose we have developed reagents allowing quantitative Western-blot analysis, and quantification of Op18 on the single cell level by flow cytometric analysis. The data demonstrates a pronounced up-regulation of the Op18 protein in most leukemia/lymphoma cell lines. The HPB-ALL cell line provided the most extreme case and expressed 7 x 10(6) Op18 molecules/cell, which compares with 0.65 x 10(6) Op18 molecules/cell in non-transformed lymphoblastoid cells. The expression of Op18 appears to be restricted to cell types with proliferative potential, but it is clear from our results that up-regulation of Op18 is uncoupled from cellular proliferation. Moreover, by employing an Epstein-Barr virus based shuttle vector, we expressed Op18 cDNA in lymphoblastoid cells. This resulted in a three to fourfold up-regulation of Op18 that did not have any detectable consequences for cell-surface phenotype or cell size. However, increased expression of Op18 resulted in a partial inhibition of cell proliferation. Taken altogether, the results suggest that up-regulation Op18 levels in leukemia/lymphoma cells are strongly associated with, but not a direct cause of tumour progression.

TGF-beta receptor type-2 expression in cancer-associated fibroblasts regulates breast cancer cell growth and survival and is a prognostic marker in pre-menopausal breast cancer.

Transforming growth factor-beta (TGF-ß) is a pleiotropic cytokine with the capability to act as tumour suppressor or tumour promoter depending on the cellular context. TGF-beta receptor type-2 (TGFBR2) is the ligand-binding receptor for all members of the TGF-ß family. Data from mouse model experiments demonstrated that loss of Tgfbr2 expression in mammary fibroblasts was linked to tumour initiation and metastasis. Using a randomised tamoxifen trial cohort including in total 564 invasive breast carcinomas, we examined TGFBR2 expression (n=252) and phosphorylation level of downstream target SMAD2 (pSMAD2) (n=319) in cancer-associated fibroblasts (CAFs) and assessed links to clinicopathological markers, prognostic and treatment-predictive values. The study revealed that CAF-specific TGFBR2 expression correlated with improved recurrence-free survival. Multivariate analysis confirmed CAF-TGFBR2 to be an independent prognostic marker (multivariate Cox regression, hazard ratio: 0.534, 95% (CI): 0.360-0.793, P=0.002). CAF-specific pSMAD2 levels, however, did not associate with survival outcome. Experimentally, TGF-ß signalling in fibroblasts was modulated using a TGF-ß ligand and inhibitor or through lentiviral short hairpin RNA-mediated TGFBR2-specific knockdown. To determine the role of fibroblastic TGF-ß pathway on breast cancer cells, we used cell contact-dependent cell growth and clonogenicity assays, which showed that knockdown of TGFBR2 in CAFs resulted in increased cell growth, proliferation and clonogenic survival. Further, in a mouse model transfected CAFs were co-injected with MCF7 and tumour weight and proportion was monitored. We found that mouse xenograft tumours comprising TGFBR2 knockdown fibroblasts were slightly bigger and displayed increased tumour cell capacity. Overall, our data demonstrate that fibroblast-related biomarkers possess clinically relevant information and that fibroblasts confer effects on breast cancer cell growth and survival. Regulation of tumour-stromal cross-talk through fibroblastic TGF-ß pathway may depend on fibroblast phenotype, emphasising the importance to characterise tumour microenvironment subtypes.

Proliferating cell nuclear antigen and Ki-67 antigen expression in human haematopoietic cells during growth stimulation and differentiation.

By flow cytometric dual parameter analysis of proliferating cell nuclear antigen (PCNA) and the Ki-67 antigen a detailed cell cycle analysis can be performed. In this study the coordinated expression of these two growth-related antigens was investigated in human haematopoietic cells at entrance into the cell cycle as well as at exit from the cycle. In mitogen-stimulated peripheral blood lymphocytes entering the first cell cycle, the Ki-67 antigen was found to be expressed in S phase cells and not in G1 cells. Thus, the Ki-67 antigen expression in PCNA-positive S phase cells differed between continuously cycling cells and cells entering the cell cycle. Based on this difference, it was possible to visualize and evaluate the recruitment of cells into the first cell cycle from a resting stage. This new cell cycle parameter can give additional information concerning tumour growth. The Ki-67 antigen was also studied during different stages of G1 and was found to be expressed at high levels in early G1 cells compared with other parts of G1.

Expression of cyclin D1 and retinoblastoma protein in colorectal cancer.

Abnormal expression of the retinoblastoma protein (pRb) and cyclin D1 have been reported in a variety of malignancies, but the frequencies of these deregulations and their relation to prognosis in colorectal cancer has not been clarified. We characterised 90 colorectal cancers with respect to immunohistochemical expression of cyclin D1, pRb and Ki-67. Two of 90 (2%) tumours lacked nuclear pRb staining, indicating inactivation of the protein, while 10 (11%) expressed high levels of pRb. Abnormal expression of pRb was significantly correlated to low levels of nuclear cyclin D1 observed in 32% of the tumours. Strong nuclear cyclin D1 expression was detected in 12% of the tumours. Cytoplasmic staining of cyclin D1 was observed in 17% of the tumours, showing an inverse relationship (P = 0.006) to the Ki-67 labelling index. Eight of 11 tumours with high nuclear overexpression of cyclin D1 and both tumours with pRb defects were located in the right colon in comparison with zero of 25 in the rectum (P = 0.009). Regarding prognosis, neither pRb nor cyclin D1 expression correlated with patient survival.

Methylation of the p16(Ink4a) tumor suppressor gene 5'-CpG island in breast cancer.

Methylation of the p16(Ink4a) tumor suppressor gene 5'CpG island was analyzed in 104 primary breast cancer specimens using Southern blotting and methylation specific polymerase chain reaction (PCR) (MSP). Eight and four tumors, respectively, showed methylation, and all MSP positive tumors were detected by Southern blotting. To investigate possible methylation not detectable by these methods, bisulphite genomic sequencing was performed in 220 clones from 14 selected tumors. Absent methylation or methylation of single CpG dinucleotides prevailed in all tumors, but of the MSP positive tumors, three contained alleles with methylation of 31 or 32 of the 32 analyzed CpG dinucleotides in the island. Partially methylated alleles were also observed. In a group with low p16(Ink4a) expression determined by Western blotting, four randomly selected tumors contained several identical clones with methylation of 15 CpG dinucleotides by bisulphite genomic sequencing but with a methylation pattern that did not support detection by either Southern blotting or MSP, increasing the potential significance of p16(Ink4a) methylation in breast cancer.

Long-term cultured IL-2-dependent T cell lines demonstrate p16(INK4a) overexpression, normal pRb/p53, and upregulation of cyclins E or D2.

Acquisition of an immortal phenotype by circumvention of the normal senescence program can be an important step in tumor development and progression. The regulation of life-span checkpoints is complex and abrogation of these processes can occur at different levels. To better understand these mechanisms in long-term cultured lymphocytes we have characterized two human long-term cultured IL-2-dependent T cell lines regarding telomere length, telomerase activity, and the expression of selected cell cycle regulators (pRb, p53, cyclin E, cyclin D1, cyclin D2, cyclin D3, cdk4, p16(INK4a), p21(WAF1), p27(KIP1), c-myc, bcl-2, and NPAT). We compared these cell lines with a primary T lymphoblast population with a limited life span from the same donor. Both T cell lines with extraordinary growth capacity showed telomere length stabilization, high telomerase activity and demonstrated wild-type pattern of pRb and p53 but strong p16(INK4a) protein expression. The growth inhibitory activity of p16(INK4a) seemed to be abrogated by enhanced expression of cyclin D2, cdk4, and c-myc in one T cell line and overexpression of cyclin E in the second T cell line.