Multiparameter DNA content analysis identifies distinct groups in primary breast cancer.

BACKGROUND: Multiparameter flow cytometry is a robust and reliable method for determining tumour DNA content applicable to formalin-fixed paraffin-embedded (FFPE) tissue. This study examined the clinical and pathological associations of DNA content in primary breast cancer using an improved multiparametric technique. METHODS: The FFPE tissue from 201 primary breast cancers was examined and the cancers categorised according to their DNA content using multiparametric flow cytometry incorporating differential labelling of stromal and tumour cell populations. Mathematical modelling software (ModFit 3.2.1) was used to calculate the DNA index (DI) and percentage S-phase fraction (SPF%) for each tumour. Independent associations with clinical and pathological parameters were sought using backward stepwise Binary Logistic Regression (BLR) and Cox's Regression (CR) analysis. RESULTS: Tumours were grouped into four categories based on the DI of the tumour cell population. Low DI tumours (DI=0.76-1.14) associated with progesterone receptor-positive status (P=0.012, BLR), intermediate DI (DI=1.18-1.79) associated with p53 mutant tumours (P=0.001, BLR), high DI (DI1.80) tumours with human epidermal growth factor receptor 2 (HER2)-positive status (P=0.004, BLR) and 'multiploid tumours' (two or more tumour DNA peaks) did not show any significant associations. Tumours with high SPF% (10%) independently associated with poor overall survival (P=0.027, CR). CONCLUSION: Multiparametric flow analysis of FFPE tissue can accurately assess tumour DNA content. Tumour sub-populations associated with biomarkers of prognosis or likely response to therapy. The alterations in DNA content present the potential for greater understanding of the mechanisms underlying clinically significant biomarker changes in primary breast cancer.

Relationship between point gene mutation, chromosomal abnormality, and tumour suppressor gene methylation status in colorectal adenomas.

Epigenetic mechanisms in carcinogenesis may have a significant role in the development of colorectal cancer. To investigate this phenomenon in early-stage disease, promoter methylation status in the tumour suppressor genes APC, MGMT, hMLH1, P14/P14ARF, and CDKN2A/P16 was investigated in 78 colorectal adenomas. These had previously been characterized for mutations of APC, KRAS, and TP53 genes and for chromosomal abnormality by comparative genomic hybridization (CGH). APC hypermethylation was seen in 52 tumours (66.7%). APC showed either methylation or mutation in 66 lesions (84.6%), but these events were not statistically associated. MGMT methylation was detected in 39 cases (50%). Adenomas with this abnormality showed a significantly lower number of chromosomal changes by CGH (p < 0.02), confirming that DNA repair defect of this type is associated with a lower level of chromosomal instability. An hMLH1 methylation defect was seen in only one adenoma (1.3%), from a patient who had a synchronous cancer showing the same defect. Methylation of P14 (P14ARF) was seen in 31 adenomas (39.7%) and CDKN2A (P16) abnormality in 25 (32.1%). DNA methylation at two or more loci was seen in 46 tumours (59%), while 11 lesions (14.1%) showed no evidence of hypermethylation at any of the loci studied. Methylation at any or all of MGMT, P14 or P16 was significantly associated with APC methylation (p = 0.01). Those neoplasms with more than two methylated genes showed significantly fewer chromosomal abnormalities than adenomas with one or no methylated loci (p < 0.001). There was no association between specific individual chromosomal abnormalities, APC, KRAS or TP53 mutations and any pattern of methylation abnormality. We conclude that methylation abnormality is very common in pre-invasive colorectal neoplasia, and that high level methylation is associated with low level chromosomal instability.

The colorectal adenoma-carcinoma sequence.

BACKGROUND: It is widely accepted that the adenoma-carcinoma sequence represents the process by which most, if not all, colorectal cancers arise. The evidence supporting this hypothesis has increased rapidly in recent years and the purpose of this article is to review this evidence critically and highlight its clinical significance. METHODS: Medline searches were used to identify recent key articles relating to the adenoma-carcinoma sequence. Further pertinent articles were obtained by manual scanning of the reference lists of identified papers. RESULTS: The evidence supporting the adenoma-carcinoma sequence can be classified as epidemiological, clinicopathological and genetic. The most recent and largest body of data relates to molecular genetic events and their cellular effects; however, many other approaches, such as cytogenetics, molecular cytogenetics and cytometry, have also yielded valuable information. CONCLUSION: Recent work continues to support the adenoma-carcinoma sequence, but there is a paucity of data on the interrelationship between different genetic mutations and on the relationship between molecular and other types of genetic abnormalities. The clinical utility of the observations described has yet to be fully realized and global genetic analysis of colorectal tumours may prove to be central in rational adenoma management.

Solid tissue culture for cytogenetic analysis: a collaborative survey for the Association of Clinical Cytogeneticists.

AIMS: To survey the diagnostic service provided by UK laboratories for the culture of solid tissue samples (excluding tumours) and in particular to examine the variation in culture success rates and the problems of maternal cell overgrowth. METHODS: Twenty seven laboratories took part in a collaborative survey during 1992. Each laboratory submitted data on up to a maximum of 60 consecutive specimens (n = 1361) over a six month period. RESULTS: Skin specimens, the largest category received (n = 520), were the most problematic (51% success rate). Culture success rates were significantly lower (43%) when skin specimens (n = 140) were transported dry to the laboratory. Success rates for skin specimens also varied, depending on the origin of the specimen, from 18% for intra-uterine deaths (IUD) (n = 94) to 85% for neonatal deaths (n = 33) and 83% for live patients (n = 54). Culture of selected extra-fetal tissues from IUD, stillbirths and following elective termination of pregnancy (TOP) gave comparable success rates to those achieved for skin samples from neonatal deaths and live births. Skewed sex ratios, female > male, were identified for products of conception (POC) (n = 298) and placental biopsy specimens (n = 97). CONCLUSIONS: By appropriate selection, transport and processing of tissues, and in particular by avoiding relying solely on skin samples from IUD, stillbirths and TOP, an increase in culture success rates for solid tissue samples submitted for cytogenetic analysis could be achieved. The high risk of maternal cell contamination from POC and placental biopsy specimens was also identified in this survey.

In vivo tumour x host cell fusion in spontaneous Syrian hamster metastasis.

The HSV-2 tumour system was originally derived from an in vitro transformation of HEF by inactivated HSV-2. When injected s.c. these cells produce spindle-cell sarcomas which are metastatic at a low level. Detailed cytogenetic studies have provided evidence of tumour x normal host cell fusion in two of seven cell lines derived from metastatic lung deposits (Met D and Met G). This is the first report, in an unselected, intraspecific system, of in vivo cell fusion in spontaneous metastasis. The cells of Met D consisted of a heterogeneous population of fused and unfused tumour cells, whereas those of Met G were a homogeneous population of hybrid cells. Fusion, therefore, is likely to have occurred after metastasis in Met D and prior to, or at, metastasis in Met G. The fused cells of Met D showed comparatively little chromosome loss, while in Met G there was loss of approaching one haploid set of chromosomes. The generation of metastatic variants by cell fusion contributes to genetic diversity and emphasizes the importance of tumour heterogeneity in malignancy.

Non-random chromosome changes in a herpes-virus-transformed Syrian hamster cell line and its metastatic derivatives.

A primary subcutaneous tumour of low spontaneous metastatic capacity, produced after inoculation of Herpes-virus hominis type-2-transformed hamster fibroblasts (parent line) and two in vivo derived highly metastatic lung deposits (Met A and Met B) were karyotyped and compared after trypsin G-banding. The parent line was cytogenetically heterogeneous with a modal chromosome number of 74. However, a number of cells were of a higher ploidy level. A large variation in both numerical and structural abnormalities was observed, the chromosome rearrangements were often complex and unstable, but all the cells contained a theme of common marker chromosomes. Met A and Met B were near diploid (mean chromosome numbers 42 and 44 respectively) with a low level of tetraploid cells. They shared many chromosome rearrangements but could be readily distinguished by an additional translocation unique to Met A. Cytogenetic homogeneity within and between metastases suggested that they were of monoclonal origin and had been derived from a karyotypically similar subpopulation within the parent tumour. We were unable to detect such cells in the parent line; thus, their numbers within the parent tumour were likely to be low. Metastasis, therefore, has been highly selective, depending on the particular phenotypic properties of Met A and Met B. All cells of the parent and metastatic lines have homogeneously staining regions (HSR) and abnormalities of chromosomes 15 (C15) which may be important in tumorigenesis. In addition, Met A and Met B cells have a number of chromosome rearrangements [translocations, deletions and a double minute chromosome (DM)] not present in the parent cells. They are retained at a high frequency in the cells of Met A and Met B and thus it seems likely that the metastatic phenotype is associated with one or more of these chromosome aberrations.

Partial trisomy 12q: a clinically recognisable syndrome. Genetic risks associated with translocations of chromosome 12q.

A newborn child with an unusual facial appearance and multiple abnormalities was found to be trisomic for a large part of 12q as a result of adjacent 1 segregation of a familial translocation, t(9;12) (p24;q21.2). A combination of cytogenetic analysis, clinical features, and enzyme marker studies allows an accurate assessment of the breakpoints. Although trisomic for a considerably larger area of 12q than other reported cases, there are many similar features suggesting that trisomy 12q is a clinically recognisable syndrome. The frequency and mode of segregation of 12q translocations and their implications for genetic counselling are discussed.