Combination heparin plus cortisone treatment of two transplanted tumors in C3H/He mice.

C3H/He inbred mice bearing either C3H mouse mammary or RIF-1 tumors of 180-mm3 volume were treated with a combination of heparin (500 anticoagulation U/ml drinking water) plus cortisone (either 250 mg/kg/day tapering to 37 mg/kg/day or a constant dose of 75 mg/kg/day). Five types of heparin were tested in this study. RIF-1 tumors shrank to approximately half the volume at the start of therapy after only 3 days of treatment; mammary tumors took longer to respond, not reaching half the starting volume until after 11 days of treatment. In both tumors response was transient, the tumors eventually regrowing. However, response to combined heparin and cortisone therapy was in fact no different from the response to cortisone used alone. Also, cortisone treatment was extremely toxic to these animals and experiments had to be terminated after about 3 weeks of therapy.

Two functional assays employed to detect an unusual mutation in the oligomerisation domain of p53 in a Li-Fraumeni like family.

Previous investigations of a Li - Fraumeni like family (Barnes et al., 1992) demonstrated that both the proband and her mother had elevated p53 protein levels in both tumour tissue and normal tissue at sites distant from the tumour, although no mutation was found in the p53 gene. In the present study two recently described functional assays for p53, an apoptotic assay and the functional assay for the separation of alleles in yeast (FASAY), have been employed to study the functional activity of p53 from this patient. The results of the apoptotic assay demonstrated that this patient had a p53 functional defect and the FASAY result suggested that this defect was in fact a germline mutation of the p53 gene. A point mutation of codon 337, which results in an amino acid substitution of a cysteine for an arginine, was demonstrated initially in cDNA and was confirmed by sequencing of genomic DNA. This is an unusual mutation as it is in the oligomerisation domain of p53, in contrast to the majority of p53 mutations which are in the core DNA binding domain. This mutation results in a protein which still retains partial transactivational activity in the FASAY. The mutation of codon 337 is only the second reported case of a germline missense mutation occurring in the oligomerisation domain of p53.

Characterization of p53 oligomerization domain mutations isolated from Li-Fraumeni and Li-Fraumeni like family members.

p53 is a tumour suppressor gene which functions as a transcription factor to upregulate genes for growth arrest and apoptosis following DNA damage. p53 mutations are associated with Li-Fraumeni and Li-Fraumeni like syndromes. Recently mutations of the oligomerization domain have been isolated from an LFS and an LFL family affecting respectively codon 344 (Leu to Pro) and 337 (Arg to Cys). The present study was designed to determine the affect of these mutations on the function of p53 protein. p53 344 Leu to Pro existed only in a monomeric form and could not bind to DNA. It was inactive at inducing apoptosis, transactivating luciferase from a bax promoter and inhibiting cell growth. In contrast, p53 337 Arg to Cys could form tetramers and could bind to DNA. However, p53 337 Arg to Cys was not fully active and could only induce apoptosis, transactivate luciferase from a bax promoter and inhibit cell growth with approximately 60% of the ability of wild-type p53. Both mutant proteins had reduced ability to bind to MDM2, p53 337 Arg to Cys being more reduced than p53 344 Leu to Pro. These results indicate that point mutations in the oligomerization domain can disrupt p53 function. In addition, the value of LFS and LFL families for the further understanding of the biological and biochemical properties of p53 is demonstrated.

Genetic and functional studies of a germline TP53 splicing mutation in a Li-Fraumeni-like family.

We report an extensive Li-Fraumeni-like family in which there is an unusual spectrum of tumours at relatively late onset. A germline TP53 splice donor mutation in exon 4 is present in all affected family members available for testing. The mutation abolishes correct splicing of intron 4 and techniques of RT-PCR have identified three different aberrant transcripts from the mutant TP53 allele. Using the yeast functional assay to analyse transcripts in cells from a number of family members with the mutant allele, TP53 appears wild-type. Functional studies have been carried out on cells from patients with and without cancer who carry the germline mutation, and on cells from unaffected individuals from the same family who do not carry the mutation. Using a number of functional endpoints known to distinguish between cells carrying mutant or wild-type TP53 alleles, we were unable to discriminate normal (wt/wt) from heterozygous (wt/mut) cells by lymphocyte apoptosis and fibroblast survival following low dose rate ionising radiation exposure. However germline mutation carriers show increased sensitivity to radiation-induced chromosome damage in the G2 phase of the cell cycle, and decreased transient and permanent G1 arrest. These studies demonstrate the importance of fully characterising the effects of TP53 germline mutations, and may explain some of the phenotypic features of this family.

Node-negative breast cancer: prognostic subgroups defined by tumor size and flow cytometry.

Adjuvant systemic therapy for women with node-negative breast cancer is most easily justified for those patients at highest risk of relapse. We have examined the impact of tumor size, histologic grade, estrogen receptor (ER) status, tumor ploidy, and S-phase fraction (SPF) on relapse-free survival (RFS) for 169 patients with node-negative breast cancer in order to identify groups of patients at high and low risk of relapse. Patients with small tumors (less than or equal to 1.0 cm) had a significantly better RFS than those with larger tumors (P = .005), with 96% remaining relapse-free at 5 years. Patients with tumors less than or equal to 1.0 cm were thus excluded from analysis when attempting to define a group with a poor prognosis. Within the group of patients with tumors greater than 1.0 cm, tumor ploidy (P = .63), ER status (P = .3), or progesterone receptor (PgR) status (P = .24) did not predict for RFS. Patients with grade 1 or 2 infiltrating ductal tumors had a significantly better prognosis than those with grade 3 tumors (P = .04). The prognostic factor that gave the widest separation between subgroups, however, was SPF. Patients whose tumors were greater than 1.0 cm with an SPF less than or equal to 10% had a 5-year RFS of 78% compared with a 5-year RFS of 52% for those with an SPF greater than 10% (P = .006). We have combined tumor size and SPF to identify three prognostic groups: (1) tumor less than or equal to 1.0 cm, 5-year RFS 96%; (2) tumor greater than 1.0 cm plus SPF less than or equal to 10%, 5-year RFS 78%; 3) tumor greater than 1.0 cm plus SPF greater than 10%, 5-year RFS 52%. These prognostic groupings may help identify patients most suitable for adjuvant therapy.

p53 functional assays: detecting p53 mutations in both the germline and in sporadic tumours.

The tumour suppressor gene p53 is the gene most often reported to be mutated in clinical cancers with something like half of all tumours harbouring mutations. Further, many studies have suggested that p53 mutations have prognostic importance and sometimes are a significant factor in determining the response of tumours to therapy. The value of knowing the p53 status of individual tumours will increase if currently researched strategies aimed at developing p53-based treatment protocols come to fruition. There are quite a number of techniques used to detect p53 defects in both tumours and in the germline of cancer-prone families, although some of these methods are indirect and each has certain drawbacks. In this brief review we will discuss the value of two assays of p53 function as a means of detecting and partly characterizing p53 mutations. The two assays are the apoptotic assay, which measures the response of peripheral blood lymphocytes to radiation-induced DNA damage and the FASAY, a yeast based assay which assesses the ability of a given p53 protein to transactivate p53 target genes. Both of these assays are rapid, yielding results within 5 days. Further, they not only offer the possibility of detecting p53 mutations but also of characterizing a given mutation in terms of two of p53's most important functions, namely the induction of apoptosis and the transactivation of target genes.

The need for dynamic methods for measuring cell cycle perturbations: a study in radiation-treated lymphoblastoid cell lines of varying p53 status.

Reports on the p53-related cell cycle and apoptotic responses of EBV-transformed lymphoblastoid cell lines to DNA damage have led to some confusion. This may be due to differences in the nature of the specific p53 mutations under examination, but it can also be partly attributed to methodological and analytical problems (e.g. the inappropriate use of static DNA histograms for cell cycle analysis). Taking seven lymphoblastoid cell lines derived from both normal individuals and Li-Fraumeni Syndrome/Li-Fraumeni-Like (LFS/LFL) patients of differing p53 status, we completed a detailed study of radiation-induced cell cycle perturbations. Using BrdUrd pulse labelling and flow cytometry it was found that, regardless of p53 status, the cells did not arrest in G1 despite all of the lines showing p53 upregulation 3 hours postirradiation. The irradiated cells did, however, show a general slowing both in S-phase entry from G1 and in movement through S-phase. These facts would not have been apparent from the analysis of static DNA histograms. The problems with the use of static methods to assess changes in the dynamics of cell cycle progression apply not only to studies involving EBV-transformed cell lines, but also to a wide range of investigations into the molecular control of cell proliferation.

Proliferative activity, histological grade and benefit from adjuvant chemotherapy in node positive breast cancer.

The influence of S-phase fraction (SPF), measured by DNA flow cytometry, and histological grade on outcome following adjuvant chemotherapy was analysed for 214 patients with node positive breast cancer treated at Guy's Hospital who were entered into the Guy's/Manchester trial of combination chemotherapy with cyclophosphamide/methotrexate/5-fluorouracil (CMF) vs. no adjuvant treatment. Adjuvant CMF significantly improved relapse-free survival (RFS) for premenopausal patients whose tumours had an SPF of 10% or less (control vs. CMF, P = 0.05) and premenopausal patients whose tumours had an SPF over 10% (control vs. CMF, P = 0.003). No significant improvement in RFS attributable to CMF was seen for either subgroup of postmenopausal patients. When patients were divided into subgroups based on histological grade of tumour, an improvement in RFS attributable to CMF was seen for premenopausal patients with well differentiated (grade 1 or 2) tumours (control vs. CMF, P = 0.03) and premenopausal patients with poorly differentiated (grade 3) tumours (control vs. CMF, P = 0.006). Again, no improvement in RFS was noted for any subgroup of postmenopausal patients defined by tumour grade.

DNA flow cytometry and response to preoperative chemotherapy for primary breast cancer.

Between October 1988 and June 1990, 22 patients with locally advanced, inoperable breast cancer entered a pilot study of four cycles of anthracycline based cytotoxic chemotherapy followed by surgery and tamoxifen. Fine needle aspirate samples of tumour were obtained for DNA flow cytometry before treatment and during the first cycle of chemotherapy. 21 patients are eligible for assessment of response and toxicity. Chemotherapy was well tolerated with greater than WHO grade 2 vomiting or stomatitis in 4 patients. Granulocytopenia less than 10(9)/l was noted in 16/21 patients but there were no episodes of neutropenic sepsis. There were 7 complete responses (CR) and 11 partial responses (PR), giving an overall response rate to chemotherapy (CR+PR) of 18/21 (86%). Responses were observed more commonly in patients who had aneuploid tumours (P = 0.06) and in patients whose tumours had a high S-phase fraction (P = 0.1). Tumours which responded to chemotherapy (CR or PR) had a significantly higher median SPF compared with tumours which did not regress (P less than 0.05). There was no consistent pattern of change in SPF values during the first cycle of chemotherapy, either for patients who responded to treatment or for those whose tumours did not regress. This combination therapy is well tolerated with a high response rate. The results of this pilot study support the recent suggestion that tumours with rapidly proliferating, aneuploid populations of cells exhibit the best short-term response to chemotherapy.

Flow cytometric characterisation of proliferating cell nuclear antigen using the monoclonal antibody PC10.

Anti-PCNA antibodies have aroused considerable interest recently as potential immunohistochemical markers of proliferation for use on clinical samples. PC10 is a monoclonal antibody which has been shown to recognise its epitope on formalin-fixed, paraffin-embedded, archival material. However, whilst PC10 gives the expected labelling pattern for growth fraction in normal tissues and some tumours, discrepant results have been obtained, for example, in carcinoma of the breast. By means of flow cytometry, we have attempted to characterise the different staining patterns that can be obtained with PC10. Intact fixed cells from proliferative mammalian cultures show 100% labelling, consistent with a growth fraction estimate. In contrast, detergent-extracted nuclei show S-phase specific staining. Nuclei extracted by treatment of fixed cells with pepsin show a different staining pattern again, with many G1 cells weakly stained and staining intensity increasing through S-phase into G2. The results demonstrate that multiparametric flow cytometry can define the cell populations which label with proliferation-related antibodies, such as PC10, under a variety of experimental conditions.

Overexpression of the c-erbB-2 oncoprotein: why does this occur more frequently in ductal carcinoma in situ than in invasive mammary carcinoma and is this of prognostic significance?

Overexpression of c-erbB-2 occurs in 60% of in situ and 25% of infiltrating ductal carcinomas. We have previously found very strong associations between immunohistochemical staining for c-erbB-2 and histological pattern and nuclear size in ductal carcinoma in situ (DCIS) and less strong correlation with proliferative activity. In a further study of infiltrating ductal carcinomas we have found that, in addition to tumours arising from c-erbB-2 positive, large celled, rapidly proliferating, comedo carcinomas and c-erbB-2 negative small celled cribriform/micropapillary carcinomas with a low proliferative rate, there is a third group of c-erbB-2 negative tumours with large nuclei and variable proliferative activity. These latter tumours are not seen in pure DCIS suggesting that they have a very transient in situ stage. Therefore, although in pure DCIS c-erbB-2 positively appears to be associated with tumours with a greater invasive potential, and c-erbB-2 negativity with tumours having a more favourable prognosis, the latter is not necessarily true in infiltrating disease.

Improving the detection of p53 mutations in breast cancer by use of the FASAY, a functional assay.

The aim of this investigation was to examine the ability of the yeast-based functional assay, the functional analysis for the separation of alleles in yeast (FASAY), to detect p53 mutations in breast cancers when compared with immunohistochemistry and automated sequencing of the whole p53 gene (exons 1-11). To achieve this, all three methods were carried out on a cohort of aggressive breast tumors. In those tumors, in which the FASAY analysis indicated the presence of a mutation, cDNA was extracted from red yeast colonies and was sequenced to identify the base change in the p53 gene. The FASAY detected all 24 mutations found in the series of 48 tumors, whereas initial automated sequencing of genomic DNA detected 18/24 mutations. A second round of automated sequencing carried out using an independent source of genomic DNA detected mutations in 3 of the 6 tumors that originally appeared to lack a mutation in genomic DNA. All but 1 of the mutations originally missed by sequencing of genomic DNA were point mutations. Five mutations in this series (21%) were outside the commonly investigated exons 5-8, reinforcing the need to extend sequencing beyond this region. Of 24 tumors, 14 had strong immunohistochemical staining, and all 14 had p53 mutations; the majority of mutations missed by immunohistochemistry produced a truncated protein. Strong staining was not seen in tumors lacking a p53 mutation. The FASAY proved to be a rapid, reliable, and effective method for identifying those breast tumors harboring p53 mutations.

Myxofibrosarcoma. Clinicopathologic analysis of 75 cases with emphasis on the low-grade variant.

Myxofibrosarcoma is one of the most common sarcomas in the extremities of elderly patients. We analysed the clinicopathologic features in a series of 75 patients. All patients were adults (range, 22-91 years; median, 66 years) with an approximately equal incidence in men and women. Thirty-five tumors arose in the lower and 25 in the upper extremities, nine on the trunk, two each in the retroperitoneum and the head and neck region, and one each in the pelvis and penis. Forty-eight cases (69.5%) were located in dermal or subcutaneous tissues. Distinctive histologic features included the following: a commonly nodular growth pattern; a myxoid matrix containing elongated, curvilinear capillaries; and fusiform, round or stellate tumor cells with indistinct cell margins, slightly eosinophilic cytoplasm, and hyperchromatic atypical nuclei. These lesions varied from a hypocellular, mainly myxoid, and purely spindle-cell appearance (low-grade neoplasms) to high-grade, pleomorphic (malignant fibrous histiocytoma-like) lesions with multinucleated giant cells, high mitotic activity, and areas of necrosis. Immunohistochemistry in 44 cases revealed only vimentin and occasional actin positivity. Ultrastructurally, tumor cells had a fibroblastic phenotype. DNA flow cytometry and proliferation analysis showed an association between aneuploidy and histologic grade. An average follow-up of 45 months (range, 5-300 months) in 60 cases has revealed local recurrence in 33 cases (54%). Thirteen patients developed metastases, and 13 tumor-related deaths occurred. A short interval to first local recurrence was associated with poor clinical outcome. The rate of local recurrence was independent of histologic grade, but only intermediate and high-grade neoplasms metastasized. The depth of the primary lesion did not influence the incidence of local recurrence. However, in deep-seated neoplasms, the incidence of metastases was higher and the percentage of tumor-related deaths was twice as high as in superficially located lesions, reflecting the fact that deep-seated lesions tended to be higher-grade, larger tumors. Myxofibrosarcoma tends to become progressively higher grade in recurrences, as demonstrated in five cases in our series. The poorly recognized low-grade myxofibrosarcoma is emphasized, as proper diagnosis and treatment and scrupulous follow-up are mandatory to avoid local recurrence and gradual tumor progression to a higher-grade neoplasm that may then metastasize.

Comparison of DNA flow cytometry from fresh and paraffin embedded samples of non-Hodgkin's lymphoma.

Cell suspensions were prepared from fresh and paraffin embedded samples of lymph nodes from nine patients with non-Hodgkin's lymphoma. DNA flow cytometry was performed on these samples and the results from fresh tissue compared with those from paraffin embedded material. Results were compared in terms of DNA index (as a measure of aneuploidy) and S phase fraction (as an indication of proliferative activity). Good agreement was found between the results from the samples prepared by the two methods. The quality of DNA flow cytometry from paraffin embedded material was comparable with or better than that from fresh samples.

Comparison of three cell cycle associated antigens as markers of proliferative activity and prognosis in breast carcinoma.

The staining patterns obtained with two antibodies against proliferating cell nuclear antigen (PC10 and 19A2) and another cell cycle associated antibody (KiS1) were compared with each other and with a number of established prognostic markers of breast carcinoma. Although PC10 and 19A2 staining patterns were similar, only the latter was significantly associated with KiS1 antibody staining. These findings suggest that the two PCNA antibodies detect different epitopes. KiS1 was the only antibody to show an association with S phase fraction measured by flow cytometry (p < 0.001). It was also associated with histological grade (p = 0.003), oestrogen receptors (p = 0.045), and DNA index (p = 0.007). PC10 showed no association with any of the markers of prognosis, while 19A2 was associated with histological grade (p = 0.017) and oestrogen receptors (p = 0.043). The two PCNA antibodies do not seem to be of value in measuring proliferative activity nor do they seem to be associated with established markers of prognosis in breast cancer.

DNA flow cytometry of follicular non-Hodgkin's lymphoma.

S-phase fraction, an index of cellular proliferation, and DNA ploidy were measured by DNA flow cytometry in a retrospective study of lymph node biopsy specimens from 83 cases (before treatment) of follicular non-Hodgkin's lymphoma, Working Formulation categories B and C. The correlations between these measures and survival, clinical stage, symptoms and histopathological factors were investigated. Aneuploidy was rare (n = 16) and had no effect on length of survival or transformation to high grade lymphoma. The overall mean S-phase fraction was 3.6%; for the whole series increasing S-phase fraction was associated with decreased survival. A high S-phase fraction (more than 5%) in initial biopsy specimens was also associated with an increased risk of subsequent high grade transformation at relapse. There was no difference between the survival or proliferative activity of tumours composed of mainly small cleaved cells compared with those composed of mixed small and large cells. There was no difference in survival or proliferative activity between tumours showing a pure follicular growth pattern and those with a mixed follicular and diffuse growth pattern. Multifactorial analysis showed that an S-phase fraction of more than 5% and B symptoms were the most important factors determining survival in these follicular non-Hodgkin's lymphomas.

Prognostic importance of DNA flow cytometry in non-Hodgkin's lymphomas.

Eighty one cases of non-Hodgkin's lymphoma were examined by DNA flow cytometry, using fixed embedded histological tissue. The frequency of detection of DNA aneuploidy and the values for S phase fractions depended on the histological subtype and grade of lymphoma. Twenty two of the patients with low grade centroblastic/centrocytic non-Hodgkin's lymphoma had repeat biopsies. Eleven of these patients remained histologically and cytometrically stable, but the remaining eleven transformed into high grade non-Hodgkin's lymphoma. The mean value for the S phase fraction in the initial biopsy specimens from patients which transformed was higher than that for patients whose lymphomas remained stable (p less than 0.001). It is proposed that estimates of S phase fraction prospectively identify patients with low grade non-Hodgkin's lymphoma at risk from transformation.

Proliferating cell nuclear antigen and S phase fraction in endometrial stromal sarcoma.

AIMS: To investigate the value of immunohistochemical staining for the cell cycle protein proliferating cell nuclear antigen (PCNA) and flow cytometric S phase fraction in determining prognosis in endometrial stromal sarcoma, graded according to mitotic count. METHODS: Seventeen endometrial stromal sarcomas from 13 patients treated at the Royal Marsden Hospital were analysed. Serial 5 microns sections were cut for haematoxylin and eosin and immunohistochemical staining for PCNA, performed using the murine monoclonal antibody PC10. PCNA positivity was expressed as a percentage of the total number of cells (PCNA index). Flow cytometric analysis was performed on nuclei extracted from paraffin wax sections. RESULTS: In the five patients who died of disease within five years, PCNA index varied between < 1% and 60% (mean 21%) and S phase fraction ranged from 11.3 and 20.1 (mean 13.8). Four patients who were apparently cured showed PCNA indices ranging from < 1% to 5% (mean 1.75%) and S phase fraction ranging from 1.4 to 3.5 (mean 2.3); and three patients alive with disease showed PCNA indices ranging from 1% to 15% (mean 8.6%) and S phase fraction ranging from 1.4 to 3.5 (mean 2.3). One patient who died from indolent local disease after nine years showed a PCNA of 1 or less and an S phase fraction of 0.9. CONCLUSIONS: PCNA staining was variable and therefore not a reliable prognostic indicator, but a high PCNA index was only found in those patients dying of disease within five years. A stronger association was seen between S phase fraction and prognosis; this also correlated well with histological grade determined by mitotic count. In individual borderline cases that are between low and high grade categories, these procedures may be useful.

Growth inhibitory activity of S12363, a novel vinca alkaloid derivative on human melanoma cell lines.

The growth inhibitory activity of S12363, a new antineoplastic agent which belongs to the vinca alkaloid group incorporating an amino-phosphonate (bioester of valine), was studied on six human melanoma cell lines with different phenotypic characteristics and in vitro growth rates. S12363 was compared with vinblastine (VBL), vincristine (VCR) and vindesine (VDS) using the MTT assay. Inhibition was time- and dose-dependent. Overall, IC50 values ranged from 24-6770nM and 4.6-11.6 nM for the reference drugs and for S12363 respectively, after exposure for 1 hr. All the vinca alkaloids were more active when cells were exposed continuously for 72 hrs, inhibition by S12363 was greater than the reference drugs (p < 0.05 in 15/18 comparisons). The activities of VDS and S12363 were also compared using the clonogenic assay. IC50 values ranged from 45-500 nM and 17-75 nM respectively. On a molar basis, S12363 was significantly more active than VDS (ANOVA p < 0.0001). The shape of the cell survival curve obtained with S12363 was exponential, whereas that of VDS was of the exponential-plateau type. Furthermore, survival with higher concentrations of S12363 was inversely related to cells seeded. Cell cycle analysis showed these compounds to block cells in G2+M after exposure to their respective IC50 concentrations for 1 hr. This effect was obtained using a lower S12363 concentration. In summary, S12363 proved to be 18-83 times more active than the reference drugs in the MTT and 3-11 times more active than VDS in the clonogenic assay. Its high potency and dissimilar cell survival profile indicate that this compound possesses different biological properties, and therefore merits further in vivo evaluation.

Flow cytometric measurement of cell proliferation.

The simplest guide to cell proliferation that can be obtained by the use of flow cytometry is the S-phase fraction (SPF) calculated from DNA histograms. Measurement of such histograms was one of the earliest applications of flow cytometry, being first reported in the late 1960s. SPF is the fraction of cells in the S phase of the cell cycle and is broadly equivalent to a tritiated thymidine labeling index ([(3)H]TdR LI). The advantages of SPF as a proliferative index include that it can be obtained rapidly from fresh, frozen, or paraffin wax-embedded tissue without the need for radioactive chemicals. The disadvantages include the need to disaggregate solid tissues, thus losing tissue morphology, and the fact that, like mitotic index and [(3)H]TdR LI, SPF is only a crude proliferative index, which gives no details of the rate of cell proliferation. Flow cytometry offers a wide range of more sophisticated methods that allow more detailed analysis of the cell cycle and the rate of cell proliferation, and one such method involving bromodeoxyuridine (BrdUrd) labeling is described in this chapter. In the section on further reading, reference is made to sources that describe the combined measurement of DNA content and cell cycle related proteins (1,2). Also beyond the scope of this chapter are applications of flow cytometry that allow the measurement of cell death and differentiation, but references to these areas are also included (3).The literature on DNA flow cytometric studies is enormous, and a considerable number of such studies have looked at various aspects of the relationship between proliferation and metastasis.