Evidence for a new tumour suppressor locus (DBM) in human B-cell neoplasia telomeric to the retinoblastoma gene.

Roughly 25% of human B-cell chronic lymphocytic leukaemias (CLL) are characterized by a chromosomal lesion involving 13q14. This region contains the retinoblastoma gene (RB1). We have used a variety of techniques to determine whether RB1 or some other locus is the critical region in 11 cases of low grade B-cell malignancy (mainly CLL), all with deletions or translocations involving 13q14. In all cases, except the one with minimal disease, there was deletion or a structural lesion in the region of D13S25, with at least 4 cases showing homozygous disruption. We conclude that D13S25 lies close to a tumour suppressor locus whose inactivation contributes to the initiation or progression of low grade B-cell malignancy. This locus is located at least 530 kilobases telomeric to RB1.

Regional distribution of collagen and haemosiderin in the lungs of horses with exercise-induced pulmonary haemorrhage.

REASONS FOR PERFORMING STUDY: Regional veno-occlusive remodelling of pulmonary veins in EIPH-affected horses, suggests that pulmonary veins may be central to pathogenesis. The current study quantified site-specific changes in vein walls, collagen and haemosiderin accumulation, and pleural vascular profiles in the lungs of horses suffering EIPH. HYPOTHESIS: In the caudodorsal lung regions of EIPH-affected horses, there is veno-occlusive remodelling with haemosiderosis, angiogenesis and fibrosis of the interstitium, interlobular septa and pleura. METHODS: Morphometric methods were used to analyse the distribution and accumulation of pulmonary collagen and haemosiderin, and to count pleural vascular profiles in the lungs of 5 EIPH-affected and 2 control horses. RESULTS: Vein wall thickness was greatest in the dorsocaudal lung and significantly correlated with haemosiderin accumulation. Increased venous, interstitial, pleural and septal collagen; lung haemosiderin; and pleural vascular profiles occurred together and changes were most pronounced in the dorsocaudal lung. Further, haemosiderin accumulation colocalised with decreased pulmonary vein lumen size. Vein wall thickening, haemosiderin accumulation and histological score were highly correlated and these changes occurred only in the caudodorsal part of the lung. CONCLUSION: The colocalisation of these changes suggests that regional (caudodorsal) venous remodelling plays an important role in the pathogenesis of EIPH. POTENTIAL RELEVANCE: The results support the hypothesis that repeated bouts of venous hypertension during strenuous exercise cause regional vein wall remodelling and collagen accumulation, venous occlusion and pulmonary capillary hypertension. Subjected to these high pressures, there is capillary stress failure, bleeding, haemosiderin accumulation and, subsequently, lung fibrosis.

Survey of trainers on the use of swimming exercise for Standardbred racehorses in Australia.

OBJECTIVES: To determine swimming training practices in Australian harness racing horses and potential targets for future research. METHODS: An online survey of Australian Standardbred trainers and telephone interviews with 20 leading trainers were conducted. Questions relating to swimming facility and protocol, perceived benefits and contraindications were included. Descriptive data analysis was performed. RESULTS: Data were collected from 270 trainers: 250 by online survey (250/1770, response rate 14.1%) and 20 by interview. Of these, 103 trainers (38.1%), including 91 surveyed trainers (91/250, 36.4%) and 12 interviewed trainers (12/20, 60.0%), used swimming exercise. The most popular reasons for swimming were to replace trackwork for horses with limb injuries (79.4%), improve or maintain fitness (62.7%) and provide mental stimulation through variety in training (40.0%). Free swimming (78.4%) was more common than tethered, but the frequency and duration for horses in full training varied widely between trainers, with a median of four swim sessions per horse each week (range 0.5-12) for a median of 7 min (range 1.5-30 min) per session, mostly as a continuous swim, but sometimes as intervals. The main reasons given by those not swimming horses were lack of an adequate facility (60.5%) and lack of perceived benefit (16.2%). Reasons for not swimming individual horses varied widely. CONCLUSIONS: Trainer opinions and protocols varied widely with respect to swimming exercise for Standardbred race horses. The role of swimming exercise requires further study so that evidence-based recommendations can be made.

Fragmentation of the dorsal distal aspect of the talus on weanling survey and pre-sale radiographs of juvenile Thoroughbreds: prevalence and 2- and 3-year-olds racing performance.

BACKGROUND: Fragmentation of the dorsal aspect of the distal talus (FDDT), at the dorsolateral articular margin of the proximal intertarsal joint (PITJ) on pre-sale radiographs of yearling Thoroughbreds has not been previously described and data to support decisions made by veterinarians to predict future racing potential of horses with these lesions are lacking. METHODS: In this retrospective case-control study we aimed to determine the prevalence of FDDT in juvenile Thoroughbreds and to report their race records. From a database of survey and repository radiographic examinations of 5709 horses, 36 with FDDT were identified. RESULTS: The prevalence of FDDT was 0.63% (36/5709; 95%CI 0.44, 0.87), compared with 5.01% (286/5709; 95%CI 4.46, 5.61) for osteochondrosis of the distal intermediate ridge of the tibia in the same population. In most cases, a single oval-shaped fragment 1-12 mm in diameter was present. When comparing cases with matched controls, there were no significant differences in mean sale price, whether horses started in a trial or race and mean number of starts, wins, places and prize money when 2- and 3-years old. CONCLUSION: FDDT did not appear to affect racing performance, although a larger-scale study is warranted to confirm this finding.

Prevalence, radiographic resolution and outcomes of slab fractures of the third and central tarsal bones in juvenile Thoroughbred horses.

BACKGROUND: Veterinarians are required to interpret the significance of radiographic findings for sale, soundness and future racing performance of weanling and yearling Thoroughbreds. We investigated the prevalence and radiographic appearance of slab fractures of the third (T3) and central tarsal (Tc) bones. METHODS: Weanling and yearling horses with complete or incomplete T3 or Tc fracture were identified by searching a database of radiographs. The prevalence and radiographic appearance at initial diagnosis and after continued pasture turnout, as well as prognosis for racing, of fractures of T3 and Tc were determined. RESULTS: Fractures were identified in 186 tarsi (184 T3 fracture only, 1 Tc fracture only, 1 Tc and T3 fracture) of 157 horses (126 unilateral T3, 29 bilateral T3, 1 contralateral Tc and T3, 1 unilateral Tc and T3) from 7676 examinations. The prevalence of T3 and Tc fractures was 2.40 (95% CI 2.07, 2.76) and 0.04 (95% CI 0.01, 0.11) per 100 radiographic examinations respectively. Fractures were identified on the D556-65°MPlLO view and occurred by survey examination at 11.1 ± 1.3 months in 85.7% horses. At initial diagnosis, 84.3% of T3 fractures appeared incomplete and involved the distal articular surface. Fracture score improved (P < 0.001), and dorsal modelling (P < 0.001) and osteoarthritis score increased in the distal intertarsal joint (P < 0.001), but not the tarsometatarsal joint, between survey and repository examinations. Fractures healed by repository examination in 71.9% of tarsi if there was > 6 months between examinations. There was no difference in sale price, and horses with T3 fractures had fewer trials when 2 and 3 years old (P = 0.023), yet no difference in other parameters of racing success when 2 or 3 years old compared with controls. CONCLUSION: Tarsal slab fractures can occur in juvenile Thoroughbreds and most heal with continued pasture turnout of > 6 months. Further investigation is required to determine risk factors and before making firm conclusions regarding the optimal management, prognosis for racing and long-term soundness.

Chromosome aberrations acquired in vitro by human B-cell lines. II. Distribution of break points.

Among 165 human B-cell lines examined, 57 were found to have karyotypic abnormalities that involved chromosome breakage. The sites of breakage were identified with quinacrine-, Giemsa-, and reverse-banding techniques, and the distribution of 239 break points was plotted. A pronounced excess of telomeric and, to a lesser extent, centromeric breaks was observed. Chromosomes No. 7, 8, 9, 11, and 14 were involved in structural rearrangements more often and chromosomes No. 2, 5, 10, 20, and X less often than was predicted on the basis of their relative lengths. Lines derived from patients with different categories of disorders varied in the distribution of break points throughout the karyotype. In this sample of cell lines, No. 8q; 14q translocations were found only in cultures derived from patients with Burkitt's lymphoma and were never observed to arise among other Epstein-Barr virus-carrying lines even after several hundred cell generations in vitro. An additional feature, which was evidently restricted to lines derived from leukemia or lymphoma patients, was the presence of interstitial insertion, deletion, or reduplication, particularly involving the long arm of chromosome No. 1.

Chromosome aberrations acquired in vitro by human B-cell lines. I. Gains and losses of material.

Gains and losses of chromosomes or chromosome arms were recorded in 45 of 165 human B-cell lines. Most aberrations were acquired in vitro, and their frequency was related to duration of culture. Gains occurred more frequently than losses and their distribution was nonrandom. Chromosomes most commonly affected were No. 3, 7, 8 (particularly 8q), 9, 12, and 21. Certain differences in the frequency of particular aberrations appeared to be related to the clinical conditions of the patients from whom the lines were derived. The distribution of chromosome gains in this material was correlated with those detected in direct preparations from human tumors.

Multifactorial analysis of differences between sporadic breast cancers and cancers involving BRCA1 and BRCA2 mutations.

BACKGROUND: We have previously demonstrated that breast cancers associated with inherited BRCA1 and BRCA2 gene mutations differ from each other in their histopathologic appearances and that each of these types differs from breast cancers in patients unselected for family history (i.e., sporadic cancers). We have now conducted a more detailed examination of cytologic and architectural features of these tumors. METHODS: Specimens of tumor tissue (5-microm-thick sections) were examined independently by two pathologists, who were unaware of the case or control subject status, for the presence of cell mitosis, lymphocytic infiltration, continuous pushing margins, and solid sheets of cancer cells; cell nuclei, cell nucleoli, cell necrosis, and cell borders were also evaluated. The resulting data were combined with previously available information on tumor type and tumor grade and further evaluated by multifactorial analysis. All statistical tests are two-sided. RESULTS: Cancers associated with BRCA1 mutations exhibited higher mitotic counts (P = .001), a greater proportion of the tumor with a continuous pushing margin (P<.0001), and more lymphocytic infiltration (P = .002) than sporadic (i.e., control) cancers. Cancers associated with BRCA2 mutations exhibited a higher score for tubule formation (fewer tubules) (P = .0002), a higher proportion of the tumor perimeter with a continuous pushing margin (P<.0001), and a lower mitotic count (P = .003) than control cancers. CONCLUSIONS: Our study has identified key features of the histologic phenotypes of breast cancers in carriers of mutant BRCA1 and BRCA2 genes. This information may improve the classification of breast cancers in individuals with a family history of the disease and may ultimately aid in the clinical management of patients.

Clinical findings, diagnosis, prevalence and predisposing factors for lameness localised to the middle carpal joint in young Standardbred racehorses.

REASONS FOR PERFORMING STUDY: Lameness related to the middle carpal joint (MCJ) occurs in up to 30% of young Standardbred horses in race training and the incidence increase with radiographic severity of third carpal bone (C3) sclerosis on DPr-DDIO (skyline) view of the carpus. Factors predisposing horses to carpal injury have not been well investigated. OBJECTIVES: To determine the importance of MCJ lameness as a cause of wastage in young Standardbred racehorses, stage of training at which it occurs and predisposing factors, and to describe clinical findings and diagnosis. METHODS: Standardbred horses (n = 114) entering their first year of race training were examined at approximately 3-month intervals over 12-18 months. For 87 of the horses, a training diary was available and these horses were trained at 3 different stables, each using a different exercise regime. At each examination, forelimb conformation, MCJ effusion, MCJ lameness and radiographic findings were graded, and training history and reasons for lost training days recorded. Nuclear scintigraphy and exploratory arthroscopy were performed on a limited selection of horses. Results for horses that developed MCJ lameness during the study period were compared statistically with results for horses that did not. RESULTS: Carpal lameness occurred in 28% of horses and was present in 56% with forelimb lameness. In most cases lameness was mild, bilateral and with little or no MCJ effusion and was attributed to subchondral bone pain associated with radiographic evidence of C3 sclerosis. Carpal lameness was the most common reason for >1 month's rest during the study period. It occurred at any stage of training but, in most cases, some speed training had begun. Of the variables studied, poor forelimb conformation and more intense speed training were predisposing factors. CONCLUSIONS AND POTENTIAL RELEVANCE: The information gained should assist in making recommendations regarding training young Standardbreds to reduce the incidence of MCJ lameness. However, further investigations to determine the optimal training regime are warranted.

p53 mutations in breast cancer.

We have identified and analyzed 41 mutations in p53 in sporadic breast tumors from 136 unselected breast cancer patients and estimate that approximately 40% of such tumors contain p53 mutations. The frequency of G-T transversions and the incidence of guanosine mutations in the nontranscribed strand of the p53 gene were found to be higher than expected, and we suggest, therefore, that exogenous carcinogens have an etiological role in sporadic breast cancers. Mutations were recorded in 44 codons of the p53 gene, with no obvious mutational hot-spots, although mutations at codons 175, 194, 273, and 280 accounted for 25% of the changes. One germ-line mutation was found in 136 patients and so we conclude that constitutional mutation of p53 may be an uncommon etiological factor in breast cancer.

Definition and refinement of a region of loss of heterozygosity at 11q23.3-q24.3 in epithelial ovarian cancer associated with poor prognosis.

Previous cytogenetic and loss of heterozygosity (LOH) data suggest that disruption of chromosome 11q23-qter occurs frequently in epithelial ovarian cancer and is associated with an adverse clinicopathological phenotype. Ten polymorphic microsatellite repeat loci were analyzed by PCR from the 11q22-q25 region between D11S35 and D11S968 in 40 ovarian tumors (including 31 epithelial ovarian cancers). Two distinct regions of loss were detected, suggesting possible sites for genes involved in epithelial ovarian neoplasia: a large centromeric region between D11S35 and D11S933 (11q22-q23.3) and a telomeric 8.5-Mb region lying between D11S934 and D11S1320 (11q23.3-24.3) not previously defined. LOH of the latter region but not the former one was significantly associated with poor survival, despite all tumors in this study having LOH somewhere on chromosome 11. This analysis provides a starting point for positional cloning.

Allele losses on chromosome 17 in human epithelial ovarian carcinoma.

16 epithelial ovarian carcinomas (12 serous, 2 mucinous and 2 endometrioid) and 2 benign ovarian adenomas were studied using recombinant DNA technology to compare loci on chromosome 17 in germ-line and tumour DNA. Four polymorphic probes mapping to 17p and one mapping to 17q were used. There was a high frequency of allele loss on 17q/(77%) and a slightly lower frequency on 17p (69%). These findings probably indicate loss of tumour suppressor genes which are of fundamental importance in the genesis or progression of epithelial ovarian carcinomas.

MCC, a candidate familial polyposis gene in 5q.21, shows frequent allele loss in colorectal and lung cancer.

MCC is a gene located within human chromosome band 5q.21 that shows somatically acquired mutations in colorectal cancer, and may be identical to the gene responsible for inheritance of familial adenomatous polyposis. Here we demonstrate that alleles contiguous with or within MCC are deleted in a high proportion of sporadic colorectal carcinomas. Of 106 carcinomas that were informative concurrently at close-flanking sites both centromeric and telomeric to MCC, 41.5% showed acquired allele loss contiguous with MCC. Evidence is presented to show that the true frequency of loss of MCC alleles is higher still. In contrast, allele losses in chromosome 5 that were incompatible with involvement of MCC were very rare (2% of a total series of 201 informative tumours). Interstitial deletion was the commonest mechanism of allele loss, and L5.71-3, a probe known to include coding sequences of MCC, marks the most consistently deleted site. Moreover mapping of chromosome breakpoints with six probes within 5q.21 sited the common critical deletion in a 2.5 Mb region which included L5.71-3. However use of L5.71-3 itself suggested that critical deleted regions may lie on either side of the probed sequence. The simplest explanation for this unexpected finding is that MCC itself is the essential deleted gene, the lost exons lying sometimes centromeric to, sometimes telomeric to and occasionally within the region detected by L5.71-3. Tumours in which MCC-related alleles were lost by interstitial deletion were in general larger than those with other mechanisms of acquired homozygosity (e.g. mitotic recombination), but there were no other obvious associations with clinicopathological features. Between 20% and 25% of lung cancers also showed acquired allele losses contiguous with MCC. The significance of this observation is still to be determined, as lung tumours show allele losses at many other sites, but the specificity of the probes used in this study does establish that the 5q.21 losses in these tumours are compatible with involvement of MCC.

High frequency of APC loss in sporadic colorectal carcinoma due to breaks clustered in 5q21-22.

Familial adenomatous polyposis is transmitted by a gene (APC) located within 5q21-22. Hemizygous loss of at least a part of 5q has been reported in 19-36% of sporadic colorectal carcinomas. This suggests that an anti-oncogene is located on that chromosome arm, but the probes used previously gave little information on the status of APC in the tumours. Using DNA probes homologous to polymorphic sequences flanking and close to the APC locus we show that more than half of a large series of carcinomas had lost at least one flanking allele. Mapping of allele losses provides data that imply clustering of breakpoints in a 10-15 megabase region around APC. The commonest chromosome defect responsible for APC loss was interstitial deletion. Mitotic recombination or partial arm loss were less frequent mechanisms. Whole chromosome loss was rare. This pattern contrasts with that reported in acquired homozygosity at other anti-oncogene loci in sporadic tumours and implies that APC loss is an early event in colorectal carcinogenesis. This view is also supported by the observations that 5q21-22 loss occurs with similar frequency in DNA diploid and DNA aneuploid tumours, and also in tumours at all clinical stages of progression.

Loss of heterozygosity at 11q22 correlates with low progesterone receptor content in epithelial ovarian cancer.

Forty-seven epithelial ovarian cancers were analyzed for loss of heterozygosity (LOH) at D11S35 (11q22), close to the progesterone receptor (PR) gene, and for tumoral estrogen receptor (ER) and PR content. Thirty-eight of 47 tumors were informative, and, of these, 14 exhibited LOH. There was a significant association (P = 0.014) between D11S35 LOH and low tumoral PR content. For all informative tumors, there was no correlation between ER and PR; however, exclusion of tumors with LOH from the informative series revealed a linear correlation between tumoral ER and PR (P = 0.013), and established ER (P = 0.025) and PR (P = 0.05) content as significant factors in relation to patient survival. Patients with ER-rich tumors with D11S35 LOH had particularly poor survival compared with ER-rich, D11S35 heterozygous, no loss patients (P = 0.014). Analysis of the same tumors using two other microsatellites, D11S935 (11p13) and NM23 (17q22), showed no statistically significant relationships, although there were nonsignificant trends for the correlation of ER and PR expression in informative tumors without allele loss at these loci. We propose that genomic structural alteration at or close to the PR gene locus has biological and clinical sequelae in ovarian cancer.

The pathology of familial breast cancer: histological features of cancers in families not attributable to mutations in BRCA1 or BRCA2.

Breast cancers arising in carriers of mutations in the breast cancer susceptibility genes, BRCA1 and BRCA2, differ histologically from each other and from breast cancers unselected for a family history. However, a substantial proportion of families with multiple cases of breast cancer is not attributable to these two genes (non-BRCA1/2 families). We have now characterized the pathology of 82 breast cancers from non-BRCA1/2 families. Breast cancers in non-BRCA1/2 families were of lower grade (P = 0.0018), showed fewer mitoses (P < 0.0001), less nuclear pleomorphism (P = 0.0014), less lymphocytic infiltrate (P < 0.0001), a lesser extent of the tumor with a continuous pushing margin (P = 0.004), a lesser extent of the tumor composed of solid sheets of cells (P = 0.0047), less necrosis (P = 0.002), and wereparison with BRCA2 tumors, non-BRCA1/2 tumors were lower grade (P = 0.017) and exhibited less pleomorphism (P = 0.01) and more tubule formation (P = 0.05). In comparison with control breast cancers unselected for a family history of the disease, non-BRCA1/2 tumors were of significantly lower grade (P = 0.001), showed less pleomorphism (P = 0.0002), and had a lower mitotic count (P = 0.003). The results indicate that non-BRCA1/2 breast cancers differ histologically from both BRCA1 and BRCA2 breast cancers and are overall of lower grade. They also suggest that non-BRCA1/2 breast cancers differ from nonfamilial breast cancers, but these differences may be attributable to various types of bias.

Measurement of radiation survival using the MTT assay.

There is increasing interest in the development of rapid assays of radiosensitivity which can be used on clinical specimens. Unfortunately, the measurement of radiation survival using clonogenic assays, which are the established standard, can be difficult and time consuming. We have used the MTT assay to measure the radiation survival of four lymphoblastoid cell lines with low plating efficiencies. We measured surviving fractions both when the irradiated cells had regained exponential growth and when the non-irradiated cells had undergone four or more doublings. The results were compared to surviving fractions measured by clonogenic assay. We found both methods could be used successfully to rank the cell lines in order of radiosensitivity. However, cells exposed to the higher radiation doses in the MTT assay did not always regain exponential growth, limiting the dose range for which the assay was useful. We also found the best correlation between the two assays was sometimes obtained by using the MTT surviving fractions from different days for different radiation dose levels. Thus, although the MTT assay can be used to measure radiation survival in relation to other cell lines, its use can be complicated by restrictions on radiation dose ranges and difficulties with data interpretation.

Current policies for surveillance and management in women at risk of breast and ovarian cancer: a survey among 16 European family cancer clinics. European Familial Breast Cancer Collaborative Group.

The recent isolation of breast cancer predisposing genes (BRCA1 and BRCA2) allows the identification of carriers within affected families. These carriers have a 50-85% risk of developing breast or ovarian cancer and need careful follow-up. The purpose of this study was to evaluate the management and screening protocols implemented in high risk families at various family cancer clinics in Europe. A questionnaire was mailed to the members of the European Familial Breast Cancer Collaborative Group (n = 30) requesting information on the following issues: indication for surveillance of breasts and ovaries, the recommended protocol, coordination of the screening examination, prophylactic surgery, the specific management of breast cancer in a mutation carrier and the use of oestrogen. 16 centres from nine countries responded. Most centres recommend surveillance of the breasts if the lifetime risk exceeds 15-20%. The surveillance protocol that is generally advised comprises monthly self breast examination, examination by a specialist every 6 months and annual mammography, all starting from an age between 25 and 35 years. Surveillance of the ovaries is recommended in BRCA1 and BRCA2-mutation carriers, in members from breast/ovarian cancer families and in some centres in 'breast cancer only' families with an early onset of breast cancer. The recommended protocol includes gynaecological examination, sonography and estimation of CA-125 at yearly intervals starting from the age 30-35 years. Prophylactic mastectomy is considered for proven mutation carriers in some centres. Most centres consider prophylactic oophorectomy in mutation carriers and some centres also consider it for members of breast/ovarian cancer families. This survey provides insight into the guidelines for surveillance and management of familial breast cancer used at various family cancer clinics in Europe; this insight may contribute to the appropriate management of these high risk women. It should be emphasised that most recommendations are based on experts' opinion rather than on any specific studies.

Glutaraldehyde and human T cell rosettes.

Blood mononuclear cells from ten healthy donors and cells from fifty cultured human lymphoblastoid lines were tested for the capacity to form rosettes with sheep erythrocytes. The effect of adding glutaralhehyde before scoring rosettes and the effect of substituting glutaraldehyde-fixed for fresh sheep cells were both examined. The results suggest that glutaraldehyde increases the apparent proportion of rosette-forming cells by altering the sheep erythrocytes in such a way that they will adhere to some B lymphocytes. Although a small number of cells carry both surface immunoglobulins and receptors for fresh sheep erythrocytes, the rosette test with fresh sheep cells identifies T lymphocytes more accurately in the absence of glutaraldehyde than in its presence.

The use of detergents in velocity sedimentation of cell culture IgM.

The biosynthetically radiolabelled secreted proteins of human lymphoblastoid cell lines have been analysed by sucrose gradient velocity sedimentation. The addition of detergent to the gradient buffer dramatically improves the recovery of immunoglobulins from cell line supernatants, allowing the gradient fractions to be analysed further both serologically and biochemically. The results of these analyses show that IgM is synthesised in the 19S form and can be clearly separated from other components which include free light chains. Immunoglobulin appears to form a much larger portion of the proteins secreted by human lymphoblastoid lines than has formerly been recognised, a finding which may be of practical importance for the development and exploitation of human monoclonal antibodies.