Lubricin/proteoglycan 4 increases in both experimental and naturally occurring equine osteoarthritis.

OBJECTIVE: The goals of this study were (1) to quantify proteoglycan 4 (PRG4) gene expression; (2) to assess lubricin immunostaining; and (3) to measure synovial fluid lubricin concentrations in clinical and experimental models of equine carpal osteoarthritis (OA). DESIGN: Lubricin synovial fluid concentrations and cartilage and synovial membrane PRG4 expression were analyzed in research horses undergoing experimental OA induction (n = 8) and in equine clinical patients with carpal OA (n = 58). Lubricin concentrations were measured using a custom sandwich enzyme-linked immunosorbent assay, and PRG4 expression was quantified using qRT-PCR. Lubricin immunostaining was assessed in synovial membrane and osteochondral sections in the experimental model. RESULTS: Lubricin concentrations increased in synovial fluid following induction of OA, peaking at 21 days post-operatively in OA joints vs sham-operated controls (331 ± 69 µg/mL vs 110 ± 19 µg/mL, P = 0.001). Lubricin concentrations also increased in horses with naturally occurring OA as compared to control joints (152 ± 32 µg/mL vs 68 ± 4 µg/mL, P = 0.003). Synovial membrane PRG4 expression increased nearly 2-fold in naturally occurring OA (P = 0.003), whereas cartilage PRG4 expression decreased 2.5-fold (P = 0.025). Lubricin immunostaining was more pronounced in synovial membrane from OA joints as compared to controls, with intense lubricin localization to sites of cartilage damage. CONCLUSIONS: Although PRG4 gene expression decreases in OA cartilage, synovial membrane PRG4 expression, synovial fluid lubricin concentrations and lubricin immunostaining all increase in an equine OA model. Lubricin may be elevated to protect joints from post-traumatic OA.

A chondrocyte infiltrated collagen type I/III membrane (MACI® implant) improves cartilage healing in the equine patellofemoral joint model.

UNLABELLED: Autologous chondrocyte implantation (ACI) has improved outcome in long-term studies of joint repair in man. However, ACI requires sutured periosteal flaps to secure the cells, which precludes minimally-invasive implantation, and introduces complications with arthrofibrosis and graft hypertrophy. This study evaluated ACI on a collagen type I/III scaffold (matrix-induced autologous chondrocyte implantation; MACI(®)) in critical sized defects in the equine model. METHODS: Chondrocytes were isolated from horses, expanded and seeded onto a collagen I/III membrane (ACI-Maix™) and implanted into one of two 15-mm defects in the femoral trochlear ridge of six horses. Control defects remained empty as ungrafted debrided defects. The animals were examined daily, scored by second look arthroscopy at 12 weeks, and necropsy examination 6 months after implantation. Reaction to the implant was determined by lameness, and synovial fluid constituents and synovial membrane histology. Cartilage healing was assessed by arthroscopic scores, gross assessment, repair tissue histology and immunohistochemistry, cartilage glycosaminoglycan (GAG) and DNA assay, and mechanical testing. RESULTS: MACI(®) implanted defects had improved arthroscopic second-look, gross healing, and composite histologic scores, compared to spontaneously healing empty defects. Cartilage GAG and DNA content in the defects repaired by MACI implant were significantly improved compared to controls. Mechanical properties were improved but remained inferior to normal cartilage. There was minimal evidence of reaction to the implant in the synovial fluid, synovial membrane, subchondral bone, or cartilage. CONCLUSIONS: The MACI(®) implant appeared to improve cartilage healing in a critical sized defect in the equine model evaluated over 6 months.

Cytokine and catabolic enzyme expression in synovium, synovial fluid and articular cartilage of naturally osteoarthritic equine carpi.

REASONS FOR PERFORMING STUDY: Understanding the expression of catabolic and anabolic genes during osteoarthritis progression should help to identify the major mediators of the disease. OBJECTIVE: To compare the cytokine and anabolic marker concentrations in synovium, synovial fluid and cartilage between normal and osteoarthritic joints. METHODS: Carpi from horses age 2-11 years were used. Tissues were harvested at the time of surgery or euthanasia, and RNA was isolated for RT-PCR analysis. Tumour necrosis factor alpha (TNFα), interleukin-1beta (IL-1ß), aggrecanase 1 (ADAMTS-4), aggrecanase 2 (ADAMTS-5), matrix metalloproteinase-13 (MMP-13), interleukin 17 (IL-17) and collagen type I alpha 1(Col-1) expression were determined in synovium. TNFα, IL-1ß, ADAMTS-4, ADAMTS-5, MMP-13, IL-17, collagen type IIB (Col-2B), and aggrecan expression were determined in cartilage. TNFα concentration in the synovial fluid was determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: Expression of TNFα, ADAMTS-5 and MMP-13 was significantly increased in synovial tissue from OA joints. Synovial membrane IL-1ß abundance showed only moderate elevations in OA, without reaching significant levels. Cytokine expression was increased significantly in OA cartilage samples, particularly TNFα, IL-1ß, ADAMTS-4 and MMP-13; and collagen type I expression was significantly increased in synovial tissues from OA groups. Collagen type II message was diminished in mild and moderate stages of OA, but rebounded to significant elevations in severely degenerate joints. Conversely, aggrecan levels significantly declined in cartilage from all OA groups. Synovial fluid TNFα peptide concentration was significantly increased in severe OA cases. CONCLUSION: TNFα was increased in all degrees of equine OA, and was abundantly expressed in synovial membrane and cartilage. IL-1ß was overexpressed in OA cartilage, but not to a significant extent in synovium. POTENTIAL RELEVANCE: Control of TNFα should be considered further as a target in the treatment of OA. ADAMTS-4 may be the primary aggrecanase causing cartilage breakdown in OA.

Development of a novel equine whole transcript oligonucleotide GeneChip microarray and its use in gene expression profiling of normal articular-epiphyseal cartilage.

REASONS FOR PERFORMING STUDY: No large scale equine microarray is available commercially to allow genomic and transcriptional profiling of the majority of genes that would define the genetic basis of equine disease. OBJECTIVES: To generate a whole transcript target labelled GeneChip to interrogate the equine transcriptome and validate chip performance using RNA samples derived from organs, articular cells and normal cartilage. METHODS: Equine mRNA and selected equine gene sequences derived from perfect cross-hybridisation of equine RNA on human microarray GeneChips, were used to design a custom equine gene microarray. Sequence data were used as a template for generation of a glass-slide based 5'-3' multi-exon-encompassing gene chip. The microarray was characterised using RNA derived from organs including spleen, liver, brain and kidney, and RNA from cultured chondrocytes, cartilage, synovial tissue and stem cells, employing a whole transcript target labelling assay to sample mRNA across the 5'-3' spectrum. RESULTS: The custom microarray simultaneously interrogated over 12,300 equine specific genes. Probing the chip with mixtures of total RNA derived from parenchymatous organs and articular tissues resulted in 61.7 and 62.8% present calls, respectively. This gene chip provided expression information on up to 90% of the key molecules in important signalling, metabolic and development pathways. Cartilage specific matrix genes were abundantly expressed in normal articular cartilage, but surprisingly high levels of collagen types I, III, V and XI, reflected expression from the epiphyseal layers of maturing articular epiphyseal cartilage. CONCLUSION: An oligonucleotide microarray with over 12,300 probe sets was generated by uniquely combining a labelling strategy incorporating expressed sequence tags from the entire transcriptome and supplementing selected human sequences that cross-hybridised with the horse. Validation showed robust performance of the microarray. POTENTIAL RELEVANCE: This array may be a useful tool to elucidate the pathogenesis of equine diseases.

Genetic modification of chondrocytes with insulin-like growth factor-1 enhances cartilage healing in an equine model.

Gene therapy with insulin-like growth factor-1 (IGF-1) increases matrix production and enhances chondrocyte proliferation and survival in vitro. The purpose of this study was to determine whether arthroscopically-grafted chondrocytes genetically modified by an adenovirus vector encoding equine IGF-1 (AdIGF-1) would have a beneficial effect on cartilage healing in an equine femoropatellar joint model. A total of 16 horses underwent arthroscopic repair of a single 15 mm cartilage defect in each femoropatellar joint. One joint received 2 x 10(7) AdIGF-1 modified chondrocytes and the contralateral joint received 2 x 10(7) naive (unmodified) chondrocytes. Repairs were analysed at four weeks, nine weeks and eight months after surgery. Morphological and histological appearance, IGF-1 and collagen type II gene expression (polymerase chain reaction, in situ hybridisation and immunohistochemistry), collagen type II content (cyanogen bromide and sodium dodecyl sulphate-polyacrylamide gel electrophoresis), proteoglycan content (dimethylmethylene blue assay), and gene expression for collagen type I, matrix metalloproteinase (MMP)-1, MMP-3, MMP-13, aggrecanase-1, tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) and TIMP-3 were evaluated. Genetic modification of chondrocytes significantly increased IGF-1 mRNA and ligand production in repair tissue for up to nine weeks following transplantation. The gross and histological appearance of IGF-1 modified repair tissue was improved over control defects. Gross filling of defects was significantly improved at four weeks, and a more hyaline-like tissue covered the lesions at eight months. Histological outcome at four and nine weeks post-transplantation revealed greater tissue filling of defects transplanted with genetically modified chondrocytes, whereas repair tissue in control defects was thin and irregular and more fibrous. Collagen type II expression in IGF-1 gene-transduced defects was increased 100-fold at four weeks and correlated with increased collagen type II immunoreaction up to eight months. Genetic modification of chondrocytes with AdIGF-1 prior to transplantation improved early (four to nine weeks), and to a lesser degree long-term, cartilage healing in the equine model. The equine model of cartilage healing closely resembles human clinical cartilage repair. The results of this study suggest that cartilage healing can be enhanced through genetic modification of chondrocytes prior to transplantation.

Prolactin delays hair regrowth in mice.

Mammalian hair growth is cyclic, with hair-producing follicles alternating between active (anagen) and quiescent (telogen) phases. The timing of hair cycles is advanced in prolactin receptor (PRLR) knockout mice, suggesting that prolactin has a role in regulating follicle cycling. In this study, the relationship between profiles of circulating prolactin and the first post-natal hair growth cycle was examined in female Balb/c mice. Prolactin was found to increase at 3 weeks of age, prior to the onset of anagen 1 week later. Expression of PRLR mRNA in skin increased fourfold during early anagen. This was followed by upregulation of prolactin mRNA, also expressed in the skin. Pharmacological suppression of pituitary prolactin advanced dorsal hair growth by 3.5 days. Normal hair cycling was restored by replacement with exogenous prolactin for 3 days. Increasing the duration of prolactin treatment further retarded entry into anagen. However, prolactin treatments, which began after follicles had entered anagen at 26 days of age, did not alter the subsequent progression of the hair cycle. Skin from PRLR-deficient mice grafted onto endocrine-normal hosts underwent more rapid hair cycling than comparable wild-type grafts, with reduced duration of the telogen phase. These experiments demonstrate that prolactin regulates the timing of hair growth cycles in mice via a direct effect on the skin, rather than solely via the modulation of other endocrine factors.

Relationship of patient age to pathologic features of the tumor and prognosis for patients with stage I or II breast cancer.

PURPOSE: This analysis was performed to clarify the relationship of young age at diagnosis to the pathologic features of the tumor and prognosis in patients with early-stage breast cancer. PATIENTS AND METHODS: We retrospectively analyzed data from 1,398 patients with American Joint Committee on Cancer Staging stage I or II breast cancer treated by breast-conserving therapy between 1968 and 1985. One hundred seven patients were younger than 35 years at the time of diagnosis. The median follow-up duration for the 1,032 survivors was 99 months. RESULTS: Patients younger than 35 years had a significantly higher overall recurrence rate (P = .002), as well as a greater risk for developing distant metastases (P = .03), when compared with older patients. The cancers in younger patients more commonly showed factors associated with a worse prognosis (including grade 3 histology, lymphatic vessel invasion [LVI], necrosis, and estrogen receptor [ER] negativity) as compared with older patients. In a proportional hazards model that included clinical and treatment-related variables, as well as these pathologic features, age younger than 35 years remained a significant predictor for time to recurrence (relative risk [RR], 1.70), time to distant failure (RR, 1.60), and overall mortality (RR, 1.50). CONCLUSION: Breast cancer patients younger than 35 years have a worse prognosis than older patients. This difference is only partially explained by a higher frequency of adverse pathologic factors seen in younger patients.

No long-term increase in cardiac-related mortality after breast-conserving surgery and radiation therapy using modern techniques.

PURPOSE: To determine whether left-breast irradiation using modern techniques after breast-conserving surgery leads to an increased risk of cardiac-related mortality. METHODS: Between 1968 and 1986, 1,624 patients were treated for unilateral stage I or II breast cancer at the Joint Center for Radiation Therapy, Harvard Medical School, Boston, MA, with conservative surgery and breast irradiation. Seven hundred forty-five patients with a potential follow-up of at least 12 years were analyzed. Clinical, pathologic, and treatment characteristics were compared between the 365 patients (49%) who received left-sided irradiation and the 380 patients (51%) who received right-sided irradiation. The relationship between left-sided breast irradiation and the risk of nonbreast cancer- and cardiac-related mortality was examined. RESULTS: There was no significant difference in the distribution of clinical, pathologic, or treatment characteristics between the two groups, with the exception of a small difference in pathologic tumor size (medians, left, 2.0 cm, right, 1.5 cm; P = .007). At 12 years, a majority of patients still were alive. Slightly more patients with left-sided tumors had died of breast cancer (31% v 27%; P = NS). Equivalent proportions from each group died of nonbreast cancer causes (11%), including nine patients (2%) from each group who died from cardiac causes. The risk of cardiac mortality did not increase as time after treatment increased for patients who received left-sided irradiation compared with right-sided irradiation. A model that controlled for clinical, pathologic, and treatment differences showed no significant increase in any category of cause of death (breast, cardiac, or other) for patients who received left-sided irradiation. CONCLUSION: These results suggest that modern breast radiotherapy is not associated with an increased risk of cardiac-related mortality within at least the first 12 years after treatment.

Direct adenovirus-mediated insulin-like growth factor I gene transfer enhances transplant chondrocyte function.

Cell-based cartilage-resurfacing procedures may be enhanced by the addition of insulin-like growth factor I (IGF-I) to the transplant biomatrix. Given the relatively short half-life of IGF-I in biological systems, however, maintenance of effective concentrations of this peptide necessitates either high initial doses, or repeated treatment. This study investigated IGF-I delivery via adenoviral gene therapy, targeting graftable articular chondrocytes. Cultured articular chondrocytes were infected with an E1-deleted adenoviral vector containing IGF-I-coding sequence under CMV promoter control. Increased adenovirus-IGF-I concentrations resulted in coordinate increase in IGF-I mRNA and ligand expression; however, chondrocyte matrix synthesis was maximized by the lower adenovirus-IGF-I concentration (100 MOI) without additional increase at 200 or 500 MOI. Using 100 MOI, infected monolayers produced medium IGF-I content of at least 10 ng/ml in each 48-hr period for 28 days, reaching a day 4 peak concentration of 66 +/- 4.0 ng/ml. These concentrations were sufficient to produce significant stimulation of normal cartilage matrix gene expression. The concentration of secreted matrix products in medium from infected monolayers was increased up to 8-fold over uninfected control cultures. Moreover, compared with uninfected cultures, cells in infected cultures were more resistant to de-differentiation over time under serum-starved conditions, maintaining a normal chondrocyte molecular phenotype for at least 28 days. These data indicate that cultured chondrocytes are readily transduced by recombinant adenoviral vectors. The adenoviral-IGF transgene is abundantly expressed and its product secreted at therapeutic concentrations for at least 28 days, resulting in increased matrix biosynthesis and maintenance of the chondrocytic phenotype. Combined, this information suggests that there may be significant value in preimplantation adenoviral-IGF gene therapy for chondrocytes destined for cartilage resurfacing.

Identification of differentially expressed genes during a wool follicle growth cycle induced by prolactin.

The wool follicles of New Zealand Wiltshire sheep can be induced to undergo growth cycles by manipulating circulating prolactin levels. Altered patterns of gene expression through this cycle were examined using differential display, and nine sequence tags for differentially expressed genes were isolated. Four of these tags were identified as fragments of known genes, encoding a wool keratin, KRTAP3.2, a desmosome component, desmoglein 1, an epithelial cell marker, stratifin, and a protein kinase, Clk3. All four genes were shown to be downregulated in telogen skin compared with anagen. In situ hybridization showed that all had localization patterns which included cells that are absent in telogen. The stratifin tag was used to clone a cDNA that incorporated a complete open-reading frame for ovine stratifin. Ovine stratifin is similar to the human form, showing only six single residue differences in the predicted amino acid sequence. Stratifin probably acts as a regulator of other proteins involved in trichocyte cell cycling and differentiation. Clk3 is involved in regulating RNA splicing. KRTAP3.2 and Dsg1 both play structural roles in hair follicles. The other five tags, including two representing genes that were upregulated during catagen, could not be identified by homology. Differential display is an effective means of identifying genes involved in follicle function and, potentially, of genes controlling the growth cycle.

Prolactin signaling influences the timing mechanism of the hair follicle: analysis of hair growth cycles in prolactin receptor knockout mice.

Pituitary PRL regulates seasonal hair follicle growth cycles in many mammals. Here we present the first evidence implicating PRL in the nonseasonal, wave-like pelage replacement of laboratory mice. In this study we show that messenger RNA transcripts encoding the one long and two short forms of PRL receptor are present in the skin of adult and neonate mice. The receptor protein was immunolocalized to the hair follicle as well as the epidermis and sebaceous glands. Furthermore, PRL messenger RNA was detected within skin extracts, suggesting a possible autocrine/paracrine role. Analysis of the hair growth phenotype of PRL gene-disrupted mice (PRLR(-/-)) revealed a change in the timing of hair cycling events. Although no hair follicle development differences were noted in PRLR(-/-) neonates, observations of the second generation of hair growth revealed PRLR(-/-) mice molted earlier than wild types (PRLR(+/+)). The advance was greater in females (29 days) than in males (4 days), resulting in the elimination of the sexual dimorphism associated with murine hair replacement. Heterozygotes were intermediate between PRLR(-/-) and PRLR(+/+) mice in molt onset. Once initiated, the pattern and progression of the molt across the body were similar in all genotypes. Although all fiber types were present and appeared structurally normal, PRLR(-/-) mice had slightly longer and coarser hair than wild types. These findings demonstrate that PRL has an inhibitory effect on murine hair cycle events. The pituitary PRL regulation of hair follicle cycles observed in seasonally responsive mammals may be a result of pituitary PRL interacting with a local regulatory mechanism.

Molecular cloning of equine transforming growth factor-beta1 reveals equine-specific amino acid substitutions in the mature peptide sequence.

This study cloned and sequenced equine transforming growth factor (TGF)-beta1, yielding a unique nucleotide structure which predicted amino acid substitutions not seen in other mammalian species. The nucleotide sequence homology was 89% to bovine, 91% to man, 90% to ovine, and 86% to rat. Derived amino acid sequence comparison showed that the equine protein was unique, differing by two residues from man, cow, sheep, pig, and dog, and by three residues in the rat. Subsequent use of the cDNA clones to examine the expression of the TGF-beta1 gene in various tissues indicated predominant expression in adult spleen and kidney, with an age-related peak in cartilage expression at 12 months, followed by a decline as the animals matured. Northern blots showed that the predominant transcript sizes were 2.5 and 1.9 kb. More sensitive mRNA detection using PCR reaction showed peak cartilage TGF-beta mRNA levels in horses 0.7 and 1 year of age, with declining expression in older animals (2.5 and 5.5 years of age). In conclusion, although the primary nucleotide sequence of equine TGF-beta was relatively homologous to that of other species, the resulting amino acid sequence was unique to the horse, differing by two residues from the majority of mammalian sequences, where the peptide structure is identical. Expression of TGF-beta was particularly evident in spleen and kidney, and showed an age-related increase in expression in cartilage as the animals approached maturity and then a decline with progressive aging.

Options in the local management of invasive breast cancer.

Newly diagnosed, early-stage breast cancer confronts the patient and her clinician with multiple treatment decisions. This review examines some of these local treatment options including the choice between breast-conserving treatment (BCT) and mastectomy, how best to treat the axilla, and the optimal sequencing of local and systemic therapy. Key elements in the selection of patients for BCT or mastectomy include preoperative mammography, careful pathological evaluation, and an assessment of patient desires in order to balance the risk of local recurrence against preservation of a cosmetically acceptable breast. Although some absolute contraindications to BCT exist, most patients are candidates for BCT. The role of axillary dissection is currently being redefined, and in the future, more limited procedures may be able to identify patients who can avoid axillary dissection. The relationship between timing of breast surgery with regard to the menstrual cycle and outcome is intriguing but not yet established. As well, the appropriate sequencing of chemotherapy and radiotherapy (RT) after conservative surgery (CS) is uncertain, although randomized trials are beginning to shed some light on this issue. Whether all patients treated with CS require treatment with RT is another question that is currently under investigation. This article addresses these issues, focusing on the specifics of treatment implementation.

Long-term outcome following breast-conserving surgery and radiation therapy.

PURPOSE: To examine the long-term pattern and frequency of recurrences after breast-conserving therapy and whether the outcome was influenced by the era of treatment. METHODS AND MATERIALS: From 1968 to 1986, 1870 patients with unilateral Stage I or II breast cancer were treated at the Joint Center for Radiation Therapy. Of these, 1628 underwent gross tumor excision and received a dose of > 60 Gy to the tumor bed and constituted the study population. Patients were classified as without evidence of disease, dead from other causes (DOC), or by their first site of recurrence. First sites of recurrent disease were categorized as distant/regional (DF/RNF) or local (LR). Local recurrence was defined as the detection of any invasive or in situ carcinoma occurring in the ipsilateral breast and was further categorized as: true recurrence (TR), marginal miss (MM), skin recurrence (S), or elsewhere in the breast (E). Median follow-up in survivors was 116 months. Eighty patients (4.9%) were lost to follow-up at 3-175 months. The population was divided into two time cohorts: 1968-1982 (n = 810), with a median follow-up time of 143 months, and 1983-1986 (n = 792), with a median follow-up time of 95 months. RESULTS: The overall crude rates of ipsilateral breast recurrence were 7.4 and 13.3% at 5 and 10 years, respectively. Crude rates at 5 and 10 years were 5.7 and 9.3% for TR/MM and were 0.9 and 2.8% for E recurrences, respectively. The annual incidence rates for all LR ranged from 0.5-2.4% and was relatively constant after the first year. The annual incidence rates for TR/MM ranged from 0.4 to 1.9%, whereas for E recurrences the range was 0.1-0.7%. The crude rates of DF/RNF were 16.6 and 23.1% at 5 and 10 years, respectively. The annual incidence rates for DF/RNF ranged from 1-5% over all years. Although the magnitude of the incidence was different, DF/RNF recurrence predominated in years 1-3 for both node-positive and node-negative patients. For the 1968-1982 and 1983-1986 cohorts, the 5-year crude rates of ipsilateral breast recurrence were 8.8 and 5.9%, respectively. CONCLUSIONS: Distant and regional nodal failures were the predominant form of recurrence. The annual incidence rate of LR was relatively constant over the first decade. True recurrence/marginal miss was the most frequent type of ipsilateral breast recurrence and was highest during years 2 through 7. The risk of a recurrence elsewhere in the breast increased with longer follow-up and was highest during years 8 through 10. The 5-year crude rate of ipsilateral breast recurrence appeared lower in the 1983-1986 patient cohort compared to the 1968-1982 patient cohort (8.8% vs. 5.9%), but the distributions of site of first failure did not differ significantly (p = 0.13). Any decrease in ipsilateral breast recurrence likely reflects improvements in mammographic and pathologic evaluation, patient selection, and the increased use of reexcision.

The relation between the surgery-radiotherapy interval and treatment outcome in patients treated with breast-conserving surgery and radiation therapy without systemic therapy.

PURPOSE: This analysis was performed to clarify the relationship between the surgery-radiotherapy interval and the risk of recurrence in patients treated with breast-conserving therapy for early stage invasive cancers. METHODS AND MATERIALS: We retrospectively analyzed data from 653 patients with American Joint Commission on Cancer Stage I or II, pathologically node-negative breast cancer treated by breast-conserving therapy without adjuvant systemic therapy between 1968 and 1985. All patients received a dose of at least 60 Gy to the tumor bed. Two hundred and eighty-three patients started radiotherapy within 4 weeks of surgery, 308 started 5-8 weeks after surgery, and 54 started 9-12 weeks after surgery. Median follow-up in the 531 survivors was 100 months. RESULTS: Pathologic features and treatment characteristics were well balanced between the groups with surgery-radiotherapy intervals of 0-4 weeks and 5-8 weeks. There was no statistically difference in the risk of overall recurrence among patients starting radiotherapy 5-8 weeks after surgery compared with those treated within 4 weeks. Analysis of the 5-year crude rates of failure further demonstrated no difference in the distribution of sites of failure in the 5-8 week group compared with the 0-4 week group. A multivariate model controlling for known risk factors, as well as potential treatment-related confounders, also failed to demonstrate an increased risk of recurrence with the longer surgery-radiotherapy interval (risk ratio = 0.89, p = 0.44). CONCLUSION: This retrospective analysis suggests that a delay of up to 8 weeks in the interval between the last breast surgery and the start of radiotherapy is not associated with an increased risk of recurrence in patients with early stage breast cancer treated with breast irradiation to at least 60 Gy without systemic therapy.

The significance of extracapsular extension of axillary lymph node metastases in early-stage breast cancer.

PURPOSE: To investigate if extracapsular extension (ECE) of axillary lymph node metastases predicts for a decreased rate of disease-free survival or an increased rate of regional recurrence of breast carcinoma. METHODS: The study population consisted of 368 patients with T1 or T2 breast cancer and pathologically-positive lymph nodes treated with breast-conserving therapy between 1968 and 1986. The median number of sampled lymph nodes was 10. Median follow-up time for the surviving patients was 139 months (range 70-244). Twenty percent of the patients were treated with supraclavicular RT, and 64% received both axillary and supraclavicular RT, with a median dose to the nodes of 45 Gy. The following factors were evaluated: presence of ECE, number of sampled lymph nodes (LN), number of involved LN, size of primary tumor, histologic grade of tumor, presence of lymphatic vessel invasion (LVI), presence of an extensive intraductal component (EIC), radiation dose, use of adjuvant chemotherapy, and age of patient. Recurrences were reported as the 5-year crude sites of first failure, and were divided into breast recurrences (LR), regional nodal failure (RNF, defined as isolated axillary, supraclavicular, or internal mammary recurrence), and distant metastases (DM). RESULTS: One hundred twenty-two patients (33%) had ECE and 246 patients did not. The median number of LN with ECE was 1 (range 1-10) and 20% of patients had ECE in > or =4 LN. Patients with ECE tended to be older (median age 51 vs. 47, p = 0.01), and had a higher number of involved LN (median 3 vs. 2, p = 0.005) than patients without ECE. Forty-three percent of patients with ECE had > or =4 involved LN compared to 15% of patients without ECE (p<0.0001). Models of ECE and the above factors revealed no significant correlation between ECE and either disease-free or overall survival. There was no statistically significant increase in local, regional nodal, or distant failures in patients with ECE as compared to patients without ECE. CONCLUSION: In this population of patients with nodal involvement, the presence of ECE correlates with the number of involved LN but does not appear to add predictive power to models of local, regional, or distant recurrence when the number of positive LN is included.

Treatment outcome after tangential radiation therapy without axillary dissection in patients with early-stage breast cancer and clinically negative axillary nodes.

PURPOSE: To determine the risk of nodal failure in patients with early-stage invasive breast cancer with clinically negative axillary lymph nodes treated with two-field tangential breast irradiation alone, without axillary lymph node dissection or use of a third nodal field. METHODS AND MATERIALS: Between 1988 and 1993, 986 evaluable women with clinical Stage I or II invasive breast cancer were treated with breast-conserving surgery and radiation therapy. Of these, 92 patients with clinically negative nodes received tangential breast irradiation (median dose, 45 Gy) followed by a boost, without axillary dissection. The median age was 69 years (range, 49-87). Eighty-three percent had T1 tumors. Fifty-three patients received tamoxifen, 1 received chemotherapy, and 2 patients received both. Median follow-up time for the 79 survivors was 50 months (range, 15-96). Three patients (3%) have been lost to follow-up after 20-32 months. RESULTS: No isolated regional nodal failures were identified. Two patients developed recurrence in the breast only (one of whom had a single positive axillary node found pathologically after mastectomy). One patient developed simultaneous local and distant failures, and six patients developed distant failures only. One patient developed a contralateral ductal carcinoma in situ, and two patients developed other cancers. CONCLUSION: Among a group of 92 patients with early-stage breast cancer (typically T1 and also typically elderly) treated with tangential breast irradiation alone without axillary dissection, with or without systemic therapy, there were no isolated axillary or supraclavicular regional failures. These results suggest that it is feasible to treat selected clinically node-negative patients with tangential fields alone. Prospective studies of this approach are warranted.

Transforming growth factor-alpha immunoreactivity during induced hair follicle growth cycles in sheep and ferrets.

Transforming growth factor-alpha (TGF-alpha) has been associated with cell proliferation of keratinocytes and implicated in hair growth. We therefore examined changes in the immunocytochemical localization of TGF-alpha and cell proliferation markers in the skin of two unrelated species in which hair cycles could be induced, to elucidate the role of this growth factor in the control of fiber growth. Skin was collected from melatonin-treated ferrets (Mustela putorius furo), untreated Romney sheep (Ovis aries), and New Zealand Wiltshire sheep in which interruption of wool growth had been photoperiodically induced. Immunostaining patterns were very similar in ferrets and sheep. TGF-alpha immunoreactivity was observed in epithelial tissues of the skin but was not co-localized with cell proliferation markers. In anagen follicles, specific staining was most intense in the innermost cells of the outer root sheath and cortical cells in the keratogenous zone but was absent from inner root sheath or dermal papilla. TGF-alpha immunostaining diminished during catagen, although faint staining was retained in all epithelial cells. In telogen and early proanagen follicles, staining remained faint or was restricted to cells on the margin of the brush end and follicle neck. Immunoreactivity in the outer root sheath was reestablished in late proanagen. Sebaceous glands and epidermis were stained intensely throughout the hair cycle. TGF-alpha-immunoreactive components of skin extracts, analyzed by Western blotting, showed mobility corresponding to approximately 32 KD, but not to the size of the fully cleaved peptide. These results are consistent with an epithelial autocrine or juxtacrine, but not a mitogenic, function of TGF-alpha.

Distribution of prolactin receptor immunoreactivity in ovine skin and changes during the wool follicle growth cycle.

Prolactin is believed to mediate seasonal cues entraining seasonal reproductive and hair follicle growth cycles. Prolactin receptor binding activity and prolactin receptor gene expression in mammalian skin have recently been described. In this report, prolactin receptor immunoreactivity is identified in sheep skin using a monoclonal antibody against the rat liver prolactin receptor. Western blotting analysis of microsomal membrane proteins from skin showed major bands corresponding to molecular weights of 87 and 71 kDa and minor bands at 101 and 21 kDa. RNase protection analysis revealed the presence of mRNA species coding for long and short forms of the prolactin receptor. Formalin-fixed sections, exposed to the monoclonal antibody and stained by an immunogold method, revealed prolactin receptor-immunoreactivity in the dermal papilla, germinal matrix, outer root sheath, lower regions of the inner root sheath and connective tissue sheath of wool follicles. Staining was absent from keratinised cell populations. In all samples, the interfollicular epidermis, sebaceous and sweat glands were positively stained. The distribution of prolactin receptor is described in both growing and inactive wool follicles and related to postulated cycle-specific actions of circulating prolactin in the control of seasonal fibre growth.

Localisation of receptors for prolactin in ovine skin.

Although prolactin (PRL) receptors are found in many mammalian tissues, specific PRL binding to mammalian skin has not been demonstrated. In view of the temporal relationships observed between photoperiod, circulating PRL and pelage replacement in seasonally responsive mammals, we sought to provide evidence of PRL receptors in ovine skin. Cryosections of skin from New Zealand Wiltshire ewes were incubated with radiolabelled human GH (125I-hGH) and ovine PRL (125I-oPRL) in the presence and absence of excess unlabelled hormones (hGH, oPRL or ovine GH (oGH)). Binding was inhibited by unlabelled oPRL and hGH but not by oGH. In microautoradiographs, both radioligands were localised most strongly in the dermal papillae of wool follicles in the anagen (growth) phase of the hair cycle and in apocrine sweat glands. Higher levels of specific binding to dermal papilla cells, compared with the follicle epithelial matrix and the surrounding dermis, were confirmed by measurement of microautoradiograph silver grain density (respectively, 34.1 +/- 3.0, 11.4 +/- 1.0 and 5.5 +/- 0.5 grains per 100 microns2 (mean +/- S.E.M., n = 10)). Total binding for 125I-hGH and 125I-oPRL radioligands to follicle dermal papilla was not significantly different (34.1 +/- 3.0 vs 43.6 +/- 2.5 grains per 100 microns2, n = 10) but the level of non-specific binding of 123I-oPRL was higher than for 125I-hGH (18.9 +/- 1.4 vs 6.1 +/- 0.6 grains per 100 microns2, n = 10; P < 0.001). Binding assays of receptors in crude microsomal membranes extracted from ovine skin were used to ascertain binding capacity and specificity.(ABSTRACT TRUNCATED AT 250 WORDS)