Pooled long-term outcomes from two randomized trials of axillary node sampling with axillary radiotherapy versus axillary node clearance in patients with operable node-positive breast cancer.

BACKGROUND: The aim was to determine long-term overall, breast cancer-specific and metastasis-free survival as well as axillary relapse rate from a pooled analysis of two randomized trials in women with operable breast cancer. These trials compared axillary node sampling (ANS), combined with axillary radiotherapy (AXRT) if the sampled nodes were involved, with axillary node clearance (ANC). METHODS: Data from two clinical trials at the Edinburgh Breast Unit that randomized patients between 1980 and 1995 were pooled. Long-term survival was analysed using Kaplan-Meier curves and Cox regression, with separate analyses for patients with node-positive (ANS + AXRT versus ANC) and node-negative (ANS versus ANC) disease. RESULTS: Of 855 women randomized, 799 were included in the present analysis after a median follow-up of 19·4 years. Some 301 patients (37·7 per cent) had node-positive disease. There was no evidence of a breast cancer survival advantage for ANS versus ANC in patients with node-negative disease (hazard ratio (HR) 0·88, 95 per cent c.i. 0·58 to 1·34; P = 0·557), or for ANS + AXRT versus ANC in those with node-positive breast cancer (HR 1·07, 0·77 to 1·50; P = 0·688). There was no metastasis-free survival advantage for ANS versus ANC in patients with node-negative tumours (HR 1·03, 0·70 to 1·51; P = 0·877), or ANS + AXRT versus ANC in those with node-positive disease (HR 1·03, 0·75 to 1·43; P = 0·847). Node-negative patients who underwent ANS had a higher risk of axillary recurrence than those who had ANC (HR 3·53, 1·29 to 9·63; P = 0·014). Similarly, among women with node-positive tumours, the risk of axillary recurrence was greater after ANS + AXRT than ANC (HR 2·64, 1·00 to 6·95; P = 0·049). CONCLUSION: Despite a higher rate of axillary recurrence with ANS combined with radiotherapy to the axilla, ANC did not improve overall, breast cancer-specific or metastasis-free survival. Axillary recurrence is thus not a satisfactory endpoint when comparing axillary treatments.

Radiotherapy or tamoxifen after conserving surgery for breast cancers of excellent prognosis: British Association of Surgical Oncology (BASO) II trial.

BACKGROUND: The incidence of local recurrence (LR) after conservative surgery for early breast cancer without adjuvant therapy is unacceptably high even with favourable tumours. The aim of this study was to examine the effect of adjuvant therapies in tumours with excellent prognostic features. METHODS: Patients with primary invasive breast cancer <2 cm diameter, grade 1 or good prognosis special type, and node negative, treated by wide local excision (WLE) with clear margins were randomised into a 2 × 2 clinical trial of factorial design with or without radiotherapy and with or without tamoxifen. Trial entry was allowed to either comparison or both. FINDINGS: The actuarial breast cancer specific survival in 1135 randomised patients at 10 years was 96%. Analysis by intention to treat showed that LR after WLE was reduced in patients randomised to radiotherapy (RT) (HR 0.37, CI 0.22-0.61 p<0.001) and to tamoxifen (HR 0.33, CI 0.15 - 0.70 p<0.004). Actuarial analysis of patients entered into the four-way randomisation showed that LR after WLE alone was 1.9% per annum (PA) versus 0.7% with RT alone and 0.8% with tamoxifen alone. No patient randomised to both adjuvant treatments developed LR. Analysis by treatment received showed LR at 2.2%PA for surgery alone versus 0.8% for either adjuvant radiotherapy or tamoxifen and 0.2% for both treatments. CONCLUSIONS: Even in these patients with tumours of excellent prognosis, LR after conservative surgery without adjuvant therapy was still very high. This was reduced to a similar extent by either radiotherapy or tamoxifen but to a greater extent by the receipt of both treatments.

Combination blue dye sentinel lymph node biopsy and axillary node sampling: the Edinburgh experience.

AIMS: The sentinel node biopsy technique which is being increasingly used to stage breast cancers has limitations regarding not being able to find the blue hot node in about 5% of cases and false negative results in 7% of cases. It has been suggested this is probably due to tumour blocking the lymphatic channels. The four node sampling technique is dependent on the surgeon's ability to find abnormal palpable nodes in the axilla without visual or radioactive signal directions. We have combined the two techniques with the expectation to improve the results. METHOD: The study evaluates the combined technique in 434 patients with early breast cancer in a single centre. RESULTS: A blue sentinel node was identified in 394 of 434 cases (91.7%), the false negative rate was 2.4%. Thirty six patients had no sentinel node identified. Thirteen of these had positive nodes in the node sample. CONCLUSION: Axillary node sampling adds to the accuracy of the sentinel node biopsy using blue dye. Pathological features suggest that the principal cause of false negative sentinel node biopsy is due to blocking of the lymphatic channels by the cancer.

TELEMAM: a cluster randomised trial to assess the use of telemedicine in multi-disciplinary breast cancer decision making.

AIM: The TELEMAM trial aimed to assess the clinical effectiveness and costs of telemedicine in conducting breast cancer multi-disciplinary meetings (MDTs). METHODS: Over 12 months 473 MDT patient discussions in two district general hospitals (DGHs) were cluster randomised (2:1) to the intervention of telemedicine linkage to breast specialists in a cancer centre or to the control group of 'in-person' meetings. Primary endpoints were clinical effectiveness and costs. Economic analysis was based on a cost-minimisation approach. RESULTS: Levels of agreement of MDT members on a scale from 1 to 5 were high and similar in both the telemedicine and standard meetings for decision sharing (4.04 versus 4.17), consensus (4.06 versus 4.20) and confidence in the decision (4.16 versus 4.07). The threshold at which the telemedicine meetings became cheaper than standard MDTs was approximately 40 meetings per year. CONCLUSION: Telemedicine delivered breast cancer multi-disciplinary meetings have similar clinical effectiveness to standard 'in-person' meetings.

A comprehensive approach for evaluating telemedicine-delivered multidisciplinary breast cancer meetings in southern Scotland.

Multidisciplinary team (MDT) meetings for decisions on cancer management are a cornerstone of UK cancer policy. We have proposed a comprehensive methodology to assess the clinical and economic effectiveness of telemedicine in this setting, which is being tested in a randomized breast cancer trial. Pre- and post-telemedicine assessment includes attitudes to and expectations of telemedicine, based on semistructured interviews. The communication content of videotapes of the MDT meeting is being scored using Borgatta's revised Interaction Process Analysis System. The technical performance of the telemedicine equipment is reported on a standardized pro forma. A short questionnaire captures key elements of professional satisfaction for each patient discussion (consensus on future management, confidence in and sharing of decision), added value of linkage, group atmosphere, overall conduct of the meeting and compliance with SIGN guidelines. A cost-minimization analysis will be used for economic assessment.

Role of routine preoperative lymphoscintigraphy in sentinel node biopsy for breast cancer.

Sentinel node biopsy (SNB) is rapidly emerging as the preferred technique for nodal staging in breast cancer. When radioactive colloid is used, a preoperative lymphoscintiscan is obtained to ease sentinel lymph node (SN) identification. This study evaluates whether preoperative lymphoscintigraphy adds diagnostic accuracy to offset the additional time and cost required. 823 breast cancer patients underwent SNB based on lymphoscintigraphy, intraoperative gamma probe detection, and blue dye mapping using 99 mTc-nanocolloid and Patent Blue V injected peritumourally. The SNB was followed by standard axillary treatment at the same operation. Preoperative lymphoscintigraphy was performed around 3 h after the radioisotope injection. Preoperative lymphoscintigraphy revealed SNs in 593 (72%) of the 823 patients imaged. SN visualisation on lymphoscintigraphy was less successful in large tumours and tumours involving the upper outer quadrant of the breast (P=0.046, P<0.001, respectively). Lymphoscintigraphy showed internal mammary sentinel nodes in 9% (62/707) patients. The SN was identified intraoperatively in 98% (581) patients who had SN visualised on preoperative lymphoscintigraphy, with a false-negative rate of 7%. In patients who did not have SN visualised on preoperative lymphoscintigraphy, the SN was identified at operation in 90% (204) patients, with a false-negative rate of 7%. The SN identification rate was significantly higher in patients with SN visualised on preoperative lymphoscintigraphy (P<0.001). SN identification rate intraoperatively using the gamma probe was significantly higher in the SN visualised group compared with the SN non-visualised group (95% vs. 68%; chi square (1 degrees of freedom (df)) P<0.001. There was no statistically significant difference in the false-negative rate and the operative time between the two groups. A mean of 2.3 (standard deviation (SD) 1.3) SNs per patient were removed in patients with SN visualised on preoperative lymphoscintigraphy compared with 1.8 (SD 1.2) in patients with no SN visualised on lymphoscintigraphy (P<0.001). Although SN visualisation on preoperative lymphoscintigraphy significantly improved the intraoperative SN localisation rate, SN was successfully identified in 90% of patients with no SN visualisation on lymphoscintigraphy. Given the time and cost required to perform routine preoperative lymphoscintigraphy, these data suggest that it may not be necessary in all cases. It may be valuable for surgeons in the learning phase to shorten the learning curve and in patients who have increased risk of intraoperative failed localisation (obese or old patients). A negative preoperative lymphoscintiscan predicts the inability to localise with the hand-held gamma probe. Therefore, if the SN is not visualised on lymphoscintigraphy then the addition of intraoperative blue dye is recommended to increase the likelihood of SN identification.

Attitudes of breast cancer professionals to conventional and telemedicine-delivered multidisciplinary breast meetings.

We surveyed the attitudes of breast cancer professionals to standard face-to-face and future telemedicine-delivered breast multidisciplinary team (MDT) meetings. Interviews, which included the Group Behaviour Inventory, were conducted face-to-face (n = 19) or by telephone (n = 26). The mean total score on the Group Behaviour Inventory was 96 (SD 19) for 33 respondents, which indicated satisfaction with standard MDT meetings, irrespective of role and base hospital. Positive attitudes to videoconferencing were more common among participants with previous experience of telemedicine (Spearman's rank correlation 0.26, P = 0.91). Common themes emerging from the interviews about telemedicine-delivered MDTs included group leadership, meeting efficiency, group interaction, group atmosphere and technical quality of communication. Most participants were satisfied with standard breast MDTs. Nurses and allied health professionals were least supportive of telemedicine.

Sentinel lymph node biopsy in male breast cancer patients.

The concept of sentinel node biopsy has been validated for female breast cancer patients whereas, ALND remains the standard of care for male breast cancer patients with similar tumours. We evaluated the results of SLN biopsy in male breast cancer patients with clinically negative axillae. This study included all male breast cancer patients who underwent SLN biopsy between February 1998 and October 2003. All patients had negative axillae on clinical examination. All patients underwent pre-operative lymphoscintigraphy. SLN biopsy was performed using a combination of Patent blue V and 99mTc-radiolabelled colloidal albumin injected peritumourally. Nine patients, 26-79 years of age, were included in the study. Pre-operative lymphoscinitgraphy identified SLNs in all patients. Intraoperatively, SLNs were successfully localised in all patients. The mean number of SLNs encountered was 2.4. Five patients had a positive SLN, four a negative SLN. Five patients (one with a negative SLN, four with a positive SLN) had been elected pre-operatively to undergo ALND regardless of findings on SLN biopsy. ALND confirmed the SLN to be negative in one patient (false-negative rate: 0%) and three of the four patients with positive SLN(s) had additional positive nodes in the axilla. SLN biopsy accurately predicted axillary lymph node status in these five patients. These findings compare favourably with findings reported in the literature regarding SLN biopsy in female breast cancer patients. SLN biopsy accurately staged the axilla in male breast cancer patients and should be considered for axillary staging in male breast cancer patients with clinically negative axillae.

Sentinel lymph node biopsy in patients with multifocal breast cancer.

BACKGROUND: Multifocal or multicentric breast cancer has been suggested as a contraindication for sentinel node biopsy (SNB). However, recent studies have demonstrated that all quadrants of the breast drain through common afferent channels to a common axillary sentinel node. This should mean that the presence of multifocal tumour should not affect the lymphatic drainage. The purpose of this study was to evaluate the feasibility and accuracy of SNB in patients with multifocal breast cancer using a peritumoural injection technique for sentinel lymph node (SN) mapping. METHODS: In the ALMANAC multicentre trial validation phase, we took SNB samples from 842 patients with node negative, invasive breast cancer with use of a blue dye and radiolabelled colloid mapping technique at the peritumoural injection site. All patients underwent standard axillary treatment after SNB. Seventy-five of the 842 patients had multifocal lesions on final histopathologic examination. The following analysis is focused on patients with multifocal lesions. RESULTS: A mean number of 2.4 SNs were identified in 71 of 75 patients (identification rate: 94.7%). Thirty-one patients had a positive SN, 40 a negative SN. Standard axillary treatment confirmed the SN to be negative in 37 of 40 patients, whereas three patients revealed positive non-sentinel lymph nodes (false-negative rate: 8.8%). Overall SN biopsy accurately predicted axillary lymph node status in 68 of 71 patients (95.8%). CONCLUSION: SNB accurately staged the axilla in multifocal breast cancer and may become an alternative to complete axillary lymph node dissection in node negative patients with multifocal breast cancer.

Imprint cytology of axillary lymph nodes as an intraoperative diagnostic tool.

AIMS: Frozen section histology of lymph nodes intraoperatively is associated with a false negative rate of twenty-five per cent. Imprint cytology is reported to have greater accuracy than frozen section. Accurate intraoperative examination of lymph nodes could help to prevent some patients from having a second axillary operation in breast cancer. This study assesses the sensitivity of imprint cytology to lymph node metastases using two different staining techniques. METHODS: Imprint cytology of 238 freshly excised axillary lymph nodes in 53 patients with stage T1-2, N0, M0 breast cancer was performed. Imprints were stained using toluidine blue dye and anti-pancytokeratin immunoglobulin to compare the two staining methods. A consultant histopathologist blinded to the routine histology results examined each set of imprints. A non-pathologist also examined each set of imprints to determine whether a technician could be used to screen slides in order to decrease the workload of the pathologist. RESULTS: Sensitivity was 82% with toluidine blue and 36.4% with anti-pancytokeratin immunoglobulin. Positive predictive values were 100% and 57% respectively for toluidine blue and anti-pancytokeratin. The false-negative rate was 18% for toluidine blue and 63.6% for anti-pancytokeratin antibody when examined by a consultant histopathologist. CONCLUSIONS: Imprint cytology using toluidine blue is as sensitive as frozen section in the detection of axillary lymph node metastases. A positive result may be acted upon with acceptable safety. Immunohistochemistry using antibody to pancytokeratin did not improve imprint sensitivity in this study. Imprint cytology could help to prevent a second operation in 80% of breast cancer patients with positive lymph nodes.

Does local surgery have a role in the management of stage IV breast cancer?

AIMS: There are no uniformly agreed guidelines regarding the treatment of local breast cancer in patients who have stable metastatic disease. The aim of this study was to define the role of breast surgery in the management of stage IV disease by reviewing the clinical outcome in patients with stage IV disease submitted to surgery in a regional breast cancer unit. METHODS: All patients who underwent breast surgery from 1993 to 1999 and had known metastatic disease or who were diagnosed with metastases within one month of surgery were identified and their clinical outcome was studied using death and local recurrence as end points. RESULTS: Median survival after breast surgery was 23 months. Ten of the 20 patients were alive with no local disease at 20 months mean follow-up. Three of 10 patients who died developed local recurrence and had local disease at the time of death. CONCLUSION: The local surgery does have a role in controlling the primary cancer and controlling local symptoms in a selected group of patients with stable metastatic disease.

Outcome after extended follow-up in a prospective study of operable breast cancer: key factors and a prognostic index.

In 1990, 215 patients with operable breast cancer were entered into a prospective study of the prognostic significance of five biochemical markers and 15 other factors (pathological/chronological/patient). After a median follow-up of 6.6 years, there were 77 recurrences and 77 deaths (59 breast cancer-related). By univariate analysis, patient outcome related significantly to 13 factors. By multivariate analysis, the most important of nine independent factors were: number of nodes involved, steroid receptors (for oestrogen or progestogen), age, clinical or pathological tumour size and grade. Receptors and grade exerted their influence only in the first 3 years. Progestogen receptors (immunohistochemical) and oestrogen receptors (biochemical) were of similar prognostic significance. The two receptors were correlated (r=+0.50, P=0.001) and displaced each other from the analytical model but some evidence for the additivity of their prognostic values was seen when their levels were discordant.

Management of the axilla in operable breast cancer treated by breast conservation: a randomized clinical trial. Edinburgh Breast Unit.

BACKGROUND: In the treatment of operable breast cancer by breast conservation, the extent of axillary dissection, the need for radiotherapy to the axilla and the morbidity associated with these procedures have not been assessed adequately. METHODS: Patients with operable breast cancer were randomized to have level III axillary node clearance (232 patients) or axillary node sample (234 patients). Radiotherapy to the axilla was given selectively. Radiotherapy was not given to those who had an axillary clearance. In the early part of the study all patients who had node sample were treated by radiotherapy (54 patients); subsequently this was modified to include only those who were node positive. The morbidity to the shoulder and arm was assessed before and after operation by measuring upper limb volume and circumference, and combined glenohumeral and scapular movement and muscle power. RESULTS: Comparing the two surgical policies, no difference was found in local (axillary clearance 14 versus sample 15), axillary (eight versus seven) or distant (29 versus 29) recurrence. There was no statistically significant difference in 5-year survival rate (clearance 82.1 versus sample 88.6 per cent). Morbidity was least in those who had a node sample and no radiotherapy to the axilla. Radiotherapy to the axilla in patients who had a node sample resulted in a significant reduction in range of movement of the shoulder, e.g. mean(s.e.) 2.2(0.6) cm reduction in lateral rotation at 3 years. Surgical axillary clearance was associated with significant lymphoedema of the upper limb, e.g. 4.1(0.7) per cent increase in arm volume at 3 years. CONCLUSION: A selective policy for the management of the axilla is associated with no increase in axillary recurrence or mortality rate compared with routine axillary node clearance. Patients who are node negative after axillary sample can avoid radiotherapy or axillary clearance.

A randomized double blind-cross over trial of soya protein for the treatment of cyclical breast pain.

Twenty patients with cyclical breast pain were enrolled in a double-blind cross-over trial in which either a soy protein drink or a flavoured cow's milk was taken orally each day for 3 months before crossing over to the alternate drink for a further 3 months. Records of pain scores were taken throughout the study. Blood was also taken before and after 3 and 6 months for the measurement of phytoestrogents to assess compliance. Two women withdrew from the study at the outset leaving 18 evaluable patients who completed the study. Of these 10 (56%) felt that soy protein improved breast pain (two of whom received soy as first treatment) and two (11%) felt that cow's milk alleviated symptoms (one receiving this as first preparation) and the remaining six (33%) experienced no relief of pain with either dietary preparation. Blood levels of diadzein and genistein were elevated after the ingestion of soy protein in only 13 patients (seven of whom felt that soy improved their breast pain); in the remaining five patients (three of whom suggested that soy protein improved breast pain) phytoestrogen levels were no higher than pretreatment values. Although the ingestion of soy protein may be associated with relief of breast pain, these results illustrate the problem of non-specific effects in studies of mastalgia in that 1) cow's milk also relieved breast pain in some patients and 2) that the benefits of soy protein were not always associated with evidence of elevated circulating levels of phyto-estrogens, indicating the difficulty of compliance in dietary intervention studies using soy foods.

A rational approach to melanoma follow-up in patients with primary cutaneous melanoma. Scottish Melanoma Group.

From the Scottish Melanoma Group database for south-east Scotland we evaluated 5-year follow-up in patients with cutaneous malignant melanoma excised between 1979 and 1994 and devised an 'evidence-based' review protocol. Of the 1568 with stage I melanoma, 293 (19%) developed a recurrence, 32 had a second primary melanoma and 97 had an in-situ melanoma. The disease-free interval shortened progressively with increasing tumour thickness. Overall, 80% of recurrences were within the first 3 years, but a few patients (< 8%) had recurrences 5 or 10 years after the initial surgery. In-situ melanomas did not recur. Almost half (47%) the recurrences were noted first by the patient, and only 26% were detected first at a follow-up clinic. One hundred and thirty-nine patients (89%) were still under review when their recurrences were detected, and 102 (65%) had been seen within the previous 3 months. Questionnaires were completed by 120 patients: sun protection and avoidance, and mole examination were more likely after melanoma excision. We recommend 3-monthly review of patients with invasive lesions for the first 3 years. Thereafter, those with lesions >/= 1.0 mm need two further annual reviews. Patients with in-situ lesions should be reviewed once, to confirm adequate excision (0.5 cm margins) and to give appropriate education. Surveillance beyond 5 years is only justified if there are special risk factors.

Allelic imbalance at chromosome 17p13.3 (YNZ22) in breast cancer is independent of p53 mutation or p53 overexpression and is associated with poor prognosis at medium-term follow-up.

Molecular and immunohistochemical studies of genetic events on chromosome 17p were prospectively compared with conventional clinical and pathological parameters and disease behaviour at a minimum of 72 months follow-up. In a series of 91 patients with primary operable breast cancer, 37 out of 91 (41%) patients had disease relapse and 23 out of 91 (25%) had died during the follow-up period. Allelic imbalance at the YNZ22 locus (17p13.3), demonstrated in 33 out of 63 (52%) informative patients, was significantly associated with disease recurrence (P < 0.01, 2 d.f. Cox analysis) and showed a trend towards impaired survival (P = 0.08, 2 d.f. Cox analysis) after a mean follow-up of 84 months for survivors. By contrast, p53 mutation (in 10 out of 60, 17% of cancers), p53 allelic imbalance (in 23 out of 56, 41% informative patients), p53 mRNA expression (in 47 out of 87, 54% patients), p53 mRNA overexpression (in 24 out of 87, 28%) or p53 protein expression (detected in 25/76, 32%) were not associated with disease behaviour. There was no significant association between allelic imbalance at YNZ22 and any abnormality of p53 DNA, RNA or protein. Allelic imbalance at 17p13.3 (YNZ22) serves as a marker of poor prognosis in breast cancer. As yet unidentified genes on 17p13.3, distinct from and telomeric to p53, are therefore likely to be of clinical importance in breast cancer.

PS2 mRNA expression adds prognostic information to node status for 6-year survival in breast cancer.

Expression of pS2, an oestrogen-regulated gene, has been associated with a good short-term prognosis and response to endocrine therapy. The aim of this study was to determine whether expression of mRNA for the pS2 gene in breast cancer could contribute useful information on disease behaviour and survival at medium-term follow-up. Northern blotting was used to detect pS2 messenger ribonucleic acid (mRNA) in the primary tumour tissue from each of 90 patients with breast cancer. Axillary node status was established by sampling or clearance, oestrogen receptor concentration by enzyme immunosorbant assay and follow-up was continued for at least 6 years or until death. At 83 months mean follow-up, 29 of 90 (32%) patients had recurrent disease and, of these, 18 (20%) had died from breast cancer. pS2 mRNA expression, present in 26 of 90 (29%) cancers, was associated with freedom from disease recurrence (P = 0.026) and was significantly associated with survival at a minimum of 6 years follow-up (P < 0.001). Pathological node status and tumour size were also significantly associated with disease recurrence (P < 0.001 and P = 0.002 respectively) and inversely with survival (P < 0.001 and P < 0.001 respectively). After multiple Cox regression analysis, pS2 expression was still a significant predictor of recurrence (but not survival) after adjusting for node status and tumour size; oestrogen receptor was an independent predictor of survival. The combination of node status and pS2 expression discriminated patients with particularly good prognosis (node negative, pS2 positive: no mortality at 6 years) or poor prognosis (node positive, pS2 negative; 41% mortality at 6 years). Evaluation of pS2 expression in breast cancer at diagnosis may provide additional useful prognostic information to conventional staging.

Silicone gel-filled breast implants and connective tissue diseases.

Recent worldwide press and media speculation that silicone implants may be linked to an increased incidence of breast cancer, other cancers, and connective tissue diseases-particularly systemic sclerosis-is a current cause for concern to the medical profession and public alike. We conducted a cross-sectional study of the prevalence of connective tissue diseases, as well as signs and symptoms associated with these conditions, in women who had received a silicone gel-filled breast implant for either breast augmentation or breast reconstruction following mastectomy for breast cancer compared with women without implants in South East Scotland. We compared 317 patients who had had a silicone gel-filled breast implant inserted with matched controls. We found no increased incidence of antinuclear antibodies or rheumatoid factor in the study groups. We detected one case of rheumatoid arthritis in the reconstruction group and one in matched controls, but no cases of any other connective tissue disease. No cases were found among the augmentation patients or their controls. No differences were found in symptoms or physical signs of connective tissue diseases between the study patients and their controls. This study has failed to find any case for a link between silicone gel-filled breast implants and connective tissue diseases.