Feasibility study of combined dynamic imaging and lymphaticovenous anastomosis surgery for breast cancer-related lymphoedema.

BACKGROUND: Breast cancer-related lymphoedema (BCRL) presents a significant healthcare burden and adversely affects quality of life of breast cancer survivors. A prospective feasibility study was performed on lymphaticovenous anastomosis (LVA) for the treatment of BCRL. METHODS: Patients with BCRL underwent near-infrared spectroscopy with indocyanine green lymphatic mapping to identify suitable lymphatic channels for LVA. End-to-end anastomoses to subdermal venules were performed and patients recommenced compression garment therapy (CGT) after surgery. Volumetric assessment of the affected limb was performed at regular intervals using infrared perometry to calculate the excess volume reduction. RESULTS: Over a 24-month interval, 27 patients with BCRL underwent LVA. The mean duration of lymphoedema was 3·5 (range 0·5-18) years, and the mean number of LVAs performed was 3 (range 2-5). Twenty-four of the 27 patients completed 12-month follow-up. Patients exhibited three patterns of volumetric response following LVA: sustained response (16 patients), transient response (5) or no response (6). Sustained responders showed an excess volume reduction of -33·2 per cent at 12 months, and this correlated positively with the number of LVAs performed (r = -0·56, P = 0·034). Overall, ten patients were able to downgrade CGT after surgery, and two patients were CGT-free at 12 months. CONCLUSION: LVA resulted in a sustained volume reduction in selected patients and may offset the burden of CGT. Further work is required to identify biomarkers that predict a favourable response to LVA surgery.

Two novel frameshift mutations in BRCA2 gene detected by next generation sequencing in a survey of Spanish patients of breast cancer

Purpose. To analyze BRCA1 and BRCA2 genes using a cost-effective and rapid approach based on next generation sequencing (NGS) technology. Methods. A population of Spanish cancer patients with a personal or familial history of breast and/or ovarian cancer was analyzed for germline mutations in BRCA1 and BRCA2 genes. The methodology relies on a 5 multiplex PCR assay coupled to NGS. Results. Ten pathogenic mutations (four in BRCA1 and six in BRCA2 gene) were identified in a Spanish population. The deletion c.1792delA, in exon 10, and the duplication c.5869dupA, in exon 11 of BRCA2 gene were not previously reported and should be considered as pathogenic due to its frameshift nature. Conclusion. Two novel frameshift mutations in BRCA2 gene were detected using the multiplex PCR-based assay following by NGS (AU)

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Two novel frameshift mutations in BRCA2 gene detected by next generation sequencing in a survey of Spanish patients of breast cancer.

PURPOSE: To analyze BRCA1 and BRCA2 genes using a cost-effective and rapid approach based on next generation sequencing (NGS) technology. METHODS: A population of Spanish cancer patients with a personal or familial history of breast and/or ovarian cancer was analyzed for germline mutations in BRCA1 and BRCA2 genes. The methodology relies on a 5 multiplex PCR assay coupled to NGS. RESULTS: Ten pathogenic mutations (four in BRCA1 and six in BRCA2 gene) were identified in a Spanish population. The deletion c.1792delA, in exon 10, and the duplication c.5869dupA, in exon 11 of BRCA2 gene were not previously reported and should be considered as pathogenic due to its frameshift nature. CONCLUSION: Two novel frameshift mutations in BRCA2 gene were detected using the multiplex PCR-based assay following by NGS.

Detection of novel genetic variation in autosomal dominant retinitis pigmentosa.

We explored an approach to detect disease-causing sequence variants in 448 candidate genes from five index cases of autosomal dominant retinitis pigmentosa (adRP) by sequence DNA capture and next-generation DNA sequencing (NGS). Detection of sequence variants was carried out by sequence capture NimbleGen and NGS in a SOLiD platform. After filtering out variants previously reported in genomic databases, novel potential adRP-causing variants were validated by dideoxy capillary electrophoresis (Sanger) sequencing and co-segregation in the families. A total of 55 novel sequence variants in the coding or splicing regions of adRP candidate genes were detected, 49 of which were confirmed by Sanger sequencing. Segregation of these variants in the corresponding adRP families showed three variants present in all the RP-affected members of the family. A novel mutation, p.L270R in IMPDH1, was found to be disease causing in one family. In another family a variant, p.M96T in the NRL gene was detected; this variant was previously reported as probably causing adRP. However, the previously reported p.A76V mutation in NRL as a cause of RP was excluded by co-segregation in the family. We discuss the benefits and limitations of our approach in the context of mutation detection in adRP patients.

Novel p.M96T variant of NRL and shRNA-based suppression and replacement of NRL mutants associated with autosomal dominant retinitis pigmentosa.

Mutations in the gene encoding the transcription factor neural retina leucine zipper (NRL) are known to cause autosomal dominant (adRP) or recessive (arRP) retinitis pigmentosa (RP). In an adRP Spanish family, we detected a novel sequence variation (c.287T>C) in the NRL gene that results in the p.M96T protein change. A functional test of the ability of NRL, in conjunction with cone-rod homeobox (CRX), to transactivate a human rhodopsin (RHO) promoter was used to evaluate the pathogenic mechanisms of NRL. We found upregulation of the RHO promoter by p.M96T protein similar to that shown by other missense NRL mutations that cause adRP. Affected RP patients of the family carry the nucleotide change, although two other family members that also carry the c.287T>C variation remain asymptomatic. This result complicates the genetic counselling of the family. The pathogenic mechanisms associated with adRP NRL mutations appear to be caused by a gain of function. To suppress the negative effect of an NRL mutant, the suppression and replacement strategy seems to be the most suitable therapeutic approach capable of overcoming the mutational heterogeneity associated with NRL-linked adRP. Thus, we evaluated this methodology in the NRL gene for the first time.

Immunomodulation by imiquimod in patients with high-risk primary melanoma.

Imiquimod is a synthetic Toll-like receptor 7 (TLR7) agonist approved for the topical treatment of actinic keratoses, superficial basal cell carcinoma, and genital warts. Imiquimod leads to an 80-100% cure rate of lentigo maligna; however, studies of invasive melanoma are lacking. We conducted a pilot study to characterize the local, regional, and systemic immune responses induced by imiquimod in patients with high-risk melanoma. After treatment of the primary melanoma biopsy site with placebo or imiquimod cream, we measured immune responses in the treated skin, sentinel lymph nodes (SLNs), and peripheral blood. Treatment of primary melanomas with 5% imiquimod cream was associated with an increase in both CD4+ and CD8+ T cells in the skin, and CD4+ T cells in the SLN. Most of the CD8+ T cells in the skin were CD25 negative. We could not detect any increases in CD8+ T cells specifically recognizing HLA-A(*)0201-restricted melanoma epitopes in the peripheral blood. The findings from this small pilot study demonstrate that topical imiquimod treatment results in enhanced local and regional T-cell numbers in both the skin and SLN. Further research into TLR7 immunomodulating pathways as a basis for effective immunotherapy against melanoma in conjunction with surgery is warranted.

Functional analysis of splicing mutations in MYO7A and USH2A genes.

Usher syndrome is defined by the association of sensorineural hearing loss, retinitis pigmentosa and variable vestibular dysfunction. Many disease-causative mutations have been identified in MYO7A and USH2A genes, which play a major role in Usher syndrome type I and type II, respectively. The pathogenic nature of mutations that lead to premature stop codons is not questioned; nevertheless, additional studies are needed to verify the pathogenicity of some changes such as those putatively involved in the splice process. Five putative splice-site variants were detected in our cohort of patients: c.2283-1G>T and c.5856G>A in MYO7A and c.1841-2A>G, c.2167+5G>A and c.5298+1G>C in the USH2A gene. In this study, we analyze these changes with bioinformatic tools and investigate the expression of MYO7A and USH2A transcripts through hybrid minigene assays. Our study showed that all five mutations abolished the consensus splice site producing the skipping of involved exons. In addition, for variant c.2167+5G>A, a new donor splice site was observed. Our data reveal the pathogenic nature of the analyzed variants. The fact that splicing mutations led to in-frame or out-of-frame alterations cannot explain phenotypic differences, thus, genotype-phenotype correlations cannot be inferred.

Axillary recurrence after sentinel lymph node biopsy for breast cancer.

Sentinel lymph node biopsy (SLNB) is routinely performed as an axillary staging procedure for breast cancer. Although the reported false-negative rate approaches 10 per cent, this does not always lead to axillary recurrence. We previously reported an axillary recurrence rate of 1 per cent at a median follow-up of 2 years. Our objective is to determine the rate of axillary recurrence with longer follow-up. A retrospective review of patients with invasive breast cancer and a negative SLNB treated between 2001 and 2005 was performed. Cases where neoadjuvant therapy was used or where isolated tumor cells (ITCs) were found were included, whereas those with fewer than 18 months of follow-up were excluded. One (0.7%) out of 139 patients had an axillary recurrence after a median follow-up of 52 months. No patient who underwent neoadjuvant chemotherapy or with ITCs had axillary recurrence. Twelve (8.6%) patients have died, with death attributed to breast cancer in three. Our study demonstrates that axillary recurrence after SLNB remains a rare event after a median follow-up of 52 months, despite including potentially higher risk scenarios such as where neoadjuvant chemotherapy is used and ITCs are found. Therefore, axillary lymph node dissection can safely be avoided in patients where SLNB is negative.

Extracorporeal shock waves, a new non-surgical method to treat severe burns.

UNLABELLED: Extracorporeal shock wave treatment (ESWT) increases perfusion in ischaemic tissues, stimulates growth factors, decreases inflammation and accelerates wound healing. It is a safe technique classically used in urology and orthopaedic surgery with success, but there is still limited literature regarding its use in the management of burns. PURPOSE: The aim of this study is to analyse the effect of ESWT on deep partial/full thickness burns in patients attended at our emergency burn unit. MATERIALS AND METHODS: We performed two ESWT sessions in 15 patients with <5% TBSA (total body surface area) deep partial/full thickness burns, on the third and fifth day after injury; prior to each session, we used laser Doppler imaging (LDI). RESULTS: Of all treated burns, 80% healed uneventfully prior to 3 weeks; as many as 15% required surgical debridement and grafting and 5% developed hypertrophic scarring. After one ESW session, burns had a significant increase in perfusion, objectivated by the LDI images. CONCLUSIONS: Extracorporeal shock wave therapy emerges as a new non-invasive, feasible, safe and cost-effective method in deep partial/full thickness burns. It may decrease the need of surgery and therefore the morbidity of the patient. There is a strong need for more studies to establish the optimal timing and dosage of treatment.

Focused microwave thermotherapy for preoperative treatment of invasive breast cancer: a review of clinical studies.

BACKGROUND: Preoperative focused microwave thermotherapy (FMT) is a promising method for targeted treatment of breast cancer cells. Results of four multi-institutional clinical studies of preoperative FMT for treating invasive carcinomas in the intact breast are reviewed. METHODS: Externally applied wide-field adaptive phased-array FMT has been investigated both as a preoperative heat-alone ablation treatment and as a combination treatment with preoperative anthracycline-based chemotherapy for breast tumors ranging in ultrasound-measured size from 0.8 to 7.8 cm. RESULTS: In phase I, eight of ten (80%) patients receiving a single low dose of FMT prior to receiving mastectomy had a partial tumor response quantified by either ultrasound measurements of tumor volume reduction or by pathologic cell kill. In phase II, the FMT thermal dose was increased to establish a threshold dose to induce 100% pathologic tumor cell kill for invasive carcinomas prior to breast-conserving surgery (BCS). In a randomized study for patients with early-stage invasive breast cancer, of those patients receiving preoperative FMT at ablative temperatures, 0 of 34 (0%) patients had positive tumor margins, whereas positive margins occurred in 4 of 41 (9.8%) of patients receiving BCS alone (P = 0.13). In a randomized study for patients with large tumors, based on ultrasound measurements the median tumor volume reduction was 88.4% (n = 14) for patients receiving FMT and neoadjuvant chemotherapy, compared with 58.8% (n = 10) reduction in the neoadjuvant chemotherapy-alone arm (P = 0.048). CONCLUSIONS: Wide-field adaptive phased-array FMT can be safely administered in a preoperative setting, and data from randomized studies suggest both a reduction in positive tumor margins as a heat-alone treatment for early-stage breast cancer and a reduction in tumor volume when used in combination with anthracycline-based chemotherapy for patients with large breast cancer tumors. Larger randomized studies are required to verify these conclusions.

Analysis of the involvement of the NR2E3 gene in autosomal recessive retinal dystrophies.

The nuclear receptor protein NR2E3 is postulated to play an important role in rod and cone photoreceptor development. NR2E3 gene mutational analyses were carried out in 103 unrelated subjects with different retinal diseases. A total of 14 different sequence variants were identified, including 3 mutations, 6 rare sequence variants and five polymorphisms. One of three mutations is novel (a frameshift mutation: c.1034_1038del5bp). Five of the six rare sequence variants and one of the polymorphisms identified are novel. Splice prediction programs and functional splicing assays were performed to study three of these variants. The c.119-2 A>C mutant allele construction produces, in addition to the normal one, an abnormal transcript of 180 bp resulting from an aberrant splicing with skipping of exon 2 and the generation of a premature stop codon in exon 3. These experimental data confirm the splice predictions made by the computer programs. The obtained results reinforce the idea that NR2E3 gene is involved in several retinal diseases without a clear genotype-phenotype correlation.

Differential cytokine and transcription factor expression in patients with allergic reactions to drugs.

BACKGROUND: Allergic drug reactions (ADR) can be either immediate reaction (IR) (IgE mediated) or delayed reaction (DR) (T-cell mediated). They follow the Th1/Th2 paradigm, with DR expressing interferon-gamma (IFN-gamma) with down-regulation of interleukin-4 (IL-4) and IR expressing IL-4 with down-regulation of IFN-gamma. We studied the extension of this polarization in DR and IR by examining the cytokine and transcription factor profile in T-cell subpopulations during the acute phase of an ADR. METHODS: Expressions of cytokines [IL-4, IFN-gamma and tumor necrosis factor-alpha (TNF-alpha)] and transcription factors (c-maf, GATA-3 and T-bet) were analysed by semi-quantitative real time-polymerase chain reaction in peripheral blood mononuclear cells and in CD4 and CD8 subpopulations from ADR patients. RESULTS: In DR, IFN-gamma, TNF-alpha and T-bet increased significantly in both CD4 and CD8 subpopulations, depending on the clinical severity. In IR, IL-4, c-Maf and GATA-3 were increased, but only significantly in CD4. A positive correlation existed between IFN-gamma and T-bet in DR and between IL-4 and c-Maf and GATA-3 in IR. In DR, IFN-gamma, TNF-alpha and T-bet were increased during the acute phase in CD4 and CD8. In IR, IL-4, c-Maf and GATA-3 were all increased in the acute phase, but only in CD4. CONCLUSIONS: These results support the Th1/Th2 paradigm in ADR, confirming previous findings that include the expression in both CD4 and CD8 T cells, and extending the observation to the transcription factors involved in the polarization of the immune response. Monitoring the reactions in the cell populations implicated, could be an important tool for assessing the mechanisms involved in ADR.

Outcomes following sentinel lymph node biopsy for breast cancer.

Evaluation of axillary lymph nodes for metastatic involvement is the most significant factor in gauging prognosis in breast cancer patients. Complete axillary dissection can be associated with significant morbidity. Therefore, sentinel node biopsy was developed to sample nodes and avoid dissection in patients without clinical evidence of nodal involvement. While most surgeons currently perform the procedure, the technique remains unstandardized. Sentinel node identification rates, false-negative rates and procedural complication rates are the main outcomes measured and can depend significantly on variations in technique. Future studies on sentinel lymph node biopsy will probably focus on clarifying accuracy of the procedure in different clinical settings, delineating standard technical practice guidelines and further achieving improved outcomes.

Evaluation of three scoring systems predicting non sentinel node metastasis in breast cancer patients with a positive sentinel node biopsy.

BACKGROUND: Completion axillary lymph node dissection (cALND), performed after a positive sentinel lymph node biopsy (SLNB) in breast cancer patients, often results in no additional positive nodes. Scoring systems have been published to aid in the prediction of nonsentinel node metastasis. Our purpose was to assess the validity of these scoring systems in our patient population. METHODS: For 39 consecutive patients who underwent cALND after a positive SLNB, scores were calculated using retrospective patient data for each of the three scoring systems used. Receiver operating characteristics (ROC) curves were drawn, and the areas under the curves were calculated to assess the discriminative power of each system. Univariate analysis was performed to assess the predictability of individual patient and tumor characteristics. RESULTS: Nonsentinel nodes were positive in 23 (59%) patients. The areas under the ROC curves were 0.63, 0.70, and 0.68, respectively. The proportion of sentinel nodes that were positive and the total number of sentinel nodes retrieved were the only individual predictors of nonsentinel node metastasis. CONCLUSIONS: Given the high incidence of retrieving no additional metastasis on cALND, individualized patient management according to risk is desirable. Scoring systems provide additional information regarding the likelihood of metastasis in nonsentinel nodes, but their predictability remains less than optimal. The use of scoring systems must be applied with caution until future studies provide a more accurate assessment of risk for patients with a positive SLNB.

Success of neoadjuvant chemotherapy in conversion of mastectomy to breast conservation surgery.

Neoadjuvant chemotherapy (NC) in patients with breast cancer results in high response rates and has been used with the purpose of reducing tumor size and achieving breast conservation (BC) in individuals who initially require mastectomy. Our objective is to determine the success of NC in achieving BC in women who initially were not candidates for BC. We conducted a cohort study of women with invasive breast cancer who required mastectomy but desired BC surgery. Outcomes measured were tumor response and rates of BC. Thirty-seven women had a mean age of 45 years. Mean tumor size was 51 mm, and 62 per cent were larger than 4 cm. Tumors were predominantly infiltrating ductal carcinoma (83.3%) and high grade (62.2%). Cyclophosphamide, doxorubicin, and 5-fluorouracil with or without taxotere were most commonly used (86%). Complete clinical and pathologic responses were seen in 32.4 per cent and 10.8 per cent of patients, respectively. BC was achieved in 56.7 per cent of cases. Only initial tumor size predicted tumor regression and success of BC (P = 0.014). Neither tumor histology nor biologic markers predicted tumor response. In conclusion, NC is an effective alternative in achieving tumor reduction and BC in selected patients who require mastectomy but desire BC surgery.

Axillary regional recurrence after sentinel lymph node biopsy for breast cancer.

The accuracy of sentinel lymph node biopsy (SLNB) staging in breast cancer has been demonstrated in studies comparing it with axillary dissection. There is a 5 per cent false-negative rate, but this does not always correlate with axillary recurrence. Our purpose was to determine the rate of axillary lymphatic recurrence in breast cancer patients who had a negative SLNB. We conducted a cohort study of breast cancer patients who underwent SLNB between 2001 and 2005. Only patients who had a negative SLNB were included. Patient demographics and tumor factors were reviewed. Outcomes measured were axillary and systemic recurrence and survival. Eighty-nine patients with a mean age of 54.4 +/- 9.9 years were included. Eighty-nine per cent of cases had infiltrating ductal carcinoma histology. Mean tumor size was 19 +/- 14 mm. Breast conservation surgery was done in 65 cases and mastectomy in 24. A mean of 2.3 +/- 2.4 SLN were found. After a median follow-up of 2.15 years, 1 (1%) patient developed a lymphatic recurrence in the axilla. SLNB provides accurate staging of breast cancer. Patients with negative SLNB do not require axillary dissection.

Percutaneous excisional biopsy of palpable breast masses under ultrasound visualization.

A palpable breast mass is a common reason for surgical consultation. Our goal was to determine whether ultrasound-guided vacuum-assisted core biopsy (US-VACB) is safe and effective in completely removing presumed benign palpable breast masses. We conducted a cohort study of 201 consecutive patients with presumed benign palpable masses who underwent removal with US-VACB. The main outcome measured was the successful removal of palpable masses. Palpable masses were successfully removed with US-VACB in 99% of cases; 2% were cancer and 7.5% were atypical ductal hyperplasia or phyllodes tumor. Two clinical recurrences representing a seroma were seen on follow-up. US-VACB is safe and effective in the initial diagnosis and management of presumed benign palpable breast masses. It provides the benefits of percutaneous biopsy and the palpable abnormality no longer remains.

Intraoperative injection of technetium-99m sulfur colloid is effective in the detection of sentinel lymph nodes in breast cancer.

BACKGROUND: Our objective was to determine if intraoperative injection of technetium-99m-labeled sulfur colloid is as effective as preoperative injection in the detection of sentinel lymph nodes (SLNs). METHODS: Two hundred consecutive patients with breast cancer underwent SLN biopsy examination. Radiocolloid was injected in the preoperative area (group A) or immediately after induction of anesthesia in the operating room (group B). RESULTS: The SLN detection rate was similar for groups A (96%) and B (100%; P = .2). Radioactive SLNs were detected in 95% of patients in group A and in 97% of patients in group B (P = .1). The mean number of SLNs harvested was 1.6 and 2.1 for groups A and B, respectively. There was no significant difference in positive SLNs between groups (P = .11). CONCLUSIONS: Intraoperative injection of sulfur colloid is highly effective in the detection of SLNs, avoiding patient discomfort and surgical schedule delays.