Eric C. Liao

Breast reconstruction following nipple-sparing mastectomy: predictors of complications, reconstruction outcomes, and 5-year trends.

Background: Nipple-sparing mastectomy is increasingly used for treatment and prevention of breast cancer. Few data exist on risk factors for complications and reconstruction outcomes. Methods: A single-institution retrospective review was performed between 2007 and 2012. Results: Two hundred eighty-five patients underwent 500 nipple-sparing mastectomy procedures for breast cancer (46 percent) or risk reduction (54 percent). The average body mass index was 24, and 6 percent were smokers. The mean follow-up was 2.17 years. Immediate breast reconstruction (reconstruction rate, 98.8 percent) was performed with direct-to-implant (59 percent), tissue expander/implant (38 percent), or autologous (2 percent) reconstruction. Acellular dermal matrix was used in 71 percent and mesh was used in 11 percent. Seventy-seven reconstructions had radiotherapy. Complications included infection (3.3 percent), skin necrosis (5.2 percent), nipple necrosis (4.4 percent), seroma (1.7 percent), hematoma (1.7 percent), and implant loss (1.9 percent). Positive predictors for total complications included smoking (OR, 3.3; 95 percent CI, 1.289 to 8.486) and periareolar incisions (OR, 3.63; 95 percent CI, 1.850 to 7.107). Increasing body mass index predicted skin necrosis (OR, 1.154; 95 percent CI, 1.036 to 1.286) and preoperative irradiation predicted nipple necrosis (OR, 4.86; 95 percent CI, 1.0197 to 23.169). An inframammary fold incision decreased complications (OR, 0.018; 95 percent CI, 0.0026 to 0.12089). Five-year trends showed increasing numbers of nipple-sparing mastectomy with immediate reconstruction and more single-stage versus two-stage reconstructions (p < 0.05). Conclusions: Nipple-sparing mastectomy reconstructions have a low number of complications. Smoking, body mass index, preoperative irradiation, and incision type were predictors of complications. CLINICAL QUESTION/LEVEL OF EVIDENCE: Risk, III.

Cell-specific mitotic defect and dyserythropoiesis associated with erythroid band 3 deficiency.

Most eukaryotic cell types use a common program to regulate the process of cell division. During mitosis, successful partitioning of the genetic material depends on spatially coordinated chromosome movement and cell cleavage. Here we characterize a zebrafish mutant, retsina (ret), that exhibits an erythroid-specific defect in cell division with marked dyserythropoiesis similar to human congenital dyserythropoietic anemia. Erythroblasts from ret fish show binuclearity and undergo apoptosis due to a failure in the completion of chromosome segregation and cytokinesis. Through positional cloning, we show that the ret mutation is in a gene (slc4a1) encoding the anion exchanger 1 (also called band 3 and AE1), an erythroid-specific cytoskeletal protein. We further show an association between deficiency in Slc4a1 and mitotic defects in the mouse. Rescue experiments in ret zebrafish embryos expressing transgenic slc4a1 with a variety of mutations show that the requirement for band 3 in normal erythroid mitosis is mediated through its protein 4.1R–binding domains. Our report establishes an evolutionarily conserved role for band 3 in erythroid-specific cell division and illustrates the concept of cell-specific adaptation for mitosis.

A zebrafish melanoma model reveals emergence of neural crest identity during melanoma initiation

Visualizing the beginnings of melanoma In cancer biology, a tumor begins from a single cell within a group of precancerous cells that share genetic mutations. Kaufman et al. used a zebrafish melanoma model to visualize cancer initiation (see the Perspective by Boumahdi and Blanpain). They used a fluorescent reporter that specifically lit up neural crest progenitors that are only present during embryogenesis or during adult melanoma tumor formation. The appearance of this tumor correlated with a set of gene regulatory elements, called super-enhancers, whose identification and manipulation may prove beneficial in detecting and preventing melanoma initiation. Science, this issue p. 10.1126/science.aad2197; see also p. 453 Melanocytes with oncogenic or tumor suppressor mutations revert to expressing the crestin gene early in melanoma formation. [Also see Perspective by Boumahdi and Blanpain] INTRODUCTION The “cancerized field” concept posits that cells in a given tissue sharing an oncogenic mutation are cancer-prone, yet only discreet clones within the field initiate tumors. Studying the process of cancer initiation has remained challenging because of (i) the rarity of these events, (ii) the difficulty of visiualizing initiating clones in living organisms, and (iii) the transient nature of a newly transformed clone emerging before it expands to form an early tumor. A more complete understanding of the molecular processes that regulate cancer initiation could provide important prognostic information about which precancerous lesions are most prone to becoming cancer and also implicate druggable molecular pathways that, when inhibited, may prevent the cancer from ever starting. RATIONALE The majority of benign nevi carry oncogenic BRAFV600E mutations and can be considered a cancerized field of melanocytes, but they only rarely convert to melanoma. In an effort to define events that initiate cancer, we used a melanoma model in the zebrafish in which the human BRAFV600E oncogene is driven by the melanocyte-specific mitfa promoter. When bred into a p53 mutant background, these fish develop melanoma tumors over the course of many months. The zebrafish crestin gene is expressed embryonically in neural crest progenitors (NCPs) and is specifically reexpressed only in melanoma tumors, making it an ideal candidate for tracking melanoma from initiation onward. RESULTS We developed a crestin:EGFP reporter that recapitulates the embryonic neural crest expression pattern of crestin and its expression in melanoma tumors. We show through live imaging of transgenic zebrafish crestin reporters that within a cancerized field (BRAFV600E-mutant; p53-deficient), a single melanocyte reactivates the NCP state, and this establishes that a fate change occurs at melanoma initiation in this model. Early crestin+ patches of cells expand and are transplantable in a manner consistent with their possessing tumorigenic activity, and they exhibit a gene expression pattern consistent with the NCP identity readout by the crestin reporter. The crestin element is regulated by NCP transcription factors, including sox10. Forced sox10 overexpression in melanocytes accelerated melanoma formation, whereas CRISPR/Cas9 targeting of sox10 delayed melanoma onset. We show activation of super-enhancers at NCP genes in both zebrafish and human melanomas, identifying an epigenetic mechanism for control of this NCP signature leading to melanoma. CONCLUSION This work using our zebrafish melanoma model and in vivo reporter of NCP identity allows us to see cancer from its birth as a single cell and shows the importance of NCP-state reemergence as a key event in melanoma initiation from a field of cancer-prone melanocytes. Thus, in addition to the typical fixed genetic alterations in oncogenes and tumor supressors that are required for cancer development, the reemergence of progenitor identity may be an additional rate-limiting step in the formation of melanoma. Preventing NCP reemergence in a field of cancer-prone melanocytes may thus prove therapeutically useful, and the association of NCP genes with super-enhancer regulatory elements implicates the associated druggable epigenetic machinery in this process. Neural crest reporter expression in melanoma. The crestin:EGFP transgene is specifically expressed in melanoma in BRAFV600E/p53 mutant melanoma-prone zebrafish. (Top) A single cell expressing crestin:EGFP expands into a small patch of cells over the course of 2 weeks, capturing the initiation of melanoma formation (bracket). (Bottom) A fully formed melanoma specifically expresses crestin:EGFP, whereas the rest of the fish remains EGFP-negative. The “cancerized field” concept posits that cancer-prone cells in a given tissue share an oncogenic mutation, but only discreet clones within the field initiate tumors. Most benign nevi carry oncogenic BRAFV600E mutations but rarely become melanoma. The zebrafish crestin gene is expressed embryonically in neural crest progenitors (NCPs) and specifically reexpressed in melanoma. Live imaging of transgenic zebrafish crestin reporters shows that within a cancerized field (BRAFV600E-mutant; p53-deficient), a single melanocyte reactivates the NCP state, revealing a fate change at melanoma initiation in this model. NCP transcription factors, including sox10, regulate crestin expression. Forced sox10 overexpression in melanocytes accelerated melanoma formation, which is consistent with activation of NCP genes and super-enhancers leading to melanoma. Our work highlights NCP state reemergence as a key event in melanoma initiation.

Infection following implant-based reconstruction in 1952 consecutive breast reconstructions: salvage rates and predictors of success.

Background: Few studies address salvage rates for infection in implant-based breast reconstruction. An understanding of success rates and clinical predictors of failure may help guide management. Method: A retrospective analysis of multisurgeon consecutive implant reconstructions from 2004 to 2010 was performed. Results: Immediate implant-based reconstructions (n = 1952) were performed in 1241 patients. Ninety-nine reconstruction patients (5.1 percent) were admitted for breast erythema and had a higher incidence of smoking (p = 0.007), chemotherapy (p = 0.007), radiation therapy (p = 0.001), and mastectomy skin necrosis (p < 0.0001). There was no difference in age, body mass index, or acellular dermal matrix usage. With intravenous antibiotics, 25 (25.3 percent) reconstruction patients cleared the infection, whereas 74 (74.7 percent) underwent attempted operative salvage (n = 18) or explantation (n = 56). Patients who failed to clear infection had a higher mean white blood cell count at admission (p < 0.0001). Of the attempted operative salvage group, 12 cleared the infection with immediate implant exchange and six eventually lost the implant. Patients who failed implant salvage were more likely to have methicillin-resistant Staphylococcus aureus (p = 0.004). The total explantation rate was 3.2 percent. Following explantation, 32 patients underwent attempted secondary tissue expander insertion. Twenty-six were successful and six had recurrent infection and implant loss. There were no differences in time interval to tissue expander insertion between successful and unsuccessful secondary operations. Conclusions: Salvage with intravenous antibiotics and implant exchange was successful in 37.3 percent of patients. Smoking, irradiation, chemotherapy, and mastectomy skin necrosis were predictors for developing infection. Patients with a higher white blood cell count at admission and methicillin-resistant S. aureus were more likely to fail implant salvage. There was no association with time interval to tissue expander insertion and secondary explantation.(Plast. Reconstr. Surg. 131: 1223, 2013.) CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, IV.

Breast reconstruction outcomes after nipple-sparing mastectomy and radiation therapy.

Background: Few studies in the literature examine outcomes of immediate breast reconstruction after mastectomy with nipple preservation and radiation therapy. Methods: Retrospective analysis of multisurgeon consecutive implant-based reconstructions after nipple-sparing mastectomy from June of 2007 to December of 2012 was conducted at a single institution. Results: Six hundred five immediate breast reconstructions were performed following nipple-sparing mastectomy, of which 88 were treated with radiation therapy. There was a trend toward more complications in patients with radiation (19.3 percent versus 12.8 percent; p = 0.099) associated with a higher rate of implant loss (6.8 percent versus 1.0 percent; p = 0.001). Preoperative radiotherapy had a higher risk of total complications (p = 0.04; OR, 2.225; 95 percent CI, 1.040 to 4.758) and postoperative radiotherapy had a higher risk of explantation (p = 0.015; OR, 5.634; 95 percent CI, 1.405 to 22.603). There were no significant differences in nipple removal secondary to malposition or positive oncologic margins in patients with radiation compared to those without radiation. Patients with radiation did have a higher incidence of secondary procedures for capsular contracture (12.5 percent versus 2.3 percent; p < 0.001) and fat grafting (13.6 percent versus 3.9 percent; p < 0.001). The total nipple retention rate in patients with radiation therapy was 90 percent (79 of 88), and the reconstruction failure rate was 8 percent. Conclusions: Nipple-sparing mastectomy and immediate reconstruction in patients who had or will receive radiation therapy is associated with a higher incidence of complications and operative revisions compared with patients without radiation. However, most patients have successful reconstructions with nipple retention and no recurrences. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, III.

Incidence of Hematoma Complication with Heparin Venous Thrombosis Prophylaxis after Tram Flap Breast Reconstruction

Background: Randomized controlled studies provide ample evidence that heparin is effective in reducing the risk of thromboembolic complications. Nevertheless, plastic surgeons are often reluctant to use heparin chemoprophylaxis for fear of postoperative bleeding. The authors investigated whether heparin chemoprophylaxis was associated with postoperative hematoma that required evacuation in patients who underwent transverse rectus abdominis myocutaneous (TRAM) flap breast reconstruction. Methods: A multicenter retrospective review of consecutive TRAM flap cases identified 679 patients, 392 in the heparin-treated group and 287 in the control group. The post hoc sample sizes were adequate to detect a 5 percent difference in hematoma rate with 89 percent power at an alpha level of 5 percent (p < 0.05). Outcome measures of reoperative hematoma, deep vein thrombosis, and pulmonary embolism were recorded. Results: Reoperative hematoma occurred in 0.5 percent of patients in the heparin-treated group and 1.0 percent of patients in the control group; this difference was not statistically significant (p = 0.66). Thromboembolic events were detected at a low rate (0.8 percent in the heparin-treated group versus 1.4 percent in the untreated group; p = 0.46). Conclusions: The use of heparin for venous thrombotic prophylaxis did not increase the risk of reoperative hematoma after breast reconstruction with abdominal tissue. The authors propose a risk assessment that balances a statistical hematoma rate of 0.5 to 5 percent (clinically observed rate, 0.5 percent) with use of heparin prophylaxis against a rare (clinically observed rate, 1.4 percent) but morbid occurrence of thromboembolic complications when chemoprophylaxis is omitted.

Haploinsufficiency of KDM6A is associated with severe psychomotor retardation, global growth restriction, seizures and cleft palate

We describe a female subject (DGAP100) with a 46,X,t(X;5)(p11.3;q35.3)inv(5)(q35.3q35.1)dn, severe psychomotor retardation with hypotonia, global postnatal growth restriction, microcephaly, globally reduced cerebral volume, seizures, facial dysmorphia and cleft palate. Fluorescence in situ hybridization and whole-genome sequencing demonstrated that the X chromosome breakpoint disrupts KDM6A in the second intron. No genes were directly disrupted on chromosome 5. KDM6A is a histone 3 lysine 27 demethylase and a histone 3 lysine 4 methyltransferase. Expression of KDM6A is significantly reduced in DGAP100 lymphoblastoid cells compared to control samples. We identified nine additional cases with neurodevelopmental delay and various other features consistent with the DGAP100 phenotype with copy number variation encompassing KDM6A from microarray databases. We evaluated haploinsufficiency of kdm6a in a zebrafish model. kdm6a is expressed in the pharyngeal arches and ethmoid plate of the developing zebrafish, while a kdm6a morpholino knockdown exhibited craniofacial defects. We conclude KDM6A dosage regulation is associated with severe and diverse structural defects and developmental abnormalities.

Distinct requirements for wnt9a and irf6 in extension and integration mechanisms during zebrafish palate morphogenesis

Development of the palate in vertebrates involves cranial neural crest migration, convergence of facial prominences and extension of the cartilaginous framework. Dysregulation of palatogenesis results in orofacial clefts, which represent the most common structural birth defects. Detailed analysis of zebrafish palatogenesis revealed distinct mechanisms of palatal morphogenesis: extension, proliferation and integration. We show that wnt9a is required for palatal extension, wherein the chondrocytes form a proliferative front, undergo morphological change and intercalate to form the ethmoid plate. Meanwhile, irf6 is required specifically for integration of facial prominences along a V-shaped seam. This work presents a mechanistic analysis of palate morphogenesis in a clinically relevant context.

Zebrafish wnt9a is expressed in pharyngeal ectoderm and is required for palate and lower jaw development

Wnt activity is critical in craniofacial morphogenesis. Dysregulation of Wnt/β-catenin signaling results in significant alterations in the facial form, and has been implicated in cleft palate phenotypes in mouse and man. In zebrafish, we show that wnt9a is expressed in the pharyngeal arch, oropharyngeal epithelium that circumscribes the ethmoid plate, and ectodermal cells superficial to the lower jaw structures. Alcian blue staining of morpholino-mediated knockdown of wnt9a results in loss of the ethmoid plate, absence of lateral and posterior parachordals, and significant abrogation of the lower jaw structures. Analysis of cranial neural crest cells in the sox10:eGFP transgenic demonstrates that the wnt9a is required early during pharyngeal development, and confirms that the absence of Alcian blue staining is due to absence of neural crest derived chondrocytes. Molecular analysis of genes regulating cranial neural crest migration and chondrogenic differentiation suggest that wnt9a is dispensable for early cranial neural crest migration, but is required for chondrogenic development of major craniofacial structures. Taken together, these data corroborate the central role for Wnt signaling in vertebrate craniofacial development, and reveal that wnt9a provides the signal from the pharyngeal epithelium to support craniofacial chondrogenic morphogenesis in zebrafish.

Assessment of Patient Factors, Surgeons, and Surgeon Teams in Immediate Implant-based Breast Reconstruction Outcomes

Background: Outcome studies of immediate implant-based breast reconstruction have focused largely on patient factors, whereas the relative impact of the surgeon as a contributing variable is not known. As the procedure requires collaboration of both a surgical oncologist and a plastic surgeon, the effect of the surgeon team interaction can have a significant impact on outcome. This study examines outcomes in implant-based breast reconstruction and the association with patient characteristics, surgeon, and surgeon team familiarity. Methods: A retrospective review of 3142 consecutive implant-based breast reconstruction mastectomy procedures at one institution was performed. Infection and skin necrosis rates were measured. Predictors of outcomes were identified by unadjusted logistic regression followed by multivariate logistic regression. Surgeon teams were grouped according to number of cases performed together. Results: Patient characteristics remain the most important predictors for outcomes in implant-based breast reconstruction, with odds ratios above those of surgeon variables. The authors observed significant differences in the rate of skin necrosis between surgical oncologists with an approximately two-fold difference between surgeons with the highest and lowest rates. Surgeon teams that worked together on fewer than 150 procedures had higher rates of infection. Conclusions: Patient characteristics are the most important predictors for surgical outcomes in implant-based breast reconstruction, but surgeons and surgeon teams are also important variables. High-volume surgeon teams achieve lower rates of infection. This study highlights the need to examine modifiable risk factors associated with optimum implant-based breast reconstruction outcomes, which include patient and provider characteristics and the surgical team treating the patient. CLINICAL QUESTION/LEVEL OF EVIDENCE: Risk, III.