The Trimeric Autotransporter Adhesin EmaA and Infective Endocarditis.

Infective endocarditis (IE), a disease of the endocardial surface of the heart, is usually of bacterial origin and disproportionally affects individuals with underlying structural heart disease. Although IE is typically associated with Gram-positive bacteria, a minority of cases are caused by a group of Gram-negative species referred to as the HACEK group. These species, classically associated with the oral cavity, consist of bacteria from the genera Haemophilus (excluding Haemophilus influenzae), Aggregatibacter, Cardiobacterium, Eikenella, and Kingella. Aggregatibacter actinomycetemcomitans, a bacterium of the Pasteurellaceae family, is classically associated with Aggressive Periodontitis and is also concomitant with the chronic form of the disease. Bacterial colonization of the oral cavity serves as a reservoir for infection at distal body sites via hematological spreading. A. actinomycetemcomitans adheres to and causes disease at multiple physiologic niches using a diverse array of bacterial cell surface structures, which include both fimbrial and nonfimbrial adhesins. The nonfimbrial adhesin EmaA (extracellular matrix binding protein adhesin A), which displays sequence heterogeneity dependent on the serotype of the bacterium, has been identified as a virulence determinant in the initiation of IE. In this chapter, we will discuss the known biochemical, molecular, and structural aspects of this protein, including its interactions with extracellular matrix components and how this multifunctional adhesin may contribute to the pathogenicity of A. actinomycetemcomitans.

Society of Surgical Oncology Breast Disease Site Working Group Statement on Contralateral Mastectomy: Indications, Outcomes, and Risks.

Rates of contralateral mastectomy (CM) among patients with unilateral breast cancer have been increasing in the United States. In this Society of Surgical Oncology position statement, we review the literature addressing the indications, risks, and benefits of CM since the society's 2017 statement. We held a virtual meeting to outline key topics and then conducted a literature search using PubMed to identify relevant articles. We reviewed the articles and made recommendations based on group consensus. Patients consider CM for many reasons, including concerns regarding the risk of contralateral breast cancer (CBC), desire for improved cosmesis and symmetry, and preferences to avoid ongoing screening, whereas surgeons primarily consider CBC risk when making a recommendation for CM. For patients with a high risk of CBC, CM reduces the risk of new breast cancer, however it is not known to convey an overall survival benefit. Studies evaluating patient satisfaction with CM and reconstruction have yielded mixed results. Imaging with mammography within 12 months before CM is recommended, but routine preoperative breast magnetic resonance imaging is not; there is also no evidence to support routine postmastectomy imaging surveillance. Because the likelihood of identifying an occult malignancy during CM is low, routine sentinel lymph node surgery is not recommended. Data on the rates of postoperative complications are conflicting, and such complications may not be directly related to CM. Adjuvant therapy delays due to complications have not been reported. Surgeons can reduce CM rates by encouraging shared decision making and informed discussions incorporating patient preferences.

Dual function of the O-antigen WaaL ligase of Aggregatibacter actinomycetemcomitans.

Protein glycosylation is critical to the quaternary structure and collagen-binding activity of the extracellular matrix protein adhesin A (EmaA) associated with Aggregatibacter actinomycetemcomitans. The glycosylation of this large, trimeric autotransporter adhesin is postulated to be mediated by WaaL, an enzyme with the canonical function to ligate the O-polysaccharide (O-PS) antigen with a terminal sugar of the lipid A-core oligosaccharide of lipopolysaccharide (LPS). In this study, we have determined that the Escherichia coli waaL ortholog (rflA) does not restore collagen binding of a waaL mutant strain of A. actinomycetemcomitans but does restore O-PS ligase activity following transformation of a plasmid expressing waaL. Therefore, a heterologous E. coli expression system was developed constituted of two independently replicating plasmids expressing either waaL or emaA of A. actinomycetemcomitans to directly demonstrate the necessity of ligase activity for EmaA collagen binding. Proper expression of the protein encoded by each plasmid was characterized, and the individually transformed strains did not promote collagen binding. However, coexpression of the two plasmids resulted in a strain with a significant increase in collagen binding activity and a change in the biochemical properties of the protein. These results provide additional data supporting the novel hypothesis that the WaaL ligase of A. actinomycetemcomitans shares a dual role as a ligase in LPS biosynthesis and is required for collagen binding activity of EmaA.

Regulation of adhesin synthesis in Aggregatibacter actinomycetemcomitans.

Aggregatibacter actinomycetemcomitans is a gram-negative bacterium associated with periodontal disease and a variety of disseminated extra-oral infections. Tissue colonization is mediated by fimbriae and non-fimbriae adhesins resulting in the formation of a sessile bacterial community or biofilm, which confers enhanced resistance to antibiotics and mechanical removal. The environmental changes experienced by A. actinomycetemcomitans during infection are detected and processed by undefined signaling pathways that alter gene expression. In this study, we have characterized the promoter region of the extracellular matrix protein adhesin A (EmaA), which is an important surface adhesin in biofilm biogenesis and disease initiation using a series of deletion constructs consisting of the emaA intergenic region and a promotor-less lacZ sequence. Two regions of the promoter sequence were found to regulate gene transcription and in silico analysis indicated the presence of multiple transcriptional regulatory binding sequences. Analysis of four regulatory elements, CpxR, ArcA, OxyR, and DeoR, was undertaken in this study. Inactivation of arcA, the regulator moiety of the ArcAB two-component signaling pathway involved in redox homeostasis, resulted in a decrease in EmaA synthesis and biofilm formation. Analysis of the promoter sequences of other adhesins identified binding sequences for the same regulatory proteins, which suggests that these proteins are involved in the coordinate regulation of adhesins required for colonization and pathogenesis.

The serotype a-EmaA adhesin of Aggregatibacter actinomycetemcomitans does not require O-PS synthesis for collagen binding activity.

Aggregatibacter actinomycetemcomitans, a causative agent of periodontitis and non-oral diseases, synthesizes a trimeric extracellular matrix protein adhesin A (EmaA) that mediates collagen binding and biofilm formation. EmaA is found as two molecular forms, which correlate with the serotype of the bacterium. The canonical protein (b-EmaA), associated with serotypes b and c, has a monomeric molecular mass of 202 kDa. The collagen binding activity of b-EmaA is dependent on the presence of O-polysaccharide (O-PS), whereas biofilm activity is independent of O-PS synthesis. The EmaA associated with serotype a strains (a-EmaA) has a monomeric molecular mass of 173 kDa and differs in the amino acid sequence of the functional domain of the protein. In this study, a-emaA was confirmed to encode a protein that forms antenna-like appendages on the surface of the bacterium, which were found to be important for both collagen binding and biofilm formation. In an O-PS-deficient talose biosynthetic (tld) mutant strain, the electrophoretic mobility of the a-EmaA monomers was altered and the amount of membrane-associated EmaA was decreased when compared to the parent strain. The mass of biofilm formed remained unchanged. Interestingly, the collagen binding activity of the mutant strain was similar to the activity associated with the parent strain, which differs from that observed with the canonical b-EmaA isoform. These data suggest that the properties of the a-EmaA isoform are like those of b-EmaA, with the exception that collagen binding activity is independent of the presence or absence of the O-PS.

The Role of Chronic Inflammation in the Development of Breast Cancer.

Chronic inflammation contributes to the malignant transformation of several malignancies and is an important component of breast cancer. The role of chronic inflammation in the initiation and development of breast cancer from normal breast tissue, however, is unclear and needs to be clarified. A review of the literature was conducted to define the chronic inflammatory processes in normal breast tissue at risk for breast cancer and in breast cancer, including the role of lymphocyte and macrophage infiltrates, chronic active adipocytes and fibroblasts, and processes that may promote chronic inflammation including the microbiome and factors related to genomic abnormalities and cellular injury. The findings indicate that in healthy normal breast tissue there is systemic evidence to suggest inflammatory changes are present and associated with breast cancer risk, and adipocytes and crown-like structures in normal breast tissue may be associated with chronic inflammatory changes. The microbiome, genomic abnormalities, and cellular changes are present in healthy normal breast tissue, with the potential to elicit inflammatory changes, while infiltrating lymphocytes are uncommon in these tissues. Chronic inflammatory changes occur prominently in breast cancer tissues, with important contributions from tumor-infiltrating lymphocytes and tumor-associated macrophages, cancer-associated adipocytes and crown-like structures, and cancer-associated fibroblasts, while the microbiome and DNA damage may serve to promote inflammatory events. Together, these findings suggest that chronic inflammation may play a role in influencing the initiation, development and conduct of breast cancer, although several chronic inflammatory processes in breast tissue may occur later in breast carcinogenesis.

Impact of Prior Treatment on the Efficacy of Adoptive Transfer of Tumor-Infiltrating Lymphocytes in Patients with Metastatic Melanoma.

PURPOSE: Adoptive cell transfer (ACT) of autologous tumor-infiltrating lymphocytes (TIL) can mediate durable responses in patients with metastatic melanoma. This retrospective analysis provides long-term follow-up and describes the effect of prior therapy on outcomes after ACT-TIL. PATIENTS AND METHODS: Patients with metastatic melanoma underwent surgical resection of a tumor for generation of TILs and were treated with a lymphodepleting preparative regimen followed by adoptive transfer of TILs and intravenous IL2. Clinical characteristics of enrolled patients and treatment characteristics of TIL infusion products over two decades of ACT were analyzed to identify predictors of objective response. RESULTS: Adoptive transfer of TILs mediated an objective response rate of 56% (108/192) and median melanoma-specific survival of 28.5 months in patients naïve to anti-programmed cell death-1 (PD-1) therapy compared with 24% (8/34) and 11.6 months in patients refractory to anti-PD-1 (aPD-1). Among patients with BRAF V600E/K-mutated disease, prior treatment with targeted molecular therapy was also associated with a decreased response rate (21% vs. 60%) and decreased survival (9.3 vs. 50.7 months) when compared with those patients naïve to targeted therapy. With a median potential follow-up of 89 months, 46 of 48 complete responders in the aPD-1-naïve cohort have ongoing responses after a single treatment and 10-year melanoma-specific survival of 96%. CONCLUSIONS: Patients previously treated with PD-1 or MAPK inhibition are significantly less likely to develop durable objective responses to ACT-TIL. While ACT-TIL is currently being investigated for treatment-refractory patients, it should also be considered as an initial treatment option for eligible patients with metastatic melanoma. See related commentary by Sznol, p. 5156.

Contribution of adhesion proteins to Aggregatibacter actinomycetemcomitans biofilm formation.

Aggregatibacter actinomycetemcomitans is a Gram-negative bacterium associated with periodontal disease and multiple disseminated extra-oral infections. Colonization of these distinct physiological niches is contingent on the expression of specific surface proteins during the initiation of developing biofilms. In this investigation, we studied fimbriae and three well-characterized nonfimbrial surface proteins (EmaA, Aae, and ApiA/Omp100) for their contribution to biofilm formation. Mutations of these proteins in multiple strains covering four different serotypes demonstrated variance in biofilm development that was strain dependent but independent of serotype. In a fimbriated background, only inactivation of emaA impacted biofilm mass. In contrast, inactivation of emaA and/or aae affected biofilm formation in nonfimbriated A. actinomycetemcomitans strains, whereas inactivation of apiA/omp100 had little effect on biofilm formation. When these genes were expressed individually in Escherichia coli, all transformed strains demonstrated an increase in biofilm mass compared to the parent strain. The strain expressing emaA generated the greatest mass of biofilm, whereas the strains expressing either aae or apiA/omp100 were greatly reduced and similar in mass. These data suggest a redundancy in function of these nonfimbrial adhesins, which is dependent on the genetic background of the strain investigated.

The Role of Immune Cells in Breast Tissue and Immunotherapy for the Treatment of Breast Cancer.

Immune cells are present in normal breast tissue and in breast carcinoma. The nature and distribution of the immune cell subtypes in these tissues are reviewed to promote a better understanding of their important role in breast cancer prevention and treatment. We conducted a review of the literature to define the type, location, distribution, and role of immune cells in normal breast tissue and in in situ and invasive breast cancer. Immune cells in normal breast tissue are located predominantly within the epithelial component in breast ductal lobules. Immune cell subtypes representing innate immunity (NK, CD68+, and CD11c+ cells) and adaptive immunity (most commonly CD8+, but CD4+ and CD20+ as well) are present; CD8+ cells are the most common subtype and are primarily effector memory cells. Immune cells may recognize neoantigens and endogenous and exogenous ligands and may serve in chronic inflammation and immunosurveillance. Progression to breast cancer is characterized by increased immune cell infiltrates in tumor parenchyma and stroma, including CD4+ and CD8+ granzyme B+ cytotoxic T cells, B cells, macrophages and dendritic cells. Tumor-infiltrating lymphocytes in breast cancer may serve as prognostic indicators for response to chemotherapy and for survival. Experimental strategies of adoptive transfer of breast tumor-infiltrating lymphocyte may allow regression of metastatic breast cancer and encourage development of innovative T-cell strategies for the immunotherapy of breast cancer. In conclusion, immune cells in breast tissues play an important role throughout breast carcinogenesis. An understanding of these roles has important implications for the prevention and the treatment of breast cancer.

Characteristics of Breast Ducts in Normal-Risk and High-risk Women and Their Relationship to Ductal Cytologic Atypia.

Breast ductal cytologic atypia is an important risk factor for sporadic breast cancer. Characterization of the associated normal breast tissue is needed to develop additional methods of risk assessment and new targets for breast cancer prevention. We conducted a prospective clinical trial evaluating women at normal-risk or at high-risk for sporadic breast cancer. Breast ductal cells were collected and studied cytologically and by gene expression profiling, and breast ductal architectural changes were studied by breast ductal endoscopy (BDE) and breast MRI. One hundred and forty subjects were studied, 70 at high risk (RR, 2.0-4.6) and 70 at normal risk. Cytologic atypia was present in 22.9% of high-risk and 25.7% of normal-risk subjects. Ductal endoscopy was performed in 89 subjects and revealed benign intraductal abnormalities, primarily intraductal fibrous webbing suggesting chronic inflammation, in 40.4% of high-risk and 5.4% of normal-risk subjects, respectively (P 2 = 0.0002). Two high-risk subjects with atypia and no normal-risk subjects with atypia developed invasive breast cancer. Gene expression profiling of ductal cells showed comparable gene expression profiles without enriched expression of previously defined oncogenic signatures in subjects with cellular atypia compared with those without atypia, and in high-risk subjects compared with normal-risk subjects (FDR > 0.5). Cytologic ductal atypia in normal-risk subjects does not appear to be of clinical significance. Atypia in women at high risk may be associated with benign and malignant breast ductal abnormalities; these characteristics of high-risk ductal cells may not be reflected in gene expression profiles.

Twenty-five-Year Follow-up of a Prospective Randomized Trial Comparing Preoperative Versus Postoperative FLAC/Granulocyte Colony-Stimulating Factor Chemotherapy for Stage II Breast Cancer.

BACKGROUND: Preoperative chemotherapy is important in the management of women with breast cancer, with the ability to downstage the breast primary tumor and axillary lymph nodes. Long-term studies are needed to identify late toxicities, recurrence patterns, and equivalency with postoperative chemotherapy for recurrence-free survival (RFS) and overall survival (OS). PATIENTS AND METHODS: We conducted a single-institution prospective randomized control trial comparing preoperative or postoperative fluorouracil, leucovorin calcium, doxorubicin, and cyclophosphamides/granulocyte colony-stimulating factor chemotherapy for women with untreated clinical stage II (T1N1, T2N0, and T2N1) breast cancer. Long-term follow-up was conducted to define toxicities, recurrence patterns and RFS and OS. RESULTS: Fifty-three women with clinical stage II breast cancer were randomized, 26 patients to receive preoperative chemotherapy and 27 to receive postoperative chemotherapy. Long-term follow-up, with a median of 25.3 years, was obtained. Local or systemic recurrence occurred in 8 women in the preoperative group and in 10 women in the postoperative group, and recurrences were predominantly within 10 years of treatment. Late toxicities included local upper extremity paresthesia's, upper extremity edema and congestive heart failure in 1 patient each. Analysis revealed no difference in RFS (20-year RFS probabilities; preoperative: 61.3%, postoperative: 54.7%, P=0.42), or in OS between the 2 treatment groups (20-year probabilities, preoperative: 64.6%, postoperative: 62.2%, P=0.44). Twenty-five of 53 patients (47%) were alive and without disease at this follow-up. CONCLUSION: Twenty-five-year follow-up for this prospective randomized trial confirms the equivalency of preoperative versus postoperative chemotherapy with fluorouracil, leucovorin calcium, doxorubicin, and cyclophosphamides/granulocyte colony-stimulating factor for stage II breast cancer for both RFS and OS.

A Nonfimbrial Adhesin of Aggregatibacter actinomycetemcomitans Mediates Biofilm Biogenesis.

Periodontitis is an inflammatory disease caused by polymicrobial biofilms. The periodontal pathogen Aggregatibacter actinomycetemcomitans displays two proteinaceous surface structures, the fimbriae and the nonfimbrial extracellular matrix binding protein A (EmaA), as observed by electron microscopy. Fimbriae participate in biofilm biogenesis and the EmaA adhesins mediate collagen binding. However, in the absence of fimbriae, A. actinomycetemcomitans still retains the potential to form robust biofilms, suggesting that other surface macromolecules participate in biofilm development. Here, isogenic mutant strains lacking EmaA structures, but still expressing fimbriae, were observed to have reduced biofilm potential. In strains lacking both EmaA and fimbriae, biofilm mass was reduced by 80%. EmaA enhanced biofilm formation in different strains, independent of the fimbriation state or serotype. Confocal microscopy revealed differences in cell density within microcolonies between the EmaA positive and mutant strains. EmaA-mediated biofilm formation was found to be independent of the glycosylation state and the precise three-dimensional conformation of the protein, and thus this function is uncorrelated with collagen binding activity. The data suggest that EmaA is a multifunctional adhesin that utilizes different mechanisms to enhance bacterial binding to collagen and to enhance biofilm formation, both of which are important for A. actinomycetemcomitans colonization and subsequent infection.

T-cell Responses to TP53 "Hotspot" Mutations and Unique Neoantigens Expressed by Human Ovarian Cancers.

Purpose: This was a study prospectively evaluating intratumoral T-cell responses to autologous somatic mutated neoepitopes expressed by human metastatic ovarian cancers.Patients and Methods: Tumor-infiltrating lymphocytes (TIL) were expanded from resected ovarian cancer metastases, which were analyzed by whole-exome and transcriptome sequencing to identify autologous somatic mutations. All mutated neoepitopes, independent of prediction algorithms, were expressed in autologous antigen-presenting cells and then cocultured with TIL fragment cultures. Secretion of IFNγ or upregulation of 41BB indicated a T-cell response.Results: Seven women with metastatic ovarian cancer were evaluated, and 5 patients had clear, dominant T-cell responses to mutated neoantigens, which were corroborated by comparison with the wild-type sequence, identification of the minimal epitope, human leukocyte antigen (HLA) restriction element(s), and neoantigen-specific T-cell receptor(s). Mutated neoantigens were restricted by HLA-B, -C, -DP, -DQ, and/or -DR alleles and appeared to principally arise from random, somatic mutations unique to each patient. We established that TP53 "hotspot" mutations (c.659A>G; p.Y220C and c.733G>A; p.G245S) expressed by two different patients' tumors were both immunogenic in the context of HLA-DRB3*02:02.Conclusions: Mutation-reactive T cells infiltrated ovarian cancer metastases at sufficient frequencies to warrant their investigation as adoptive cell therapy. In addition, transfer of TP53 "hotspot" mutation-reactive T-cell receptors into peripheral blood T cells could be evaluated as a gene therapy for a diverse range of tumor histologies. Clin Cancer Res; 24(22); 5562-73. ©2018 AACR See related commentary by McNeish, p. 5493.

Molecular profile of atypical hyperplasia of the breast.

PURPOSE: Atypical ductal and atypical lobular hyperplasia (AH) of the breast are important proliferative lesions which are associated with a significantly increased risk for breast cancer. The breast cancer which develops in association with AH may occur synchronously, representing local progression, or metachronously at a later date in either the ipsilateral or contralateral breast. These high-risk characteristics of AH suggest they contain significant genomic changes. METHODS: To define the genomic changes in AH, a comprehensive review of the literature was conducted to identify the numerical chromosomal and structural chromosomal changes, DNA methylation, and gene expression abnormalities in atypical ductal and atypical lobular hyperplasia. RESULTS: AHs are characterized by advanced genomic changes including aneuploidy, loss of heterozygosity, gross chromosomal rearrangements such as amplifications and large-scale deletions, DNA methylation of tumor suppressor and other genes, and gene expression differences between AH and surrounding normal breast tissue including significant estrogen receptor expression. Many of these changes are shared by an associated synchronous breast cancer, consistent with an important precursor role for AH. At the same time, many of the genomic changes of AHs are also shared by common sporadic breast cancer, consistent with a high risk for future development of metachronous breast cancer. CONCLUSIONS: This molecular profile should help clarify the genomic characteristics and malignant predisposition of AH, and aid in the identification of new targets for the prevention of breast cancer.

Treatment of metastatic uveal melanoma with adoptive transfer of tumour-infiltrating lymphocytes: a single-centre, two-stage, single-arm, phase 2 study.

BACKGROUND: Uveal melanoma is a rare tumour with no established treatments once metastases develop. Although a variety of immune-based therapies have shown efficacy in metastatic cutaneous melanoma, their use in ocular variants has been disappointing. Recently, adoptive T-cell therapy has shown salvage responses in multiple refractory solid tumours. Thus, we sought to determine if adoptive transfer of autologous tumour-infiltrating lymphocytes (TILs) could mediate regression of metastatic uveal melanoma. METHODS: In this ongoing single-centre, two-stage, phase 2, single-arm trial, patients (aged ≥16 years) with histologically confirmed metastatic ocular melanoma were enrolled. Key eligibility criteria were an Eastern Cooperative Oncology Group performance status of 0 or 1, progressive metastatic disease, and adequate haematological, renal, and hepatic function. Metastasectomies were done to procure tumour tissue to generate autologous TIL cultures, which then underwent large scale ex-vivo expansion. Patients were treated with lymphodepleting conditioning chemotherapy (intravenous cyclophosphamide [60 mg/kg] daily for 2 days followed by fludarabine [25 mg/m2] daily for 5 days, followed by a single intravenous infusion of autologous TILs and high-dose interleukin-2 [720 000 IU/kg] every 8 h). The primary endpoint was objective tumour response in evaluable patients per protocol using Response to Evaluation Criteria in Solid Tumors, version 1.0. An interim analysis of this trial is reported here. The trial is registered at ClinicalTrials.gov, number NCT01814046. FINDINGS: From the completed first stage and ongoing expansion stage of this trial, a total of 21 consecutive patients with metastatic uveal melanoma were enrolled between June 7, 2013, and Sept 9, 2016, and received TIL therapy. Seven (35%, 95% CI 16-59) of 20 evaluable patients had objective tumour regression. Among the responders, six patients achieved a partial response, two of which are ongoing and have not reached maximum response. One patient achieved complete response of numerous hepatic metastases, currently ongoing at 21 months post therapy. Three of the responders were refractory to previous immune checkpoint blockade. Common grade 3 or worse toxic effects were related to the lymphodepleting chemotherapy regimen and included lymphopenia, neutropenia, and thrombocytopenia (21 [100%] patients for each toxicity); anaemia (14 [67%] patients); and infection (six [29%] patients). There was one treatment-related death secondary to sepsis-induced multiorgan failure. INTERPRETATION: To our knowledge, this is the first report describing adoptive transfer of autologous TILs to mediate objective tumour regression in patients with metastatic uveal melanoma. These initial results challenge the belief that metastatic uveal melanoma is immunotherapy resistant and support the further investigation of immune-based therapies for this cancer. Refinement of this T-cell therapy is crucial to improve the frequency of clinical responses and the general applicability of this treatment modality. FUNDING: Intramural Research Program of the National Institutes of Health, National Cancer Institute, Center for Cancer Research.

Genomic Changes in Normal Breast Tissue in Women at Normal Risk or at High Risk for Breast Cancer.

Sporadic breast cancer develops through the accumulation of molecular abnormalities in normal breast tissue, resulting from exposure to estrogens and other carcinogens beginning at adolescence and continuing throughout life. These molecular changes may take a variety of forms, including numerical and structural chromosomal abnormalities, epigenetic changes, and gene expression alterations. To characterize these abnormalities, a review of the literature has been conducted to define the molecular changes in each of the above major genomic categories in normal breast tissue considered to be either at normal risk or at high risk for sporadic breast cancer. This review indicates that normal risk breast tissues (such as reduction mammoplasty) contain evidence of early breast carcinogenesis including loss of heterozygosity, DNA methylation of tumor suppressor and other genes, and telomere shortening. In normal tissues at high risk for breast cancer (such as normal breast tissue adjacent to breast cancer or the contralateral breast), these changes persist, and are increased and accompanied by aneuploidy, increased genomic instability, a wide range of gene expression differences, development of large cancerized fields, and increased proliferation. These changes are consistent with early and long-standing exposure to carcinogens, especially estrogens. A model for the breast carcinogenic pathway in normal risk and high-risk breast tissues is proposed. These findings should clarify our understanding of breast carcinogenesis in normal breast tissue and promote development of improved methods for risk assessment and breast cancer prevention in women.

Randomized, Prospective Evaluation Comparing Intensity of Lymphodepletion Before Adoptive Transfer of Tumor-Infiltrating Lymphocytes for Patients With Metastatic Melanoma.

PURPOSE: Adoptive cell transfer, the infusion of large numbers of activated autologous lymphocytes, can mediate objective tumor regression in a majority of patients with metastatic melanoma (52 of 93; 56%). Addition and intensification of total body irradiation (TBI) to the preparative lymphodepleting chemotherapy regimen in sequential trials improved objective partial and complete response (CR) rates. Here, we evaluated the importance of adding TBI to the adoptive transfer of tumor-infiltrating lymphocytes (TIL) in a randomized fashion. PATIENTS AND METHODS: A total of 101 patients with metastatic melanoma, including 76 patients with M1c disease, were randomly assigned to receive nonmyeloablative chemotherapy with or without 1,200 cGy TBI before transfer of tumor-infiltrating lymphcytes. Primary end points were CR rate (as defined by Response Evaluation Criteria in Solid Tumors v1.0) and overall survival (OS). Clinical and laboratory data were analyzed for correlates of response. RESULTS: CR rates were 24% in both groups (12 of 50 v 12 of 51), and OS was also similar (median OS, 38.2 v 36.6 months; hazard ratio, 1.11; 95% CI, 0.65 to 1.91; P = .71). Thrombotic microangiopathy was an adverse event unique to the TBI arm and occurred in 13 of 48 treated patients. With a median potential follow-up of 40.9 months, only one of 24 patients who achieved a CR recurred. CONCLUSION: Adoptive cell transfer can mediate durable complete regressions in 24% of patients with metastatic melanoma, with median survival > 3 years. Results were similar using chemotherapy preparative regimens with or without addition of TBI.

An Improved Breast Epithelial Sampling Method for Molecular Profiling and Biomarker Analysis in Women at Risk for Breast Cancer.

BACKGROUND: There is a strong need to define the molecular changes in normal at-risk breast epithelium to identify biomarkers and new targets for breast cancer prevention and to develop a molecular signature for risk assessment. Improved methods of breast epithelial sampling are needed to promote whole-genome molecular profiling, increase ductal epithelial cell yield, and reduce sample cell heterogeneity. METHODS: We developed an improved method of breast ductal sampling with ductal lavage through a 22-gauge catheter and collection of ductal samples with a microaspirator. Women at normal risk or increased risk for breast cancer were studied. Ductal epithelial samples were analyzed for cytopathologic changes, cellular yield, epithelial cell purity, quality and quantity of DNA and RNA, and use in multiple downstream molecular applications. RESULTS: We studied 50 subjects, including 40 subjects at normal risk for breast cancer and 37 subjects with non-nipple aspirate fluid-yielding ducts. This method provided multiple 1.0 mL samples of high ductal epithelial cell content (median ≥8 samples per subject of ≥5,000 cells per sample) with 80%-100% epithelial cell purity. Extraction of a single intact ductal sample (fluid and cells) or the separate frozen cellular component provided DNA and RNA for multiple downstream studies, including quantitative reverse transcription- polymerase chain reaction (PCR) for microRNA, quantitative PCR for the human telomerase reverse transcriptase gene, whole-genome DNA amplification, and array comparative genomic hybridization analysis. CONCLUSION: An improved breast epithelial sampling method has been developed, which should significantly expand the acquisition and biomarker analysis of breast ductal epithelium in women at risk for breast cancer.

Commentary on the Canadian National Breast Screening study.

In the setting of the 25-year follow-up of the Canadian National Breast Screening Study, the Society of Surgical Oncology continues to endorse mammographic screening for women beginning at 40 years of age, while acknowledging that mammography has both risks and benefits. Further investigation is warranted to develop better screening methods and to determine optimal screening schedules for women based on their risk of future breast cancer and their imaging characteristics.

Appendicitis secondary to metastatic melanoma: review of the National Institutes of Health experience.

IMPORTANCE: Malignant melanoma has an unusual propensity to metastasize to the small bowel; however, malignant melanoma with metastatic spread to the appendix presenting as acute appendicitis has rarely been reported. We describe cases of melanoma of the appendix presenting with appendicitis and review our institutional experience with this entity. OBSERVATIONS: Medical records were reviewed in patients with melanoma at the National Cancer Institute between January 1, 1953, and December 31, 2010, who underwent appendectomy. Of 5822 cases of melanoma treated at the National Institutes of Health, appendectomies were performed on 31 patients, 2 of whom had acute appendicitis secondary to malignant obstruction and presented with symptoms of vague abdominal pain. Both patients had been heavily pretreated for metastatic melanoma and had multiple sites of intraperitoneal and extraperitoneal disease. On exploratory laparotomy, both patients showed clinical evidence of acute appendicitis, and an appendectomy was performed. Both patients recovered fully from the operation and proceeded to further systemic therapy. CONCLUSIONS AND RELEVANCE: Although rare, the diagnosis of appendicitis should be considered in patients with melanoma and acute abdominal pain. Timely surgical intervention may allow palliation and the ability to pursue subsequent systemic treatment.