DNA Methylation Identifies Epigenetic Subtypes of Triple-Negative Breast Cancers With Distinct Clinicopathologic and Molecular Features.

Triple-negative breast cancers (TNBC) include diverse carcinomas with heterogeneous clinical behavior. DNA methylation is a useful tool in classifying a variety of cancers. In this study, we analyzed TNBC using DNA methylation profiling and compared the results to those of mutational analysis. DNA methylation profiling (Infinium MethylationEPIC array, Illumina) and 50-gene panel-targeted DNA sequencing were performed in 44 treatment-naïve TNBC. We identified 3 distinct DNA methylation clusters with specific clinicopathologic and molecular features. Cluster 1 (phosphoinositide 3-kinase/protein kinase B-enriched cluster; n = 9) patients were significantly older (mean age, 71 years; P = .008) with tumors that were more likely to exhibit apocrine differentiation (78%; P < .001), a lower grade (44% were grade 2), a lower proliferation index (median Ki-67, 15%; P = .002), and lower tumor-infiltrating lymphocyte fractions (median, 15%; P = .0142). Tumors carried recurrent PIK3CA and AKT1 mutations and a higher percentage of low HER-2 expression (89%; P = .033). Cluster 3 (chromosomal instability cluster; n = 28) patients were significantly younger (median age, 57 years). Tumors were of higher grade (grade 3, 93%), had a higher proliferation index (median Ki-67, 75%), and were with a high fraction of tumor-infiltrating lymphocytes (median, 30%). Ninety-one percent of the germline BRCA1/2 mutation carriers were in cluster 3, and these tumors showed the highest level of copy number alterations. Cluster 2 represented cases with intermediate clinicopathologic characteristics and no specific molecular profile (no specific molecular profile cluster; n = 7). There were no differences in relation to stage, recurrence, and survival. In conclusion, DNA methylation profiling is a promising tool to classify patients with TNBC into biologically relevant groups, which may result in better disease characterization and reveal potential targets for emerging therapies.

Macrophage density is an adverse prognosticator for ipsilateral recurrence in ductal carcinoma in situ.

INTRODUCTION: There is evidence that supports the association of dense tumor infiltrating lymphocyte (TILs) with an increased risk of ipsilateral recurrence in ductal carcinoma in situ (DCIS). However, the association of cellular composition of DCIS immune microenvironment with the histopathologic parameters and outcome is not well understood. METHODS: We queried our institutional database for patients with pure DCIS diagnosed between 2010 and 2019. Immunohistochemical studies for CD8, CD4, CD68, CD163, and FOXP3 were performed and evaluated in the DCIS microenvironment using tissue microarrays. Statistical methods included Fisher's exact test for categorical variables and the two-sample t-test or the Wilcoxon Rank-Sum test for continuous variables. RESULTS: The analytic sample included 67 patients. Median age was 62 years (range = 53 to 66) and median follow up was 6.7 years (range = 5.3 to 7.8). Thirteen patients had ipsilateral recurrence. Of all the clinicopathologic variables, only the DCIS size and TIL density were significantly associated with recurrence (p = 0.023 and 0.006, respectively). After adjusting for age and TIL density, only high CD68 (>50) and high CD68/CD163 ratio (>0.46) correlated with ipsilateral recurrence (p = 0.026 and 0.013, respectively) and shorter time to recurrence [hazard ratio 4.87 (95% CI: 1.24-19, p = 0.023) and 10.32 (95% CI: 1.34-80, p = 0.025), respectively]. CONCLUSIONS: In addition to DCIS size and TIL density, high CD68+ tumor-associated macrophages predict ipsilateral recurrence in DCIS. High CD68+ macrophage density and CD68/CD163 ratio also predict a shorter time to recurrence.

Sentinel lymph node positivity in patients undergoing mastectomies for ductal carcinoma in situ (DCIS).

Current guidelines recommend sentinel lymph node biopsy (SLNB) for patients undergoing mastectomy for a preoperative diagnosis of ductal carcinoma in situ (DCIS). We examined the factors associated with sentinel lymph node positivity for patients undergoing mastectomy for a diagnosis of DCIS on preoperative core biopsy (PCB). The Institutional Breast Cancer Database was queried for patients with PCB demonstrating pure DCIS followed by mastectomy and SLNB from 2010 to 2018. Patients were divided according to final pathology (DCIS or invasive cancer). Clinico-pathologic variables were analyzed using Pearson's chi-squared, Wilcoxon Rank-Sum and logistic regression. Of 3145 patients, 168(5%) had pure DCIS on PCB and underwent mastectomy with SLNB. On final mastectomy pathology, 120(71%) patients had DCIS with 0 positive sentinel lymph nodes (PSLNs) and 48(29%) patients had invasive carcinoma with 5(10%) cases of ≥1 PSLNs. Factors positively associated with upstaging to invasive cancer in univariate analysis included age (P = .0289), palpability (P < .0001), extent of disease on imaging (P = .0121), mass on preoperative imaging (P = .0003), multifocality (P = .0231) and multicentricity (P = .0395). In multivariate analysis, palpability (P = .0080), extent of disease on imaging (P = .0074) and mass on preoperative imaging (P = .0245) remained significant (Table 2). In a subset of patients undergoing mastectomy for DCIS with limited disease on preoperative evaluation, SLNB may be omitted as the risk of upstaging is low. However, patients who present with clinical findings of palpability, large extent of disease on imaging and mass on preoperative imaging have a meaningful risk of upstaging to invasive cancer, and SLNB remains important for management.

Pregnancy-associated breast cancer in a contemporary cohort of newly diagnosed women.

Pregnancy-associated breast cancer (PABC) refers to breast cancer (BC) diagnosed during pregnancy, lactation, or in the postpartum period. There is evidence that PABC is associated with a poorer prognosis, and that the development of the disease is influenced by the unique hormonal milieu of pregnancy. The purpose of this study was to investigate the clinicopathologic characteristics associated with PABC in a contemporary cohort of women with newly diagnosed BC. Our institutional Breast Cancer Database was queried for women diagnosed with BC between 2009-2018 who had at least one full-term pregnancy (FTP). Variables of interest included patient demographics and clinical and tumor characteristics. PABC was defined as breast cancer diagnosed within 24 months of delivery. Statistical analyses included Pearson's chi-square and logistic regression. Out of a total of 2202 women, 46 (2.1%) had PABC. Median follow-up in the total cohort was 5.5 years. After adjusting for age at first FTP, PABC was associated with younger age at diagnosis, older age at first FTP, non-Caucasian race, BRCA positivity, presentation with a palpable mass, higher pathologic stage, higher histologic grade, and ER-negative and triple-negative receptor status. The association of PABC with non-Caucasian race may be reflected in the increased proportion of triple-negative breast cancers in the PABC group. PABC was also associated with older age at first FTP. As more women delay childbearing, risk for PABC may increase. Our findings suggest that women who become pregnant at older ages should be followed carefully during pregnancy and the postpartum period, especially if they are BRCA mutation carriers. The optimal approach for monitoring older women during pregnancy and the postpartum period is unclear.

Regional Node Basin Recurrence in Melanoma Patients: More Common After Node Dissection for Macroscopic Rather than Clinically Occult Nodal Disease.

BACKGROUND: Recommended treatment for patients with sentinel lymph node (SLN)-positive melanoma has recently changed. Randomized trials demonstrated equivalent survival with close observation versus completion lymph node dissection (CLND), but increased regional node recurrence. We evaluated factors related to in-basin nodal recurrence after lymphadenectomy (LND) for SLN-positive or macroscopic nodal metastases. METHODS: An institutional database and the first Multicenter Selective Lymphadenectomy Trial (MSLT-I) were analyzed independently. Exclusions were multiple primaries, multi-basin involvement, or in-transit metastases. Patient demographics, primary tumor thickness and ulceration, lymph nodes retrieved, and use of adjuvant radiotherapy were analyzed. Multivariate analyses were performed to determine factors predicting in-basin nodal recurrence (significance p ≤ 0.05). RESULTS: The retrospective cohort (577 patients) showed an in-basin failure rate of 6.6% after CLND for a positive SLN and 13.1% after LND for palpable disease (p = 0.001). This recurrence risk persisted after adjustment for patient, tumor, and LND factors [hazard ratio (HR) 2.32; p = 0.004]. In the MSLT-I cohort (326 patients), the failure rate after CLND following SLNB was 6.2%, but 10.1% after LND for palpable recurrence in observation patients. After adjustment for other factors, macroscopic disease was associated with an increased risk of recurrence after LND (HR 2.24; p = 0.05). CONCLUSION: After LND for melanoma, in-basin recurrence is infrequent, but a clinically significant fraction will fail. Failure is less likely if dissection is performed for clinically occult disease. Further research is warranted to evaluate the long-term regional control and quality of life associated with nodal basin observation, which has now become standard practice.

A Nomogram to Predict Factors Associated with Lymph Node Metastasis in Ductal Carcinoma In Situ with Microinvasion.

INTRODUCTION: Ductal carcinoma in situ (DCIS) with foci of invasion measuring ≤ 1 mm (DCISM), represents < 1% of all invasive breast cancers. Sentinel lymph node biopsy (SLNB) has been a standard component of surgery for patients with invasive carcinoma or extensive DCIS. We hypothesize that selective performance of SLNB may be appropriate given the low incidence of sentinel node (SN) metastasis for DCISM. We investigated the clinicopathologic predictors for SN positivity in DCISM, to identify which patients might benefit from SLNB. METHODS: A retrospective review of the National Cancer Database was performed for cases from 2012 to 2015. Clinical and tumor characteristics, including SN results, were evaluated, and Pearson's Chi square tests and logistic regression were performed. RESULTS: Of 7803 patients with DCISM, 306 (4%) had at least one positive SN. Patients with positive SNs were younger, more often of Black race, had higher-grade histology and larger tumor size, and were more likely to have lymphovascular invasion (LVI; all p < 0.001). In an adjusted model, the presence of LVI was associated with the highest odds ratio (OR) for node positivity (OR 8.80, 95% confidence interval 4.56-16.96). CONCLUSIONS: Among women with DCISM, only 4% had a positive SN. Node positivity was associated with more extensive and higher-grade DCIS, and the presence of LVI was strongly correlated with node positivity. Our data suggest that LVI is the most important factor in determining which patients with DCISM will benefit from SN biopsy.

Tumor-Infiltrating Lymphocytes in a Contemporary Cohort of Women with Ductal Carcinoma In Situ (DCIS).

BACKGROUND: Growing evidence suggests that the tumor immune microenvironment influences breast cancer development and prognosis. Density of tumor-infiltrating lymphocytes (TILs) within invasive breast cancer is correlated with response to therapy, especially in triple-negative disease. The clinical relevance and outcomes of TILs within ductal carcinoma in situ (DCIS) are less understood. METHODS: Our institutional database of 668 patients with pure DCIS from 2010 to 2018 was queried. TILs were evaluated by International TILs Working Group guidelines. Percentage of TILs was assessed from the densest focus (hotspot) in one high-power field of stroma touching the basement membrane. Statistical methods included cluster analyses (to define sparse versus dense TILs), logistic, and Cox regression models. RESULTS: Sixty-nine patients with DCIS and TILs were evaluated, of whom 54 (78%) were treated by breast-conserving surgery. Thirteen (19%) patients had ipsilateral recurrence. Each recurrence (n = 13) was matched to four controls (n = 56) based on date of surgery. Median follow-up was 6.7 years. TILs were defined as sparse (< 45%) or dense (≥ 45%). Dense TILs were associated with younger age (p = 0.045), larger tumor size (p < 0.001), high nuclear grade (p = 0.010), comedo histology (p = 0.033), necrosis (p = 0.027), estrogen receptor (ER) negativity (p = 0.037), and ipsilateral recurrence (p = 0.001). Nine patients with dense TILs had mean time to recurrence of 73.5 months compared with four patients with sparse TILs with mean time to recurrence of 97.9 months (p = 0.003). CONCLUSIONS: Dense TILs were significantly associated with age, tumor size, nuclear grade, comedo histology, necrosis, and ER status and was a significant predictor of recurrence in patients with pure DCIS.

A genomic ruler to assess oncogenic transition between breast tumor and stroma.

BACKGROUND: Cancers induce gene expression alterations in stroma surrounding tumors that supports cancer progression. However, it is actually not at all known the extent of altered stromal gene expression enacted by tumors nor the extent to which altered stromal gene expression penetrates the stromal tissue. Presently, post-surgical "tumor-free" stromal tissue is determined to be cancer-free based on solely on morphological normality-a criteria that has not changed in more than 100 years despite the existence of sophisticated gene expression data to the contrary. We therefore investigated the extent to which breast tumors alter stromal gene expression in three dimensions in women undergoing mastectomy with the intent of providing a genomic determination for development of future risk of recurrence criteria, and to inform the need for adjuvant full-breast irradiation. METHODS AND FINDINGS: Genome-wide gene expression changes were determined in histopathologically normal breast tissue in 33 women undergoing mastectomy for stage II and III primary invasive ductal carcinoma at serial distances in three dimensions from the tumor. Gene expression was determined by genome-wide mRNA analysis and subjected to metagene mRNA characterization. Tumor-like gene expression signatures in stroma were identified that surprisingly transitioned to a plastic, normalizing homeostatic signature with distance from tumor. Stroma closest to tumor displayed a pronounced tumor-like signature enriched in cancer-promoting pathways involved in disruption of basement membrane, cell migration and invasion, WNT signaling and angiogenesis. By 2 cm from tumor in all dimensions, stromal tissues were in transition, displaying homeostatic and tumor suppressing gene activity, while also expressing cancer supporting pathways. CONCLUSIONS: The dynamics of gene expression in the post-tumor breast stroma likely co-determines disease outcome: reversion to normality or transition to transformation in morphologically normal tissue. Our stromal genomic signature may be important for personalizing surgical and adjuvant therapeutic decisions and risk of recurrence.

A novel immunization strategy using cytokine/chemokines induces new effective systemic immune responses, and frequent complete regressions of human metastatic melanoma.

Immune responses have been elicited by a variety of cancer vaccines, but seldom induce regressions of established cancers in humans. As a novel therapeutic immunization strategy, we tested the hypothesis that multiple cytokines/chemokines secreted early in secondary responses ex-vivo might mimic the secretory environment guiding new immune responses. The early development of immune responses is regulated by multiple cytokines/chemokines acting together, which at physiologic concentrations act locally in concert with antigen to have non-specific effects on adjacent cells, including the maturation of dendritic cells, homing and retention of T cells at the site of antigen, and the differentiation and expansion of T cell clones with appropriate receptors. We postulated that repeated injections into a metastasis of an exogenous chemokine/cytokine mixture might establish the environment of an immune response and allow circulating T cell clones to self- select for mutant neo-epitopes in the tumor and generate systemic immune responses. To test this idea we injected some metastases in patients with multiple cutaneous melanoma nodules while never injecting other control metastases in the same patient. New immune responses were identified by the development of dense lymphocytic infiltrates in never-injected metastases, and the frequent complete regression of never-injected metastases, a surprising observation. 70% of subjects developed dense infiltrates of cytotoxic CD8 cells in the center and margin of never-injected metastases; 38% of subjects had complete and often durable regressions of all metastases, without the use of check-point inhibitors, suggesting that, as a proof-of-principle, an immunization strategy can control advanced human metastatic melanoma.

The Impact of Smoking on Sentinel Node Metastasis of Primary Cutaneous Melanoma.

BACKGROUND: Although a well-established causative relationship exists between smoking and several epithelial cancers, the association of smoking with metastatic progression in melanoma is not well studied. We hypothesized that smokers would be at increased risk for melanoma metastasis as assessed by sentinel lymph node (SLN) biopsy. METHODS: Data from the first international Multicenter Selective Lymphadenectomy Trial (MSLT-I) and the screening-phase of the second trial (MSLT-II) were analyzed to determine the association of smoking with clinicopathologic variables and SLN metastasis. RESULTS: Current smoking was strongly associated with SLN metastasis (p = 0.004), even after adjusting for other predictors of metastasis. Among 4231 patients (1025 in MSLT-I and 3206 in MSLT-II), current or former smoking was also independently associated with ulceration (p < 0.001 and p < 0.001, respectively). Compared with current smoking, never smoking was independently associated with decreased Breslow thickness in multivariate analysis (p = 0.002) and with a 0.25 mm predicted decrease in thickness. CONCLUSION: The direct correlation between current smoking and SLN metastasis of primary cutaneous melanoma was independent of its correlation with tumor thickness and ulceration. Smoking cessation should be strongly encouraged among patients with or at risk for melanoma.

Prospective Randomized Trial of Prone Accelerated Intensity Modulated Breast Radiation Therapy With a Daily Versus Weekly Boost to the Tumor Bed.

PURPOSE: To report the results of a prospective randomized trial comparing a daily versus weekly boost to the tumor cavity during the course of accelerated radiation to the breast with patients in the prone position. METHODS AND MATERIALS: From 2009 to 2012, 400 patients with stage 0 to II breast cancer who had undergone segmental mastectomy participated in an institutional review board-approved trial testing prone breast radiation therapy to 40.5 Gy in 15 fractions 5 d/wk to the whole breast, after randomization to a concomitant daily boost to the tumor bed of 0.5 Gy, or a weekly boost of 2 Gy, on Friday. The present noninferiority trial tested the primary hypothesis that a weekly boost produced no more acute toxicity than did a daily boost. The recurrence-free survival was estimated for both treatment arms using the Kaplan-Meier method; the relative risk of recurrence or death was estimated, and the 2 arms were compared using the log-rank test. RESULTS: At a median follow-up period of 45 months, no deaths related to breast cancer had occurred. The weekly boost regimen produced no more grade ≥2 acute toxicity than did the daily boost regimen (8.1% vs 10.4%; noninferiority Z = -2.52; P=.006). No statistically significant difference was found in the cumulative incidence of long-term fibrosis or telangiectasia of grade ≥2 between the 2 arms (log-rank P=.923). Two local and two distant recurrences developed in the daily treatment arm and three local and one distant developed in the weekly arm. The 4-year recurrence-free survival rate was not different between the 2 treatment arms (98% for both arms). CONCLUSIONS: A tumor bed boost delivered either daily or weekly was tolerated similarly during accelerated prone breast radiation therapy, with excellent control of disease and comparable cosmetic results.

Nipple-sparing Mastectomy and Sub-areolar Biopsy: To Freeze or not to Freeze? Evaluating the Role of Sub-areolar Intraoperative Frozen Section.

Use of nipple-sparing mastectomy (NSM) for risk-reduction and therapeutic breast cancer resection is growing. The role for intraoperative frozen section of the nipple-areolar complex remains controversial. Records of patients undergoing NSM at our institution from 2006 to 2013 were reviewed. Records from 501 nipple-sparing mastectomies were reviewed (216 therapeutic, 285 prophylactic). Of the 480 breasts with sub-areolar biopsies, 307 had intraoperative frozen sections and 173 were evaluated with permanent paraffin section only. Among the 307 intraoperative frozen sections, 12 biopsies were positive on permanent paraffin section (3.9% or 12/307). Of the 12 positive permanent biopsies, five were false negative and the remaining seven concordant intraoperatively. Sensitivity and specificity of sub-areolar frozen section were 0.58 and 1, respectively. Positive sub-areolar biopsies consisted primarily of ductal carcinoma in situ (62% or 13/21). The nipples or nipple-areolar complex were resected in a separate procedure following mastectomy (10/21), intraoperatively following frozen section results (7/21) or during second-stage breast reconstruction (3/21; 1 additional scheduled). Only 30% (6/20) of resected specimens had abnormal residual pathology. Intraoperative frozen section is highly specific and moderately sensitive for the detection of positive sub-areolar biopsies in NSM. Its use can help guide intraoperative reconstructive planning. The presence of positive sub-areolar biopsies in both contralateral and high-risk prophylactic mastectomy specimens emphasizes the need to perform sub-areolar biopsies in all nipple-sparing mastectomies.

Pro-oncogenic cytokines and growth factors are differentially expressed in the post-surgical wound fluid from malignant compared to benign breast lesions.

PURPOSE: The accumulation of wound fluid known as seroma in the chest cavity following breast surgery is a common occurrence that can persist for many weeks. While the pro-inflammatory composition of seroma is well established, there has been remarkably little research to determine whether seroma contains pro-oncogenic factors, and whether this is influenced by previous malignant disease. METHODS: We developed a clinical trial in which we obtained post-surgical seroma fluids from women with benign or malignant disease 1 or 2 weeks following lumpectomy or mastectomy. We conducted an analysis of more than 80 different cytokines, chemokines and growth factors. RESULTS: We found that surgical cavity seroma from breast cancer patients has a higher expression of key tumor-promoting cytokines and lower expression of important tumor-inhibiting factors when compared to benign lesions from non-cancer patients. Patients with high body mass index also had higher levels of leptin regardless of malignancy. CONCLUSIONS: We conclude that the breast post-surgical tumor cavity contains factors that are pro-inflammatory regardless of malignant or benign disease, but in malignant disease there is significant enrichment of additional pro-oncogenic chemokines, cytokines and growth factors, and reduction in tumor-inhibiting factors. These results are consistent with tumor conditioning of surrounding normal stromal tissue and creation of a pro-oncogenic environment that persists long after surgical removal of the tumor.

Prone breast intensity modulated radiation therapy: 5-year results.

PURPOSE: To report the 5-year results of a technique of prone breast radiation therapy delivered by a regimen of accelerated intensity modulated radiation therapy with a concurrent boost to the tumor bed. METHODS AND MATERIALS: Between 2003 and 2006, 404 patients with stage I-II breast cancer were prospectively enrolled into 2 consecutive protocols, institutional trials 03-30 and 05-181, that used the same regimen of 40.5 Gy/15 fractions delivered to the index breast over 3 weeks, with a concomitant daily boost to the tumor bed of 0.5 Gy (total dose 48 Gy). All patients were treated after segmental mastectomy and had negative margins and nodal assessment. Patients were set up prone: only if lung or heart volumes were in the field was a supine setup attempted and chosen if found to better spare these organs. RESULTS: Ninety-two percent of patients were treated prone, 8% supine. Seventy-two percent had stage I, 28% stage II invasive breast cancer. In-field lung volume ranged from 0 to 228.27 cm(3), mean 19.65 cm(3). In-field heart volume for left breast cancer patients ranged from 0 to 21.24 cm(3), mean 1.59 cm(3). There was no heart in the field for right breast cancer patients. At a median follow-up of 5 years, the 5-year cumulative incidence of isolated ipsilateral breast tumor recurrence was 0.82% (95% confidence interval [CI] 0.65%-1.04%). The 5-year cumulative incidence of regional recurrence was 0.53% (95% CI 0.41%-0.69%), and the 5-year overall cumulative death rate was 1.28% (95% CI 0.48%-3.38%). Eighty-two percent (95% CI 77%-85%) of patients judged their final cosmetic result as excellent/good. CONCLUSIONS: Prone accelerated intensity modulated radiation therapy with a concomitant boost results in excellent local control and optimal sparing of heart and lung, with good cosmesis. Radiation Therapy Oncology Group protocol 1005, a phase 3, multi-institutional, randomized trial is ongoing and is evaluating the equivalence of a similar dose and fractionation approach to standard 6-week radiation therapy with a sequential boost.

Final trial report of sentinel-node biopsy versus nodal observation in melanoma.

BACKGROUND: Sentinel-node biopsy, a minimally invasive procedure for regional melanoma staging, was evaluated in a phase 3 trial. METHODS: We evaluated outcomes in 2001 patients with primary cutaneous melanomas randomly assigned to undergo wide excision and nodal observation, with lymphadenectomy for nodal relapse (observation group), or wide excision and sentinel-node biopsy, with immediate lymphadenectomy for nodal metastases detected on biopsy (biopsy group). Results No significant treatment-related difference in the 10-year melanoma-specific survival rate was seen in the overall study population (20.8% with and 79.2% without nodal metastases). Mean (± SE) 10-year disease-free survival rates were significantly improved in the biopsy group, as compared with the observation group, among patients with intermediate-thickness melanomas, defined as 1.20 to 3.50 mm (71.3 ± 1.8% vs. 64.7 ± 2.3%; hazard ratio for recurrence or metastasis, 0.76; P=0.01), and those with thick melanomas, defined as >3.50 mm (50.7 ± 4.0% vs. 40.5 ± 4.7%; hazard ratio, 0.70; P=0.03). Among patients with intermediate-thickness melanomas, the 10-year melanoma-specific survival rate was 62.1 ± 4.8% among those with metastasis versus 85.1 ± 1.5% for those without metastasis (hazard ratio for death from melanoma, 3.09; P<0.001); among patients with thick melanomas, the respective rates were 48.0 ± 7.0% and 64.6 ± 4.9% (hazard ratio, 1.75; P=0.03). Biopsy-based management improved the 10-year rate of distant disease-free survival (hazard ratio for distant metastasis, 0.62; P=0.02) and the 10-year rate of melanoma-specific survival (hazard ratio for death from melanoma, 0.56; P=0.006) for patients with intermediate-thickness melanomas and nodal metastases. Accelerated-failure-time latent-subgroup analysis was performed to account for the fact that nodal status was initially known only in the biopsy group, and a significant treatment benefit persisted. CONCLUSIONS: Biopsy-based staging of intermediate-thickness or thick primary melanomas provides important prognostic information and identifies patients with nodal metastases who may benefit from immediate complete lymphadenectomy. Biopsy-based management prolongs disease-free survival for all patients and prolongs distant disease-free survival and melanoma-specific survival for patients with nodal metastases from intermediate-thickness melanomas. (Funded by the National Cancer Institute, National Institutes of Health, and the Australia and New Zealand Melanoma Trials Group; ClinicalTrials.gov number, NCT00275496.).

Multiple breast cancer cell-lines derived from a single tumor differ in their molecular characteristics and tumorigenic potential.

BACKGROUND: Breast cancer cell lines are widely used tools to investigate breast cancer biology and to develop new therapies. Breast cancer tissue contains molecularly heterogeneous cell populations. Thus, it is important to understand which cell lines best represent the primary tumor and have similarly diverse phenotype. Here, we describe the development of five breast cancer cell lines from a single patient's breast cancer tissue. We characterize the molecular profiles, tumorigenicity and metastatic ability in vivo of all five cell lines and compare their responsiveness to 4-hydroxytamoxifen (4-OHT) treatment. METHODS: Five breast cancer cell lines were derived from a single patient's primary breast cancer tissue. Expression of different antigens including HER2, estrogen receptor (ER), CK8/18, CD44 and CD24 was determined by flow cytometry, western blotting and immunohistochemistry (IHC). In addition, a Fluorescent In Situ Hybridization (FISH) assay for HER2 gene amplification and p53 genotyping was performed on all cell lines. A xenograft model in nude mice was utilized to assess the tumorigenic and metastatic abilities of the breast cancer cells. RESULTS: We have isolated, cloned and established five new breast cancer cell lines with different tumorigenicity and metastatic abilities from a single primary breast cancer. Although all the cell lines expressed low levels of ER, their growth was estrogen-independent and all had high-levels of expression of mutated non-functional p53. The HER2 gene was rearranged in all cell lines. Low doses of 4-OHT induced proliferation of these breast cancer cell lines. CONCLUSIONS: All five breast cancer cell lines have different antigenic expression profiles, tumorigenicity and organ specific metastatic abilities although they derive from a single tumor. None of the studied markers correlated with tumorigenic potential. These new cell lines could serve as a model for detailed genomic and proteomic analyses to identify mechanisms of organ-specific metastasis of breast cancer.

Genetic variants associated with breast cancer risk for Ashkenazi Jewish women with strong family histories but no identifiable BRCA1/2 mutation.

The ability to establish genetic risk models is critical for early identification and optimal treatment of breast cancer. For such a model to gain clinical utility, more variants must be identified beyond those discovered in previous genome-wide association studies (GWAS). This is especially true for women at high risk because of family history, but without BRCA1/2 mutations. This study incorporates three datasets in a GWAS analysis of women with Ashkenazi Jewish (AJ) homogeneous ancestry. Two independent discovery cohorts comprised 239 and 238 AJ women with invasive breast cancer or preinvasive ductal carcinoma in situ and strong family histories of breast cancer, but lacking the three BRCA1/2 founder mutations, along with 294 and 230 AJ controls, respectively. An independent, third cohort of 203 AJ cases with familial breast cancer history and 263 healthy controls of AJ women was used for validation. A total of 19 SNPs were identified as associated with familial breast cancer risk in AJ women. Among these SNPs, 13 were identified from a panel of 109 discovery SNPs, including an FGFR2 haplotype. In addition, six previously identified breast cancer GWAS SNPs were confirmed in this population. Seven of the 19 markers were significant in a multivariate predictive model of familial breast cancer in AJ women, three novel SNPs [rs17663555(5q13.2), rs566164(6q21), and rs11075884(16q22.2)], the FGFR2 haplotype, and three previously published SNPs [rs13387042(2q35), rs2046210(ESR1), and rs3112612(TOX3)], yielding moderate predictive power with an area under the curve (AUC) of the ROC (receiver-operator characteristic curve) of 0.74. Population-specific genetic variants in addition to variants shared with populations of European ancestry may improve breast cancer risk prediction among AJ women from high-risk families without founder BRCA1/2 mutations.

Metastasectomy for distant metastatic melanoma: analysis of data from the first Multicenter Selective Lymphadenectomy Trial (MSLT-I).

BACKGROUND: For stage IV melanoma, systemic medical therapy (SMT) is used most frequently; surgery is considered an adjunct in selected patients. We retrospectively compared survival after surgery with or without SMT versus SMT alone for melanoma patients developing distant metastases while enrolled in the first Multicenter Selective Lymphadenectomy Trial. METHODS: Patients were randomized to wide excision and sentinel node biopsy, or wide excision and nodal observation. We evaluated recurrence site, therapy (selected by treating clinician), and survival after stage IV diagnosis. RESULTS: Of 291 patients with complete data for stage IV recurrence, 161 (55 %) underwent surgery with or without SMT. Median survival was 15.8 versus 6.9 months, and 4-year survival was 20.8 versus 7.0 % for patients receiving surgery with or without SMT versus SMT alone (p < 0.0001; hazard ratio 0.406). Surgery with or without SMT conferred a survival advantage for patients with M1a (median > 60 months vs. 12.4 months; 4-year survival 69.3 % vs. 0; p = 0.0106), M1b (median 17.9 vs. 9.1 months; 4-year survival 24.1 vs. 14.3 %; p = 0.1143), and M1c (median 15.0 vs. 6.3 months; 4-year survival 10.5 vs. 4.6 %; p = 0.0001) disease. Patients with multiple metastases treated surgically had a survival advantage, and number of operations did not reduce survival in the 67 patients (42 %) who had multiple surgeries for distant melanoma. CONCLUSIONS: Our findings suggest that over half of stage IV patients are candidates for resection and exhibit improved survival over patients receiving SMT alone, regardless of site and number of metastases. We have begun a multicenter randomized phase III trial comparing surgery versus SMT as initial treatment for resectable distant melanoma.

Five year outcome of 145 patients with ductal carcinoma in situ (DCIS) after accelerated breast radiotherapy.

BACKGROUND: Accelerated whole-breast radiotherapy (RT) with tumor bed boost in the treatment of early invasive breast cancer has demonstrated equivalent local control and cosmesis when compared with standard RT. Its efficacy in the treatment of ductal carcinoma in situ (DCIS) remains unknown. METHODS AND MATERIALS: Patients treated for DCIS with lumpectomy and negative margins were eligible for 2 consecutive hypofractionated whole-breast RT clinical trials. The first trial (New York University [NYU] 01-51) prescribed to the whole breast 42 Gy (2.8 Gy in 15 fractions) and the second trial (NYU 05-181) 40.5 Gy (2.7 Gy in 15 fractions) with an additional daily boost of 0.5 Gy to the surgical cavity. RESULTS: Between 2002 and 2009, 145 DCIS patients accrued, 59 to the first protocol and 86 to the second trial. Median age was 56 years and 65% were postmenopausal at the time of treatment. Based on optimal sparing of normal tissue, 79% of the patients were planned and treated prone and 21% supine. At 5 years' median follow-up (60 months; range 2.6-105.5 months), 6 patients (4.1%) experienced an ipsilateral breast recurrence in all cases of DCIS histology. In 3/6 patients, recurrence occurred at the original site of DCIS and in the remaining 3 cases outside the original tumor bed. New contralateral breast cancers arose in 3 cases (1 DCIS and 2 invasive carcinomas). Cosmetic self-assessment at least 2 years after treatment is available in 125 patients: 91% reported good-to-excellent and 9% reported fair-to-poor outcomes. CONCLUSIONS: With a median follow-up of 5 years, the ipsilateral local recurrence rate is 4.1%, comparable to that reported from the NSABP (National Surgical Adjuvant Breast and Bowel Project) trials that employed 50 Gy in 25 fractions of radiotherapy for DCIS. There were no invasive recurrences. These results provide preliminary evidence that accelerated hypofractionated external beam radiotherapy is a viable option for DCIS.