Magee Equation 3 predicts pathologic response to neoadjuvant systemic chemotherapy in estrogen receptor positive, HER2 negative/equivocal breast tumors.

Magee Equations were derived as an inexpensive, rapid alternative to Oncotype DX. The Magee Equation 3 utilizes immunohistochemical and FISH data for estrogen receptor (ER), progesterone receptor (PR), HER2 and Ki-67 for its calculation (24.30812+ERIHC × (-0.02177)+PRIHC × (-0.02884)+(0 for HER2 negative, 1.46495 for equivocal, 12.75525 for HER2 positive)+Ki-67 × 0.18649). We hypothesize that Magee Equation 3 scores from pre-therapy core biopsy can predict response to neoadjuvant systemic chemotherapy. A prospectively-maintained database of patients who received neoadjuvant systemic therapy from 2010 to 2014 at a single institution was retrospectively reviewed. Pathologic complete response was defined as absence of invasive tumor in the breast and regional lymph nodes. Of the 614 cases, tumors with missing immunohistochemical results and those that were ER negative or HER2 positive were excluded. This resulted in 237 ER positive, HER2 negative/equivocal tumors that formed the basis of this study. Magee Equation 3 scores were divided into 3 categories similar to Oncotype DX, ie, 0 to <18 (low), 18 to <31 (intermediate), and 31 or higher (high) scores. The pathologic complete response rate for low, intermediate and high Magee Equation 3 scores was 0%, 4%, and 36%, respectively. Patients with high Magee Equation 3 scores were 13 times more likely to achieve pathologic complete response compared to those with Magee Equation 3 scores less than 31 (95% CI 5.09-32.87, P<0.0001). For patients that did not achieve pathologic complete response, high Magee Equation 3 correlated with higher recurrence rate, with the majority occurring in patients with positive lymph nodes in the resection specimen. Magee Equation 3 score ≥31 predicts pathologic complete response in the neoadjuvant setting and for tumor recurrence, when pathologic complete response is not achieved. These results show the utility of Magee Equation 3 in predicting patients who will benefit from chemotherapy but warrant prospective multi-institutional validation.

Secondary mutations as a mechanism of cisplatin resistance in BRCA2-mutated cancers.

Ovarian carcinomas with mutations in the tumour suppressor BRCA2 are particularly sensitive to platinum compounds. However, such carcinomas ultimately develop cisplatin resistance. The mechanism of that resistance is largely unknown. Here we show that acquired resistance to cisplatin can be mediated by secondary intragenic mutations in BRCA2 that restore the wild-type BRCA2 reading frame. First, in a cisplatin-resistant BRCA2-mutated breast-cancer cell line, HCC1428, a secondary genetic change in BRCA2 rescued BRCA2 function. Second, cisplatin selection of a BRCA2-mutated pancreatic cancer cell line, Capan-1 (refs 3, 4), led to five different secondary mutations that restored the wild-type BRCA2 reading frame. All clones with secondary mutations were resistant both to cisplatin and to a poly(ADP-ribose) polymerase (PARP) inhibitor (AG14361). Finally, we evaluated recurrent cancers from patients whose primary BRCA2-mutated ovarian carcinomas were treated with cisplatin. The recurrent tumour that acquired cisplatin resistance had undergone reversion of its BRCA2 mutation. Our results suggest that secondary mutations that restore the wild-type BRCA2 reading frame may be a major clinical mediator of acquired resistance to platinum-based chemotherapy.

Detection of splicing aberrations caused by BRCA1 and BRCA2 sequence variants encoding missense substitutions: implications for prediction of pathogenicity.

Missense substitutions in high-risk cancer susceptibility genes create clinical uncertainty in the genetic counseling process. Multifactorial likelihood classification approaches and in vitro assays are useful for the classification of exonic sequence variants in BRCA1 and BRCA2, but these currently rely on the assumption that changes in protein function are the major biological mechanism of pathogenicity. This study investigates the potentially pathogenic role of aberrant splicing for exonic variants predicted to encode missense substitutions using patient-derived RNA. No splicing aberrations were identified for BRCA1c.5054C>T and BRCA2c.7336A>G, c.8839G>A, and c.9154C>T. However, RT-PCR analysis identified a major splicing aberration for BRCA1c.4868C>G(p.Ala1623Gly), a variant encoding a missense substitution considered likely to be neutral. Splicing aberrations were also observed for BRCA2c.7988A>T(p.Glu2663Val) and c.8168A>G(p.Asp2723Gly), but both variant and wildtype alleles were shown to be present in full-length mRNA transcripts, suggesting that variant protein may be translated. BRCA2 protein function assays indicated that BRCA2p.Glu2663Val, p.Asp2723Gly and p.Arg3052Trp missense proteins have abrogated function consistent with pathogenicity. Multifactorial likelihood analysis provided evidence for pathogenicity for BRCA1 c.5054C>T(p.Thr1685Ile) and BRCA2c.7988A>T(p.Glu2663Val), c.8168A>G(p.Asp2723Gly) and c.9154C>T(p.Arg3052Trp), supporting experimentally derived evidence. These findings highlight the need for improved bioinformatic prediction of splicing aberrations and to refine multifactorial likelihood models used to assess clinical significance.

Is completion axillary lymph node dissection necessary in patients who are underrepresented in the ACOSOG Z0011 trial?

PURPOSE: The American College of Surgeons Oncology Group trial Z0011 demonstrated that axillary node dissection (ALND) can be omitted in patients managed with breast conserving surgery and 1 to 2 positive sentinel lymph nodes (SLNs) without adverse effects on locoregional recurrence or disease-free survival (DFS). We investigated patients with breast cancer for whom clinicopathologic features were underrepresented in the Z0011 trial and analyzed radiation therapy treatment patterns and clinical outcomes. METHODS AND MATERIALS: We retrospectively reviewed records of patients who underwent a lumpectomy and SLN biopsy with positive SLNs but not an ALND and completed adjuvant radiation therapy. Eligible patients had T3 tumors, >2 positive SLNs, invasive lobular carcinoma, estrogen receptor negative status, extranodal extension, Nottingham Grade 3, or were age <50 years. RESULTS: We identified 105 women treated between July 2011 and July 2016 with a median follow-up time of 48.5 months (Range, 11-83 months). There were 40 women with an extranodal extension (38.9%) and 42 women with grade 3 disease (40.0%). Nineteen patients received whole breast irradiation alone (18.1%) and 86 patients were treated with modified tangent fields including the superior axilla level I/II (81.9%). Thirty-three patients (31.4%) also received a 3rd supraclavicular, nodal-directed field. Among the 86 patients who received axillary nodal irradiation, nodal volume contouring was performed in 77 patients (89.5%). Fifty-one patients (48.6%) also received adjuvant chemotherapy. The overall rates of 4-year DFS and locoregional control (LRC) were 94.3% and 98.1%, respectively. Off all patients, 1 patient experienced an internal mammary nodal recurrence, another patient a contralateral breast tumor, and two patients distant metastases. There were no axillary or ipsilateral breast tumor recurrences. CONCLUSIONS: This retrospective analysis of women who were underrepresented or excluded from the Z11 trial and underwent a lumpectomy and SLN biopsy with positive SLNs demonstrated comparable rates of LRC and DFS. The high rates of LRC and DFS suggest that completion ALND may be safely omitted in this patient population but larger data sets and longer follow-up times are needed to confirm this finding.

Prognostic Significance of Modified Residual Disease in Breast and Nodes (mRDBN) Algorithm After Neoadjuvant Chemotherapy for Breast Cancer.

OBJECTIVES: We hypothesized that prognostic accuracy of the residual disease in breast and lymph nodes (RDBN) method, which is calculated using residual tumor size, nodal involvement, and tumor grade, may be improved by incorporating residual tumor cellularity. METHODS: Cases included 614 patients who underwent neoadjuvant therapy for breast cancer. Tumor size was adjusted for residual cellularity of invasive carcinoma and used to calculate modified RDBN (mRDBN) and compared with unmodified gross tumor size (gRDBN). RESULTS: RDBN could be calculated in 428 cases. Relative risks of recurrence and death were significantly higher for RDBN-3 and RDBN-4 compared with RDBN-1. Kaplan-Meier analysis showed significant differences in disease-free survival and overall survival for estrogen receptor (ER)-negative/human epidermal growth factor receptor 2 (HER2)-negative and ER-positive/HER2-negative subgroups (P < .0001). CONCLUSIONS: Both mRDBN and gRDBN provide prognostic information, particularly in HER2-negative carcinoma; however, mRDBN showed better stratification of RDBN-3 and RDBN-4 patients.

Low Estrogen Receptor (ER)-Positive Breast Cancer and Neoadjuvant Systemic Chemotherapy: Is Response Similar to Typical ER-Positive or ER-Negative Disease?

OBJECTIVES: Pathologic complete response (pCR) rate after neoadjuvant chemotherapy was compared between 141 estrogen receptor (ER)-negative (43%), 41 low ER+ (13%), 47 moderate ER+ (14%), and 98 high ER+ (30%) tumors. METHODS: Human epidermal growth factor receptor 2-positive cases, cases without semiquantitative ER score, and patients treated with neoadjuvant endocrine therapy alone were excluded. RESULTS: The pCR rate of low ER+ tumors was similar to the pCR rate of ER- tumors (37% and 26% for low ER and ER- respectively, P = .1722) but significantly different from the pCR rate of moderately ER+ (11%, P = .0049) and high ER+ tumors (4%, P < .0001). Patients with pCR had an excellent prognosis regardless of the ER status. In patients with residual disease (no pCR), the recurrence and death rate were higher in ER- and low ER+ cases compared with moderate and high ER+ cases. CONCLUSIONS: Low ER+ breast cancers are biologically similar to ER- tumors. Semiquantitative ER H-score is an important determinant of response to neoadjuvant chemotherapy.

Identification of BRCA1 missense substitutions that confer partial functional activity: potential moderate risk variants?

INTRODUCTION: Many of the DNA sequence variants identified in the breast cancer susceptibility gene BRCA1 remain unclassified in terms of their potential pathogenicity. Both multifactorial likelihood analysis and functional approaches have been proposed as a means to elucidate likely clinical significance of such variants, but analysis of the comparative value of these methods for classifying all sequence variants has been limited. METHODS: We have compared the results from multifactorial likelihood analysis with those from several functional analyses for the four BRCA1 sequence variants A1708E, G1738R, R1699Q, and A1708V. RESULTS: Our results show that multifactorial likelihood analysis, which incorporates sequence conservation, co-inheritance, segregation, and tumour immunohistochemical analysis, may improve classification of variants. For A1708E, previously shown to be functionally compromised, analysis of oestrogen receptor, cytokeratin 5/6, and cytokeratin 14 tumour expression data significantly strengthened the prediction of pathogenicity, giving a posterior probability of pathogenicity of 99%. For G1738R, shown to be functionally defective in this study, immunohistochemistry analysis confirmed previous findings of inconsistent 'BRCA1-like' phenotypes for the two tumours studied, and the posterior probability for this variant was 96%. The posterior probabilities of R1699Q and A1708V were 54% and 69%, respectively, only moderately suggestive of increased risk. Interestingly, results from functional analyses suggest that both of these variants have only partial functional activity. R1699Q was defective in foci formation in response to DNA damage and displayed intermediate transcriptional transactivation activity but showed no evidence for centrosome amplification. In contrast, A1708V displayed an intermediate transcriptional transactivation activity and a normal foci formation response in response to DNA damage but induced centrosome amplification. CONCLUSION: These data highlight the need for a range of functional studies to be performed in order to identify variants with partially compromised function. The results also raise the possibility that A1708V and R1699Q may be associated with a low or moderate risk of cancer. While data pooling strategies may provide more information for multifactorial analysis to improve the interpretation of the clinical significance of these variants, it is likely that the development of current multifactorial likelihood approaches and the consideration of alternative statistical approaches will be needed to determine whether these individually rare variants do confer a low or moderate risk of breast cancer.

Mcl-1 mediates tumor necrosis factor-related apoptosis-inducing ligand resistance in human cholangiocarcinoma cells.

Cholangiocarcinomas are usually fatal neoplasms originating from bile duct epithelia. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising agent for cancer therapy, including cholangiocarcinoma. However, many cholangiocarcinoma cells are resistant to TRAIL-mediated apoptosis. Thus, our aim was to examine the intracellular mechanisms responsible for TRAIL resistance in human cholangiocarcinoma cell lines. Three TRAIL-resistant human cholangiocarcinoma cell lines were identified. All of the cell lines expressed TRAIL receptor 1/death receptor 4 (TRAIL-R1/DR4) and TRAIL-R2/DR5. Expression of TRAIL decoy receptors and the antiapoptotic cellular FLICE-inhibitory protein (cFLIP) was inconsistent across the cell lines. Of the antiapoptotic Bcl-2 family of proteins profiled (Bcl-2, Bcl-x(L), and Mcl-1), Mcl-1 was uniquely overexpressed by the cell lines. When small-interfering-RNA (siRNA) technology was used to knock down expression of Bcl-2, Bcl-x(L), and Mcl-1, only the Mcl-1-siRNA sensitized the cells to TRAIL-mediated apoptosis. In a cell line stably transfected with Mcl-1-small-hairpin-RNA (Mcl-1-shRNA), Mcl-1 depletion sensitized cells to TRAIL-mediated apoptosis despite Bcl-2 expression. TRAIL-mediated apoptosis in the stably transfected cells was associated with mitochondrial depolarization, Bax activation, cytochrome c release from mitochondria, and caspase activation. Finally, flavopiridol, an anticancer drug that rapidly down-regulates Mcl-1, also sensitized cells to TRAIL cytotoxicity. In conclusion, these studies not only demonstrate that Mcl-1 mediates TRAIL resistance in cholangiocarcinoma cells by blocking the mitochondrial pathway of cell death but also identify two strategies for circumventing this resistance.

Genotoxic stress leads to centrosome amplification in breast cancer cell lines that have an inactive G1/S cell cycle checkpoint.

Centrosome amplification plays a key role in the origin of chromosomal instability during cancer development and progression. In this study, breast cancer cell lines with different p53 backgrounds were used to investigate the relationship between genotoxic stress, G(1)/S cell cycle checkpoint integrity, and the development of centrosome amplification. Introduction of DNA damage in the MCF-7 cell line by treatment with hydroxyurea (HU) or daunorubicin (DR) resulted in the arrest of both G(1)/S cell cycle progression and centriole duplication. In these cells, which carry functional p53, HU treatment also led to nuclear accumulation of p53 and p21(WAF1), retinoblastoma hypophosphorylation, and downregulation of cyclin A. MCF-7 cells carrying a recombinant dominant-negative p53 mutant (vMCF-7(DNp53)) exhibited a shortened G(1) phase of the cell cycle and retained a normal centrosome phenotype. However, these cells developed amplified centrosomes following HU treatment. The MDA-MB 231 cell line, which carries mutant p53 at both alleles, showed amplified centrosomes at the outset, and developed a hyperamplified centrosome phenotype following HU treatment. In cells carrying defective p53, the development of centrosome amplification also occurred following treatment with another DNA damaging agent, DR. Taken together, these findings demonstrate that loss of p53 function alone is not sufficient to drive centrosome amplification, but plays a critical role in this process following DNA damage through abrogation of the G(1)/S cell cycle checkpoint. Furthermore, these studies have important clinical implications because they suggest that breast cancers with compromised p53 function may develop centrosome amplification and consequent chromosomal instability following treatment with genotoxic anticancer drugs.

Improved Breast Cancer Care Quality Metrics After Implementation of a Standardized Tumor Board Documentation Template.

PURPOSE: Cancer treatment requires a coordinated multidisciplinary treatment approach, which led to the development of the Rapid Quality Reporting System by the Commission on Cancer. However, the lack of immediate availability of documented treatment plans and the inefficiency of global medical record reviews represent significant barriers to adherence reporting and the timely implementation of quality improvement measures. METHODS: Adherence to national guidelines in the areas of radiation treatment, chemotherapy, and hormone therapy was assessed after breast conservation surgery (BCS). Adherence rates within 1 year of BCS were analyzed 10 weeks before and after the implementation of a standardized documentation template at weekly multidisciplinary breast cancer conferences. RESULTS: Documented adherence rates increased postimplementation in patients undergoing consideration for both radiation treatment and hormone therapy within 1 year of BCS (89% v 65%; P = .045% and 85% v 62%; P = .002, respectively). No change was observed in patients undergoing evaluation for cytotoxic chemotherapy (80% v 85%; P = 1.00). CONCLUSION: The addition of a documentation template to multidisciplinary breast cancer conferences resulted in increased recorded adherence rates to national guidelines. This template provided a means of both accurate and efficient documentation of evidence-based practice, which represents a concept with broad application in quality improvement. Although evaluation of the project was not continued beyond the pilot stage, current quality measure scores remain within the same range.

Functional assays for classification of BRCA2 variants of uncertain significance.

The assessment of the influence of many rare BRCA2 missense mutations on cancer risk has proved difficult. A multifactorial likelihood model that predicts the odds of cancer causality for missense variants is effective, but is limited by the availability of family data. As an alternative, we developed functional assays that measure the influence of missense mutations on the ability of BRCA2 to repair DNA damage by homologous recombination and to control centriole amplification. We evaluated 22 missense mutations from the BRCA2 DNA binding domain (DBD) that were identified in multiple breast cancer families using these assays and compared the results with those from the likelihood model. Thirteen variants inactivated BRCA2 function in at least one assay; two others truncated BRCA2 by aberrant splicing; and seven had no effect on BRCA2 function. Of 10 variants with odds in favor of causality in the likelihood model of 50:1 or more and a posterior probability of pathogenicity of 0.99, eight inactivated BRCA2 function and the other two caused splicing defects. Four variants and four controls displaying odds in favor of neutrality of 50:1 and posterior probabilities of pathogenicity of at least 1 x 10(-3) had no effect on function in either assay. The strong correlation between the functional assays and likelihood model data suggests that these functional assays are an excellent method for identifying inactivating missense mutations in the BRCA2 DBD and that the assays may be a useful addition to models that predict the likelihood of cancer in carriers of missense mutations.