Clinicopathologic and genetic analysis of invasive breast carcinomas in women with germline CHEK2 variants.

PURPOSE: Germline pathogenic variants in checkpoint kinase 2 (CHEK2) are associated with a moderately increased risk of breast cancer (BC). The spectrum of clinicopathologic features and genetics of these tumors has not been fully established. METHODS: We characterized the histopathologic and clinicopathologic features of 44 CHEK2-associated BCs from 35 women, and assessed responses to neoadjuvant chemotherapy. A subset of cases (n = 23) was additionally analyzed using targeted next-generation DNA sequencing (NGS). RESULTS: Most (94%, 33/35) patients were heterozygous carriers for germline CHEK2 variants, and 40% had the c.1100delC allele. Two patients were homozygous, and five had additional germline pathogenic variants in ATM (2), PALB2 (1), RAD50 (1), or MUTYH (1). CHEK2-associated BCs occurred in younger women (median age 45 years, range 25-75) and were often multifocal (20%) or bilateral (11%). Most (86%, 38/44) were invasive ductal carcinomas of no special type (IDC-NST). Almost all (95%, 41/43) BCs were ER + (79% ER + HER2-, 16% ER + HER2 + , 5% ER-HER2 +), and most (69%) were luminal B. Nottingham grade, proliferation index, and results of multiparametric molecular testing were heterogeneous. Biallelic CHEK2 alteration with loss of heterozygosity was identified in most BCs (57%, 13/23) by NGS. Additional recurrent alterations included GATA3 (26%), PIK3CA (226%), CCND1 (22%), FGFR1 (22%), ERBB2 (17%), ZNF703 (17%), TP53 (9%), and PPM1D (9%), among others. Responses to neoadjuvant chemotherapy were variable, but few patients (21%, 3/14) achieved pathologic complete response. Most patients (85%) were without evidence of disease at time of study (n = 34). Five patients (15%) developed distant metastasis, and one (3%) died (mean follow-up 50 months). CONCLUSION: Almost all CHEK2-associated BCs were ER + IDC-NST, with most classified as luminal B with or without HER2 overexpression. NGS supported the luminal-like phenotype and confirmed CHEK2 as an oncogenic driver in the majority of cases. Responses to neoadjuvant chemotherapy were variable but mostly incomplete.

Targeted DNA Sequencing in Diagnosis of Malignant Phyllodes Tumors With Emphasis on Tumors With Keratin and p63 Expression.

The differential diagnosis of malignant spindle cell neoplasms in the breast most frequently rests between malignant phyllodes tumor (MPT) and metaplastic carcinoma (MBC). Diagnosis of MPT can be challenging due to diffuse stromal overgrowth, keratin (CK) and/or p63 immunopositivity, and absent CD34 expression, which can mimic MBC, especially in core biopsies. Distinction of MPT from MBC has clinical implications, with differences in surgical approach, chemotherapy, and radiation. In this study, we evaluated MPTs (78 tumors, 64 patients) for stromal CK, p63, and CD34 expression and profiled a subset (n = 31) by targeted next-generation DNA sequencing, with comparison to MBC (n = 44). Most MPTs (71%) were CK+ and/or p63+, including 32% CK+ (25/77 focal) and 65% p63+ (32/66 focal, 10/66 patchy, and 1/66 diffuse). Thirty percent of MPTs expressed both CK and p63 (20/66), compared with 95% of MBCs (40/42, P < .001). CK and/or p63 were positive in CD34+ and CD34- MPTs. Recurrent genetic aberrations in MPTs involved TERT, TP53, MED12, CDKN2A, chromatin modifiers, growth factor receptors/ligands, and phosphoinositide-3 kinase (PI-3K) and MAPK pathway genes. Only MED12 (39%, 12/31) and SETD2 (13%, 4/31) were exclusively mutated in MPTs and not MBCs (P < .001 and P = .044, respectively), whereas PIK3R1 mutations were only found in MBCs (37%, 13/35, P < .001). Comparative literature review additionally identified ARID1B, EGFR, FLNA, NRAS, PDGFRB, RAD50, and RARA alterations enriched or exclusively in MPTs vs MBCs. MED12 was mutated in MPTs with diffuse stromal overgrowth (53%, 9/17), CD34- MPTs (41%, 7/17), and CK+ and/or p63+ MPTs (39%, 9/23), including 36% of CD34- MPTs with CK and/or p63 expression. Overall, MED12 mutation and/or CD34 expression were observed in 68% (21/31) MPTs, including 61% (14/23) of CK+ and/or p63+ tumors. Our results emphasize the prevalence of CK and p63 expression in MPTs and demonstrate the diagnostic utility of next-generation DNA sequencing, especially in MPTs with confounding factors that can mimic MBC.

RB1 Genetic Alterations in Estrogen Receptor-Positive Breast Carcinomas: Correlation With Neuroendocrine Differentiation.

Genetic alterations in the retinoblastoma susceptibility gene (RB1) are present in up to 40% of triple-negative breast cancers (BCs) and frequent in tumors with neuroendocrine differentiation, including small cell neuroendocrine carcinoma. Data on RB1 genetic alterations in estrogen receptor (ER)-positive BCs are scarce. In this study, we sought to define the morphologic, immunohistochemical, and genetic features of ER-positive BCs harboring somatic alterations in RB1, with emphasis on neuroendocrine differentiation. ER-positive BCs with pathogenic RB1 genetic alterations were identified in <1% of cases (N = 55) from a cohort of 6026 BCs previously subjected to targeted next-generation sequencing, including 23 primary BCs (pBCs) and 32 recurrent/metastatic BCs (mBCs). In cases where loss of heterozygosity of the wild-type RB1 allele could be assessed (93%, 51/55), most pBCs (82%, 18/22) and mBCs (90%, 26/29) exhibited biallelic RB1 inactivation, primarily through loss-of-function mutation and loss of heterozygosity (98%, 43/44). Upon histologic review, a subset of RB1-altered tumors exhibited neuroendocrine morphology (13%, 7/55), which correlated with expression of neuroendocrine markers (39%, 9/23) in both pBCs (27%, 3/11) and mBCs (50%, 6/12). Loss of Rb protein expression was observed in BCs with biallelic RB1 loss only, with similar frequency in pBCs (82%, 9/11) and mBCs (75%, 9/12). All cases with neuroendocrine marker expression (n = 9) and/or neuroendocrine morphology (n = 7) harbored biallelic genetic inactivation of RB1 and exhibited Rb loss of expression. TP53 (53%, 29/55) and PIK3CA (45%, 25/55) were the most frequently comutated genes across the cohort. Overall, these findings suggest that ER-positive BCs with biallelic RB1 genetic alterations frequently exhibit Rb protein loss, which correlates with neuroendocrine differentiation in select BCs. This study provides insights into the molecular and phenotypic heterogeneity of BCs with RB1 genetic inactivation, underscoring the need for further research into the potential clinical implications associated with these tumors.

Malignant phyllodes tumour with lymph node metastasis: a diagnostic conundrum resolved by next generation DNA sequencing.

A malignant neoplasm with spindle cell and chondroid differentiation in the breast, metastatic to lymph node. In this context, a metaplastic carcinoma is typically favored given the exceptional nature of lymph node metastases in malignant phyllodes tumors (MPT). However, we demonstrate pathognomonic hotspot mutations in MED12 and the promoter of the TERT gene by targeted next-generation DNA sequencing, supporting a diagnosis of MPT.

Salivary gland-like tumors of the breast: Histopathologic and genetic features with clinical implications.

Salivary gland-like tumors of the breast are rare neoplasms that share morphologic, immunophenotypic, and/or genetic features with their salivary gland counterparts, highlighting a shared underlying histopathogenesis in most cases. Salivary gland-like carcinomas included in the World Health Organization classification of breast tumors are adenoid cystic carcinoma, secretory carcinoma, mucoepidermoid carcinoma, acinic cell carcinoma, and the exceedingly rare polymorphous adenocarcinoma. These carcinomas are usually triple negative for estrogen receptor and progesterone receptor expression and HER2 overexpression, yet generally have favorable prognosis, in contrast to high-grade triple negative carcinomas of no special type. On the other hand, a small subset, such as solid-basaloid adenoid cystic carcinoma, rare high-grade carcinomas, and those associated with transformation to other types of high-grade invasive carcinoma can behave more aggressively. Other salivary gland-like tumors of the breast, such as pleomorphic adenoma and adenomyoepithelioma, are usually benign but can rarely undergo malignant transformation. Although clinical experience with salivary gland-like breast tumors is overall limited, their recognition and accurate classification has important implications for prognosis and clinical management, especially to avoid overtreatment of salivary gland-like carcinomas. The identification of characteristic genetic alterations and/or immunohistochemical surrogates in many of these tumors has practical applications to establishing an accurate diagnosis and directing clinical management. This review highlights the histopathologic and genetic characteristics of salivary gland-like breast tumors and the implications of the diagnosis for current clinical management.

HER-2 low status in early-stage invasive lobular carcinoma of the breast: associated factors and outcomes in an institutional series.

PURPOSE: HER2 overexpression has a central role in breast cancer carcinogenesis and is associated with poor prognosis if untreated. Lately, identification of HER2-low breast cancer has been proposed to select patients for novel HER2-directed chemotherapy and includes cancers with immunohistochemistry 1 + or 2 + with negative FISH, encompassing approximately 55-60% of all breast carcinomas. In early-stage breast cancer, the prognostic significance of HER2 low-disease is less well understood, with a particular paucity of data evaluating the prevalence and implications of HER2-low status in invasive lobular carcinoma (ILC). METHODS: We evaluated 666 stage I-III ILC tumors from a prospectively maintained institutional database, comparing clinicopathologic features and disease-free survival (DFS) using a multivariable Cox proportional hazards model. RESULTS: HER2-low status was common in this cohort of patients with ILC, but most clinicopathologic features did not differ between HER2-low and HER2-negative cases. However, when adjusting for tumor size, number of positive nodes, ER/PR status, and local therapy received, patients with HER2-low status had worse disease-free survival (DFS) than those with HER2-negative tumors (hazard ratio 2.0, 95% confidence interval 1.0-4.1, p = 0.05). CONCLUSION: This difference in DFS supports the notion that HER2-low and HER2-negative early stage ILC may differ clinically, despite similar clinicopathologic features. Further investigation into the potential benefit of HER2 targeted therapy in HER2-low early-stage breast cancer, and specifically lobular cancer, is warranted to ensure optimal outcomes in this distinct tumor subtype.

Quantifying hormone receptor status in lobular breast cancer in an institutional series: the relationship between estrogen and progesterone receptor status and outcomes.

PURPOSE: Recent guidelines defined a new reporting category of ER-low-positive breast cancer based on immunohistochemistry (IHC). While low positivity of either hormone receptor is uncommon in invasive lobular carcinoma (ILC), we sought to investigate whether relatively low hormone receptor positivity was associated with tumor characteristics and patient outcomes in a single institutional cohort. METHODS: We searched an institutional database for cases of stage I-III ILC with available IHC reports. Based on prior published categories in ILC, ER was classified as low, medium, or high as defined by ER staining of 10-69%, 70-89%, and ≥ 90% respectively. PR low and high tumors were defined by < 20%, or ≥ 20% staining respectively. We used chi-squared tests, t-tests, and Cox proportional hazards models to evaluate associations between ER/PR categories and tumor characteristics or disease-free survival (DFS). RESULTS: The cohort consisted of 707 ILC cases, with 11% of cases categorized as ER low, 15.1% as medium, and 73.8% as high. The majority (67.6%) were PR high. Patients with ER low/medium expression were significantly younger, and more likely to also have PR low and/or HER2 positive tumors compared to those that were ER high. In a Cox proportional hazards model adjusting for age, stage, grade, pleomorphic histology, and treatment, ER category was not prognostic for DFS, but PR negative and PR low status each had significantly worse DFS compared to PR high status (HR 3.5, 95% CI 1.8-6.7, p < 0.001; and HR 2.0, 95% CI 1.1-3.5, p = 0.015, respectively). CONCLUSION: These findings highlight the relevance of quantifying ER and PR within ILC.

Solid-Basaloid Adenoid Cystic Carcinoma of the Breast: An Aggressive Subtype Enriched for Notch Pathway and Chromatin Modifier Mutations With MYB Overexpression.

Adenoid cystic carcinoma (AdCC) is a rare triple-negative breast cancer analogous to its extramammary counterparts. Diagnosis of the more aggressive solid-basaloid variant of AdCC (SB-AdCC) can be challenging due to poorly defined histopathologic and molecular features. We characterized 22 invasive and in situ basaloid carcinomas by morphology, immunohistochemistry, genetics, and MYB status using multiple platforms and assessed clinical behavior and neoadjuvant chemotherapy responses. After consensus review, 16/22 cases were classified as SB-AdCC. All SB-AdCC had predominantly solid growth and at least focal myxohyaline stroma and were immune-poor. Eosinophilic squamoid cells (69%, 11/16) and basement membrane-like secretions (69%, 11/16) were common, and intercalated ducts (31%, 5/16) were less frequent. SB-AdCC typically expressed SOX10 (100%, 16/16) and luminal markers (100%, 16/16 CK7; 88%, 14/16 CD117; 93%, 13/14 CAM5.2). SMA (40%, 6/15) expression was less common, and SMM (27%, 3/11), GATA3 (20%, 3/15), and p63 (25%, 4/16) were mostly negative. MYB protein and/or MYB RNA overexpression was universal in evaluable cases (13/13), with RNA in situ hybridization (10/10) more reliable than immunohistochemistry (10/11, plus 4 excisions inconclusive). Fluorescence in situ hybridization and/or next-generation sequencing identified MYB rearrangements (20%, 3/15) and amplifications/copy gains (60%, 9/15) but no MYB::NFIB fusions. SB-AdCC often had aberrations in Notch pathway (60%, including 40% NOTCH1 and 20% NOTCH2) and/or chromatin modifier (60%, including 33% CREBBP) genes, with relatively infrequent TP53 mutations (27%). Unclassified invasive basaloid carcinomas lacking described histologic features of SB-AdCC (n = 4) and basaloid ductal carcinoma in situ (n = 2) showed similar immunoprofiles and genetics as SB-AdCC, including Notch aberrations and MYB overexpression with MYB rearrangements/amplifications. Overall, nodal (22%) and distant (33%) metastases were common, and 23% of patients died of disease (mean follow-up, 35 months; n = 22). Responses were poor in all 7 neoadjuvant chemotherapy-treated patients, without any achieving pathologic complete response. The data highlight the histopathologic spectrum of basaloid carcinomas including SB-AdCC and reveal shared genetics and MYB activation, which can be diagnostically useful. Aggressive behavior and poor treatment responses emphasize a need for additional treatment approaches.

Triple-Negative Apocrine Carcinomas: Toward a Unified Group With Shared Molecular Features and Clinical Behavior.

Triple-negative apocrine carcinomas (TNACs) are rare breast tumors with limited studies evaluating their molecular characteristics and clinical behavior. We performed a histologic, immunohistochemical, genetic, and clinicopathologic assessment of 42 invasive TNACs (1 with a focal spindle cell component) from 41 patients, 2 pure apocrine ductal carcinomas in situ (A-DCIS), and 1 A-DCIS associated with spindle cell metaplastic carcinoma (SCMBC). All TNACs had characteristic apocrine morphology and expressed androgen receptor (42/42), gross cystic disease fluid protein 15 (24/24), and CK5/6 (16/16). GATA3 was positive in most cases (16/18, 89%), and SOX10 was negative (0/22). TRPS1 was weakly expressed in a minority of tumors (3/14, 21%). Most TNACs had low Ki67 proliferation (≤10% in 67%, 26/39), with a median index of 10%. Levels of tumor infiltrating lymphocytes were low (≤10% in 93%, 39/42, and 15% in 7%, 3/42). Eighteen percent of TNACs presented with axillary nodal metastasis (7/38). No patients treated with neoadjuvant chemotherapy achieved pathologic complete response (0%, 0/10). Nearly all patients with TNAC (97%, n = 32) were without evidence of disease at the time of study (mean follow-up of 62 months). Seventeen invasive TNACs and 10 A-DCIS (7 with paired invasive TNAC) were profiled by targeted capture-based next-generation DNA sequencing. Pathogenic mutations in phosphatidylinositol 3-kinase pathway genes PIK3CA (53%) and/or PIK3R1 (53%) were identified in all TNACs (100%), including 4 (24%) with comutated PTEN. Ras-MAPK pathway genes, including NF1 (24%), and TP53 were mutated in 6 tumors each (35%). All A-DCIS shared mutations, such as phosphatidylinositol 3-kinase aberrations and copy number alterations with paired invasive TNACs or SCMBC, and a subset of invasive carcinomas showed additional mutations in tumor suppressors (NF1, TP53, ARID2, and CDKN2A). Divergent genetic profiles between A-DCIS and invasive carcinoma were identified in 1 case. In summary, our findings support TNAC as a morphologically, immunohistochemically, and genetically homogeneous subgroup of triple-negative breast carcinomas and suggest overall favorable clinical behavior.

The clinical behavior and genomic features of the so-called adenoid cystic carcinomas of the solid variant with basaloid features.

Classic adenoid cystic carcinomas (C-AdCCs) of the breast are rare, relatively indolent forms of triple negative cancers, characterized by recurrent MYB or MYBL1 genetic alterations. Solid and basaloid adenoid cystic carcinoma (SB-AdCC) is considered a rare variant of AdCC yet to be fully characterized. Here, we sought to determine the clinical behavior and repertoire of genetic alterations of SB-AdCCs. Clinicopathologic data were collected on a cohort of 104 breast AdCCs (75 C-AdCCs and 29 SB-AdCCs). MYB expression was assessed by immunohistochemistry and MYB-NFIB and MYBL1 gene rearrangements were investigated by fluorescent in-situ hybridization. AdCCs lacking MYB/MYBL1 rearrangements were subjected to RNA-sequencing. Targeted sequencing data were available for 9 cases. The invasive disease-free survival (IDFS) and overall survival (OS) were assessed in C-AdCC and SB-AdCC. SB-AdCCs have higher histologic grade, and more frequent nodal and distant metastases than C-AdCCs. MYB/MYBL1 rearrangements were significantly less frequent in SB-AdCC than C-AdCC (3/14, 21% vs 17/20, 85% P < 0.05), despite the frequent MYB expression (9/14, 64%). In SB-AdCCs lacking MYB rearrangements, CREBBP, KMT2C, and NOTCH1 alterations were observed in 2 of 4 cases. SB-AdCCs displayed a shorter IDFS than C-AdCCs (46.5 vs 151.8 months, respectively, P < 0.001), independent of stage. In summary, SB-AdCCs are a molecularly heterogeneous but clinically aggressive group of tumors. Less than 25% of SB-AdCCs display the genomic features of C-AdCC. Defining whether these tumors represent a single entity or a collection of different cancer types with a similar basaloid histologic appearance is warranted.

Histologic and genomic features of breast cancers with alterations affecting the SWI/SNF (SMARC) genes.

The SWI/SNF family of proteins is a multisubunit ATPase complex frequently altered in human cancer. Inactivating mutations in SWI/SNF-related matrix-associated actin-dependent regulator of chromatin (SMARCs) underpin a subset of tumors such as the malignant rhabdoid tumor and small cell carcinoma of the ovary, hypercalcemic type. Here, we investigated the genotypic and phenotypic characteristics of breast cancers harboring somatic genetic alterations affecting genes of the SMARC family. We analyzed a series of 6026 primary and metastatic breast cancers subjected to targeted-capture sequencing. SMARC core subunit (SMARCA4, SMARCB1, and SMARCA2) alterations were identified in <1% of all breast cancers, consisting of 27 primary and 30 recurrent/metastatic tumors. The majority of SMARC alterations were monoallelic mutations (47/57, 82%) and thus categorized into two groups: Class 1 alterations consisting of potentially pathogenic mutations and rearrangements and Class 2 alterations consisting of missense mutations and small in-frame deletions of unknown significance. Biallelic events in a SMARC gene were present in a minority of cases (10/57, 18%). Histologic patterns in the form of rhabdoid, composite rhabdoid, sarcomatoid or anaplastic features were observed in a subset of Class 1 primary and metastatic tumors (7/57, 12%). SMARC protein was preserved in nearly all tumors analyzed with immunohistochemistry (26/30, 87%). Four Class 1 tumors demonstrated altered SMARC protein expression in the form of loss (1/30, 3%) or mosaic pattern (3/30, 10%). Complete loss of SMARCA2 (BRM) was observed in a sole tumor with composite rhabdoid morphology, and biallelic hits in the SMARCA2 gene. The genomic landscape of both primary Class 1 and 2 breast cancers did not reveal any characteristic findings. In summary, SMARC alterations likely contribute to the biology of a rare subset of breast cancers in the form of biallelic or pathogenic alterations in SMARC, as evidenced by SMARC-deficient phenotype or altered expression of SMARC protein.

Gynaecological perivascular epithelioid cell tumour (PEComa): comparative analysis of proposed algorithms for prediction of clinical outcome.

AIMS: Perivascular epithelioid cell tumours (PEComas) are rare mesenchymal tumours that coexpress smooth muscle and melanocytic markers. They have a predilection for gynaecological organs, where they present a unique diagnostic challenge, because of morphological and immunohistochemical overlap with more common smooth muscle and stromal tumours. Limited information regarding the natural history, owing to the rarity of this tumour, makes accurate risk stratification difficult. We aimed to review clinicopathological features of gynaecological PEComa and compare accuracy of five different classification systems for prediction of prognosis. METHODS AND RESULTS: We have described the clinicopathological features of 13 new cases and tested five prognostic algorithms in a total of 67 cases of gynaecological PEComa. Receiver operating characteristic curves were constructed and areas under the curve (AUCs) were calculated to evaluate predictive accuracy. The modified gynaecological-specific algorithm showed high sensitivity and specificity and yielded the highest AUC (0.864). It's earlier version, the gynaecological-specific algorithm, suffered from lower specificity (AUC = 0.843). The post-hoc McNemar test confirmed significant differences between the performances of the modified gynaecological-specific algorithm and the gynaecological-specific algorithm (P = 0.008). The original Folpe algorithm for PEComas of all sites showed low specificity, had a lower AUC (0.591), and was inapplicable in 18% of cases. Its two later versions (the revised Folpe algorithm and the modified Folpe algorithm) also yielded lower AUCs (0.690 and 0.591, respectively). CONCLUSION: We have shown that the modified gynaecological-specific algorithm predicts the clinical outcome of gynaecological PEComa with high accuracy, and have validated its use for prognostic stratification of gynaecological PEComa.

Sympathetic neural responsiveness to sleep deprivation in older adults: sex differences.

Our laboratory has previously reported that total sleep deprivation (TSD) modifies muscle sympathetic neural activity (MSNA) differently in young men and women. Because postmenopausal women are among the highest risk for hypertension, this study compares MSNA responses with TSD in older men and women. We hypothesized that TSD would alter MSNA in older adults, with greater sympathoexcitation in postmenopausal women. Twenty-seven participants (14 men and 13 women) between the ages of 55 and 75 yr were tested twice, once after 24-h TSD and once after normal sleep (randomized, crossover design). Our primary outcome measure of MSNA (microneurography) was successful across both conditions in 20 participants (10 men and 10 women). Secondary outcome measures included seated blood pressure, heart rate, and fasting plasma testosterone, estradiol, and progesterone. Age (60 ± 1 vs. 61 ± 2 yr) and BMI (27 ± 1 vs. 26 ± 1 kg/m2) were not different between groups. TSD increased systolic blood pressure in both men (124 ± 5 to 130 ± 4 mmHg) and women (107 ± 5 to 116 ± 4 mmHg), but the increases were not different between groups (condition, P = 0.014; condition × sex, P > 0.05). In contrast, TSD elicited divergent MSNA responses in older men and women. Specifically, MSNA burst frequency increased in postmenopausal women (28 ± 3 to 34 ± 3 burst/min), but not older men (38 ± 3 to 35 ± 3 bursts/min; condition × sex, P = 0.032). In conclusion, TSD elicited sympathoexcitation in postmenopausal women but not age-matched men. These findings provide new mechanistic insight into reported links between sleep deprivation and hypertension.NEW & NOTEWORTHY Epidemiological studies report that sleep deprivation is more strongly associated with hypertension in women than in men. In the present study, 24-h total sleep deprivation (TSD) increased blood pressure in postmenopausal women and age-matched men. In contrast, only women demonstrated increases in muscle sympathetic nerve activity after TSD. The sympathoexcitation observed in postmenopausal women suggests a potential contributing mechanism for epidemiological observations and advances our understanding of the complex relations between sleep, sex, and hypertension.

Validating genome-wide CRISPR-Cas9 function improves screening in the oleaginous yeast Yarrowia lipolytica.

Genome-wide mutational screens are central to understanding the genetic underpinnings of evolved and engineered phenotypes. The widespread adoption of CRISPR-Cas9 genome editing has enabled such screens in many organisms, but identifying functional sgRNAs still remains a challenge. Here, we developed a methodology to quantify the cutting efficiency of each sgRNA in a genome-scale library, and in doing so improve screens in the biotechnologically important yeast Yarrowia lipolytica. Screening in the presence and absence of native DNA repair enabled high-throughput quantification of sgRNA function leading to the identification of high efficiency sgRNAs that cover 94% of genes. Library validation enhanced the classification of essential genes by identifying inactive guides that create false negatives and mask the effects of successful disruptions. Quantification of guide effectiveness also creates a dataset from which determinants of CRISPR-Cas9 can be identified. Finally, application of the library identified novel mutations for metabolic engineering of high lipid accumulation.

Impact of biomarkers and genetic profiling on breast cancer prognostication: A comparative analysis of the 8th edition of breast cancer staging system.

The 8th edition of the American Joint Committee on Cancer (AJCC) staging guidelines combine traditional TNM system with biomarkers to reflect our current understanding of tumor biology and targeted therapy. In this study, we investigated the impact of the TNM + Biomarkers staging system and the additive value of Oncotype Dx™ genomic profile recurrence score (RS) (TNM + Biomarkers+RS <11) for the staging of breast cancer (BC) using data from two tertiary referral cancer centers. Compared to TNM alone, the TNM + Biomarkers system changed the stage group in 32.7% of BCs (27% downstage, 5.7% upstage). Most (98.3%) of the downstaged BCs were estrogen receptor (ER)+/progesterone receptor (PR)+, whereas 78% of the upstaged BCs were ER-/PR-/human epidermal growth factor receptor 2 (HER2)-. Compared to TNM + Biomarkers staging, the addition of genetic profile data (TNM + Biomarker+RS <11) downstaged only <1% BCs. Our analysis suggests that for T1-T2N0 ER+/HER2- BCs, Oncotype Dx™ RS <11 provides added value as a staging parameter only in a very small group of cases compared to TNM + Biomarkers alone.

Natural allelic variation underlying a major fitness trade-off in Arabidopsis thaliana.

Plants can defend themselves against a wide array of enemies, from microbes to large animals, yet there is great variability in the effectiveness of such defences, both within and between species. Some of this variation can be explained by conflicting pressures from pathogens with different modes of attack. A second explanation comes from an evolutionary 'tug of war', in which pathogens adapt to evade detection, until the plant has evolved new recognition capabilities for pathogen invasion. If selection is, however, sufficiently strong, susceptible hosts should remain rare. That this is not the case is best explained by costs incurred from constitutive defences in a pest-free environment. Using a combination of forward genetics and genome-wide association analyses, we demonstrate that allelic diversity at a single locus, ACCELERATED CELL DEATH 6 (ACD6), underpins marked pleiotropic differences in both vegetative growth and resistance to microbial infection and herbivory among natural Arabidopsis thaliana strains. A hyperactive ACD6 allele, compared to the reference allele, strongly enhances resistance to a broad range of pathogens from different phyla, but at the same time slows the production of new leaves and greatly reduces the biomass of mature leaves. This allele segregates at intermediate frequency both throughout the worldwide range of A. thaliana and within local populations, consistent with this allele providing substantial fitness benefits despite its marked impact on growth.

Interaction of photoperiod and vernalization determines flowering time of Brachypodium distachyon.

Timing of flowering is key to the reproductive success of many plants. In temperate climates, flowering is often coordinated with seasonal environmental cues such as temperature and photoperiod. Vernalization is an example of temperature influencing the timing of flowering and is defined as the process by which a prolonged exposure to the cold of winter results in competence to flower during the following spring. In cereals, three genes (VERNALIZATION1 [VRN1], VRN2, and FLOWERING LOCUS T [FT]) have been identified that influence the vernalization requirement and are thought to form a regulatory loop to control the timing of flowering. Here, we characterize natural variation in the vernalization and photoperiod responses in Brachypodium distachyon, a small temperate grass related to wheat (Triticum aestivum) and barley (Hordeum vulgare). Brachypodium spp. accessions display a wide range of flowering responses to different photoperiods and lengths of vernalization. In addition, we characterize the expression patterns of the closest homologs of VRN1, VRN2 (VRN2-like [BdVRN2L]), and FT before, during, and after cold exposure as well as in different photoperiods. FT messenger RNA levels generally correlate with flowering time among accessions grown in different photoperiods, and FT is more highly expressed in vernalized plants after cold. VRN1 is induced by cold in leaves and remains high following vernalization. Plants overexpressing VRN1 or FT flower rapidly in the absence of vernalization, and plants overexpressing VRN1 exhibit lower BdVRN2L levels. Interestingly, BdVRN2L is induced during cold, which is a difference in the behavior of BdVRN2L compared with wheat VRN2 during cold.

Design and implementation of practical bidirectional texture function measurement devices focusing on the developments at the University of Bonn.

Understanding as well as realistic reproduction of the appearance of materials play an important role in computer graphics, computer vision and industry. They enable applications such as digital material design, virtual prototyping and faithful virtual surrogates for entertainment, marketing, education or cultural heritage documentation. A particularly fruitful way to obtain the digital appearance is the acquisition of reflectance from real-world material samples. Therefore, a great variety of devices to perform this task has been proposed. In this work, we investigate their practical usefulness. We first identify a set of necessary attributes and establish a general categorization of different designs that have been realized. Subsequently, we provide an in-depth discussion of three particular implementations by our work group, demonstrating advantages and disadvantages of different system designs with respect to the previously established attributes. Finally, we survey the existing literature to compare our implementation with related approaches.

Disruption of phase synchronization between blood pressure and muscle sympathetic nerve activity in postural vasovagal syncope.

Withdrawal of muscle sympathetic nerve activity (MSNA) may not be necessary for the precipitous fall of peripheral arterial resistance and arterial pressure (AP) during vasovagal syncope (VVS). We tested the hypothesis that the MSNA-AP baroreflex entrainment is disrupted before VVS regardless of MSNA withdrawal using the phase synchronization between blood pressure and MSNA during head-up tilt (HUT) to measure reflex coupling. We studied eight VVS subjects and eight healthy control subjects. Heart rate, AP, and MSNA were measured during supine baseline and at early, mid, late, and syncope stages of HUT. Phase synchronization indexes, measuring time-dependent differences between MSNA and AP phases, were computed. Directionality indexes, indicating the influence of AP on MSNA (neural arc) and MSNA on AP (peripheral arc), were computed. Heart rate was greater in VVS compared with control subjects during early, mid, and late stages of HUT and significantly declined at syncope (P = 0.04). AP significantly decreased during mid, late, and syncope stages of tilt in VVS subjects only (P = 0.001). MSNA was not significantly different between groups during HUT (P = 0.700). However, the phase synchronization index significantly decreased during mid and late stages in VVS subjects but not in control subjects (P < .001). In addition, the neural arc was significantly affected more than the peripheral arc before syncope. In conclusion, VVS is accompanied by a loss of the synchronous AP-MSNA relationship with or without a loss in MSNA at faint. This provides insight into the mechanisms behind the loss of vasoconstriction and drop in AP independent of MSNA at the time of vasovagal faint.