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Purpose:Breast cancers with BRCA1/2 alterations have a relatively high mutational load, suggesting that immune checkpoint blockade may be a potential treatment option. However, the degree of immune cell infiltration varies widely, and molecular features contributing to this variability remain unknown.Experimental Design:We hypothesized that genomic signatures might predict immunogenicity in BRCA1/2 breast cancers. Using The Cancer Genome Atlas (TCGA) genomic data, we compared breast cancers with (89) and without (770) either germline or somatic BRCA1/2 alterations. We also studied 35 breast cancers with germline BRCA1/2 mutations from Penn using WES and IHC.Results:We found that homologous recombination deficiency (HRD) scores were negatively associated with expression-based immune indices [cytolytic index (P = 0.04), immune ESTIMATE (P = 0.002), type II IFN signaling (P = 0.002)] despite being associated with a higher mutational/neoantigen burden, in BRCA1/2 mutant breast cancers. Further, absence of allele-specific loss of heterozygosity (LOH negative; P = 0.01) or subclonality (P = 0.003) of germline and somatic BRCA1/2 mutations, respectively, predicted for heightened cytolytic activity. Gene set analysis found that multiple innate and adaptive immune pathways that converge on NF-κB may contribute to this heightened immunogenicity. IHC of Penn breast cancers demonstrated increased CD45+ (P = 0.039) and CD8+ infiltrates (P = 0.037) and increased PDL1 expression (P = 0.012) in HRD-low or LOH-negative cancers. Triple-negative cancers with low HRD had far greater CD8+ T cells (P = 0.0011) and Perforin 1 expression (P = 0.014) compared with hormone receptor-positive HRD-high cancers.Conclusions:HRD scores and hormone receptor subtype are predictive of immunogenicity in BRCA1/2 breast cancers and may inform the design of optimal immune therapeutic strategies.

Supplementary Figures S1-S4, Tables S4, S6.

Characteristics of breast cancers ascertained from Penn Medicine hospitals

The tumor microenvironment (TME) plays important roles in tumor progression, therapy response and patient survival. In this comprehensive study, we performed a detailed multiomic single cell analysis of breast cancers with inherited mutations in different genes (BRCA1, BRCA2, PALB2, ATM, CHEK2), and wild type mutation negative (WT) patients, stratified by hormone receptor status, to understand the relationship between inherited mutation and immunogenicity. Using spatial tissue multiplexing (PhenoCycler), single cell RNAseq (10X Genomics) and bulk RNAseq, we evaluated cellular markers within the TME associated with DNA damage as well as phenotypes with tumor suppressive (M1, CD8 T cells, NK cells) or tumor promotive (M2, Treg, CAF) properties. We developed a 43-plex antibody panel and characterized eight tissue microarrays containing 443 patient samples from mutation carriers (BRCA1: n=142, BRCA2: n=118, CHEK2: n=2, TP53: n=3, PALB2: n=7) and WT tumors (n=171). We addressed how the TME differs in mutation carriers versus WT tumors stratified by receptor status. To further investigate immune cell function (cytolytic function, exhaustion, immune checkpoint) as well as pathways involved in immune suppression and/or promotion, we performed scRNA sequencing using isolated single cells from fresh collected BC samples (WT ER: n=3, BRCA2 ER: n=1, CHEK2 ER: n=1, VUS ER: n=1) and bulk RNA (BRCA1: n=17, BRCA2: n=9). To determine immune cell (IC) frequency, we analyzed CD45+ cells detectable in each cohort and their co-expression of markers to determine specific phenotypes. Our data suggest that within BRCA mutation-associated BC, there are two groups: 1) with increased tumor infiltrating lymphocytes (TILs) including CD8+ cytotoxic T cells with high cytolytic function; and 2) with decreased TILs and cytolytic function. The frequency of CD8+ T cells is significantly decreased in breast cancers with BRCA2 mutations compared to BRCA1. Characterizing ER+ CHEK2 BC reveals T cell exhaustion compared to WT ER+ BC. Furthermore, preliminary scRNA results reveal increased fibroblast markers in ER+ CHEK2 and ER+ WT compared to ER+ BRCA2 tumors. Interestingly, cancer associated fibroblast (CAFs) markers (ACTA2, COL1A1, FAP, PDGFRA, PDGFRB, PDPN, THY1) as well as TAMs (CCL2, CD163, CD206, CD68, IL10, LOX, PLOD2, SIGLEC1) are increased in ER+ CHEK2 tumors compared to ER+ WT. These data could provide insights into how the TME associated with ER+ CHEK2 mutations might be driven by CAFs and TAMs with a frequency much higher than in ER+ WT. The detection of immune suppressive (Treg, M2) and immune promotive (M1) phenotypes differs in BRCA1 compared to BRCA2 mutated and WT TNBC. Our findings could decipher the role of DNA repair gene mutations and their effect on cells within the TME that might influence the design of treatment options in patients with inherited mutations based on their receptor status. Citation Format: Dana Pueschl, Derek A. Oldridge, Jonathan Belman, William Chandler, Anupma Nayak, Bradley Wubbenhorst, John Pluta, Michael Feldman, E. John Wherry, Heather Thorne, Georgia Chenevix-Trench, Kathleen Cuningham Foundation Consortium for Resea kConFab, Susan M. Domchek, Katherine L. Nathanson. How inherited mutations affect single cells within the tumor microenvironment in breast tumors stratified by receptor status. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4648.

PURPOSE Ovarian cancers can exhibit a prominent immune infiltrate, but clinical trials have not demonstrated substantive response rates to immune checkpoint blockade monotherapy. We aimed to understand genomic features associated with immunogenicity in BRCA1/2 mutation–associated cancers. MATERIALS AND METHODS Using the Cancer Genome Atlas whole-exome sequencing, methylation, and expression data, we analyzed 66 ovarian cancers with either germline or somatic loss of BRCA1/2 and whole-exome sequencing, immunohistochemistry, and CyTOF in 20 ovarian cancers with germline BRCA1/2 pathogenic variants from Penn. RESULTS We found two groups of BRCA1/2 ovarian cancers differing in their immunogenicity: (1) 37 tumors significantly enriched for PTEN loss (11, 30%) and BRCA1 promoter–hypermethylated (10, 27%; P = .0016) and (2) PTEN wild-type (28 of 29 tumors) cancers, with the latter group having longer overall survival (OS; P = .0186, median OS not reached v median OS = 66.1 months). BRCA1/2-mutant PTEN loss and BRCA1 promoter–hypermethylated cancers were characterized by the decreased composition of lymphocytes estimated by gene expression ( P = .0030), cytolytic index ( P = .034), and cytokine expression but higher homologous recombination deficiency scores ( P = .00013). Large-scale state transitions were the primary discriminating feature ( P = .001); neither mutational burden nor neoantigen burden could explain differences in immunogenicity. In Penn tumors, PTEN loss and high homologous recombination deficiency cancers exhibited fewer CD3+ ( P = .05), CD8+ ( P = .012), and FOXP3+ ( P = .0087) T cells; decreased PRF1 expression ( P = .041); and lower immune costimulatory and inhibitory molecule expression. CONCLUSION Our study suggests that within ovarian cancers with genetic loss of BRCA1/2 are two subsets exhibiting differential immunogenicity, with lower levels associated with PTEN loss and BRCA hypermethylation. These genomic features of BRCA1/2-associated ovarian cancers may inform considerations around how to optimally deploy immune checkpoint inhibitors in the clinic.

Purpose: Breast cancers with BRCA1/2 alterations have a relatively high mutational load, suggesting that immune checkpoint blockade may be a potential treatment option. However, the degree of immune cell infiltration varies widely, and molecular features contributing to this variability remain unknown. Experimental Design: We hypothesized that genomic signatures might predict immunogenicity in BRCA1/2 breast cancers. Using The Cancer Genome Atlas (TCGA) genomic data, we compared breast cancers with (89) and without (770) either germline or somatic BRCA1/2 alterations. We also studied 35 breast cancers with germline BRCA1/2 mutations from Penn using WES and IHC. Results: We found that homologous recombination deficiency (HRD) scores were negatively associated with expression-based immune indices [cytolytic index (P = 0.04), immune ESTIMATE (P = 0.002), type II IFN signaling (P = 0.002)] despite being associated with a higher mutational/neoantigen burden, in BRCA1/2 mutant breast cancers. Further, absence of allele-specific loss of heterozygosity (LOH negative; P = 0.01) or subclonality (P = 0.003) of germline and somatic BRCA1/2 mutations, respectively, predicted for heightened cytolytic activity. Gene set analysis found that multiple innate and adaptive immune pathways that converge on NF-κB may contribute to this heightened immunogenicity. IHC of Penn breast cancers demonstrated increased CD45+ (P = 0.039) and CD8+ infiltrates (P = 0.037) and increased PDL1 expression (P = 0.012) in HRD-low or LOH-negative cancers. Triple-negative cancers with low HRD had far greater CD8+ T cells (P = 0.0011) and Perforin 1 expression (P = 0.014) compared with hormone receptor-positive HRD-high cancers. Conclusions: HRD scores and hormone receptor subtype are predictive of immunogenicity in BRCA1/2 breast cancers and may inform the design of optimal immune therapeutic strategies.

Recurrence is a major cause of death among BRCA1/2 mutation carriers with breast (BrCa) and ovarian cancers (OvCa). We performed multi-omic sequencing on 67 paired primary and recurrent BrCa and OvCa from 27 BRCA1/2 mutation carriers to identify potential recurrence-specific drivers. PARP1 amplifications were identified in recurrences (FDR q = 0.05), and PARP1 was significantly overexpressed across primary BrCa and recurrent BrCa and OvCa, independent of amplification status. RNA-seq analysis found two BRCA2 isoforms, BRCA2-201/Long and BRCA2-001/Short, predicted sensitive and insensitive to nonsense-mediated decay, respectively. BRCA2-001/Short was expressed more frequently in recurrences and associated with reduced overall survival in breast cancer (87 vs. 121 months; HR = 2.5 [1.18–5.5]). Loss of heterozygosity (LOH) status was discordant in 25% of patient’s primary and recurrent tumors, with switching between both LOH and lack of LOH found. Our study revealed multiple potential drivers of recurrent disease in BRCA1/2 mutation-associated cancer, improving our understanding of tumor evolution and suggesting potential biomarkers.

Background: Studies evaluating the association between peripheral blood leukocyte telomere length (LTL) and testicular germ cell tumor (TGCT) risk have produced conflicting results. Methods: Using available genotype data from the Testicular Cancer Consortium (TECAC), polygenic risk score and Mendelian randomization analyses of genetic variants previously associated with LTL were used to assess potential etiologic associations between telomere length and TGCT risk. Results: Genetically inferred telomere length was not associated with TGCT risk among 2,049 cases and 6,921 controls with individual-level genotype data (OR, 1.02; 95% confidence interval, 0.97–1.07). Mendelian randomization analyses using summary statistic data further indicated no evidence for an association between telomere length and TGCT risk among all available TECAC participants (3,558 cases and 13,971 controls). Conclusions: Our analyses in the largest molecular genetic testicular cancer study to date provide no evidence for an association between genetically inferred peripheral blood LTL and TGCT risk. Impact: The lack of evidence for an overall association indicates that peripheral blood LTL is likely not a strong biomarker for TGCT risk.

Testicular germ cell tumors (TGCT) are the most common tumor in young white men and have a high heritability. In this study, the international Testicular Cancer Consortium assemble 10,156 and 179,683 men with and without TGCT, respectively, for a genome-wide association study. This meta-analysis identifies 22 TGCT susceptibility loci, bringing the total to 78, which account for 44% of disease heritability. Men with a polygenic risk score (PRS) in the 95th percentile have a 6.8-fold increased risk of TGCT compared to men with median scores. Among men with independent TGCT risk factors such as cryptorchidism, the PRS may guide screening decisions with the goal of reducing treatment-related complications causing long-term morbidity in survivors. These findings emphasize the interconnected nature of two known pathways that promote TGCT susceptibility: male germ cell development within its somatic niche and regulation of chromosomal division and structure, and implicate an additional biological pathway, mRNA translation., Testicular germ cell tumors are highly heritable, and the authors present the largest genome association study, identifying 22 novel loci, which account for a third of those identified to date. Implicated pathways include male germ cell development and differentiation, and chromosomal segregation.

Medial temporal lobe (MTL) subregions play integral roles in memory function and are differentially affected in various neurological and psychiatric disorders. The ability to structurally and functionally characterize these subregions may be important to understanding MTL physiology and diagnosing diseases involving the MTL. In this study, we characterized network architecture of the MTL in healthy subjects (n = 31) using both resting state functional MRI and MTL-focused T2-weighted structural MRI at 7 tesla. Ten MTL subregions per hemisphere, including hippocampal subfields and cortical regions of the parahippocampal gyrus, were segmented for each subject using a multi-atlas algorithm. Both structural covariance matrices from correlations of subregion volumes across subjects, and functional connectivity matrices from correlations between subregion BOLD time series were generated. We found a moderate structural and strong functional inter-hemispheric symmetry. Several bilateral hippocampal subregions (CA1, dentate gyrus, and subiculum) emerged as functional network hubs. We also observed that the structural and functional networks naturally separated into two modules closely corresponding to (a) bilateral hippocampal formations, and (b) bilateral extra-hippocampal structures. Finally, we found a significant correlation in structural and functional connectivity (r = 0.25). Our findings represent a comprehensive analysis of network topology of the MTL at the subregion level. We share our data, methods, and findings as a reference for imaging methods and disease-based research.

Recent advances in MRI and increasing knowledge on the characterization and anatomical variability of medial temporal lobe (MTL) anatomy have paved the way for more specific subdivisions of the MTL in humans. In addition, recent studies suggest that early changes in many neurodegenerative and neuropsychiatric diseases are better detected in smaller subregions of the MTL rather than with whole structure analyses. Here, we developed a new protocol using 7 Tesla (T) MRI incorporating novel anatomical findings for the manual segmentation of entorhinal cortex (ErC), perirhinal cortex (PrC; divided into area 35 and 36), parahippocampal cortex (PhC), and hippocampus; which includes the subfields subiculum (Sub), CA1, CA2, as well as CA3 and dentate gyrus (DG) which are separated by the endfolial pathway covering most of the long axis of the hippocampus. We provide detailed instructions alongside slice-by-slice segmentations to ease learning for the untrained but also more experienced raters. Twenty-two subjects were scanned (19–32 yrs, mean age = 26 years, 12 females) with a turbo spin echo (TSE) T2-weighted MRI sequence with high-resolution oblique coronal slices oriented orthogonal to the long axis of the hippocampus (in-plane resolution 0.44 × 0.44 mm2) and 1.0 mm slice thickness. The scans were manually delineated by two experienced raters, to assess intra- and inter-rater reliability. The Dice Similarity Index (DSI) was above 0.78 for all regions and the Intraclass Correlation Coefficients (ICC) were between 0.76 to 0.99 both for intra- and inter-rater reliability. In conclusion, this study presents a fine-grained and comprehensive segmentation protocol for MTL structures at 7 T MRI that closely follows recent knowledge from anatomical studies. More specific subdivisions (e.g. area 35 and 36 in PrC, and the separation of DG and CA3) may pave the way for more precise delineations thereby enabling the detection of early volumetric changes in dementia and neuropsychiatric diseases., Highlights • We present a new segmentation protocol for medial temporal lobe subregions at 7 T MRI. • Novel neuroanatomical knowledge from the work of Ding and Van Hoesen is incorporated. • Individual anatomical variability is covered by digitation- and depth-specific rules. • We provide detailed descriptions and slice-by-slice plots to guide the novice rater. • Reliability analyses provide compelling evidence that the rules can be replicated.

Genome-wide association studies have identified multiple novel genomic loci associated with vascular diseases. Many of these loci are common non-coding variants that affect the expression of disease-relevant genes within coronary vascular cells. To identify such genes on a genome-wide level, we performed deep transcriptomic analysis of genotyped primary human coronary artery smooth muscle cells (HCASMCs) and coronary endothelial cells (HCAECs) from the same subjects, including splicing Quantitative Trait Loci (sQTL), allele-specific expression (ASE), and colocalization analyses. We identified sQTLs for TARS2, YAP1, CFDP1, and STAT6 in HCASMCs and HCAECs, and 233 ASE genes, a subset of which are also GTEx eGenes in arterial tissues. Colocalization of GWAS association signals for coronary artery disease (CAD), migraine, stroke and abdominal aortic aneurysm with GTEx eGenes in aorta, coronary artery and tibial artery discovered novel candidate risk genes for these diseases. At the CAD and stroke locus tagged by rs2107595 we demonstrate colocalization with expression of the proximal gene TWIST1. We show that disrupting the rs2107595 locus alters TWIST1 expression and that the risk allele has increased binding of the NOTCH signaling protein RBPJ. Finally, we provide data that TWIST1 expression influences vascular SMC phenotypes, including proliferation and calcification, as a potential mechanism supporting a role for TWIST1 in CAD., Author summary Genome-wide association studies (GWAS) have identified hundreds of genetic variants that are associated with human vascular disease including coronary artery disease. These are predominantly common single nucleotide polymorphisms (SNPs) in non-coding regions, which makes the identification of the causal genes and their underlying connection to pathophysiology challenging. Mapping of expression quantitative trait loci (eQTLs) has been performed to associate GWAS SNPs with risk genes in vascular cells and tissues. However, atherosclerotic vascular tissues contain multiple cell types. We perform deep transcriptomic profiling of genotyped human-derived vascular cells–endothelial cells and smooth muscle cells–and use splicing quantitative trait locus, allele-specific expression, and colocalization analyses to annotate genetic variants associated with vascular diseases and gain insight into their potential function in a cell-type specific manner. Based on these analyses, we identified computationally and then validate experimentally an association between the CAD risk locus rs2107595 and the gene TWIST1. We propose that the minor allele for this locus can affect transcription factor binding and provide data supporting a role for TWIST1 in modulating smooth muscle cell phenotype.

Introduction Heterogeneity of segmentation protocols for medial temporal lobe regions and hippocampal subfields on in vivo magnetic resonance imaging hinders the ability to integrate findings across studies. We aim to develop a harmonized protocol based on expert consensus and histological evidence. Methods Our international working group, funded by the EU Joint Programme–Neurodegenerative Disease Research (JPND), is working toward the production of a reliable, validated, harmonized protocol for segmentation of medial temporal lobe regions. The working group uses a novel postmortem data set and online consensus procedures to ensure validity and facilitate adoption. Results This progress report describes the initial results and milestones that we have achieved to date, including the development of a draft protocol and results from the initial reliability tests and consensus procedures. Discussion A harmonized protocol will enable the standardization of segmentation methods across laboratories interested in medial temporal lobe research worldwide., Highlights • Harmonization of MRI-based segmentation of medial temporal regions is needed. • The Hippocampal Subfield Group includes >200 imaging and anatomy experts worldwide. • Reliable and valid protocol based on specialized histology data set in development. • A modified Delphi procedure is used to determine consensus on protocol rules. • Final protocol will provide subfield delineation in hippocampal body, head, and tail.

Hippocampal subfield segmentation on in vivo MRI is of great interest for cognition, aging, and disease research. Extant subfield segmentation protocols have been based on neuroanatomical references, but these references often give limited information on anatomical variability. Moreover, there is generally a mismatch between the orientation of the histological sections and the often anisotropic coronal sections on in vivo MRI. To address these issues, we provide a detailed description of hippocampal anatomy using a postmortem dataset containing nine specimens of subjects with and without dementia, which underwent a 9.4 T MRI and histological processing. Postmortem MRI matched the typical orientation of in vivo images and segmentations were generated in MRI space, based on the registered annotated histological sections. We focus on the following topics: the order of appearance of subfields, the location of subfields relative to macroanatomical features, the location of subfields in the uncus and tail and the composition of the dark band, a hypointense layer visible in T2-weighted MRI. Our main findings are that: (a) there is a consistent order of appearance of subfields in the hippocampal head, (b) the composition of subfields is not consistent in the anterior uncus, but more consistent in the posterior uncus, (c) the dark band consists only of the CA-stratum lacunosum moleculare, not the strata moleculare of the dentate gyrus, (d) the subiculum/CA1 border is located at the middle of the width of the hippocampus in the body in coronal plane, but moves in a medial direction from anterior to posterior, and (e) the variable location and composition of subfields in the hippocampal tail can be brought back to a body-like appearance when reslicing the MRI scan following the curvature of the tail. Our findings and this publicly available dataset will hopefully improve anatomical accuracy of future hippocampal subfield segmentation protocols.

[EN] Medial temporal lobe (MTL) substructures are the earliest regions affected by neurofibrillary tangle pathology-and thus are promising biomarkers for Alzheimer's disease (AD). However, automatic segmentation of the MTL using only T1-weighted (T1w) magnetic resonance imaging (MRI) is challenging due to the large anatomical variability of the MTL cortex and the confound of the dura mater, which is commonly segmented as gray matter by state-of-the-art algorithms because they have similar intensity in T1w MRI. To address these challenges, we developed a novel atlas set, consisting of 15 cognitively normal older adults and 14 patients with mild cognitive impairment with a label explicitly assigned to the dura, that can be used by the multiatlas automated pipeline (Automatic Segmentation of Hippocampal Subfields [ASHS-T1]) for the segmentation of MTL subregions, including anterior/posterior hippocampus, entorhinal cortex (ERC), Brodmann areas (BA) 35 and 36, and parahippocampal cortex on T1w MRI. Cross-validation experiments indicated good segmentation accuracy of ASHS-T1 and that the dura can be reliably separated from the cortex (6.5% mislabeled as gray matter). Conversely, FreeSurfer segmented majority of the dura mater (62.4%) as gray matter and the degree of dura mislabeling decreased with increasing disease severity. To evaluate its clinical utility, we applied the pipeline to T1w images of 663 ADNI subjects and significant volume/thickness loss is observed in BA35, ERC, and posterior hippocampus in early prodromal AD and all subregions at later stages. As such, the publicly available new atlas and ASHS-T1 could have important utility in the early diagnosis and monitoring of AD and enhancing brain-behavior studies of these regions., Northern California Institute for Research and Education; Foundation for the National Institutes of Health; Canadian Institutes of Health Research; Transition Therapeutics; Takeda Pharmaceutical Company; Servier; Piramal Imaging; Pfizer Inc.; Novartis Pharmaceuticals Corporation; Neurotrack Technologies; NeuroRx Research; Meso Scale Diagnostics, LLC.; Lundbeck and Merck & Co., Inc.; Lumosity; Johnson & Johnson Pharmaceutical Research & Development LLC.; Janssen Alzheimer Immunotherapy Research & Development, LLC.; IXICO Ltd.; GE Healthcare; Fujirebio; Genentech, Inc.; F. Hoffmann-La Roche Ltd.; EuroImmun; Eli Lilly and Company; Elan Pharmaceuticals, Inc.; Cogstate and Eisai Inc.; CereSpir, Inc.; Bristol-Myers Squibb Company; Biogen; BioClinica, Inc.; Araclon Biotech; Alzheimer's Drug Discovery Foundation; Alzheimer's Association; AbbVie; National Institute of Biomedical Imaging and Bioengineering; National Institute on Aging; Department of Defense ADNI, Grant/Award Number: W81XWH-12-2-0012; Alzheimer's Disease Neuroimaging Initiative, Grant/Award Number: U01 AG024904; Spain Ministry of Economy, Industry and Competitiveness, Grant/Award Number: DPI2017-87743-R; Foundation Philippe Chatrier; BrightFocus Foundation; National Institutes of Health, Grant/Award Numbers: R01-AG055005, R01-EB017255, P30-AG010124, R01-AG040271, R01-AG056014

Breast cancer type 1 and 2 susceptibility proteins (BRCA1/BRCA2) are well known breast cancer genes, mutations in which lead to defective homologous recombination repair (HRR). HR-based DNA repair deficiency (HRD) scores can be used to indicate DNA damage, genomic instability and may predict response to DNA damaging agents in BRCA1/2 mutated tumors. Tumors with a high HRD score caused by complete loss of BRCA1 or BRCA2 function locus-specific LOH are sensitive to DNA damage agents including platinum-based chemotherapy and poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi). To understand the relationship between BRCA1/2 mutations and DNA damage in the tumor microenvironment (TME), we have characterized 107 BRCA1/2 tumors to determine HRD score using whole exome sequencing (WES) and simultaneously measured markers of DNA damage, PARP expression, tumor-infiltrating lymphocytes (TILs) and immune checkpoints to identify potential treatment targets on matching samples (n=47) using highly multiplexed fluorescence microscopy CO-Detection by indEXing (CODEX). We have established and validated a 40-plex breast cancer specific antibody panel consisting of markers to detect DNA damage, TILs and immune checkpoints to deeply profile how the TME is affected by BRCA1/2 mutations using CODEX. Computational image processing of CODEX data was performed to interrogate changes in number, size, morphology, and marker expression in tumor and immune cells. We have characterized BRCA1/2 tumors (n=47) on Tissue Microarrays (TMAs), and we have detected cytotoxic CD8+T and CD107a+NK cells in HRD low ( Citation Format: Dana Pueschl, Derek A. Oldrige, Jonathan Belman, Jake S. Shilan, Anupma Nayak, Bradley Wubbenhorst, John Pluta, Robert H. Vonderheide, Michael Feldman, Kara N. Maxwell, E. John Wherry, Susan M. Domchek, Katherine L. Nathanson. How BRCA1/2 mutations in TNBC affect TME and subsequently immune cell functions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2723.

Background: Ipilimumab is an immune checkpoint inhibitor used to treat melanoma. Although the development of immune-related adverse events (irAEs) among patients treated with ipilimumab is well documented, little is known about factors that may increase risk of irAEs. We conducted a genome-wide association study (GWAS) to examine the genetic susceptibility to irAEs in response to ipilimumab monotherapy. Methods: In partnership with Bristol Myers Squibb BMS, extant genotype data and clinical information were obtained on melanoma patients treated with ipilimumab monotherapy from three clinical trials. Only patients who were treatment naïve were included in our analyses. We defined our outcome as the occurrence of a serious irAE, grade 3 or higher. We first analyzed data from 294 subjects, 79 with severe irAE, enrolled on CA184-169 for whom genotyping was completed using the Affymetrix 6 array. After appropriate quality control, SNP associations were determined using logistic regression models that were adjusted for ancestry, ECOG status, ipilimumab dosage (3 mg/kg vs 10 mg/kg), and number of doses ( Results: The most statistically significant marker (rs55981606, p=1.39 × 10-7) and a second independent marker (rs72712605, p=6.33 × 10-6) mapped to a non-coding region on chromosome 9. We identified a marker (rs65949485, p = 9.38 × 10-6) intragenic between the SHQ1 and GXYLT2 genes, both of which are involved in the Notch signaling pathway. Markers proximal to NR2F2 (rs13270533, p=7.8 × 10-6) and within SAMD12 (rs13270533, p = 9.23 × 10-6) were also identified; the latter two genes have been implicated as being oncogenic. Additionally, we identified several markers implicating genes involved in inflammation, specifically macrophage activation, including TLE1 (rs3739581, p = 9.25 × 10-7), SLC16A4 (rs2271885, p=9.23 × 10-6) and CYP2J2 (rs427970, p=2.03 × 10-6). Conclusions: Results from our meta-analysis suggest that genes related to inflammation processes and those with known contributions to oncogenesis may play a role in the development of severe irAEs resulting from ipilimumab monotherapy. If further validated, these findings may provide the foundation to advance models to discriminate patients with a high likelihood of suffering irAE allowing for heightened surveillance of symptom onset or joint decision making for alternative therapies. Citation Format: John Pluta, Lu Qian, Kurt D'Andrea, Chunzhe Duan, Benita Weathers, Megan Wind-Rotolo, Peter Kanetsky, Katherine Nathanson. Genetic susceptibility to immune-related adverse events among melanoma patients treated with ipilimumab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1352.

Rational The human perirhinal cortex (PRC) plays critical roles in episodic and semantic memory and visual perception. The PRC consists of Brodmann areas 35 and 36 (BA35, BA36). In Alzheimer's disease (AD), BA35 is the first cortical site affected by neurofibrillary tangle pathology, which is closely linked to neural injury in AD. Large anatomical variability, manifested in the form of different cortical folding and branching patterns, makes it difficult to segment the PRC in MRI scans. Pathology studies have found that in ~97% of specimens, the PRC falls into one of three discrete anatomical variants. However, current methods for PRC segmentation and morphometry in MRI are based on single-template approaches, which may not be able to accurately model these discrete variants Methods A multi-template analysis pipeline that explicitly accounts for anatomical variability is used to automatically label the PRC and measure its thickness in T2-weighted MRI scans. The pipeline uses multi-atlas segmentation to automatically label medial temporal lobe cortices including entorhinal cortex, PRC and the parahippocampal cortex. Pairwise registration between label maps and clustering based on residual dissimilarity after registration are used to construct separate templates for the anatomical variants of the PRC. An optimal path of deformations linking these templates is used to establish correspondences between all the subjects. Experimental evaluation focuses on the ability of single-template and multi-template analyses to detect differences in the thickness of medial temporal lobe cortices between patients with amnestic mild cognitive impairment (aMCI, n=41) and age-matched controls (n=44). Results The proposed technique is able to generate templates that recover the three dominant discrete variants of PRC and establish more meaningful correspondences between subjects than a single-template approach. The largest reduction in thickness associated with aMCI, in absolute terms, was found in left BA35 using both regional and summary thickness measures. Further, statistical maps of regional thickness difference between aMCI and controls revealed different patterns for the three anatomical variants.

The advent of high-resolution magnetic resonance imaging (MRI) has enabled in vivo research in a variety of populations and diseases on the structure and function of hippocampal subfields and subdivisions of the parahippocampal gyrus. Because of the many extant and highly discrepant segmentation protocols, comparing results across studies is difficult. To overcome this barrier, the Hippocampal Subfields Group was formed as an international collaboration with the aim of developing a harmonized protocol for manual segmentation of hippocampal and parahippocampal subregions on high-resolution MRI. In this commentary we discuss the goals for this protocol and the associated key challenges involved in its development. These include differences among existing anatomical reference materials, striking the right balance between reliability of measurements and anatomical validity, and the development of a versatile protocol that can be adopted for the study of populations varying in age and health. The commentary outlines these key challenges, as well as the proposed solution of each, with concrete examples from our working plan. Finally, with two examples, we illustrate how the harmonized protocol, once completed, is expected to impact the field by producing measurements that are quantitatively comparable across labs and by facilitating the synthesis of findings across different studies. © 2016 Wiley Periodicals, Inc.

Background: Volumetry of medial temporal lobe (MTL) structures to diagnose Alzheimer’s disease (AD) in its earliest symptomatic stage could be of great importance for interventions or disease modifying pharmacotherapy. Objective: This study aimed to demonstrate the first application of an automatic segmentation method of MTL subregions in a clinical population. Automatic segmentation of magnetic resonance images (MRIs) in a research population has previously been shown to detect evidence of neurodegeneration in MTL subregions and to help discriminate AD and mild cognitive impairment (MCI) from a healthy comparison group. Methods: Clinical patients were selected and T2-weighted MRI scan quality was checked. An automatic segmentation method of hippocampal subfields (ASHS) was applied to scans of 67 AD patients, 38 amnestic MCI patients, and 57 healthy controls. Hippocampal subfields, entorhinal cortex (ERC), and perirhinal cortex were automatically labeled and subregion volumes were compared between groups. Results: One fourth of all scans were excluded due to bad scan quality. There were significant volume reductions in all subregions, except BA36, in aMCIs (p < 0.001), most prominently in Cornu Ammonis 1 (CA1) and ERC, and in all subregions in AD. However, sensitivity of CA1 and ERC hardly differed from sensitivity of WH in aMCI and AD. Conclusion: Applying automatic segmentation of MTL subregions in a clinical setting as a potential biomarker for prodromal AD is feasible, but issues of image quality due to motion remain to be addressed. CA1 and ERC provided strongest group discrimination in differentiating aMCIs from controls, but discriminatory power of different subfields was low overall.

Carriers of pathogenic germline mutations in BRCA1/2 are highly predisposed to breast and ovarian cancers. Although their primary tumors respond to DNA-damaging agents, such as platinum-based chemotherapy and poly (ADP-ribose) polymerase inhibitors (PARPi), these malignancies often return as therapy-resistant recurrences. To identify genetic mechanisms of therapeutic resistance, we performed whole exome sequencing on 41 pairs of matched primary/recurrent breast and ovarian tumors from 27 BRCA1/2 mutation carriers. The cohort consisted of 14 ovarian cancer patients (nine BRCA1, five BRCA2) and 13 breast cancer patients (nine BRCA1, four BRCA2). Five patients received PARPi, 17 patients received platinums, and all patients received some type of chemotherapy prior to recurrence. First, we performed a segmentation analysis to assess copy number variation (CNVs) in each tumor. We calculated homologous recombination deficiency (HRD) scores for each tumor, but found no significant differences between matched primary/recurrent pairs. We noted that primary tumors had a relatively high average HRD score (55/100), which remained high in recurrences (52/100). Next, we totaled arm-level CNVs to generate aneuploidy scores for each tumor. Using a Wilcoxon signed rank test, we found that aneuploidy scores were significantly higher in recurrences than in matched primary tumors (p=0.007). This finding suggests that recurrences have more arm-level CNVs than primary tumors, but that acquired genomic abnormalities are not caused by HRD per se. Instead, arm-level CNVs indicate that replication errors or genome doubling events are more common in recurrences than in primary tumors. Lastly, we annotated all tumors' CNVs with genes to interrogate potential effects on signaling pathways, including CNVs exclusive to each recurrence. Using Gene Set Enrichment Analysis with Hallmark gene sets, we identified pathways significantly (FDR q Citation Format: Jennifer Brady Shah, Bradley Wubbenhorst, John Pluta, Caitlin Feltcher, Lauren Schmucker, Kurt D'Andrea, Heather Symecko, Catherine Ruan, Anupma Nayak, Kara N. Maxwell, Susan Domchek, Katherine L. Nathanson. Copy number variation in recurrent BRCA1/2 germline mutation-associated breast and ovarian cancers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2500.

Although temporal lobe epilepsy (TLE) and resection (TLR) impact olfactory eloquent brain structures, their influences on olfaction remain enigmatic. We sought to more definitively assess the influences of TLE and TLR on olfaction using three well-validated olfactory tests and measuring the tests’ associations with the volume of numerous temporal lobe brain structures. The University of Pennsylvania Smell Identification Test and an odor detection threshold test were administered to 71 TLE patients and 71 age- and sex-matched controls; 69 TLE patients and controls received an odor discrimination/memory test. Fifty-seven patients and 57 controls were tested on odor identification and threshold before and after TLR; 27 patients and 27 controls were similarly tested for odor detection/discrimination. Scores were compared using analysis of variance and correlated with pre- and post-operative volumes of the target brain structures. TLE was associated with bilateral deficits in all test measures. TLR further decreased function on the side ipsilateral to resection. The hippocampus and other structures were smaller on the focus side of the TLE subjects. Although post-operative volumetric decreases were evident in most measured brain structures, modest contralateral volumetric increases were observed in some cases. No meaningful correlations were evident pre- or post-operatively between the olfactory test scores and the structural volumes. In conclusion, we demonstrate that smell dysfunction is clearly a key element of both TLE and TLR, impacting odor identification, detection, and discrimination/memory. Whether our novel finding of significant post-operative increases in the volume of brain structures contralateral to the resection side reflects plasticity and compensatory processes requires further study.

Although the hippocampus is one of the most studied structures in the human brain, limited quantitative data exist on its 3D organization, anatomical variability, and effects of disease on its subregions. Histological studies provide restricted reference information due to their 2D nature. In this paper, high-resolution (∼200 × 200 × 200 μm3) ex vivo MRI scans of 31 human hippocampal specimens are combined using a groupwise diffeomorphic registration approach into a 3D probabilistic atlas that captures average anatomy and anatomic variability of hippocampal subfields. Serial histological imaging in 9 of the 31 specimens was used to label hippocampal subfields in the atlas based on cytoarchitecture. Specimens were obtained from autopsies in patients with a clinical diagnosis of Alzheimer's disease (AD; 9 subjects, 13 hemispheres), of other dementia (nine subjects, nine hemispheres), and in subjects without dementia (seven subjects, nine hemispheres), and morphometric analysis was performed in atlas space to measure effects of age and AD on hippocampal subfields. Disproportional involvement of the cornu ammonis (CA) 1 subfield and stratum radiatum lacunosum moleculare was found in AD, with lesser involvement of the dentate gyrus and CA2/3 subfields. An association with age was found for the dentate gyrus and, to a lesser extent, for CA1. Three-dimensional patterns of variability and disease and aging effects discovered via the ex vivo hippocampus atlas provide information highly relevant to the active field of in vivo hippocampal subfield imaging.

BACKGROUND AND PURPOSE: High resolution 7T MRI is increasingly used to investigate hippocampal subfields in vivo, but most studies rely on manual segmentation which is labor intensive. We aimed to evaluate an automated technique to segment hippocampal subfields and the entorhinal cortex at 7T MRI. MATERIALS AND METHODS: The cornu ammonis (CA)1, CA2, CA3, dentate gyrus, subiculum, and entorhinal cortex were manually segmented, covering most of the long axis of the hippocampus on 0.70-mm 3 T2-weighted 7T images of 26 participants (59 ± 9 years, 46% men). The automated segmentation of hippocampal subfields approach was applied and evaluated by using leave-one-out cross-validation. RESULTS: Comparison of automated segmentations with corresponding manual segmentations yielded a Dice similarity coefficient of >0.75 for CA1, the dentate gyrus, subiculum, and entorhinal cortex and >0.54 for CA2 and CA3. Intraclass correlation coefficients were >0.74 for CA1, the dentate gyrus, and subiculum; and >0.43 for CA2, CA3, and the entorhinal cortex. Restricting the comparison of the entorhinal cortex segmentation to a smaller range along the anteroposterior axis improved both intraclass correlation coefficients (left: 0.71; right: 0.82) and Dice similarity coefficients (left: 0.78; right: 0.77). The accuracy of the automated segmentation versus a manual rater was lower, though only slightly for most subfields, than the intrarater reliability of an expert manual rater, but it was similar to or slightly higher than the accuracy of an expert-versus-manual rater with ∼170 hours of training for almost all subfields. CONCLUSIONS: This work demonstrates the feasibility of using a computational technique to automatically label hippocampal subfields and the entorhinal cortex at 7T MRI, with a high accuracy for most subfields that is competitive with the labor-intensive manual segmentation. The software and atlas are publicly available: http://www.nitrc.org/projects/ashs/.

ObjectiveAn increasing number of human in vivo magnetic resonance imaging (MRI) studies have focused on examining the structure and function of the subfields of the hippocampal formation (the dentate gyrus, CA fields 1 − 3, and the subiculum) and subregions of the parahippocampal gyrus (entorhinal, perirhinal, and parahippocampal cortices). The ability to interpret the results of such studies and to relate them to each other would be improved if a common standard existed for labeling hippocampal subfields and parahippocampal subregions. Currently, research groups label different subsets of structures and use different rules, landmarks, and cues to define their anatomical extents. This paper characterizes, both qualitatively and quantitatively, the variability in the existing manual segmentation protocols for labeling hippocampal and parahippocampal substructures in MRI, with the goal of guiding subsequent work on developing a harmonized substructure segmentation protocol.MethodMRI scans of a single healthy adult human subject were acquired both at 3 T and 7 T. Representatives from 21 research groups applied their respective manual segmentation protocols to the MRI modalities of their choice. The resulting set of 21 segmentations was analyzed in a common anatomical space to quantify similarity and identify areas of agreement.ResultsThe differences between the 21 protocols include the region within which segmentation is performed, the set of anatomical labels used, and the extents of specific anatomical labels. The greatest overall disagreement among the protocols is at the CA1/subiculum boundary, and disagreement across all structures is greatest in the anterior portion of the hippocampal formation relative to the body and tail.ConclusionsThe combined examination of the 21 protocols in the same dataset suggests possible strategies towards developing a harmonized subfield segmentation protocol and facilitates comparison between published studies. published

There is currently some debate as to whether hippocampus mediates contextual cueing. In the present study, we examined contextual cueing in patients diagnosed with mild cognitive impairment (MCI) and healthy older adults, with the main goal of investigating the role of hippocampus in this form of learning. Amnestic MCI (aMCI) patients and healthy controls completed the contextual cueing task, in which they were asked to search for a target (a horizontal T) in an array of distractors (rotated L’s). Unbeknownst to them, the spatial arrangement of elements on some displays was repeated thus making the configuration a contextual cue to the location of the target. In contrast, the configuration for novel displays was generated randomly on each trial. The difference in response times between repeated and novel configurations served as a measure of contextual learning. aMCI patients, as a group, were able to learn spatial contextual cues as well as healthy older adults. However, better learning on this task was associated with higher hippocampal volume, particularly in right hemisphere. Furthermore, contextual cueing performance was significantly associated with hippocampal volume, even after controlling for age and MCI status. These findings support the role of the hippocampus in learning of spatial contexts, and also suggest that the contextual cueing paradigm can be useful in detecting neuropathological changes associated with the hippocampus. (JINS, 2015, 21, 285–296)

The most recent Genome-wide Association Study (GWAS) meta-analysis has reported a total of 58 genomic loci to be statistically significantly associated with genetic susceptibility to Coronary Artery Disease (CAD) (Consortium, 2015). Many of these loci also associate with other phenotypes, with the majority being lipid traits (Tada et al., 2014). But also hypertension, stroke (Dichgans et al., 2014) and migraine (Pickrell et al., 2016) appear to share genetic determinants with CAD. To functionally annotate the genomic loci harboring these association SNPs we sequenced the transcriptomes of 20 same donor human coronary artery endothelial (EC) and smooth muscle cell (SMC) lines. Deep RNA-Sequencing was used to assess Differential Gene Expression, Differential Splicing and Allele-Specific Expression. Focusing on GWAS loci for vascular phenotypes (CAD, stroke, migraine) we identified genes which display allele-specific differences in mRNA expression or splicing. We propose these genes as suitable targets for follow up studies. Consortium, C.A.D. (2015). A comprehensive 1000 Genomes-based genome-wide association meta-analysis of coronary artery disease. Nature genetics 47, 1121-1130. Tada, H., Won, H.H., Melander, O., Yang, J., Peloso, G.M., and Kathiresan, S. (2014). Multiple associated variants increase the heritability explained for plasma lipids and coronary artery disease. Circulation Cardiovascular genetics 7, 583-587. Dichgans, M., Malik, R., Konig, I.R., Rosand, J., Clarke, R., Gretarsdottir, S., Thorleifsson, G., Mitchell, B.D., Assimes, T.L., Levi, C., et al. (2014). Shared genetic susceptibility to ischemic stroke and coronary artery disease: a genome-wide analysis of common variants. Stroke; a journal of cerebral circulation 45, 24-36. Pickrell, J.K., Berisa, T., Liu, J.Z., Segurel, L., Tung, J.Y., and Hinds, D.A. (2016). Detection and interpretation of shared genetic influences on 42 human traits. Nature genetics 48, 709-717.

Traumatic brain injury (TBI) is likely to disrupt structural network properties due to diffuse white matter pathology. The present study aimed to detect alterations in structural network topology in TBI and relate them to cognitive and real-world behavioral impairment. Twenty-two people with moderate to severe TBI with mostly diffuse pathology and 18 demographically matched healthy controls were included in the final analysis. Graph theoretical network analysis was applied to diffusion tensor imaging (DTI) data to characterize structural connectivity in both groups. Neuropsychological functions were assessed by a battery of psychometric tests and the Frontal Systems Behavior Scale (FrSBe). Local connection-wise analysis demonstrated reduced structural connectivity in TBI arising from subcortical areas including thalamus, caudate, and hippocampus. Global network metrics revealed that shortest path length in participants with TBI was longer compared to controls, and that this reduced network efficiency was associated with worse performance in executive function and verbal learning. The shortest path length measure was also correlated with family-reported FrSBe scores. These findings support the notion that the diffuse form of neuropathology caused by TBI results in alterations in structural connectivity that contribute to cognitive and real-world behavioral impairment. (JINS, 2014,20, 1–10)

We evaluate a fully automatic technique for labeling hippocampal subfields and cortical subregions in the medial temporal lobe in in vivo 3 Tesla MRI. The method performs segmentation on a T2-weighted MRI scan with 0.4 × 0.4 × 2.0 mm(3) resolution, partial brain coverage, and oblique orientation. Hippocampal subfields, entorhinal cortex, and perirhinal cortex are labeled using a pipeline that combines multi-atlas label fusion and learning-based error correction. In contrast to earlier work on automatic subfield segmentation in T2-weighted MRI [Yushkevich et al., 2010], our approach requires no manual initialization, labels hippocampal subfields over a greater anterior-posterior extent, and labels the perirhinal cortex, which is further subdivided into Brodmann areas 35 and 36. The accuracy of the automatic segmentation relative to manual segmentation is measured using cross-validation in 29 subjects from a study of amnestic mild cognitive impairment (aMCI) and is highest for the dentate gyrus (Dice coefficient is 0.823), CA1 (0.803), perirhinal cortex (0.797), and entorhinal cortex (0.786) labels. A larger cohort of 83 subjects is used to examine the effects of aMCI in the hippocampal region using both subfield volume and regional subfield thickness maps. Most significant differences between aMCI and healthy aging are observed bilaterally in the CA1 subfield and in the left Brodmann area 35. Thickness analysis results are consistent with volumetry, but provide additional regional specificity and suggest nonuniformity in the effects of aMCI on hippocampal subfields and MTL cortical subregions.

Testicular germ cell tumors (TGCT) are ideally suited to agnostic genome-wide association methods because of their known high heritability and homogeneous cell of origin. Here we report the most updated results from the international Testicular Cancer Consortium (TECAC) that extends published findings from our recent meta-analysis, which included 3,558 cases and 13,970 controls from five centers. To these existing samples, we added summary statistics from 300 cases and 151,991 controls provided by deCODE combined with imputed genotypes from an additional 5,602 cases and 5,006 controls assembled from 15 TECAC centers, almost all (97%) of which were centrally genotyped using the Illumina Human Core array. A total of 9,458 TGCT cases were included in our current meta-analysis, representing a 66% increase since our last publication. We identified 14 new and confirmed 41 previously reported loci that surpassed genome-wide significance (5 × 10-8). Fifteen previously reported loci did not reach this threshold, although one did so in subgroup analysis. In addition, 57 new loci attained nominal significance (5 × 10-8 ≤ p ≤ 1 × 10-5). As expected, a substantial proportion of top hits continue to map to genetic regions belonging to biological pathways essential to male germ cell development, sex determination, and chromosomal segregation. For example, newly identified genes include anaphase promoting complex subunit 2 (ANAPC2), BCL2 like 11 (BCL211), anti-Mullerian hormone receptor type 2 (AMHR2), and DEP domain containing mTOR interacting protein (DEPTOR). Our findings also further implicate genes located on the X chromosome, specifically the androgen receptor (AR). In analyses stratified by tumor subtype, we identified GATA-binding protein 4 (GATA4) as a susceptibility locus among men diagnosed with seminoma. Despite a similar effect size observed among men diagnosed solely with non-seminoma (i.e. excluding cases with a mixture of both seminoma and non-seminoma tumors), this marker did not surpass genome-wide significance in this subtype likely because of the smaller case sample size. GATA4 previously has been associated with overall risk of TGCT (Litchfield et al., Nat Genet. 2017). Biological mechanisms for all genome-wide significant loci are currently being explored using publicly available data sets, ATAC-seq of four TGCT cell lines, and SPATIaL-Seq (a novel high-resolution chromatin conformation capture (3C) assay) of the NTERA2 pluripotent human embryonal carcinoma cell line. Additional genotyping of top hits in an independent sample set of 1,103 TGCT cases and 1,210 controls is currently ongoing. Citation Format: Louise C. Pyle, John Pluta, Kevin T. Nead, Nandita Mitra, Javier Benitez, D. Timothy Bishop, Victoria Cortessis, Alberto Ferlin, Jourik Gietema, Mark Greene, Tom Grotmol, Ramneek Gupta, Rob Hamilton, Michelle A. Hildebrandt, Trine B. Haugen, Lambertus Kiemeney, Christian Kubisch, Davor Lessel, Paloma Martin, Thorunn Rafnar, Lorenzo Richiardi, Rolf Skotheim, Clare Turnbull, Fredrik Wiklund, Tongzhang Zheng, Ewa Rajpert-De Meyts, Stephen M. Schwartz, Katherine A. McGlynn, Peter A. Kanetsky, Katherine L. Nathanson, Testicular Cancer Consortium (TECAC). Identification of 14 novel genetic loci for testicular germ cell tumor susceptibility [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2684.

Recently, there has been a growing effort to analyze the morphometry of hippocampal subfields using both in vivo and postmortem magnetic resonance imaging (MRI). However, given that boundaries between subregions of the hippocampal formation (HF) are conventionally defined on the basis of microscopic features that often lack discernible signature in MRI, subfield delineation in MRI literature has largely relied on heuristic geometric rules, the validity of which with respect to the underlying anatomy is largely unknown. The development and evaluation of such rules is challenged by the limited availability of data linking MRI appearance to microscopic hippocampal anatomy, particularly in three dimensions (3D). The present paper, for the first time, demonstrates the feasibility of labeling hippocampal subfields in a high resolution volumetric MRI dataset based directly on microscopic features extracted from histology. It uses a combination of computational techniques and manual post-processing to map subfield boundaries from a stack of histology images (obtained with 200 μm spacing and 5 μm slice thickness; stained using the Kluver-Barrera method) onto a postmortem 9.4 Tesla MRI scan of the intact, whole hippocampal formation acquired with 160 μm isotropic resolution. The histology reconstruction procedure consists of sequential application of a graph-theoretic slice stacking algorithm that mitigates the effects of distorted slices, followed by iterative affine and diffeomorphic co-registration to postmortem MRI scans of approximately 1 cm-thick tissue sub-blocks acquired with 200 μm isotropic resolution. These 1 cm blocks are subsequently co-registered to the MRI of the whole HF. Reconstruction accuracy is evaluated as the average displacement error between boundaries manually delineated in both the histology and MRI following the sequential stages of reconstruction. The methods presented and evaluated in this single-subject study can potentially be applied to multiple hippocampal tissue samples in order to construct a histologically informed MRI atlas of the hippocampal formation.

Multi-atlas segmentation is an effective approach for automatically labeling objects of interest in biomedical images. In this approach, multiple expert-segmented example images, called atlases, are registered to a target image, and deformed atlas segmentations are combined using label fusion. Among the proposed label fusion strategies, weighted voting with spatially varying weight distributions derived from atlas-target intensity similarity have been particularly successful. However, one limitation of these strategies is that the weights are computed independently for each atlas, without taking into account the fact that different atlases may produce similar label errors. To address this limitation, we propose a new solution for the label fusion problem, in which weighted voting is formulated in terms of minimizing the total expectation of labeling error, and in which pairwise dependency between atlases is explicitly modeled as the joint probability of two atlases making a segmentation error at a voxel. This probability is approximated using intensity similarity between a pair of atlases and the target image in the neighborhood of each voxel. We validate our method in two medical image segmentation problems: hippocampus segmentation and hippocampus subfield segmentation in magnetic resonance (MR) images. For both problems, we show consistent and significant improvement over label fusion strategies that assign atlas weights independently.

Automatic segmentation of cortical and subcortical structures is commonplace in brain MRI literature and is frequently used as the first step towards quantitative analysis of structural and functional neuroimaging. Most approaches to brain structure segmentation are based on propagation of anatomical information from example MRI datasets, called atlases or templates, that are manually labeled by experts. The accuracy of automatic segmentation is usually validated against the “bronze” standard of manual segmentation of test MRI datasets. However, good performance vis-a-vis manual segmentation does not imply accuracy relative to the underlying true anatomical boundaries. In the context of segmentation of hippocampal subfields and functionally related medial temporal lobe cortical subregions, we explore the challenges associated with validating existing automatic segmentation techniques against underlying histologically-derived anatomical “gold” standard; and, further, developing automatic in vivo MRI segmentation techniques informed by histological imaging.

Complete loss of BRCA1 or BRCA2 function is associated with sensitivity to DNA damaging agents. However, not all BRCA1 and BRCA2 germline mutation-associated tumors respond. Herein we report analyses of 160 BRCA1 and BRCA2 germline mutation-associated breast and ovarian tumors. Retention of the normal BRCA1 or BRCA2 allele (absence of locus-specific loss of heterozygosity (LOH)) is observed in 7% of BRCA1 ovarian, 16% of BRCA2 ovarian, 10% of BRCA1 breast, and 46% of BRCA2 breast tumors. These tumors have equivalent homologous recombination deficiency scores to sporadic tumors, significantly lower than scores in tumors with locus-specific LOH (ovarian, P = 0.0004; breast P, Most tumours associated with germline BRCA1/BRCA2 loss of function mutations respond to DNA damaging agents, however, some do not. Herein, the authors identify that a subset of breast/ovarian tumors retain a normal allele, which is associated with decreased overall survival after DNA damage-inducing platinum chemotherapy.

Objectives: To assess risk factors for cerebrospinal fluid (CSF) leak after microvascular reconstruction of extensive cranio-orbitofacial resection with orbital exenteration (CFOE). Study Design: Retrospective Case Series Methods: 70 consecutive patients at a tertiary hospital underwent 76 procedures with microvascular reconstruction of CFOE defects. Patients were stratified by extent of skull base exposure and presence or absence of dural resection. Patients with exposure of the orbital apex and roof alone were classified as minimal skullbase exposure (MSE, n=32). Those with exposure beyond the orbital apex and roof were classified as significant skullbase exposure (SSE, n=38), including those with dural resection (n=23). The main outcome measure was incidence of postoperative CSF leak according to univariate and multivariate analysis of risk factors. Results: Five patients developed a postoperative CSF leak, and 3 required operative management. All 5 were SSE with dural resection and had middle fossa exposure, previous radiation and 4 had previous surgery. None of the MSE group or SSE without dural resection or SSE with anterior fossa exposure alone developed a CSF leak. Multivariate analysis revealed middle fossa exposure to be the only significant predictor of CSF leak (p=0.03). The overall complication rate was 31.6%. Major complications were greater in the SSE group (p=0.05). Conclusion: Middle fossa exposure increases the risk of CSF leak in microvascular reconstruction of CFOE defects.

The measurement of hippocampal volumes using MRI is a useful in-vivo biomarker for detection and monitoring of early Alzheimer's disease (AD), including during the amnestic mild cognitive impairment (a-MCI) stage. The pathology underlying AD has regionally selective effects within the hippocampus. As such, we predict that hippocampal subfields are more sensitive in discriminating prodromal AD (i.e., a-MCI) from cognitively normal controls than whole hippocampal volumes, and attempt to demonstrate this using a semi-automatic method that can accurately segment hippocampal subfields. High-resolution coronal-oblique T2-weighted images of the hippocampal formation were acquired in 45 subjects (28 controls and 17 a-MCI (mean age: 69.5 ± 9.2; 70.2 ± 7.6)). CA1, CA2, CA3, and CA4/DG subfields, along with head and tail regions, were segmented using an automatic algorithm. CA1 and CA4/DG segmentations were manually edited. Whole hippocampal volumes were obtained from the subjects' T1-weighted anatomical images. Automatic segmentation produced significant group differences in the following subfields: CA1 (left: p = 0.001, right: p = 0.038), CA4/DG (left: p = 0.002, right: p = 0.043), head (left: p = 0.018, right: p = 0.002), and tail (left: p = 0.019). After manual correction, differences were increased in CA1 (left: p0.001, right: p = 0.002), and reduced in CA4/DG (left: p = 0.029, right: p = 0.221). Whole hippocampal volumes significantly differed bilaterally (left: p = 0.028, right: p = 0.009). This pattern of atrophy in a-MCI is consistent with the topography of AD pathology observed in postmortem studies, and corrected left CA1 provided stronger discrimination than whole hippocampal volume (p = 0.03). These results suggest that semi-automatic segmentation of hippocampal subfields is efficient and may provide additional sensitivity beyond whole hippocampal volumes.

Purpose: To assess test–retest stability of four functional magnetic resonance imaging (fMRI)-derived resting brain activity metrics: the seed-region-based functional connectivity (SRFC), independent component analysis (ICA)-derived network-based FC (NTFC), regional homogeneity (ReHo), and the amplitude of low frequency fluctuation (ALFF). Methods: Simulations were used to assess the sensitivity of SRFC, ReHo, and ALFF to noise interference. Repeat resting blood oxygen level-dependent (BOLD) fMRI were acquired from 32 healthy subjects. The intraclass correlation coefficient (ICC) was used to assess the stability of the four metrics. Results: Random noise yielded small random SRFC, small but consistent ReHo and ALFF. A neighborhood size greater than 20 voxels should be used for calculating ReHo in order to reduce the noise interference. Both the anterior cingulate cortex (ACC) and posterior cingulate cortex (PCC)-based SRFC were reproducible in more spatially extended regions than ICA NTFC. The two regional spontaneous brain activity (SBA) measures, ReHo and ALFF, showed test–retest reproducibility in almost the whole gray matter. Conclusion: SRFC, ReHo, and ALFF are robust to random noise interference. The neighborhood size for calculating ReHo should be larger than 20 voxels. ICC > 0.5 and cluster size > 11 should be used to assess the ICC maps for ACC/PCC SRFC, ReHo, and ALFF. BOLD fMRI-based SBA can be reliably measured using ACC/PCC SRFC, ReHo, and ALFF after 2 months. J. Magn. Reson. Imaging 2012;36:344–354. ©2012 Wiley Periodicals, Inc.

Tumors with germline or somatic BRCA1 or BRCA2 alterations are sensitive to DNA-damaging agents but often develop therapeutic resistance. Breast cancers associated with BRCA1/2 alterations have a relatively high mutational load, defined as the number of nonsynonymous single nucleotide mutations, relative to BRCA1/2 wild-type tumors, leading to suggestions that patients with BRCA1/2 mutations are possible candidates for immune checkpoint blockade. However, immune infiltration can range widely in tumors with BRCA1/2 alterations, and molecular determinants that account for this variability remain unclear. Our goal was to delineate the molecular features associated with immunogenicity in breast cancers associated with germline or somatic BRCA1/2 alterations through genetic/genomic and histopathologic analyses in 115 tumors from the TCGA (n=89) and Penn (n=26). In the TCGA dataset, we found that the level of homologous recombination deficiency (HRD), as measured by the copy number-based HRD score (high vs. low, dichotomized by median), correlated positively with neoantigen load in BRCA1/2 tumors (p=0.02), but unexpectedly inversely with immunogenicity as measured by cytolytic index (p=0.04) (geometric mean of PRF1 and GZMA expression) and immune ESTIMATE (p=0.002), an RNA-seq-based measure of immune cell infiltration. In prior studies, we had found that a surprisingly high percentage of BRCA1 and BRCA2 germline mutation-associated breast cancers lacked allele-specific loss of heterozygosity (LOH), 10% and 46%, respectively. We thus evaluated whether allele-specific LOH of germline BRCA2 mutations and somatic BRCA1/2 mutation clonality were drivers of the negative association between HRD level and cytolytic index/immune ESTIMATE. We found that allele-specific LOH was associated with higher HRD score (p=0.015) but lower enrichment of the Type II IFN signaling immune metagene (p=0.01), lower cytolytic index (p=0.04), and lower immune ESTIMATE score (p=0.01); tumors with clonal somatic BRCA1/2 mutations had significantly higher HRD score (p=2.4E-07) and mutational burden (p=0.05) but lower cytolytic index (p=0.003) and immune ESTIMATE scores (p=5E-05) than tumors with subclonal somatic BRCA1/2 mutations. Stratifying by both hormone receptor expression and median HRD score, we found that triple-negative breast cancers with low HRD were the most immunogenic and hormone receptor-positive tumors with high HRD the least (p=0.001). We confirmed these TCGA findings by histopathology in tumors associated with germline mutations in BRCA1/2 from Penn. We found that CD45+ leukocyte (p=0.03) and CD8+ cytotoxic T-cell infiltration (p=0.03) as well as expression of the immune effector PRF1 (p=0.048) were significantly lower with high HRD. Further, tumors with allele-specific LOH of the germline BRCA1/2 mutation had significantly lower PRF1 staining (p=0.004), lower membrane levels of the immune checkpoint protein PDL1 (p=0.05), and lower CD8+ (p=0.001), FoxP3+ (p=0.03), and CD20+ (p=0.04) immune cells, suggesting an immunosuppressive role for allele-specific LOH leading to high HRD. These data are consistent with a prior pan-TCGA analysis demonstrating that increased tumor chromosome/arm level copy number alterations suppress immunogenicity. We have found, for the first time, that genomic instability in BRCA1/2 tumors is associated with lower antitumor immune activity, and that the immunogenicity of BRCA1/2 mutation-associated breast cancers is directly related to their HRD level (driven by allele-specific LOH and clonality) and receptor status, both clinically evaluable biomarkers. These findings have immediate implications for the stratification of patients with BRCA1/2 mutation-associated breast cancer for checkpoint blockade therapy. Citation Format: Adam A. Kraya, Kara N. Maxwell, Bradley Wubbenhorst, Brandon M. Wenz, John Pluta, Andrew J. Rech, Nicole Lunceford, Amanda Barrett, Nandita Mitra, Jennifer J.D. Morrissette, Anupma Nayak, Michael Feldman, Susan M. Domchek, Robert H. Vonderheide, Katherine L. Nathanson. Homologous recombination deficiency negatively predicts for immune infiltration and antitumor immune activity in breast tumors with BRCA1/2 alterations [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A22.

Measurement of brain change due to neurodegenerative disease and treatment is one of the fundamental tasks of neuroimaging. Deformation-based morphometry (DBM) has been long recognized as an effective and sensitive tool for estimating the change in the volume of brain regions over time. This paper demonstrates that a straightforward application of DBM to estimate the change in the volume of the hippocampus can result in substantial bias, i.e., an overestimation of the rate of change in hippocampal volume. In ADNI data, this bias is manifested as a non-zero intercept of the regression line fitted to the 6 and 12 month rates of hippocampal atrophy. The bias is further confirmed by applying DBM to repeat scans of subjects acquired on the same day. This bias appears to be the result of asymmetry in the interpolation of baseline and followup images during longitudinal image registration. Correcting this asymmetry leads to bias-free atrophy estimation.

We evaluate the impact of template choice on template-based segmentation of the hippocampus in epilepsy. Four dataset-specific strategies are quantitatively contrasted: the “closest to average” individual template, the average shape version of the closest to average template, a best appearance template and the best appearance and shape template proposed here and implemented in the open source toolkit Advanced Normalization Tools (ANTS). The cross-correlation similarity metric drives the correspondence model and is used consistently to determine the optimal appearance. Minimum shape distance in the diffeomorphic space determines optimal shape. Our evaluation results show that, with respect to gold-standard manual labeling of hippocampi in epilepsy, optimal shape and appearance template construction outperforms the other strategies for gaining data-derived templates. Our results also show the improvement is most significant on the diseased side and insignificant on the healthy side. Thus, the importance of the template increases when used to study pathology and may be less critical for normal control studies. Furthermore, explicit geometric optimization of the shape component of the unbiased template positively impacts the study of diseased hippocampi.

This study examined the utility of structural and functional MRI at 1.5 and 3 T in the presurgical evaluation and prediction of postsurgical cognitive outcome in temporal lobe epilepsy (TLE). Forty-nine patients undergoing presurgical evaluation for temporal lobe (TL) resection and 25 control subjects were studied. Patients completed standard presurgical evaluations, including the intracarotid amobarbital test (IAT) and neuropsychological testing. During functional imaging, subjects performed a complex visual scene-encoding task. High-resolution structural MRI scans were used to quantify hippocampal volumes. Both structural and functional imaging successfully lateralized the seizure focus and correlated with IAT memory lateralization, with improvement for functional imaging at 3 T as compared with 1.5 T. Ipsilateral structural and functional MRI data were related to cognitive outcome, and greater functional asymmetry was related to earlier age at onset. These findings support continued investigation of the utility of MRI and fMRI in the presurgical evaluation of TLE.

Diffusion tensor (DT) and T1 structural magnetic resonance images provide unique and complementary tools for quantifying the living brain. We leverage both modalities in a diffeomorphic normalization method that unifies analysis of clinical datasets in a consistent and inherently multivariate (MV) statistical framework. We use this technique to study MV effects of traumatic brain injury (TBI).We contrast T1 and DT image-based measurements in the thalamus and hippocampus of 12 TBI survivors and nine matched controls normalized to a combined DT and T1 template space. The normalization method uses maps that are topology-preserving and unbiased. Normalization is based on the full tensor of information at each voxel and, simultaneously, the similarity between high-resolution features derived from T1 data. The technique is termed symmetric normalization for MV neuroanatomy (SyNMN). Voxel-wise MV statistics on the local volume and mean diffusion are assessed with Hotelling's T(2) test with correction for multiple comparisons.TBI significantly (false discovery rate P.05) reduces volume and increases mean diffusion at coincident locations in the mediodorsal thalamus and anterior hippocampus.SyNMN reveals evidence that TBI compromises the limbic system. This TBI morphometry study and an additional performance evaluation contrasting SyNMN with other methods suggest that the DT component may aid normalization quality.