Pathognomonic Alterations Research Articles

PATH-46. COMPUTATIONAL HISTOPATHOLOGY INFORMED RAPID TARGETED NANOPORE SEQUENCING ENABLES AFFORDABLE NEXT DAY REPORTING OF COMPREHENSIVE MOLECULAR MARKERS FOR CNS TUMOUR DIAGNOSTICS

Abstract BACKGROUND Integrative brain tumour diagnostics indisputably requires comprehensive reporting of molecular markers. The 2021 WHO classification of central nervous system (CNS) tumours substantially increased the set of markers for routine evaluation, with greater significance to DNA methylation analysis in diagnostics. Limited by investment and batching, smaller labs and clinics might suffer major delays in delivering clinical decisions. To make precision diagnostics accessible, we introduce an integrated computational histopathology and adaptive nanopore sequencing workflow for next day CNS tumour diagnostics. METHODS We used CNS-CHiP- a multitask deep transfer learning model to predict key molecular alterations and methylation classification from H&E stained CNS tumour slides. For further characterisation and subtyping, we used the predictions to formulate a custom panel for each patient. Targeted sequencing and analyses were performed using Rapid-CNS2- a custom neurooncology nanopore sequencing pipeline for parallel copy-number, mutational and methylation analysis that is flexible in target selection with no additional library preparation and can be initiated upon receipt of frozen sections. Sequencing was performed on a portable MinION or GridION. RESULTS We demonstrate our workflow on diagnostic samples received by the Department of Neuropathology, University Hospital Heidelberg. CNS-CHiP predicted multiple pathognomonic alterations (eg. IDH mutation, 7 gain/10 loss) with reasonable accuracy. This provided basic information regarding the tumor type instantly. Personalised panels enabled small target sizes, resulting in low sequencing time (up to 24h) and competitive costs. The GPU-accelerated bioinformatics pipeline reduced analysis time from > 24h to < 3h. CONCLUSIONS Our workflow harnessing histology-based molecular predictions to instruct targeted nanopore sequencing can be set up with low initial investment and has the potential to facilitate reporting of molecular results on the next day of sample collection. CNS-CHiP combined with Rapid-CNS2 thus aims to make CNS tumour diagnostics affordable and accessible to smaller hospitals and labs especially in low- and middle-income countries.

P04.03.B Rapid DNA methylation-based classification of pediatric brain tumors from ultrasonic aspirate specimens

Abstract Background Cavitating ultrasonic aspirator (CUSA) devices are commonly used in neurosurgical procedures to carefully debulk tumor from adjacent healthy brain tissue. Here, we explore the feasibility of using ultrasonic minced tumor tissue to classify otherwise discarded sample material by DNA methylation according to the respective World Health Organization (WHO) Classification of Tumors of the Central Nervous System (CNS) using low pass nanopore whole genome sequencing. Material and Methods 21 ultrasonic aspirated specimens from patients undergoing surgery in the department of pediatric neurosurgery at the Charité-Universitätsmedizin Berlin with either newly diagnosed cerebral lesions or pre-treated lesions were processed by nanopore sequencing to generate copy number profiles and ad-hoc random forest classification. Results were compared to microarray-based routine profiling. Tumor purity was assessed. Results In 19/21 (90.5 %) samples the minimum amount of 1,000 CpG sites were sequenced. In 20/21 (95.2 %) cases copy number variation profiles could be generated and matched microarray derived copy number profiles, allowing for identification of diagnostically or therapeutically relevant pathognomonic alterations. 12/17 (70.6 %) samples were concordantly classified to the corresponding microarray-based diagnosis by routine neuropathological workup. Applying recently defined thresholds for nanopore-based classification resulted in sensitivity of 64.7 % and specificity of 100 %. Conclusion CUSA referred sample material of pediatric brain tumors allows for methylation-based classification according to the respective WHO classification of CNS tumors with acceptable sensitivity and high specificity. Hereby, a promising opportunity for accurate classification of pediatric brain tumors by a time- and cost-efficient advanced molecular technique is offered using otherwise discarded tumor tissue.

PATH-48. RAPID-CNS2: RAPID COMPREHENSIVE ADAPTIVE NANOPORE-SEQUENCING OF CNS TUMORS, A PROOF OF CONCEPT STUDY

Abstract BACKGROUND The WHO classification 2021 includes multiple molecular markers for routine diagnostics in addition to histology. Sequencing setup for complete molecular profiling requires considerable investment, while batching samples for sequencing and methylation profiling can delay turnaround time. We introduce RAPID-CNS2, a nanopore adaptive sequencing pipeline that enables comprehensive mutational, methylation and copy number profiling of CNS tumours with a single third generation sequencing assay. It can be run for single samples and offers highly flexible target selection requiring no additional library preparation. METHODS Utilising ReadFish, a toolkit enabling targeted nanopore sequencing, we sequenced DNA from 22 diffuse glioma patient samples on a MinION device. Target regions comprised our Heidelberg brain tumour NGS panel and pre-selected CpG sites for methylation classification by an adapted random forest classifier. Pathognomonic alterations, copy number profiles, and methylation classes were called using a custom bioinformatics pipeline. Results were compared to their corresponding NGS panel-seq and EPIC array outputs. RESULTS Complete concordance with the EPIC array was found for copy number profiles from RAPID-CNS2. 94% pathognomonic mutations were congruent with NGS panel-seq. MGMT promoter status was correctly identified in all samples. Methylation families were detected with 96% congruence. Among the alterations decisive for rendering a classification-compatible integrated diagnosis, 97% of the alterations were consistent over the entire cohort (completely congruent in 19/22 cases and sufficient for unequivocal diagnosis in all). CONCLUSIONS RAPID-CNS2 provides a swift and highly flexible alternative to conventional NGS and array- based methods for SNV/Indel analysis, detection of copy number alterations and methylation classification. The turnaround time of ~4 days can be further shortened to < 12h by altering target sizes. It offers a low-capital approach that would be cost-efficient for low throughput settings and invaluable in cases requiring immediate diagnoses.

Bildgebende Diagnostik bei chronischer Pankreatitis

Chronic pancreatitis is characterized by recurrent inflammatory episodes. The diagnosis is based on the detection of pathognomonic morphological alterations by ultrasound-based methods as well as computed tomography and magnetic resonance imaging. In addition, imaging techniques play an important role for the differential diagnostics of potential complications in patients withchronic pancreatitis and for regular monitoring of patients with increased cancer risk. This article summarizes the current guideline recommendations for medical imaging procedures in chronic pancreatitis.

P07.04 Rapid-CNS2: Rapid comprehensive adaptive nanopore-sequencing of CNS tumors, a proof of concept study

Abstract BACKGROUND The WHO classification 2021 includes multiple molecular markers for routine diagnostics in addition to histology. Sequencing setup for complete molecular profiling requires considerable investment, while batching samples for sequencing and methylation profiling can delay turnaround time. We introduce RAPID-CNS2, a nanopore adaptive sequencing pipeline that enables comprehensive mutational, methylation and copy number profiling of CNS tumours with a single third generation sequencing assay. It can be run for single samples and offers highly flexible target selection requiring no additional library preparation. MATERIAL AND METHODS Utilising ReadFish, a toolkit enabling targeted nanopore sequencing, we sequenced DNA from 22 diffuse glioma patient samples on a MinION device. Target regions comprised our Heidelberg brain tumour NGS panel and pre-selected CpG sites for methylation classification by an adapted random forest classifier. Pathognomonic alterations, copy number profiles, and methylation classes were called using a custom bioinformatics pipeline. Results were compared to their corresponding NGS panel-seq and EPIC array outputs. RESULTS Complete concordance with the EPIC array was found for copy number profiles from RAPID-CNS2. 94% pathognomonic mutations were congruent with NGS panel-seq. MGMT promoter status was correctly identified in all samples. Methylation families were detected with 96% congruence. Among the alterations decisive for rendering a classification-compatible integrated diagnosis, 97% of the alterations were consistent over the entire cohort (completely congruent in 19/22 cases and sufficient for unequivocal diagnosis in all). CONCLUSION RAPID-CNS2 provides a swift and highly flexible alternative to conventional NGS and array-based methods for SNV/Indel analysis, detection of copy number alterations and methylation classification. The turnaround time of ~4 days can be further shortened to <12h by altering target sizes. It offers a low-capital approach that would be cost-efficient for low throughput settings and invaluable in cases requiring immediate diagnoses.

Impact of KRAS alterations in pancreatic ductal adenocarcinoma (PDAC).

4136 Background: The genomic alterations which characterize PDAC holds great promise for novel therapeutic interventions. Constitutive signaling via mutated KRAS is considered the signature pathognomonic alteration in PDAC, less than 10% of patients (pts) have tumors which are KRAS wild type (WT). We retrospectively reviewed our institutional genomic database to characterize PDAC pts with KRAS WT tumors. Methods: We reviewed electronic medical records of PDAC pts who underwent comprehensive genomic profiling (CPG) utilizing Foundation One CDx (50.6%) or TEMPUS (49.4%) between 2015-2020. Demographic and disease characteristics were compared between cohorts using Wilcoxon rank-sum test or chi-square tests. Left truncation at the time of CGP was used to account for the time of entry into the study cohort. Kaplan-Meier method was used for survival curve estimation, and log-rank test was used for between-group comparison. Cox regression was used to adjust for confounders. Results: We identified 235 patients: median age at diagnosis was 65 years and 52% were male. Clinical stages at diagnosis were localized (resectable/borderline resectable), locally advanced, or metastatic in 105 (44.7%), 61 (26.0%), and 69 (29.4%) patients, respectively. KRAS status was mutated in 212 (90%) patients: the most common alterations being G12D (48%), G12V (28%) and G12R (14%). KRAS WT status was noted in 23 (9.8%) pts, actionable genomic alterations in this subgroup are summarized in the table. Baseline demographic and treatment characteristics were similar between patients with KRAS mutated and WT tumors. Of the 23 patients with KRAS WT tumors, 16 (69.6%) completed all planned curative intent therapy compared to 121 (57.3%) of the 212 KRAS mutated pts (p=0.26). Median Overall Survival of patients with KRAS mutated tumors was 18.6 months compared to 44.1 months for WT pts (p=0.03). Adjusting for stage, WT vs. mutated status was associated with a 62% decreased hazard of death (HR 0.38 [0.18-0.83]; p=0.016). Conclusions: Patients with KRAS WT PDAC appear to have a distinct biology compared to those with KRAS mutations, meriting exploration in larger data sets. Further, comprehensive whole genome or transcriptomic characterization of KRAS WT tumors is necessary to identify putative driver alterations as well as actionable therapeutic targets.[Table: see text]

Genomic characterization of bladder cancer with variant histology.

470 Background: Up to 25% of tumors are of pure variant or mixed urothelial and variant histology. The presence of variant histology may be associated with more advanced stage at presentation and a poorer response to systemic therapy. While histomorphologic assessment provides important prognostic information, genomic analysis of tumors can provide important insight into the biology of disease and inform treatment. In this analysis we performed genomic sequencing of tumors from patients with bladder cancer with variant histology. Methods: Our prospectively generated institutional cohort of molecularly profiled bladder and upper tract tumors contains over 2,000 samples including nearly 300 primary bladder tumor samples from patients with variant histology. Targeted sequencing with MSK-IMPACT was used to identify alterations in cancer-associated genes and to describe trends across variant subtypes. To explore and compare tumor and immune cell heterogeneity, single-cell RNA sequencing (scRNA-seq) was performed on a subset of specimens. Results: Our cohort included patients with pure urothelial carcinoma not otherwise specified (NOS), as well as squamous, small cell, pure adenocarcinoma and urothelial carcinoma with glandular differentiation, micropapillary, nested, and plasmacytoid variants. Compared with urothelial carcinoma NOS, nearly all small cell tumors had mutations in TP53, RB1, and TERT. Squamous tumors had similar mutational frequencies as urothelial carcinoma NOS. Pure adenocarcinoma had frequent mutations in TP53, KRAS, and PIK3CA, resembling colorectal adenocarcinomas, while urothelial carcinoma with glandular differentiation resembled NOS. Micropapillary variant commonly had ERBB2 amplifications. Nested variant was more commonly found to have RHOA mutations and FOXA1 amplifications. Finally, nearly all plasmacytoid variants had pathognomonic alterations in CDH1. To further explore heterogeneity in tumor and immune cell populations, scRNA-seq was performed on four samples from patients with urothelial carcinoma NOS, squamous, micropapillary, and nested variants, showing distinct tumor cell clusters and varying contributions of immune cells from each variant. Conclusions: While the distribution of oncogenic mutations differed among distinct histologic variants, a pathognomonic DNA alteration was not found for most variant histologic subtypes. Within the context of a larger effort to characterize bladder cancer with variant histology, scRNA-seq may reveal differences in immune cell population infiltrates. Further efforts will aim to characterize these cohorts with whole exome sequencing and mutational signatures, and increase the number of samples per histology and representation of variant histologies for scRNA-seq.

Abstract P4-05-07: Whole-exome and RNA-sequencing analyses of acinic cell carcinomas of the breast

Abstract Introduction: Acinic cell carcinoma of the breast (ACC) is an exceedingly rare histologic form of triple-negative breast cancer (TNBC) with a generally good clinical behavior. Despite its unique histology, targeted sequencing analysis of ACC has failed to identify recurrent genetic alterations other than those found in common forms of TNBC. Here, we subjected three breast ACCs to whole-exome and RNA-sequencing, seeking to define whether they would be underpinned by a pathognomonic genetic alteration that would distinguish them from common forms of TNBC. Materials and methods: Three breast ACCs were subjected to whole-exome sequencing (WES) and RNA-sequencing. Somatic mutations, copy number alterations, mutational signatures and fusion genes were determined using a validated bioinformatics pipeline. We also estimated DNA-based measures of genomic instability including mutational signatures (using SigMA), large-scale state transition (LST) and telomeric allelic imbalance (NtAI). Results: No oncogenic in-frame fusion transcript was identified in the breast ACCs analyzed. ACCs displayed a median of 173 (range, 92-230) non-synonymous somatic mutations as defined by WES. We detected clonal TP53 hotspot mutations associated with loss of heterozygosity (LOH) of the wild-type allele in two ACCs (ACC1 and ACC18). In contrast, the TP53 wild-type ACC (ACC12) harbored a pathogenic MLH1 germline mutation (p.Glu227_Ser295del) and a clonal hotspot mutation in CTNNB1 (p.Lys335Ile). No cancer gene other than TP53 was found to be mutated in >1 case. Copy number analysis revealed that ACC18 harbored a BRCA1 homozygous deletion and focal amplification of 20p12.3 encompassing PCNA. Moreover, the TP53-mutant cases, ACC1 and ACC18, displayed complex copy number profiles, with multiple gains and losses and focal high-level amplifications. Consistent with the BRCA1 homozygous deletion, ACC18 displayed genomic features of homologous recombination DNA repair deficiency (HRD), such as a dominant mutational signature 3, a high LST score (24), a high NtAI score (23) and a high number of ‘small deletions’ >5bps. In contrast, and consistent with its DNA mismatch repair (MMR) deficiency, ACC12 had a simple genome without detectable copy number alterations, displayed a dominant mutational signature 6 associated with defective MMR, microsatellite instability (MSI) as determined by MSIsensor and loss of MLH1 expression by immunohistochemical analysis. Conclusions: Our findings lend further support to the notion that ACCs of the breast are genetically heterogeneous and display genomic features overlapping with those of common forms of TNBCs. Although no pathognomonic genetic alterations were detected in these cases, our data suggest that at least some ACCs of the breast may arise in the setting of HRD or MSI through distinct molecular mechanisms. Our data also suggest the possibility that both BRCA1 and TP53 loss of function may not be sufficient for a TNBC to display high-grade features and that inactivation of these two genes may not necessarily result in the development of high-grade TNBC. Citation Format: Francisco Beca, Simon SK Lee, Fresia Pareja, Arnaud Da Cruz Paula, Pier Selenica, Lorenzo Ferrando, Hannah Y Wen, Hong Zhang, Elena Guerini-Rocco, Emad A Rakha, Britta Weigelt, Jorge S Reis-Filho. Whole-exome and RNA-sequencing analyses of acinic cell carcinomas of the breast [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-05-07.

Abstract P4-05-11: Methylation profiling of mucinous breast cancer

Abstract Introduction: Mucinous carcinoma of the breast (MCB) is a rare histologic form of estrogen receptor (ER)-positive/HER2-negative breast cancer characterized by tumor cells floating in lakes of mucin. As compared to invasive ductal carcinoma of no special type (IDC-NST), we have recently shown that MCBs harbor a lower frequency of PIK3CA mutations and lack concurrent 1q gains and 16q losses, hallmark genetic alterations of ER-positive breast cancer. Despite their distinctive phenotype and previous efforts to characterize their genomic landscape by whole-genome, whole-exome and RNA-sequencing, no pathognomonic somatic mutation or fusion gene underpinning MCBs or the mucinous phenotype have been identified. In this study we sought to determine whether MCBs would be defined by specific epigenetic changes. Materials and methods: Thirty-six MCBs were subjected to DNA methylation profiling using Infinium MethylationEPIC arrays. Following quantile normalization, low quality probe filtering, and data background and dye bias correction using the ‘noob’ algorithm, methylation profiling data of MCBs were compared to those of ER-positive/HER2-negative IDC-NSTs from The Cancer Genome Atlas (TCGA) lacking concurrent 1q gains/16q losses or PIK3CA hotspot mutations and matched according to age and menopausal status at a 1:2 ratio (n=72). An enrichment analysis of the differentially methylated targets in gene promoters and enhancers was conducted using Minfi and MethylGSEA R packages. A subset of 12 MCBs of this cohort was subjected to RNA-sequencing and compared to age, menopausal status and molecular features-matched ER-positive/HER2-negative IDC-NSTs from TCGA (1:2 ratio; n=24) using gene set enrichment analysis (GSEA). Results: Enrichment analysis of differentially methylated probes revealed a significant enrichment of targets in promoters and enhancers of mucin-encoding genes and in genes of the mTOR signaling pathway between MCBs and matched IDC-NSTs from TCGA (P<0.01). Compared to matched IDC-NSTs, MCBs displayed promoter/enhancer hypomethylation of mucin-encoding genes, such as MUC1, MUC2 and MUCL1. We also observed promoter/enhancer hypomethylation of mTOR signaling pathway genes including MTOR, which encodes for the catalytic subunit of the mTORC complex, RPTOR, which codes for an mTOR binding protein that positively regulates the downstream effector S6 Kinase 1, EIF4E and EIF4B, key downstream effectors of the mTOR signaling pathway, and the oncogenes PIK3R2 and PIK3R3, which encode for regulatory isoforms of PI3K, in MCBs. Our gene expression analysis revealed that the mucin-encoding genes MUC1, MUC2 and MUCL1, and the mTOR signaling genes MTOR, RPTOR, EIF4E, EPIF4B, PIK3R1 and PIK3R2, found to be hypomethylated in MCBs, displayed a significantly higher gene expression in MCBs (P<0.001, each) compared to clinically and molecularly matched IDC-NSTs. Conclusions: Taken together, our data suggest that MCBs display a high expression of mucin-encoding genes, due to hypomethylation of promoters and enhancers and could provide the basis for their distinctive phenotype. Moreover, our findings suggest that despite the lack of genetic alterations affecting genes of the PI3K/mTOR signaling pathway in MCBs, mTOR signaling might be constitutively active in these tumors via epigenetic mechanisms, supporting the notion that, in the absence of pathognomonic genetic alterations, a disruption of the epigenetic landscape is a critical driver in the development of this rare breast cancer type. Citation Format: Fresia Pareja, Lorenzo Ferrando, Edaise M da Silva, Arnaud Da Cruz Paula, Anthe Stylianou, David N Brown, Pier Selenica, Jonathan Serrano, Hannah Y Wen, Hong Zhang, Edi Brogi, Larry Norton, Matjia Snuderl, Jorge S Reis-Filho, Britta Weigelt. Methylation profiling of mucinous breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-05-11.

MORPHOFUNCTIONAL ALTERATIONS IN ENDOTHELIUM IN THE PATHOGENESIS OF ESSENTIAL HYPERTENSION

Endothelium is a multilevel cellular structure that permeates all organs and systems of the body. A disorder of the regulation of the arterial tone underlies essential hypertension. However, its pathogenesis basis, despite intense efforts, remains unclear. The unfavorable role of emotional stress, hypodynamia, obesity and disorder of water-salt metabolism is obvious. However, the exact mechanisms and predictors of the development of arterial hypertension (HTN) are not currently defined. This opposes the prevention and detection of essential hypertension at an early stage. The investigation of endothelial function as a target and a predisposing factor for HTN development is promising and implies both scientific and applied clinical significance. Indeed, understanding of pathognomonic endothelial alterations for HTN development will clarify its pathogenesis and will help the development of the adequate treatment protocols. The paper reviews current data on the involvement of endothelial cells (EC) in the development of HTN. The role of lipid disorders in the physiological state of the endothelium is shown. The role of endothelial dysfunction in increasing production of active oxygen species and disorders in the nitric oxide metabolism is highlighted. The activity of the following enzyme is reviewed: NADPH (nicotinamide adenine dinucleotide phosphate) oxidase, cyclooxygenase, xantinoxydoreductase and endothelial NO synthase. The interaction of the endothelium and the extracellular matrix, as well as endothelium and smooth muscle cells, is also given according to the literature data. The role of ghrelin, produced by endothelium, in the regulation of vascular tone is highlighted. Methods of the EC assessment in vitro under hypoxia are presented. Based on the literature review, it is clear that the assessment of the endothelium under hypoxia is highly important, as well as the investigation of the influence of tissue and hemic hypoxia in vivo. These studies will help to establish the contribution of functional endothelial disturbances to the development of HTN.

A systematic review of the oral and craniofacial manifestations of cri du chat syndrome.

Cri du chat syndrome is an autosomal disorder. Because it affects few people in the population it is considered a rare disease, yet it is one of the most common autosomal chromosomal syndromes in humans. It entails pathognomonic alterations that affect the craniofacial and oral anatomy of patients. The aim of this study is to review these craniofacial and oral abnormalities in patients with Cri du chat syndrome. The PubMed Medline database was searched using two different strategies. First, we used "Dentistry" and "Cri du chat" as keywords; second, we used "Cri du chat" and "craniofacial." Seven articles in which the main orofacial and cranio-skeletal characteristics of patients with Cri du chat syndrome were described were selected according to the inclusion and exclusion criteria. Cri du Chat syndrome entails pathognomonic characteristics in the craniofacial area (epicanthus, short philtrum, and wide nasal bridge), the oral area (mandibular retrognathism and anterior open bite) and the cranial region (alterations at the cranial base angle and a small upper airway). However, more studies on larger samples are needed to specify the orofacial and craniofacial characteristics of patients with Cri du chat syndrome more accurately. Clin. Anat. 29:555-560, 2016. © 2015 Wiley Periodicals, Inc.

Abstract P2-03-08: Mutational landscape of metaplastic breast carcinomas

Abstract Introduction: Metaplastic breast carcinoma (MBC) is an aggressive histologic type of breast cancer, which preferentially displays a triple-negative phenotype. These tumors are characterized by the presence of malignant cells exhibiting differentiation towards squamous epithelium or mesenchymal elements, including spindle, chondroid, osseous and rhabdoid differentiation. Unlike other rare histologic types of breast cancer such as adenoid cystic and secretory carcinomas, which are underpinned by the MYB-NFIB and ETV6-NTRK3 fusion genes respectively, pathognomonic genetic alterations have not been identified in MBC. It has been suggested, however, that the frequency of PIK3CA somatic mutations would be significantly higher in MBCs than in other forms of triple-negative disease. Here we sought to characterize the mutational landscape of MBCs by means of high-depth whole exome sequencing analysis. Material and Methods: Twenty-one triple-negative MBCs were retrieved from the authors’ institutions. Representative sections from frozen blocks were microdissected to ensure tumor cell content greater than 50%. DNA samples extracted from microdissected tumor and matched peripheral blood leukocytes were subjected to high-depth (250x) whole exome sequencing on an Illumina GAIIx or HiSeq2000. Somatic point mutations were called using MuTect and somatic insertions and deletions (indels) were called using Strelka, Varscan2 and Haplotype Caller. Potentially pathogenic mutations were predicted using computational algorithms including PolyPhen-2, Mutation Taster, Mutation Assessor, CHASM and FATHMM. Significantly mutated genes were identified using MutSigCV. Pathway and network enrichment analysis of mutations was performed with Ingenuity Pathway Analysis and HOTNET. The genomic landscape of MBCs was compared with that of triple-negative breast cancers (TNBCs) analyzed as part of The Cancer Genome Atlas project. Results: A mean of 135 somatic non-synonymous point mutations and indels were identified per MBC. The most frequently mutated gene was TP53, found in 12/21 cases (57%), and the only significantly mutated gene as defined by MutSigCV (q<0.01). The repertoire of somatic mutations found in MBCs was qualitatively similar to that of TNBCs of no special type, and recurrently mutated genes were altered at similar frequencies in MBCs and TNBCs of no special type. When somatic mutations were annotated in pathways and networks, MBCs were found to have potentially pathogenic mutations affecting genes directly related to the PI3K pathway, including pathogenic non-synonymous mutations affecting PIK3CA, PIK3R1, PIK3R2, PIK3C2B, PIK3C2G and PTEN, significantly more frequently than TNBCs of no special type (10 out of 21 MBCs vs. 11 out of 62 TNBCs; Fisher's exact test p-value=0.0099). Conclusion: The majority (57%) of MBCs harbored non-synonymous mutations affecting TP53. While the frequencies of mutations affecting recurrently mutated genes in MBCs are similar to those found in other forms of TNBCs, MBCs significantly more frequently harbor mutations affecting PI3K pathway-related genes than TNBCs of no special type. Citation Format: Charlotte KY Ng, Britta Weigelt, Salvatore Piscuoglio, Y Hannah Wen, Maria R De Filippo, Luciano G Martelotto, Rachael Natrajan, Raymond Lim, Edi Brogi, Larry Norton, Anne Vincent-Salomon, Jorge S Reis-Filho. Mutational landscape of metaplastic breast carcinomas [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-03-08.

Poppers Maculopathy: Complete Restitution of Macular Changes in OCT after Drug Abstinence

Background: “Poppers” is a slang term for a group of alkyl nitrites that are used as recreational drugs. Their inhalative intoxication leads to muscle relaxation, analgesia, and euphoria. Maculopathy is a rare but serious side-effect. Patients/Methods: Clinical, imaging, and electrophysiological findings of seven patients with maculopathy after consumption of poppers were presented. Results: All seven patients were male with a median age of 35 years (range 28–45 years), the median duration of periodical poppers use until the onset of symptoms was 9.8 years (one day to 25 years). Five of seven patients were HIV-positive, one patient was negative, and the HIV-status of one patient was unknown. Median average of visual acuity at presentation was 20/30 in each eye. In all patients, optical coherence tomography (OCT) showed pathognomonic alterations of the outer foveal retina. One patient showed an almost complete restitution of the maculopathy six months after cessation of drug use and following the oral intake of Lutein. Imaging alterations returned to normal and visual acuity recovered from 20/50 and 20/30 (right and left eye, respectively) to 20/20 on both eyes. Follow up of two other cases showed no relevant functional decline or improvement. Discussion: Toxic maculopathy due to the consumption of poppers is an important differential diagnosis in acute visual loss without clinico-morphological correlate. Optical coherence tomography is the only reliable diagnostic tool in these cases. Complete recovery of visual function and macular morphology is rare, even after cessation of drug abuse. Oral lutein therapy may have a beneficial effect.

Nagelfalzkapillarmikroskopie

Capillaroscopy is performed to evaluate the morphology, frequency and blood flow of nailfold capillaries as well as relevant extracapillary changes in rheumatic conditions. The main indication is the differentiation between primary and secondary Raynaud's phenomenon. Various rheumatic conditions show different and partially typical pathognomonic alterations of the microcirculation or capillary patterns. This simple, noninvasive, inexpensive and effective technique is well suited for early diagnosis, especially of connective tissue diseases. The specific significance is highest for systemic sclerosis which is the most frequently evaluated entity. The corresponding microscopic alterations allow stage-adapted conclusions concerning the acuity of inflammation to be formed. That is not the only reason why capillaroscopy represents an important tool in follow-up controls and together with other techniques, such as flow measurement by laser Doppler, further conclusions can be drawn.

Canine Angiostrongylosis in Naturally Infected Dogs: Clinical Approach and Monitoring of Infection after Treatment

Canine angiostrongylosis is an increasingly reported disease in Europe which can be fatal if left untreated. The wide range of clinical presentation along with the absence of pathognomonic alterations can make the diagnosis challenging; thus any additional information that may provide clues to an early diagnosis may be of value, in order to ensure adequate anthelmintic treatment. Aim of the study was to assess a clinicopathological scoring system associated with natural Angiostrongylus vasorum infection diagnosed in canine patients during clinical practice, to clinically and paraclinically monitor infected dogs after treatment, and to monitor the presence of L1 larvae in faecal samples by Baermann's test. Of the total 210 enrolled animals A. vasorum infection was diagnosed in 7 dogs. These dogs were clinically and paraclinically investigated and monitored after specific treatment. Further 3 symptomatic dogs were retrospectively included in the monitoring. Results suggest that the computed scoring system can help to increase the clinical suspicion of infection particularly in asymptomatic dogs before the onset of potentially lethal lesions. Data of faecal monitoring suggested that treatment may control parasite burden but be unable to eradicate infection. Thus, a continued faecal monitoring after treatment is advisable for identification of still infected or reinfected dogs.

Detection of Papillary Thyroid Carcinoma by Analysis of BRAF and RET/PTC1 Mutations in Fine‐needle Aspiration Biopsies of Thyroid Nodules

Activating mutations of the oncogene BRAF or rearrangements of the tyrosine kinase receptor RET are observed in up to 80% of papillary thyroid carcinomas (PTCs). The predominant BRAF V600E mutation has not been detected in benign thyroid tissue so far, so consequently, this assumedly pathognomonic alteration is qualified to improve the preoperative diagnosis of PTC. Two hundred ninety preoperatively harvested fine-needle aspiration biopsies (FNABs) underwent routine cytologic assessment. BRAF V600E mutation analysis was performed by mutation-specific PCR using the same cell material; a hybrid-specific RT-PCR assay was used for detection of RET/PTC1 rearrangements. Detected genetic alterations were verified by direct sequencing. Definitive histopathology was obtained in 93/290 lesions following surgery of the respective thyroid nodule. While cytology alone diagnosed 13/30 malignancies (22 PTCs, 4 FTCs, 1 MTC, 1 UTC, 2 metastases), five additional malignancies were identified by supplementary mutation analysis. Cytology classified eight FNABs as benign, while postoperative histology demonstrated a thyroid malignancy (6 PTCs, 1 FTC, 1 metastasis). In four of these eight cases, the genetic analysis detected a BRAF V600E mutation or a RET/PTC1 rearrangement. Classifying both suspicious and malignant FNAB results as positive cytology results, supplementary genetic testing increased the overall sensitivity of FNAB from 70.4 to 85.7%, the positive predictive value (PPV) from 59.4 to 64.9%, and the negative predictive value (NPV) from 84.0 to 91.3%. Supplementary mutation analysis of RET and especially of the BRAF V600E mutation in FNABs is a fast and probably cost-effective assay in routine diagnostic setting. Mutation analyses of PTC-specific genetic alterations improve the preoperative identification and prognostic assessment of thyroid malignancies and therefore enable an optimized surgical strategy.

The Role of Ultrastructural Examination in Storage Diseases

Storage diseases (SDs) are rare metabolic disorders characterized by the intra- or extralysosomal accumulation of unmetabolized compounds. Different causes determine the buildup of undigested material, resulting in typical histochemical and ultrastructural changes. Ultrastructural examination of tissue from patients with clinically suspected SDs may disclose pathognomonic alterations or suggest a differential diagnosis even in the absence of clinically evident involvement of the biopsied tissue. Accurate diagnosis of SDs requires a continuous integration of clinical, biochemical, ultrastructural, and, when available, molecular data. It is also important for the pathologist to be familiar with the morphological variability characterizing each SD, because some morphologies are often the early stages of undeveloped forms and morphologically similar diseases are easily confused. The major advantages of transmission electron microscopy (TEM) techniques are discussed, emphasizing the current role of TEM as a rapid, cost-effective, and efficient diagnostic tool.

Proteomics translational

The proteome is a complex but precise reflection of cellular vitality and function. Proteomic techniques such as 2D gel electrophoresis (2DE) and mass spectrometry (MS) are employed to investigate pathognomonic processes and discover differentially expressed proteins when comparing normal and diseased entities. On the one hand proteomic research provides valuable findings on dysregulations in disease, while on the other it provides diagnostically relevant information which could form the basis for early detection of dysregulation and disease predisposition. 2DE is an excellent technique to detect differentially expressed proteins which are subjected to tandem mass spectrometry (MS-MS) for protein identification. Moreover, MS is applied for complex protein pattern profiling to analyze samples by means of “high throughput” screening and allocation to specific pathognomonic alterations. A series of scientific publications reporting on initial clinical applications appear promising in the search of prediction markers.

Systemic therapy of soft tissue sarcomas – “nothing new under the sun?” – ASCO 2009

The systemic treatment of patients with soft tissue sarcomas is an area of controversies where small progress has been made over the past years. The rarity of the disease in addition to the huge diversity of subtypes is probably to be responsible for the sometimes conflicting data obtained in clinical trials. Data presented on this year’s ASCO meeting dealt with the benefit from adjuvant treatment and with the concept of highdose consolidation therapy after complete surgical resection of a metastatic disease. The results of these trials lend support to the notion that systemic adjuvant treatment requires a rigorous patient selection and that dose escalation does not confer to a survival benefit in a situation of minimal residual disease. The more detailed understanding of pathognomonic alterations in sarcomas initiated early clinical trials with molecular directed therapies, which will probably invigorate our therapeutical possibilities.

Diagnostic Challenges in Celiac Disease and the Role of the Tissue Transglutaminase–Neo-Epitope

The diagnosis of celiac disease (CD) remains a clinical challenge based on the incomplete specificity and sensitivity rates of current non-invasive tests. Furthermore, histological assessments fail to identify all overt cases and, in particular, do not manifest pathognomonic alterations in silent cases. Accordingly, the majority of CD cases are diagnosed with great delay. Recent research into the pathogenesis of CD, allowed us to identify a neo-antigen that appears to be the most promising serological tool for the detection of anti-tissue transglutaminase as well as anti-gliadin antibodies.