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Current assays fail to address breast cancer's complex biology and accurately predict treatment response. On a retrospective cohort of 1082 female breast tissues, we develop and validate mFISHseq, which integrates multiplexed RNA fluorescent in situ hybridization with RNA-sequencing, guided by laser capture microdissection. This technique ensures tumor purity, unbiased whole transcriptome profiling, and explicitly quantifies intratumoral heterogeneity. Here we show mFISHseq has 93% accuracy compared to immunohistochemistry. Our consensus subtyping and risk groups mitigate single sample discordance, provide early and late prognostic information, and identify high risk patients with enriched immune signatures, which predict response to neoadjuvant immunotherapy in the multicenter, phase II, prospective I-SPY2 trial. We identify putative antibody-drug conjugate (ADC)-responsive patients, as evidenced by a 19-feature T-DM1 classifier, validated on I-SPY2. Deploying mFISHseq as a research-use only test on 48 patients demonstrates clinical feasibility, revealing insights into the efficacy of targeted therapies, like CDK4/6 inhibitors, immunotherapies, and ADCs., Competing Interests: Competing interests: E.D.P., B.H., N.Val., N.M., D.G., S.G., H.I., T.O., M.G., L.B., S.B., J.B., K.B., D.D., Z.K., M.K., D.L., V.Mam., V.Man., N.Voj., M.R. and P.Č. are current, or former employees of MultiplexDX, a biotechnology company that is developing a lab developed diagnostic test called Multiplex8+ ( https://www.multiplexdx.com/products/multiplex-eight-plus ), which is based on the research presented in the manuscript. P.Č. and E.D.P. are inventors and MultiplexDX, s.r.o is the assignee on patent applications that were filed in relation to the technology and research outlined in the manuscript. These include a family of patents entitled “METHOD FOR DIAGNOSING DISEASES USING MULTIPLEX FLUORESCENCE AND SEQUENCING” (WO/2020/070325, EP3775277, CA3114689, AU2019354863, SG11202103466T, KR1020210071003, CN113366118, BR112021006454, US20230037279, JP2022513333, IL282067, and NZ774986) as well as submitted EPO and PCT patents that are not published. T.T., F.P., and J.N.K. are members of the Scientific Advisory board at MultiplexDX. F.P. reports consulting services and serving on the advisory board for AstraZeneca. J.N.K. declares consulting services for Owkin, France, DoMore Diagnostics, Norway, Panakeia, UK, Scailyte, Switzerland, Cancilico, Germany, Mindpeak, Germany, and Histofy, UK; furthermore, he holds shares in StratifAI GmbH, Germany, has received a research grant by GSK, and has received honoraria by AstraZeneca, Bayer, Eisai, Janssen, MSD, BMS, Roche, Pfizer and Fresenius. All other authors declare no competing interests. Ethics & Inclusions: This study complies with all relevant ethical regulations for human research participants in accordance with the Declaration of Helsinki. The collection and use of human tissue in the retrospective clinical validation study on 1082 patients and the RUO testing cohort of 48 patients were approved by the Ethics Committee of the Bratislava Self-Governing Region (Ref. No. 05320/2020/HF). Additionally, the retrospective cohort received approval from the Ethics Commission of the Medical University of Graz on behalf of Biobank Graz (No. 34-354 ex 21/21, 1158−2022). Informed consent was obtained for tissues in the retrospective study from tissue sources including biobanks (PATH Biobank and Biobank Graz), a hospital (Malaga), and commercial companies (AMSBio and Precision for Medicine). For the RUO cohort, all 48 patients signed informed consent forms, with their oncologists approving them prior to tissue processing. The RUO testing occurred through routine testing with collaborating hospitals in Slovakia and included consultations and participation of local oncologists and researchers for patient selection, collecting clinicopathological data, obtaining informed consent, and explaining results. No discrimination occurred in the selection of patients for RUO testing and both clinical partners and MultiplexDX ensured sensitive patient information was anonymized when appropriate and secured both physically and electronically. A local pathologist (Dr. Karol Kajo) was also consulted with in both the retrospective and RUO cohorts to identify invasive breast cancer or unique histology in more challenging cases., (© 2025. The Author(s).)

Hodgkin lymphoma (HL) represents a neoplasm primarily affecting adolescents and young adults, necessitating the development of precise diagnostic and monitoring tools. Specifically, classical Hodgkin lymphoma (cHL), comprising 90% of cases, necessitating tailored treatments to minimize late toxicities. Although positron emission tomography/computed tomography (PET/CT) has enhanced response assessment, its limitations underscore the urgency for more reliable progression predictive tools. Genomic characterisation of rare Hodgkin Reed-Sternberg (HRS) cells is challenging but essential. Recent studies employ single-cell molecular analyses, mass cytometry, and Next-Generation Sequencing (NGS) to unveil mutational landscapes. The integration of liquid biopsies, particularly circulating tumor DNA (ctDNA), extracellular vesicles (EVs), miRNAs and cytokines, emerge as groundbreaking approaches. Recent studies demonstrate ctDNA's potential in assessing therapy responses and predicting relapses in HL. Despite cHL-specific ctDNA applications being relatively unexplored, studies emphasize its value in monitoring treatment outcomes. Overall, this review underscores the imperative role of liquid biopsies in advancing HL diagnosis and monitoring., (© 2024. The Author(s).)

Early breast cancer patients often experience relapse due to residual disease after treatment. Liquid biopsy is a methodology capable of detecting tumor components in blood, but low concentrations at early stages pose challenges. To detect them, next-generation sequencing has promise but entails complex processes. Exploring larger blood volumes could overcome detection limitations. Herein, a total of 282 high-volume plasma and blood-cell samples were collected for dual ctDNA/CTCs detection using a single droplet-digital PCR assay per patient. ctDNA and/or CTCs were detected in 100% of pre-treatment samples. On the other hand, post-treatment positive samples exhibited a minimum variant allele frequency of 0.003% for ctDNA and minimum cell number of 0.069 CTCs/mL of blood, surpassing previous investigations. Accurate prediction of residual disease before surgery was achieved in patients without a complete pathological response. A model utilizing ctDNA dynamics achieved an area under the ROC curve of 0.92 for predicting response. We detected disease recurrence in blood in the three patients who experienced a relapse, anticipating clinical relapse by 34.61, 9.10, and 7.59 months. This methodology provides an easily implemented alternative for ultrasensitive residual disease detection in early breast cancer patients., (© 2024. The Author(s).)

Background: The variability in responses to neoadjuvant treatment with anti-HER2 antibodies prompts to personalized clinical management and the development of innovative treatment strategies. Tumor-infiltrating Natural Killer (TI-NK) cells can predict the efficacy of HER2-targeted antibodies independently from clinicopathological factors in primary HER2-positive breast cancer patients. Understanding the mechanism/s underlying this association would contribute to optimizing patient stratification and provide the rationale for combinatorial approaches with immunotherapy., Methods: We sought to uncover processes enriched in NK cell-infiltrated tumors as compared to NK cell-desert tumors by microarray analysis. Findings were validated in clinical trial-derived transcriptomic data. In vitro and in vivo preclinical models were used for mechanistic studies. Findings were analysed in clinical samples (tumor and serum) from breast cancer patients., Results: NK cell-infiltrated tumors were enriched in CCL5/IFNG-CXCL9/10 transcripts. In multivariate logistic regression analysis, IFNG levels underlie the association between TI-NK cells and pathological complete response to neoadjuvant treatment with trastuzumab. Mechanistically, the production of IFN-ɣ by CD16 +  NK cells triggered the secretion of CXCL9/10 from cancer cells. This effect was associated to tumor growth control and the conversion of CD16 into CD16 - CD103 +  NK cells in humanized in vivo models. In human breast tumors, the CD16 and CD103 markers identified lineage-related NK cell subpopulations capable of producing CCL5 and IFN-ɣ, which correlated with tissue-resident CD8 +  T cells. Finally, an early increase in serum CCL5/CXCL9 levels identified patients with NK cell-rich tumors showing good responses to anti-HER2 antibody-based neoadjuvant treatment., Conclusions: This study identifies specialized NK cell subsets as the source of IFN-ɣ influencing the clinical efficacy of anti-HER2 antibodies. It also reveals the potential of serum CCL5/CXCL9 as biomarkers for identifying patients with NK cell-rich tumors and favorable responses to anti-HER2 antibody-based neoadjuvant treatment., (© 2024. The Author(s).)

Background: Analysis of circulating tumor DNA (ctDNA) is increasingly used for clinical decision-making in oncology. However, ctDNA could represent ≤ 0.1 % of cell-free DNA in early-stage tumors and its detection requires high-sensitive techniques such as digital PCR (dPCR)., Methods: In 46 samples from patients with early-stage breast cancer, we compared two leading dPCR assays for ctDNA analysis: QX200 droplet digital PCR (ddPCR) system from Bio-Rad which is the gold-standard in the field, and Absolute Q plate-based digital PCR (pdPCR) system from Thermo Fisher Scientific which has not been reported before. We analyzed 5 mL of baseline plasma samples prior to any treatment., Results: Both systems displayed a comparable sensitivity with no significant differences observed in mutant allele frequency. In fact, ddPCR and pdPCR possessed a concordance > 90 % in ctDNA positivity. Nevertheless, ddPCR exhibited higher variability and a longer workflow. Finally, we explored the association between ctDNA levels and clinicopathological features. Significantly higher ctDNA levels were present in patients with a Ki67 score > 20 % or with estrogen receptor-negative or triple-negative breast cancer subtypes., Conclusion: Both ddPCR and pdPCR may constitute sensitive and reliable tools for ctDNA analysis with an adequate agreement in early-stage breast cancer patients., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Breast cancer (BC) is the most prevalent cancer in women. While usually detected when localized, invasive procedures are still required for diagnosis. Herein, we developed a novel ultrasensitive pipeline to detect circulating tumor DNA (ctDNA) in a series of 75 plasma samples from localized BC patients prior to any medical intervention. We first performed a tumor-informed analysis to correlate the mutations found in tumor tissue and plasma. Disregarding the tumor data next, we developed an approach to detect tumor mutations in plasma. We observed a mutation concordance between the tumor and plasma of 29.50% with a sensitivity down to 0.03% in mutant variant allele frequency (VAF). We detected mutations in 33.78% of the samples, identifying eight patients with plasma-only mutations. Altogether, we determined a specificity of 86.36% and a positive predictive value of 88.46% for BC detection. We demonstrated an association between higher ctDNA median VAF and higher tumor grade, multiple plasma mutations with a likelihood of relapse and more frequent TP53 plasma mutations in hormone receptor-negative tumors. Overall, we have developed a unique ultra-sensitive sequencing workflow with a technology not previously employed in early BC, paving the way for its application in BC screening.

Invasive breast cancer (BC) is the most common cancer in women with a slightly increasing yearly incidence. BC immunohistochemical characterisation is a crucial tool to define the intrinsic nature of each tumour and personalise BC patients' clinical management. In this regard, the characterisation of human epidermal growth factor receptor 2 (HER2) status guides physicians to treat with therapies tailored to this membrane receptor. Standardly, a tumour solid biopsy is therefore required, which is an invasive procedure and has difficulties to provide the complete molecular picture of the tumour. To complement these standard-of-care approaches, liquid biopsy is a validated methodology to obtain circulating tumour components such as circulating tumour DNA (ctDNA) and circulating tumour cells (CTCs) from body fluids in an easy-to-perform minimal-invasive manner. However, its clinical validity in cancer is still to be demonstrated. This review focusses on the utilisation of both ctDNA and CTCs in early and metastatic HER2-positive BC tumours. We discuss recently published studies deciphering the capacity of liquid biopsy to determine the response to neoadjuvant and adjuvant therapies as well as to predict patients' outcomes.

Breast cancer is the most common cancer type in women worldwide and its early detection is crucial to curing the disease. Tissue biopsy, currently the method of choice to obtain tumour molecular information, is invasive and might be affected by tumour heterogeneity rendering it incapable to portray the complete molecular picture. Liquid biopsy permits to study disease features in a more comprehensive manner by sampling biofluids and extracting tumour components such as circulating-tumour DNA (ctDNA), circulating-tumour cells (CTCs), and/or circulating-tumour RNA (ctRNA) amongst others in a monitoring-compatible manner. In this review, we describe the recent progress in the utilization of the circulating tumour components using early breast cancer samples. We review the most important analytes and technologies employed for their study., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

In breast cancer (BC) the employment of sequencing technologies for metagenomic analyses has allowed not only the description of the overall metagenomic landscape but also the specific microbial changes and their functional implications. Most of the available data suggest that BC is related to bacterial dysbiosis in both the gut microenvironment and breast tissue. It is hypothesized that changes in the composition and functions of several breast and gut bacterial taxa may contribute to BC development and progression through several pathways. One of the most prominent roles of gut microbiota is the regulation of steroid-hormone metabolism, such as estrogens, a component playing an important role as risk factor in BC development, especially in postmenopausal women. On the other hand, breast and gut resident microbiota are the link in the reciprocal interactions between cancer cells and their local environment, since microbiota are capable of modulating mucosal and systemic immune responses. Several in vivo and in vitro studies show remarkable evidence that diet, probiotics and prebiotics could exert important anticarcinogenic effects in BC. Moreover, gut microbiota have an important role in the metabolism of chemotherapeutic drugs and in the activity of immunogenic chemotherapies since they are a potential dominant mediator in the response to cancer therapy. Then, the microbiome impact in BC is multi-factorial, and the gut and breast tissue bacteria population could be important in regulating the local immune system, in tumor formation and progression and in therapy response and/or resistance.

Importance: Current treatment cures most cases of early-stage, primary breast cancer. However, better techniques are required to identify which patients are at risk of relapse., Objective: To assess the clinical validity of molecular relapse detection with circulating tumor DNA (ctDNA) analysis in early-stage breast cancer., Design, Setting, and Participants: This prospective, multicenter, sample collection, validation study conducted at 5 United Kingdom medical centers from November 24, 2011, to October 18, 2016, assessed patients with early-stage breast cancer irrespective of hormone receptor and ERBB2 (formerly HER2 or HER2/neu) status who were receiving neoadjuvant chemotherapy followed by surgery or surgery before adjuvant chemotherapy. The study recruited 170 women, with mutations identified in 101 patients forming the main cohort. Secondary analyses were conducted on a combined cohort of 144 patients, including 43 patients previously analyzed in a proof of principle study., Interventions: Primary tumor was sequenced to identify somatic mutations, and personalized tumor-specific digital polymerase chain reaction assays were used to monitor these mutations in serial plasma samples taken every 3 months for the first year of follow-up and subsequently every 6 months., Main Outcomes and Measures: The primary end point was relapse-free survival analyzed with Cox proportional hazards regression models., Results: In the main cohort of 101 female patients (mean [SD] age, 54 [11] years) with a median follow-up of 35.5 months (interquartile range, 27.9-43.0 months), detection of ctDNA during follow-up was associated with relapse (hazard ratio, 25.2; 95% CI, 6.7-95.6; P < .001). Detection of ctDNA at diagnosis, before any treatment, was also associated with relapse-free survival (hazard ratio, 5.8; 95% CI, 1.2-27.1; P = .01). In the combined cohort, ctDNA detection had a median lead time of 10.7 months (95% CI, 8.1-19.1 months) compared with clinical relapse and was associated with relapse in all breast cancer subtypes. Distant extracranial metastatic relapse was detected by ctDNA in 22 of 23 patients (96%). Brain-only metastasis was less commonly detected by ctDNA (1 of 6 patients [17%]), suggesting relapse sites less readily detectable by ctDNA analysis., Conclusions and Relevance: The findings suggest that detection of ctDNA during follow-up is associated with a high risk of future relapse of early-stage breast cancer. Prospective studies are needed to assess the potential of molecular relapse detection to guide adjuvant therapy.

Purpose: MDH2 (malate dehydrogenase 2) has recently been proposed as a novel potential pheochromocytoma/paraganglioma (PPGL) susceptibility gene, but its role in the disease has not been addressed. This study aimed to determine the prevalence of MDH2 pathogenic variants among PPGL patients and determine the associated phenotype., Methods: Eight hundred thirty patients with PPGLs, negative for the main PPGL driver genes, were included in the study. Interpretation of variants of unknown significance (VUS) was performed using an algorithm based on 20 computational predictions, by implementing cell-based enzymatic and immunofluorescence assays, and/or by using a molecular dynamics simulation approach., Results: Five variants with potential involvement in pathogenicity were identified: three missense (p.Arg104Gly, p.Val160Met and p.Ala256Thr), one in-frame deletion (p.Lys314del), and a splice-site variant (c.429+1G>T). All were germline and those with available biochemical data, corresponded to noradrenergic PPGL., Conclusion: This study suggests that MDH2 pathogenic variants may play a role in PPGL susceptibility and that they might be responsible for less than 1% of PPGLs in patients without pathogenic variants in other major PPGL driver genes, a prevalence similar to the one recently described for other PPGL genes. However, more epidemiological data are needed to recommend MDH2 testing in patients negative for other major PPGL genes.

Purpose: The high percentage of patients carrying germline mutations makes pheochromocytomas/paragangliomas the most heritable of all tumors. However, there are still cases unexplained by mutations in the known genes. We aimed to identify the genetic cause of disease in patients strongly suspected of having hereditary tumors., Methods: Whole-exome sequencing was applied to the germlines of a parent-proband trio. Genome-wide methylome analysis, RNA-seq, CRISPR/Cas9 gene editing, and targeted sequencing were also performed., Results: We identified a novel de novo germline mutation in DNMT3A, affecting a highly conserved residue located close to the aromatic cage that binds to trimethylated histone H3. DNMT3A-mutated tumors exhibited significant hypermethylation of homeobox-containing genes, suggesting an activating role of the mutation. CRISPR/Cas9-mediated knock-in in HeLa cells led to global changes in methylation, providing evidence of the DNMT3A-altered function. Targeted sequencing revealed subclonal somatic mutations in six additional paragangliomas. Finally, a second germline DNMT3A mutation, also causing global tumor DNA hypermethylation, was found in a patient with a family history of pheochromocytoma., Conclusion: Our findings suggest that DNMT3A may be a susceptibility gene for paragangliomas and, if confirmed in future studies, would represent the first example of gain-of-function mutations affecting a DNA methyltransferase gene involved in cancer predisposition.

Purpose: Resistance to platinum-based chemotherapy or PARP inhibition in germline BRCA1 or BRCA2 mutation carriers may occur through somatic reversion mutations or intragenic deletions that restore BRCA1 or BRCA2 function. We assessed whether BRCA1/2 reversion mutations could be identified in circulating cell-free DNA (cfDNA) of patients with ovarian or breast cancer previously treated with platinum and/or PARP inhibitors. Experimental Design: cfDNA from 24 prospectively accrued patients with germline BRCA1 or BRCA2 mutations, including 19 patients with platinum-resistant/refractory ovarian cancer and five patients with platinum and/or PARP inhibitor pretreated metastatic breast cancer, was subjected to massively parallel sequencing targeting all exons of 141 genes and all exons and introns of BRCA1 and BRCA2 Functional studies were performed to assess the impact of the putative BRCA1/2 reversion mutations on BRCA1/2 function. Results: Diverse and often polyclonal putative BRCA1 or BRCA2 reversion mutations were identified in cfDNA from four patients with ovarian cancer (21%) and from two patients with breast cancer (40%). BRCA2 reversion mutations were detected in cfDNA prior to PARP inhibitor treatment in a patient with breast cancer who did not respond to treatment and were enriched in plasma samples after PARP inhibitor therapy. Foci formation and immunoprecipitation assays suggest that a subset of the putative reversion mutations restored BRCA1/2 function. Conclusions: Putative BRCA1/2 reversion mutations can be detected by cfDNA sequencing analysis in patients with ovarian and breast cancer. Our findings warrant further investigation of cfDNA sequencing to identify putative BRCA1/2 reversion mutations and to aid the selection of patients for PARP inhibition therapy. Clin Cancer Res; 23(21); 6708-20. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Purpose: Mutations in Krebs cycle genes are frequently found in patients with pheochromocytomas/paragangliomas. Disruption of SDH, FH or MDH2 enzymatic activities lead to accumulation of specific metabolites, which give rise to epigenetic changes in the genome that cause a characteristic hypermethylated phenotype. Tumors showing this phenotype, but no alterations in the known predisposing genes, could harbor mutations in other Krebs cycle genes. Experimental Design: We used downregulation and methylation of RBP1, as a marker of a hypermethylation phenotype, to select eleven pheochromocytomas and paragangliomas for targeted exome sequencing of a panel of Krebs cycle-related genes. Methylation profiling, metabolite assessment and additional analyses were also performed in selected cases. Results: One of the 11 tumors was found to carry a known cancer-predisposing somatic mutation in IDH1 A variant in GOT2 , c.357A>T, found in a patient with multiple tumors, was associated with higher tumor mRNA and protein expression levels, increased GOT2 enzymatic activity in lymphoblastic cells, and altered metabolite ratios both in tumors and in GOT2 knockdown HeLa cells transfected with the variant. Array methylation-based analysis uncovered a somatic epigenetic mutation in SDHC in a patient with multiple pheochromocytomas and a gastrointestinal stromal tumor. Finally, a truncating germline IDH3B mutation was found in a patient with a single paraganglioma showing an altered α-ketoglutarate/isocitrate ratio. Conclusions: This study further attests to the relevance of the Krebs cycle in the development of PCC and PGL, and points to a potential role of other metabolic enzymes involved in metabolite exchange between mitochondria and cytosol. Clin Cancer Res; 23(20); 6315-24. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Pheochromocytomas (PCCs) and paragangliomas (PGLs) are tumors arising from the adrenal medulla and sympathetic/parasympathetic paraganglia, respectively. Approximately 40% of PCCs/PGLs are due to germline mutations in one of 16 susceptibility genes, and a further 30% are due to somatic alterations in 5 main genes. Recently, somatic ATRX mutations have been found in succinate dehydrogenase (SDH)-associated hereditary PCCs/PGLs. In the present study we applied whole-exome sequencing to the germline and tumor DNA of a patient with metastatic composite PCC and no alterations in known PCC/PGL susceptibility genes. A somatic loss-of-function mutation affecting ATRX was identified in tumor DNA. Transcriptional profiling analysis classified the tumor within cluster 2 of PCCs/PGLs (without SDH gene mutations) and identified downregulation of genes involved in neuronal development and homeostasis (NLGN4, CD99 and CSF2RA) as well as upregulation of Drosha, an important gene involved in miRNA and rRNA processing. CpG island methylator phenotype typical of SDH gene-mutated tumors was ruled out, and SNP array data revealed a unique profile of gains and losses. Finally, we demonstrated the presence of alternative lengthening of telomeres in the tumor, probably associated with the failure of ATRX functions. In conclusion, somatic variants affecting ATRX may play a driver role in sporadic PCC/PGL., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Unlabelled: The presence of germline mutations affecting the MYC-associated protein X (MAX) gene has recently been identified as one of the now 11 major genetic predisposition factors for the development of hereditary pheochromocytoma and/or paraganglioma. Little is known regarding how missense variants of unknown significance (VUS) in MAX affect its pivotal role in the regulation of the MYC/MAX/MXD axis. In the present study, we propose a consensus computational prediction based on five "state-of-the-art" algorithms. We also describe a PC12-based functional assay to assess the effects that 12 MAX VUS may have on MYC's E-box transcriptional activation. For all but two of these 12 VUS, the functional assay and the consensus computational prediction gave consistent results; we classified seven variants as pathogenic and three as nonpathogenic. The introduction of wild-type MAX cDNA into PC12 cells significantly decreased MYC's ability to bind to canonical E-boxes, while pathogenic MAX proteins were not able to fully repress MYC activity. Further clinical and molecular evaluation of variant carriers corroborated the results obtained with our functional assessment. In the absence of clear heritability, clinical information, and molecular data, consensus computational predictions and functional models are able to correctly classify VUS affecting MAX., Key Messages: A functional assay assesses the effects of MAX VUS over MYC transcriptional activity. A consensus computational prediction and the functional assay show high concordance. Variant carriers' clinical and molecular data support the functional assessment.

Background: Nowadays, 65-80% of pheochromocytoma and paraganglioma (PPGL) cases are explained by germline or somatic mutations in one of 22 genes. Several genetic testing algorithms have been proposed, but they usually exclude sporadic-PPGLs (S-PPGLs) and none include somatic testing. We aimed to genetically characterise S-PPGL cases and propose an evidence-based algorithm for genetic testing, prioritising DNA source., Methods: The study included 329 probands fitting three criteria: single PPGL, no syndromic and no PPGL family history. Germline DNA was tested for point mutations in RET and for both point mutation and gross deletions in VHL, the SDH genes, TMEM127, MAX and FH. 99 tumours from patients negative for germline screening were available and tested for RET, VHL, HRAS, EPAS1, MAX and SDHB., Results: Germline mutations were found in 46 (14.0%) patients, being more prevalent in paragangliomas (PGLs) (28.7%) than in pheochromocytomas (PCCs) (4.5%) (p=6.62×10(-10)). Somatic mutations were found in 43% of those tested, being more prevalent in PCCs (48.5%) than in PGLs (32.3%) (p=0.13). A quarter of S-PPGLs had a somatic mutation, regardless of age at presentation. Head and neck PGLs (HN-PGLs) and thoracic-PGLs (T-PGLs) more commonly had germline mutations (p=2.0×10(-4) and p=0.027, respectively). Five of the 29 metastatic cases harboured a somatic mutation, one in HRAS., Conclusions: We recommend prioritising testing for germline mutations in patients with HN-PGLs and T-PGLs, and for somatic mutations in those with PCC. Biochemical secretion and SDHB-immunohistochemistry should guide genetic screening in abdominal-PGLs. Paediatric and metastatic cases should not be excluded from somatic screening., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Purpose: Pheochromocytoma and paraganglioma (PPGL) are rare neuroendocrine tumors, associated with highly variable postoperative evolution. The scarcity of reliable PPGL prognostic markers continues to complicate patient management. In this study, we explored genome-wide DNA methylation patterns in the context of PPGL malignancy to identify novel prognostic markers., Experimental Design: We retrospectively investigated DNA methylation patterns in PPGL with and without metastases using high-throughput DNA methylation profiling data (Illumina 27K) from two large, well-characterized discovery (n = 123; 24 metastatic) and primary validation (n = 154; 24 metastatic) series. Additional validation of candidate CpGs was performed by bisulfite pyrosequencing in a second independent set of 33 paraffin-embedded PPGLs (19 metastatic)., Results: Of the initial 86 candidate CpGs, we successfully replicated 52 (47 genes), associated with metastatic PPGL. Of these, 48 CpGs showed significant associations with time to progression even after correcting for SDHB genotype, suggesting their value as prognostic markers independent of genetic background. Hypermethylation of RDBP (negative elongation factor complex member E) in metastatic tumors was further validated by bisulfite pyrosequencing [Δβmetastatic-benign = 0.29, P = 0.003; HR, 1.4; 95% confidence interval (CI), 1.1-2.0; P = 0.018] and may alter transcriptional networks involving (RERG, GPX3, and PDZK1) apoptosis, invasion, and maintenance of DNA integrity., Conclusions: This is the first large-scale study of DNA methylation in metastatic PPGL that identifies and validates prognostic markers, which could be used for stratifying patients according to risk of developing metastasis. Of the three CpGs selected for further validation, one (RDBP) was clearly confirmed and could be used for stratifying patients according to the risk of developing metastases., (©2015 American Association for Cancer Research.)

Cytochrome P450 3A4 (CYP3A4) is a key drug-metabolizing enzyme. Loss-of-function variants have been reported as rare events, and the first demonstration of a CYP3A4 protein lacking functional activity is caused by CYP3A4*20 allele. Here we characterized the world distribution and origin of CYP3A4*20 mutation. CYP3A4*20 was determined in more than 4000 individuals representing different populations, and haplotype analysis was performed using CYP3A polymorphisms and microsatellite markers. CYP3A4*20 allele was present in 1.2% of the Spanish population (up to 3.8% in specific regions), and all CYP3A4*20 carriers had a common haplotype. This is compatible with a Spanish founder effect and classifies CYP3A4 as a polymorphic enzyme. This constitutes the first description of a CYP3A4 loss-of-function variant with high frequency in a population. CYP3A4*20 results together with the key role of CYP3A4 in drug metabolism support screening for rare CYP3A4 functional alleles among subjects with adverse drug events in certain populations.

Disruption of the Krebs cycle is a hallmark of cancer. IDH1 and IDH2 mutations are found in many neoplasms, and germline alterations in SDH genes and FH predispose to pheochromocytoma/paraganglioma and other cancers. We describe a paraganglioma family carrying a germline mutation in MDH2, which encodes a Krebs cycle enzyme. Whole-exome sequencing was applied to tumor DNA obtained from a man age 55 years diagnosed with multiple malignant paragangliomas. Data were analyzed with the two-sided Student's t and Mann-Whitney U tests with Bonferroni correction for multiple comparisons. Between six- and 14-fold lower levels of MDH2 expression were observed in MDH2-mutated tumors compared with control patients. Knockdown (KD) of MDH2 in HeLa cells by shRNA triggered the accumulation of both malate (mean ± SD: wild-type [WT] = 1±0.18; KD = 2.24±0.17, P = .043) and fumarate (WT = 1±0.06; KD = 2.6±0.25, P = .033), which was reversed by transient introduction of WT MDH2 cDNA. Segregation of the mutation with disease and absence of MDH2 in mutated tumors revealed MDH2 as a novel pheochromocytoma/paraganglioma susceptibility gene., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Background: Tyrosine kinase inhibitors (TKIs) have achieved remarkable clinical results in medullary thyroid carcinoma (MTC) patients. However, the considerable variability in patient response to treatment with TKIs remains largely unexplained. There is evidence that it could be due, at least in part, to alterations in genes associated with the disease via their effect on the expression of TKI targets. The objective of this study was to evaluate the influence of RAS mutations on the expression levels in MTC tumors of eight key TKI target proteins., Methods: We assessed by immunohistochemistry the expression of EGFR, KIT, MET, PDGFRB, VEGF, VEGFR1, VEGFR2, and VEGFR3 in a series of 84 primary MTC tumors that had previously been molecularly characterized, including 14 RAS-positive, 18 RET(M918T)-positive, and 24 RET(C634)-positive tumors, as well as 15 wild-type tumors with no mutations in the RET or RAS genes., Results: In contrast to RET-positive tumors, RAS-positive tumors expressed neither PDGFRB nor MET (p=0.0060 and 0.047, respectively). Similarly, fewer RAS-positive than RET-related tumors expressed VEGFR3 (p=0.00062). Finally, wild-type tumors expressed VEGF more often than both RAS- and RET-positive tumors (p=0.0082 and 0.011, respectively)., Conclusions: This is the first study identifying that the expression of TKI targets differs according to the presence of RAS mutations in MTC. This information could potentially be used to select the most beneficial TKI treatment for these patients.

The therapeutic options for patients with metastatic medullary thyroid carcinoma (MTC) have recently increased due to the development of tyrosine kinase inhibitors (TKIs), some of which have achieved remarkable clinical responses in MTC patients. However, the molecular basis for the large variability in TKI responses is unknown. In this exploratory study, we investigated the expression of eight key TKI target proteins (EGFR, KIT, MET, PDGFRB, VEGF (VEGFA), VEGFR1 (FLT1), VEGFR2 (KDR), and VEGFR3 (FLT4)) by immunohistochemistry in 103 molecularly characterized MTC samples and identified the associated clinical and molecular features. A number of MTC samples exhibited a high expression of VEGFR2 and VEGFR3, which were overexpressed in 57 and 43% of the MTC samples respectively. VEGFR1, PDGFRB, VEGF, KIT, and MET were present in 34-20% of the cases, while EGFR was highly expressed in only 10% of the MTC samples. Some proteins exhibited large differences in expression between sporadic and familial cases, suggesting that different RET mutations may be associated with the immunohistochemical profiles. MTC samples with the C634 RET mutation exhibited a higher expression of VEGFR3 and KIT than the M918T RET-mutated and non-mutated RET tumor samples (P=0.005 and P=0.007 respectively) and a lower expression of VEGFR1 (P=0.04). Non-mutated RET MTC cases exhibited a lower expression of PDGFRB (P=0.04). Overall, this is the first study, to our knowledge, to show that multiple TKI targets are highly expressed in a subset of MTCs, suggesting that molecular stratification of patients may have the potential to improve TKI therapies for MTC.

Pheochromocytomas (PCCs) and paragangliomas (PGLs) are rare neuroendocrine neoplasias of neural crest origin that can be part of several inherited syndromes. Although their mRNA profiles are known to depend on genetic background, a number of questions related to tumor biology and clinical behavior remain unanswered. As microRNAs (miRNAs) are key players in the modulation of gene expression, their comprehensive analysis could resolve some of these issues. Through characterization of miRNA profiles in 69 frozen tumors with germline mutations in the genes SDHD, SDHB, VHL, RET, NF1, TMEM127, and MAX, we identified miRNA signatures specific to, as well as common among, the genetic groups of PCCs/PGLs. miRNA expression profiles were validated in an independent series of 30 composed of VHL-, SDHB-, SDHD-, and RET-related formalin-fixed paraffin-embedded PCC/PGL samples using quantitative real-time PCR. Upregulation of miR-210 in VHL- and SDHB-related PCCs/PGLs was verified, while miR-137 and miR-382 were confirmed as generally upregulated in PCCs/PGLs (except in MAX-related tumors). Also, we confirmed overexpression of miR-133b as VHL-specific miRNAs, miR-488 and miR-885-5p as RET-specific miRNAs, and miR-183 and miR-96 as SDHB-specific miRNAs. To determine the potential roles miRNAs play in PCC/PGL pathogenesis, we performed bioinformatic integration and pathway analysis using matched mRNA profiling data that indicated a common enrichment of pathways associated with neuronal and neuroendocrine-like differentiation. We demonstrated that miR-183 and/or miR-96 impede NGF-induced differentiation in PC12 cells. Finally, global proteomic analysis in SDHB and MAX tumors allowed us to determine that miRNA regulation occurs primarily through mRNA degradation in PCCs/PGLs, which partially confirmed our miRNA-mRNA integration results.

Pheochromocytomas (PCCs) and paragangliomas (PGLs) are chromaffin-cell tumors that arise from the adrenal medulla and extra-adrenal paraganglia, respectively. The dysfunction of genes involved in the cellular response to hypoxia, such as VHL, EGL nine homolog 1, and the succinate dehydrogenase (SDH) genes, leads to a direct abrogation of hypoxia inducible factor (HIF) degradation, resulting in a pseudo-hypoxic state implicated in PCC/PGL development. Recently, somatic post-zygotic mutations in EPAS1 (HIF2A) have been found in patients with multiple PGLs and congenital erythrocytosis. We assessed 41 PCCs/PGLs for mutations in EPAS1 and herein describe the clinical, molecular and genetic characteristics of the 7 patients found to carry somatic EPAS1 mutations; 4 presented with multiple PGLs (3 of them also had congenital erythrocytosis), whereas 3 were single sporadic PCC/PGL cases. Gene expression analysis of EPAS1-mutated tumors revealed similar mRNA EPAS1 levels to those found in SDH-gene- and VHL-mutated cases and a significant up-regulation of two hypoxia-induced genes (PCSK6 and GNA14). Interestingly, single nucleotide polymorphism array analysis revealed an exclusive gain of chromosome 2p in three EPAS1-mutated tumors. Furthermore, multiplex-PCR screening for small rearrangements detected a specific EPAS1 gain in another EPAS1-mutated tumor and in three non-EPAS1-mutated cases. The finding that EPAS1 is involved in the sporadic presentation of the disease not only increases the percentage of PCCs/PGLs with known driver mutations, but also highlights the relevance of studying other hypoxia-related genes in apparently sporadic tumors. Finally, the detection of a specific copy number alteration affecting chromosome 2p in EPAS1-mutated tumors may guide the genetic diagnosis of patients with this disease.

Cellular microtubules composed of α-β-tubulin heterodimers that are essential for cell shape, division, and intracellular transport are valid targets for anticancer therapy. However, not all the conserved but differentially expressed members of the β-tubulin gene superfamily have been investigated for their role in these settings. In this study, we examined roles for the hematologic isoform β-tubulin VI and functional genetic variants in the gene. β-tubulin VI was highly expressed in blood cells with a substantial interindividual variability (seven-fold variation in mRNA). We characterized DNA missense variations leading to Q43P, T274M, and R307H, and a rare nonsense variant, Y55X. Because variations in the hematologic target of microtubule-binding drugs might alter their myelosuppressive action, we tested their effect in cell lines stably expressing the different β-tubulin VI full-length variants, finding that the T274M change significantly decreased sensitivity to paclitaxel-induced tubulin polymerization. Furthermore, patients treated with paclitaxel and carrying β-tubulin VI T274M exhibited a significantly lower thrombocytopenia than wild-type homozygous patients (P = 0.031). Together, our findings define β-tubulin VI as a hematologic isotype with significant genetic variation in humans that may affect the myelosuppresive action of microtubule-binding drugs. A polymorphism found in a tubulin isoform expressed only in hemapoietic cells may contribute to the patient variation in myelosuppression that occurs after treatment with microtubule-binding drugs.

Purpose: Peripheral neuropathy is the dose-limiting toxicity of paclitaxel, a chemotherapeutic drug widely used to treat several solid tumors such as breast, lung, and ovary. The cytotoxic effect of paclitaxel is mediated through β-tubulin binding in the cellular microtubules. In this study, we investigated the association between paclitaxel neurotoxicity risk and regulatory genetic variants in β-tubulin genes., Experimental Design: We measured variation in gene expression of three β-tubulin isotypes (I, IVb, and IIa) in lymphocytes from 100 healthy volunteers, sequenced the promoter region to identify polymorphisms putatively influencing gene expression and assessed the transcription rate of the identified variants using luciferase assays. To determine whether the identified regulatory polymorphisms were associated with paclitaxel neurotoxicity, we genotyped them in 214 patients treated with paclitaxel. In addition, paclitaxel-induced cytotoxicity in lymphoblastoid cell lines was compared with β-tubulin expression as measured by Affymetrix exon array., Results: We found a 63-fold variation in β-tubulin IIa gene (TUBB2A) mRNA content and three polymorphisms located at -101, -112, and -157 in TUBB2A promoter correlated with increased mRNA levels. The -101 and -112 variants, in total linkage disequilibrium, conferred TUBB2A increased transcription rate. Furthermore, these variants protected from paclitaxel-induced peripheral neuropathy [HR, 0.62; 95% confidence interval (CI), 0.42-0.93; P = 0.021, multivariable analysis]. In addition, an inverse correlation between TUBB2A and paclitaxel-induced apoptosis (P = 0.001) in lymphoblastoid cell lines further supported that higher TUBB2A gene expression conferred lower paclitaxel sensitivity., Conclusions: This is the first study showing that paclitaxel neuropathy risk is influenced by polymorphisms regulating the expression of a β-tubulin gene.

Purpose: Pheochromocytomas (PCC) and paragangliomas (PGL) are genetically heterogeneous neural crest-derived neoplasms. Recently we identified germline mutations in a new tumor suppressor susceptibility gene, MAX (MYC-associated factor X), which predisposes carriers to PCC. How MAX mutations contribute to PCC/PGL and associated phenotypes remain unclear. This study aimed to examine the prevalence and associated phenotypic features of germline and somatic MAX mutations in PCC/PGL., Design: We sequenced MAX in 1,694 patients with PCC or PGL (without mutations in other major susceptibility genes) from 17 independent referral centers. We screened for large deletions/duplications in 1,535 patients using a multiplex PCR-based method. Somatic mutations were searched for in tumors from an additional 245 patients. The frequency and type of MAX mutation was assessed overall and by clinical characteristics., Results: Sixteen MAX pathogenic mutations were identified in 23 index patients. All had adrenal tumors, including 13 bilateral or multiple PCCs within the same gland (P < 0.001), 15.8% developed additional tumors at thoracoabdominal sites, and 37% had familial antecedents. Age at diagnosis was lower (P = 0.001) in MAX mutation carriers compared with nonmutated cases. Two patients (10.5%) developed metastatic disease. A mutation affecting MAX was found in five tumors, four of them confirmed as somatic (1.65%). MAX tumors were characterized by substantial increases in normetanephrine, associated with normal or minor increases in metanephrine., Conclusions: Germline mutations in MAX are responsible for 1.12% of PCC/PGL in patients without evidence of other known mutations and should be considered in the genetic work-up of these patients., (©2012 AACR.)

Hereditary primary hyperparathyroidism (HPT) may develop as a solitary endocrinopathy (FIHP) or as part of multiple endocrine neoplasia Type 1, multiple endocrine neoplasia Type 2A, or hereditary HPT-jaw tumor syndrome. Inactivating germline mutations of the tumor suppressor gene CDC73 account for 14 and 50% of all FIHP and HPT-JT patients, respectively, and have also been found in almost 20% of apparently sporadic parathyroid carcinoma patients. Although more than 60 independent germline mutations have been described, to date no rearrangement affecting the CDC73 locus has been identified. By means of multiplex-PCR we found a large germline deletion affecting the whole gene in a two-generation HPT-JT family. Subsequently array-CGH and specific PCR analysis determined that the mutation spanned ∼ 547 kb, and included four additional genes: TROVE2, GLRX2, B3GALT2, and UCHL5. Although no clear mutation-specific phenotype was found associated to the presence of the mutation, further studies are needed to assess whether the loss of the neighboring genes could modify the phenotype of carriers. There was complete absence of nuclear staining in the two HPT-JT-related tumors available. The finding of the first rearrangement affecting the CDC73 gene warrants screening for this tumor suppressor gene inactivation mechanism not only in high-risk CDC73 point mutation-negative HPT-JT families, but also in FIHP patients., (Copyright © 2011 Wiley-Liss, Inc.)

Hereditary pheochromocytoma (PCC) is often caused by germline mutations in one of nine susceptibility genes described to date, but there are familial cases without mutations in these known genes. We sequenced the exomes of three unrelated individuals with hereditary PCC (cases) and identified mutations in MAX, the MYC associated factor X gene. Absence of MAX protein in the tumors and loss of heterozygosity caused by uniparental disomy supported the involvement of MAX alterations in the disease. A follow-up study of a selected series of 59 cases with PCC identified five additional MAX mutations and suggested an association with malignant outcome and preferential paternal transmission of MAX mutations. The involvement of the MYC-MAX-MXD1 network in the development and progression of neural crest cell tumors is further supported by the lack of functional MAX in rat PCC (PC12) cells and by the amplification of MYCN in neuroblastoma and suggests that loss of MAX function is correlated with metastatic potential.