Comprehensive Validation of Diagnostic Next-Generation Sequencing Panels for Acute Myeloid Leukemia Patients.

Next-generation sequencing has greatly advanced the molecular diagnostics of malignant hematological diseases and provides useful information for clinical decision making. Studies have shown that certain mutations are associated with prognosis and have a direct impact on treatment of affected patients. Therefore, reliable detection of pathogenic variants is critically important. Here, we compared four sequencing panels with different characteristics, from number of genes covered to technical aspects of library preparation and data analysis workflows, to find the panel with the best clinical utility for myeloid neoplasms with a special focus on acute myeloid leukemia. Using the Acrometrix Oncology Hotspot Control DNA and DNA from acute myeloid leukemia patients, panel performance was evaluated in terms of coverage, precision, recall, and reproducibility and different bioinformatics tools that can be used for the evaluation of any next-generation sequencing panel were tested. Taken together, our results support the reliability of the Acrometrix Oncology Hotspot Control to validate and compare sequencing panels for hematological diseases and show which panel-software combination (platform) has the best performance.

Immunohistochemical detection of PAX-FOXO1 fusion proteins in alveolar rhabdomyosarcoma using breakpoint specific monoclonal antibodies.

Alveolar Rhabdomyosarcoma (ARMS) is an aggressive pediatric cancer with about 80% of cases characterized by either a t(1;13)(p36;q14) or t(2;13)(q35;q14), which results in the formation of the fusion oncogenes PAX7-FOXO1 and PAX3-FOXO1, respectively. Since patients with fusion-positive ARMS (FP-RMS) have a poor prognosis and are treated with an aggressive therapeutic regimen, correct classification is of clinical importance. Detection of the translocation by different molecular methods is used for diagnostics, including fluorescence in situ hybridization and RT-PCR or NGS based approaches. Since these methods are complex and time consuming, we developed specific monoclonal antibodies (mAbs) directed to the junction region on the PAX3-FOXO1 fusion protein. Two mAbs, PFM.1 and PFM.2, were developed and able to immunoprecipitate in vitro-translated PAX3-FOXO1 and cellular PAX3-FOXO1 from FP-RMS cells. Furthermore, the mAbs recognized a 105 kDa band in PAX3-FOXO1-transfected cells and in FP-RMS cell lines. The mAbs did not recognize proteins in fusion-negative embryonal rhabdomyosarcoma cell lines, nor did they recognize PAX3 or FOXO1 alone when compared to anti-PAX3 and anti-FOXO1 antibodies. We next evaluated the ability of mAb PFM.2 to detect the fusion protein by immunohistochemistry. Both PAX3-FOXO1 and PAX7-FOXO1 were detected in HEK293 cells transfected with the corresponding cDNAs. Subsequently, we stained 26 primary tumor sections and a rhabdomyosarcoma tissue array and detected both fusion proteins with a positive predictive value of 100%, negative predictive value of 98%, specificity of 100% and a sensitivity of 91%. While tumors are stained homogenously in PAX3-FOXO1 cases, the staining pattern is heterogenous with scattered positive cells only in tumors expressing PAX7-FOXO1. No staining was observed in stromal cells, embryonal rhabdomyosarcoma, and fusion-negative rhabdomyosarcoma. These results demonstrate that mAbs specific for the chimeric oncoproteins PAX3-FOXO1 and PAX7-FOXO1 can be used efficiently for simple and fast subclassification of rhabdomyosarcoma in routine diagnostics via immunohistochemical detection.

Autoantibodies against the prion protein in individuals with PRNP mutations.

OBJECTIVE: To determine whether naturally occurring autoantibodies against the prion protein are present in individuals with genetic prion disease mutations and controls, and if so, whether they are protective against prion disease. METHODS: In this case-control study, we collected 124 blood samples from individuals with a variety of pathogenic PRNP mutations and 78 control individuals with a positive family history of genetic prion disease but lacking disease-associated PRNP mutations. Antibody reactivity was measured using an indirect ELISA for the detection of human immunoglobulin G1-4 antibodies against wild-type human prion protein. Multivariate linear regression models were constructed to analyze differences in autoantibody reactivity between (1) PRNP mutation carriers vs controls and (2) asymptomatic vs symptomatic PRNP mutation carriers. Robustness of results was examined in matched cohorts. RESULTS: We found that antibody reactivity was present in a subset of both PRNP mutation carriers and controls. Autoantibody levels were not influenced by PRNP mutation status or clinical manifestation of prion disease. Post hoc analyses showed anti-PrPC autoantibody titers to be independent of personal history of autoimmune disease and other immunologic disorders, as well as PRNP codon 129 polymorphism. CONCLUSIONS: Pathogenic PRNP variants do not notably stimulate antibody-mediated anti-PrPC immunity. Anti-PrPC immunoglobulin G autoantibodies are not associated with the onset of prion disease. The presence of anti-PrPC autoantibodies in the general population without any disease-specific association suggests that relatively high titers of naturally occurring antibodies are well-tolerated. CLINICALTRIALSGOV IDENTIFIER: NCT02837705.

HPV-Related Multiphenotypic Sinonasal Carcinoma: Four Cases that Expand the Morpho-Molecular Spectrum and Include Occupational Data.

HPV-related multiphenotypic sinonasal carcinoma (HMSC) is a recently described distinct tumor entity of the sinonasal tract associated with high-risk subtypes of human papilloma virus (HPV), predominantly type 33. The biological behavior seems to be less aggressive than the often high-grade, highly proliferative morphology implies; however, recurrences are frequent. Most of the cases present as polypoid tumors within the nasal cavity. Microscopic morphology frequently encompasses adenoid cystic-like features or features reminiscent of other salivary gland tumors. Here, we describe four cases of this rare entity, all observed in women. The polypoid tumors were within the nasal cavity, leading to obstruction, facial pain and epistaxis. The morphology was predominantly basaloid, solid and adenoid cystic-like in two of four cases, one with additional glomeruloid features. Another case showed basaloid tumor cells with prominent mature squamous differentiation and extensive keratinization. A single case showed a predominantly solid and reticular growth pattern. All cases were diffusely positive for p16 (100%), expressed SOX10, LEF-1 and partially S-100, and harbored HPV high-risk types 33, 56 (2×) and 82. No recurrences or metastases were detectable after 3-50 months of follow-up. Of note, three of four patients were nurses/nursing assistant. We expand the morphological spectrum by describing a glomeruloid growth pattern and extensive mature keratinization, and add HPV type 82 to the molecular spectrum. The finding of HMSC among predominantly nurses in our cohort warrants further epidemiological studies in larger cohorts.

Value of immunohistochemistry in the detection of BRAF(V600E) mutations in fine-needle aspiration biopsies of papillary thyroid carcinoma.

BACKGROUND: Fine-needle aspiration biopsy (FNAB) is important in the diagnostic establishment of suspicious thyroid nodules. In thyroid neoplasms, mutation of the BRAF gene occurs rather exclusively in papillary thyroid carcinoma (PTC) and results in>98% of the cases in V600E amino acid substitution. In the current study, the authors investigated the diagnostic value of a recently described monoclonal antibody that detects this specific mutation on FNAB specimens from patients with PTC. METHODS: BRAF(V600E) status of FNAB cell blocks from 55 patients with PTC was analyzed by immunohistochemistry (IHC) with the new BRAF(V600E) antibody (clone VE1) and by Sanger sequencing (SaS). In discrepant cases, ultra-deep sequencing was also performed. Available corresponding histological specimens were investigated by IHC and, in selected cases, with SaS as well. RESULTS: All cases yielded evaluable IHC staining results of the cell block sections with good interobserver agreement (kappa value, 0.650). Ten tumors (18.2%) demonstrated no staining, 10 tumors (18.2%) demonstrated equivocal staining, 25 tumors (45.4%) demonstrated moderate staining, and 10 tumors (18.2%) demonstrated strong staining. SaS was able to be performed in 48 cases. Nineteen cases demonstrated wild-type BRAF and 29 cases were found to have the BRAF(V600E) mutation. After performing ultra-deep sequencing 1 false-positive and 2 false-negative VE1 IHC cases remained, resulting in a sensitivity of 93.8% and a specificity of 93.8%. CONCLUSIONS: BRAF(V600E) mutations in FNAB specimens from patients with PTC can be reliably detected in most cases by IHC with a new mutation-specific antibody. Interpretation of VE1 IHC staining results on cell block slides of PTC can be difficult in some cases.

Dynamic two-stage mechanism of versatile DNA damage recognition by xeroderma pigmentosum group C protein.

The recognition and subsequent repair of DNA damage are essential reactions for the maintenance of genome stability. A key general sensor of DNA lesions is xeroderma pigmentosum group C (XPC) protein, which recognizes a wide variety of helix-distorting DNA adducts arising from ultraviolet (UV) radiation, genotoxic chemicals and reactive metabolic byproducts. By detecting damaged DNA sites, this unique molecular sensor initiates the global genome repair (GGR) pathway, which allows for the removal of all the aforementioned lesions by a limited repertoire of excision factors. A faulty GGR activity causes the accumulation of DNA adducts leading to mutagenesis, carcinogenesis, neurological degeneration and other traits of premature aging. Recent findings indicate that XPC protein achieves its extraordinary substrate versatility by an entirely indirect readout strategy implemented in two clearly discernible stages. First, the XPC subunit uses a dynamic sensor interface to monitor the double helix for the presence of non-hydrogen-bonded bases. This initial screening generates a transient nucleoprotein intermediate that subsequently matures into the ultimate recognition complex by trapping undamaged nucleotides in the abnormally oscillating native strand, in a way that no direct contacts are made between XPC protein and the offending lesion itself. It remains to be elucidated how accessory factors like Rad23B, centrin-2 or the UV-damaged DNA-binding complex contribute to this dynamic two-stage quality control process.

Two-stage dynamic DNA quality check by xeroderma pigmentosum group C protein.

Xeroderma pigmentosum group C (XPC) protein initiates the DNA excision repair of helix-distorting base lesions. To understand how this versatile subunit searches for aberrant sites within the vast background of normal genomic DNA, the real-time redistribution of fluorescent fusion constructs was monitored after high-resolution DNA damage induction. Bidirectional truncation analyses disclosed a surprisingly short recognition hotspot, comprising approximately 15% of human XPC, that includes two beta-hairpin domains with a preference for non-hydrogen-bonded bases in double-stranded DNA. However, to detect damaged sites in living cells, these DNA-attractive domains depend on the partially DNA-repulsive action of an adjacent beta-turn extension that promotes the mobility of XPC molecules searching for lesions. The key function of this dynamic interaction surface is shown by a site-directed charge inversion, which results in increased affinity for native DNA, retarded nuclear mobility and diminished repair efficiency. These studies reveal a two-stage discrimination process, whereby XPC protein first deploys a dynamic sensor interface to rapidly interrogate the double helix, thus forming a transient recognition intermediate before the final installation of a more static repair-initiating complex.

Role of DNA repair in the protection against genotoxic stress.

The genome of all organisms is constantly attacked by a variety of environmental and endogenous mutagens that cause cell death, apoptosis, senescence, genetic diseases and cancer. To mitigate these deleterious endpoints of genotoxic reactions, living organisms have evolved one or more mechanisms for repairing every type of naturally occurring DNA lesion. For example, double-strand breaks are rapidly religated by non-homologous end-joining. Homologous recombination is used for the high-fidelity repair of interstrand cross-links, double-strand breaks and other DNA injuries that disrupt the replication fork. Some genotoxic lesions inflicted by alkylating agents can be repaired by direct reversal of DNA damage. The base excision repair pathway takes advantage of multiple DNA glycosylases to remove modified or incorrect bases. Finally, the nucleotide excision repair machinery provides a versatile strategy to monitor DNA quality and eliminate all forms of helix-distorting DNA lesions, including a wide diversity of carcinogen adducts. The efficiency of DNA repair responses is enhanced by their coupling to transcription and coordination with the cell cycle circuit.

Versatile protection from mutagenic DNA lesions conferred by bipartite recognition in nucleotide excision repair.

Nucleotide excision repair is a cut-and-patch pathway that eliminates potentially mutagenic DNA lesions caused by ultraviolet light, electrophilic chemicals, oxygen radicals and many other genetic insults. Unlike antigen recognition by the immune system, which employs billions of immunoglobulins and T-cell receptors, the nucleotide excision repair complex relies on just a few generic factors to detect an extremely wide range of DNA adducts. This molecular versatility is achieved by a bipartite strategy initiated by the detection of abnormal strand fluctuations, followed by the localization of injured residues through an enzymatic scanning process coupled to DNA unwinding. The early recognition subunits are able to probe the thermodynamic properties of nucleic acid substrates but avoid direct contacts with chemically altered bases. Only downstream subunits of the bipartite recognition process interact more closely with damaged bases to delineate the sites of DNA incision. Thus, consecutive factors expand the spectrum of deleterious genetic lesions conveyed to DNA repair by detecting distinct molecular features of target substrates.

XPA gene, its product and biological roles.

The 31 kDa XPA protein is part of the core incision complex of the mammalian nucleotide excision repair (NER) system and interacts with DNA as well as with many other NER subunits. In the absence of XPA, no incision complex can form and no excision of damaged DNA damage occurs. A comparative analysis of the DNA-binding properties in the presence of different substrate conformations indicated that XPA protein interacts preferentially with kinked DNA backbones. The DNA-binding domain of XPA protein displays a positively charged deft that is involved in an indirect readout mechanism, presumably by detecting the increased negative potential encountered at sharp DNA bends. We propose that this indirect recognition function contributes to damage verification by probing the susceptibility of the DNA substrate to be kinked during the assembly of NER complexes.

DNA repair triggered by sensors of helical dynamics.

Nucleotide excision repair is a constitutive stress response that eliminates DNA lesions induced by multiple genotoxic agents. Unlike the immune system, which generates billions of immunoglobulins and T cell receptors for antigen recognition, the nucleotide excision repair complex uses only a few generic factors to detect an astounding diversity of DNA modifications. New data favor an unexpected strategy whereby damage recognition is initiated by the detection of abnormal oscillations in the undamaged strand opposite to DNA lesions. Another core subunit recognizes the increased susceptibility of DNA to be kinked at injured sites. We suggest that early nucleotide excision repair factors gain substrate versatility by avoiding direct contacts with modified residues and exploiting instead the altered dynamics of damaged DNA duplexes.

Xeroderma pigmentosum complementation group A protein is driven to nucleotide excision repair sites by the electrostatic potential of distorted DNA.

The presumed DNA-binding cleft of xeroderma pigmentosum group A (XPA) protein, a key regulatory subunit of the eukaryotic nucleotide excision repair complex, displays a distinctive array of 6 positively charged amino acid side chains. Here, the molecular function of these closely spaced electropositive residues has been tested by systematic site-directed mutagenesis. After the introduction of single amino acid substitutions, the mutants were probed for protein-DNA interactions in electrophoretic mobility shift and photochemical crosslinking assays. This analysis led to the identification of a critical hot-spot for DNA substrate recognition composed of two neighboring lysines at codons 141 and 179 of the human XPA sequence. The replacement of other basic side chains in the DNA interaction domain conferred more moderate defects of substrate binding. When the function of XPA was tested as a fusion product with either mCherry or green-fluorescent protein, a glutamate substitution of one of the positively charged residues at positions 141 and 179 was sufficient to decrease DNA repair activity in human fibroblasts. Thus, the removal of a single cationic side chain abolished DNA-binding activity and significant excision repair defects could be induced by single charge inversions on the XPA surface, indicating that this molecular sensor participates in substrate recognition by monitoring the electrostatic potential of distorted DNA repair sites.

Recognition of helical kinks by xeroderma pigmentosum group A protein triggers DNA excision repair.

The function of human XPA protein, a key subunit of the nucleotide excision repair pathway, has been examined with site-directed substitutions in its putative DNA-binding cleft. After screening for repair activity in a host-cell reactivation assay, we analyzed mutants by comparing their affinities for different substrate architectures, including DNA junctions that provide a surrogate for distorted reaction intermediates, and by testing their ability to recruit the downstream endonuclease partner. Normal repair proficiency was retained when XPA mutations abolished only the simple interaction with linear DNA molecules. By contrast, results from a K141E K179E double mutant revealed that excision is crucially dependent on the assembly of XPA protein with a sharp bending angle in the DNA substrate. These findings show how an increased deformability of damaged sites, leading to helical kinks recognized by XPA, contributes to target selectivity in DNA repair.

Mechanisms of DNA damage recognition and strand discrimination in human nucleotide excision repair.

Using only a limited repertoire of recognition subunits, the nucleotide excision repair (NER) system is able to detect a nearly infinite variety of bulky DNA lesions. This extraordinary substrate versatility has generally been ascribed to an indirect readout mechanism, whereby particular distortions of the double helix, induced by a damaged nucleotide, provide the molecular determinants not only for lesion recognition but also for subsequent verification or demarcation processes. Here, we discuss the evidence in support of a bipartite mechanism of substrate discrimination that is initiated by the detection of thermodynamically unstable base pairs followed by direct localization of the lesion through an enzymatic proofreading activity. This bipartite discrimination mechanism is part of a dynamic reaction cycle that confers high levels of selectivity to avoid futile repair events on undamaged DNA and also protect the intact complementary strand from inappropriate cleavage.