Prospective evaluation of NGS-based sequencing in epilepsy patients: results of seven NASGE-associated diagnostic laboratories.

Background: Epilepsy is one of the most common and disabling neurological disorders. It is highly prevalent in children with neurodevelopmental delay and syndromic diseases. However, epilepsy can also be the only disease-determining symptom. The exact molecular diagnosis is essential to determine prognosis, comorbidity, and probability of recurrence, and to inform therapeutic decisions. Methods and materials: Here, we describe a prospective cohort study of patients with epilepsy evaluated in seven diagnostic outpatient centers in Germany. Over a period of 2 months, 07/2022 through 08/2022, 304 patients (317 returned result) with seizure-related human phenotype ontology (HPO) were analyzed. Evaluated data included molecular results, phenotype (syndromic and non-syndromic), and sequencing methods. Results: Single exome sequencing (SE) was applied in half of all patients, followed by panel (P) testing (36%) and trio exome sequencing (TE) (14%). Overall, a pathogenic variant (PV) (ACMG cl. 4/5) was identified in 22%; furthermore, a significant number of patients (12%) carried a reported clinically meaningful variant of unknown significance (VUS). The average diagnostic yield in patients ≤ 12 y was higher compared to patients >12 y cf. Figure 2B vs. Figure 3B. This effect was more pronounced in cases, where TE was applied in patients ≤ 12 vs. >12 y [PV (PV + VUS): patients ≤ 12 y: 35% (47%), patients > 12 y: 20% (40%)]. The highest diagnostic yield was achieved by TE in syndromic patients within the age group ≤ 12 y (ACMG classes 4/5 40%). In addition, TE vs. SE had a tendency to result in less VUS in patients ≤ 12 y [SE: 19% (22/117) VUS; TE: 17% (6/36) VUS] but not in patients >12 y [SE: 19% (8/42) VUS; TE: 20% (2/10) VUS]. Finally, diagnostic findings in patients with syndromic vs. non-syndromic symptoms revealed a significant overlap of frequent causes of monogenic epilepsies, including SCN1A, CACNA1A, and SETD1B, confirming the heterogeneity of the associated conditions. Conclusion: In patients with seizures-regardless of the detailed phenotype-a monogenic cause can be frequently identified, often implying a possible change in therapeutic action (36.7% (37/109) of PV/VUS variants); this justifies early and broad application of genetic testing. Our data suggest that the diagnostic yield is highest in exome or trio-exome-based testing, resulting in a molecular diagnosis within 3 weeks, with profound implications for therapeutic strategies and for counseling families and patients regarding prognosis and recurrence risk.

The miR-34 family and its clinical significance in ovarian cancer.

The tumor suppressor miR-34 family is transcriptionally induced by p53. Clinical significance of the various miR-34 family members has not been studied in ovarian cancer. In 228 ovarian cancers and in 19 non-neoplastic fallopian tube samples we analysed miR-34 a/b/c expression in relation to clinicopathological characteristics and clinical outcome. We found significantly lower levels of miR-34 a/b/c in ovarian cancers as compared to control-tissues (P=0.002, P<0.001, P<0.001, respectively). Expression of miR-34 b/c revealed an inverse correlation with BRCA1/2 mRNA-expression (BRCA1: miR34 b/c P=0.002 each; BRCA2: miR-34 b/c P<0.001 each), the same was true for miR-34a and BRCA2 mRNA-expression (P<0.001). The miR-34 family expression was found to be significantly lower in type 2 in comparison to type 1 cancers (P<0.001) and in TP53-mutated compared with TP53-wild-type ovarian cancers (P<0.001, P=0.002, P=0.004, respectively). When low grade serous ovarian cancers were compared with high grade serous cancers the respective miR-34 a/b/c expression was 2.6-, 40.8- and 32.3-fold higher. The expression of each of the miR-34 family members was revealed to be of independent prognostic relevance regarding progression free survival (PFS); miR-34a: HR 0.6, P=0.033; miR-34b: HR 0.2, P=0.001 and miR-34c: HR 0.3, P=0.002, respectively). For overall survival (OS) independency of the prognostic value was confined to miR-34b (HR 0.4, P=0.016) and miR-34c (HR 0.6, P=0.049). The independency of the prognostic value of our identified thresholds was confirmed for PFS for miR-34c in a publicly available dataset (NCBI Gene Expression Omnibus GSE73582). Our findings suggest that downregulation of miR-34 family is a crucial part in ovarian cancer development. Low miR-34 levels are linked to a worse overall survival and progression free survival and may indicate a more aggressive disease.

LinkedSV for detection of mosaic structural variants from linked-read exome and genome sequencing data.

Linked-read sequencing provides long-range information on short-read sequencing data by barcoding reads originating from the same DNA molecule, and can improve detection and breakpoint identification for structural variants (SVs). Here we present LinkedSV for SV detection on linked-read sequencing data. LinkedSV considers barcode overlapping and enriched fragment endpoints as signals to detect large SVs, while it leverages read depth, paired-end signals and local assembly to detect small SVs. Benchmarking studies demonstrate that LinkedSV outperforms existing tools, especially on exome data and on somatic SVs with low variant allele frequencies. We demonstrate clinical cases where LinkedSV identifies disease-causal SVs from linked-read exome sequencing data missed by conventional exome sequencing, and show examples where LinkedSV identifies SVs missed by high-coverage long-read sequencing. In summary, LinkedSV can detect SVs missed by conventional short-read and long-read sequencing approaches, and may resolve negative cases from clinical genome/exome sequencing studies.

A sensitive and scalable microsatellite instability assay to diagnose constitutional mismatch repair deficiency by sequencing of peripheral blood leukocytes.

Constitutional mismatch repair deficiency (CMMRD) is caused by germline pathogenic variants in both alleles of a mismatch repair gene. Patients have an exceptionally high risk of numerous pediatric malignancies and benefit from surveillance and adjusted treatment. The diversity of its manifestation, and ambiguous genotyping results, particularly from PMS2, can complicate diagnosis and preclude timely patient management. Assessment of low-level microsatellite instability in nonneoplastic tissues can detect CMMRD, but current techniques are laborious or of limited sensitivity. Here, we present a simple, scalable CMMRD diagnostic assay. It uses sequencing and molecular barcodes to detect low-frequency microsatellite variants in peripheral blood leukocytes and classifies samples using variant frequencies. We tested 30 samples from 26 genetically-confirmed CMMRD patients, and samples from 94 controls and 40 Lynch syndrome patients. All samples were correctly classified, except one from a CMMRD patient recovering from aplasia. However, additional samples from this same patient tested positive for CMMRD. The assay also confirmed CMMRD in six suspected patients. The assay is suitable for both rapid CMMRD diagnosis within clinical decision windows and scalable screening of at-risk populations. Its deployment will improve patient care, and better define the prevalence and phenotype of this likely underreported cancer syndrome.

BRCA1 and BRCA2 mRNA-expression prove to be of clinical impact in ovarian cancer.

BACKGROUND: Mutations in BRCA1 and BRCA2 are associated with better survival in ovarian cancer (OC) patients due to a better response to platinum-based chemotherapy. However, the impact of the BRCA1/2 mRNA-expression is not well characterized in OC. PATIENTS AND METHODS: We investigated BRCA1/2 mRNA-expression in 12 non-neoplastic fallopian tubes and 201 epithelial OCs in relation to their clinical characteristics. RESULTS: We found higher BRCA1/2 mRNA-expression in OCs compared to controls (P = 0.011, P < 0.001, respectively). BRCA1 mutated OCs exhibited lower BRCA1 (P = 0.014) but higher BRCA2 mRNA-expression (P = 0.001). Low BRCA1-expression was associated with favorable overall survival (OS) (P = 0.012) and low BRCA2-expression with better progression-free survival (PFS) and OS (P = 0.004, P = 0.001, respectively). A subgroup-analysis showed that this effect was confined only to the BRCA1-wildtype cancers. Cox-regression confirmed the prognostic significance of BRCA1-expression for OS (P = 0.028). Independency of the prognostic value of BRCA2-expression for PFS (P = 0.045) and OS (P = 0.015) was restricted to high-grade serous OCs. Fully platinum-sensitivity was characterized by lower BRCA1/2 mRNA-expression in BRCA1-wildtype cancers in comparison to platinum-refractory OC. CONCLUSION: Our findings may reflect higher platinum-sensitivity due to reduced capacity of DNA damage repair in tissues with low BRCA1/2-expression. In this context, especially in BRCA-wildtype cancers both parameters could also be potential predictors for PARP-sensitivity.

Mapping of transcription factor motifs in active chromatin identifies IRF5 as key regulator in classical Hodgkin lymphoma.

Deregulated transcription factor (TF) activities are commonly observed in hematopoietic malignancies. Understanding tumorigenesis therefore requires determining the function and hierarchical role of individual TFs. To identify TFs central to lymphomagenesis, we identified lymphoma type-specific accessible chromatin by global mapping of DNaseI hypersensitive sites and analyzed enriched TF-binding motifs in these regions. Applying this unbiased approach to classical Hodgkin lymphoma (HL), a common B-cell-derived lymphoma with a complex pattern of deregulated TFs, we discovered interferon regulatory factor (IRF) sites among the top enriched motifs. High-level expression of the proinflammatory TF IRF5 was specific to HL cells and crucial for their survival. Furthermore, IRF5 initiated a regulatory cascade in human non-Hodgkin B-cell lines and primary murine B cells by inducing the TF AP-1 and cooperating with NF-κB to activate essential characteristic features of HL. Our strategy efficiently identified a lymphoma type-specific key regulator and uncovered a tumor promoting role of IRF5.

IκB-ζ controls the constitutive NF-κB target gene network and survival of ABC DLBCL.

Constitutive activation of the nuclear factor-κ B (NF-κB) pathway is a hallmark of the activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL). Recurrent mutations of NF-κB regulators that cause constitutive activity of this oncogenic pathway have been identified. However, it remains unclear how specific target genes are regulated. We identified the atypical nuclear IκB protein IκB-ζ to be upregulated in ABC compared with germinal center B-cell-like (GCB) DLBCL primary patient samples. Knockdown of IκB-ζ by RNA interference was toxic to ABC but not to GCB DLBCL cell lines. Gene expression profiling after IκB-ζ knockdown demonstrated a significant downregulation of a large number of known NF-κB target genes, indicating an essential role of IκB-ζ in regulating a specific set of NF-κB target genes. To further investigate how IκB-ζ mediates NF-κB activity, we performed immunoprecipitations and detected a physical interaction of IκB-ζ with both p50 and p52 NF-κB subunits, indicating that IκB-ζ interacts with components of both the canonical and the noncanonical NF-κB pathway in ABC DLBCL. Collectively, our data demonstrate that IκB-ζ is essential for nuclear NF-κB activity in ABC DLBCL, and thus might represent a promising molecular target for future therapies.

PTEN loss defines a PI3K/AKT pathway-dependent germinal center subtype of diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) represents a heterogeneous diagnostic category with distinct molecular subtypes that can be defined by gene expression profiling. However, even within these defined subtypes, heterogeneity prevails. To further elucidate the pathogenesis of these entities, we determined the expression of the tumor suppressor phosphatase and tensin homolog (PTEN) in 248 primary DLBCL patient samples. These analyses revealed that loss of PTEN was detectable in 55% of germinal center B-cell-like (GCB) DLBCLs, whereas this abnormality was found in only 14% of non-GCB DLBCL patient samples. In GCB DLBCL, the PTEN status was inversely correlated with activation of the oncogenic PI3K/protein kinase B (AKT) pathway in both DLBCL cell lines and primary patient samples. Reexpression of PTEN induced cytotoxicity in PTEN-deficient GCB DLBCL cell line models by inhibiting PI3K/AKT signaling, indicating an addiction to this pathway in this subset of GCB DLBCLs. PI3K/AKT inhibition induced down-regulation of the transcription factor MYC. Reexpression of MYC rescued GCB DLBCL cells from PTEN-induced toxicity, identifying a regulatory mechanism of MYC expression in DLBCL. Finally, pharmacologic PI3K inhibition resulted in toxicity selectively in PTEN-deficient GCB DLBCL lines. Collectively, our results indicate that PTEN loss defines a PI3K/AKT-dependent GCB DLBCL subtype that is addicted to PI3K and MYC signaling and suggest that pharmacologic inhibition of PI3K might represent a promising therapeutic approach in these lymphomas.

Expression of cancer-testis antigens as possible targets for antigen-specific immunotherapy in head and neck squamous cell carcinoma.

Cancer-Testis (CT) antigens are by definition expressed in tumor but not in healthy tissue except testis and might represent ideal targets for antigen-specific immunotherapy. Here, we present the first comprehensive analysis of CT antigen expression in patients with head and neck squamous cell carcinoma (HNSCC). Tumor samples (N = 51), and adjacent healthy tissue from patients with HNSCC were analyzed for the expression of 23 genes designated CT antigens using RT-PCR. Patient sera (N = 39) were screened for IgG antibody responses against NY-ESO-1, MAGEA3, and SSX2. According to their expression pattern antigens were divided into four groups. ADAM2, LIP1, SLLP1, AKAP3, CTAGE, ZNF165, CAGE, and FTHL17 were expressed in tumor and healthy tissue at comparable frequencies. NY-TLU-57, GAGE1, SAGE1 were expressed more frequently in tumor samples than in healthy tissues. TPTE, LDHC, SPO11 were expressed neither in tumor samples nor in healthy tissue. 9 CT antigens were expressed only in the tumor tissue and may represent ideal candidates for active immunotherapy in HNSCC: MAGEA3 was expressed in 72%, SSX1 in 45%, MAGEC2 in 33%, MAGEC1 in 28%, BAGE in 17%, SSX2 in 16%, SCP1 in 12%, NY-ESO-1 in 6%, and HOM-TES-85 in 4% of cases. 86% of tumor samples expressed at least one, 69% expressed at least two, and 43% expressed at least three of these antigens. Three patients showed an antibody response against NY-ESO-1. In conclusion, we demonstrate here that HNSCC frequently express CT antigens. Furthermore, a relatively high percentage of tumors express more than one CT antigen opening the perspective for polyvalent antigen-specific immunotherapy.

Pathophysiology of the Eustachian tube--relevant new aspects for the head and neck surgeon.

OBJECTIVES: The impact of cranio-maxillofacial procedures upon Eustachian tube function is still largely unknown. The aim of this presentation is to depict new aspects of Eustachian tube function and to demonstrate its importance to cranio-maxillofacial surgery. METHODS: Two different groups of patients were examined both clinically and by MRI of the Eustachian tube region. One of these groups comprised 15 adult patients with a history of cleft palate; another consisted of 32 patients with a history of a so-called patulous Eustachian tube. RESULTS: Clinical and MRI-findings revealed that the problem of persistent chronic middle ear disease with cleft palate patients depends crucially on the integrity of the pterygoid hamulus and of the tensor veli palatini muscle after cleft palate repair. The masticatory muscles on the other hand also play an important role in Eustachian tube function in non-cleft patients. CONCLUSION: The maxillofacial surgeon should be aware that he holds a key position for preventing as well as treating Eustachian tube pathophysiology.

Oxygenation of tumor recurrences following fractionated radiotherapy of primary tumors. Studies on the rhabdomyosarcoma R1H of the rat.

BACKGROUND AND PURPOSE: Tumor oxygenation is well recognized as a major factor of tumor response to radiotherapy. In this respect, a number of studies have examined the response of primary tumors, whereas little is known about the oxygenation of tumor recurrences after radiotherapy. It was the aim of this study to investigate the oxygenation of tumor recurrences after preceding irradiation of the primary tumor. MATERIAL AND METHODS: Tumor oxygenation in primary tumors and recurrences of rat rhabdomyosarcomas R1H was measured by using pO(2) probes and Eppendorf pO(2) histography. Primary tumors were irradiated at a (60)Co radiotherapy facility with a total dose of 75 Gy, given in 30 fractions over 6 weeks. Oxygenation was measured in R1H tumors before and directly after completion of irradiation. In R1H recurrences oxygenation was determined, when they reached the same size as the previously treated primary tumors (V(o) = 3.1 +/- 0.5 cm(3)). Additionally, tumor microvessel density and the intercapillary distance of tumor blood vessels were determined on histological sections using a counting grid. RESULTS: Tumor oxygenation in R1H recurrences was significantly lower when compared to primary R1H tumors. In primary tumors a median pO(2) of 17 +/- 7 mmHg was measured. By contrast, the median pO(2) in R1H recurrences was only 5 +/- 5 mmHg (p < 0.05). The high frequency of pO(2) values < 5 mmHg indicated that R1H recurrences were significantly more hypoxic (58 +/- 5%) in comparison to primary tumors (22 +/- 4%). The histological sections of the R1H recurrences showed a higher heterogeneity in their tissue structure than primary nonirradiated tumors. The morphometric studies demonstrated a reduced microvessel density (91 +/- 21/9.04 mm(2) in the tumor periphery; p = 0.0001) compared with recurrent tumors (68 +/- 26) and an enhanced mean distance of tumor blood vessels, especially in the center of the R1H recurrences (184 +/- 20 vs. 243 +/- 70 mm; p = 0.0001). CONCLUSION: In R1H rhabdomyosarcomas tumor oxygenation in recurrent tumors following radiation therapy is significantly lower than in primary tumors. This observation has to be taken into account in cases of tumor recurrences where repeated radiotherapy, chemotherapy or combined treatment modalities are used.

Tumor oxygenation under combined whole-body-hyperthermia and polychemotherapy in a case of recurrent carcinoma of the oral cavity.

BACKGROUND: Previous studies have reported synergistic effects of combined hyperthermia and chemotherapy and/or irradiation. The discussed underlying mechanism for this effect is an synergistic cytotoxic and radiosensitizing effect of hyperthermia. In addition, tumor blood-flow and, consequently, tumor oxygenation are increased during hyperthermia. Tumor response to irradiation and chemotherapy of well-oxygenated and vascularized tumors, in general, is superior to that of hypoxic tumors. Therefore, tumor oxygenation is recognized as an important predictive factor in the therapy of malignant tumors. Technically, the head-neck area remains outside the hyperthermia chamber during whole-body hyperthermia (WBH) as currently applied in a number of cancer treatment regimens. The aim of this therapeutic approach was to evaluate whether the blood flow during WBH also increased in the head-neck region and, if so, whether tumor oxygenation increase accordingly. METHODS: A 60-year-old male Caucasian patient, with the original diagnosis of pT3 pN2b M0 squamous cell carcinoma of the oral cavity, who had undergone primary surgery and irradiation (total dose 60 Gy), developed three local recurrences with consecutive surgical resection, presenting now with another recurrent local tumor (histologically confirmed) without surgical or radiotherapeutical options due to lymphangiosis carcinomatosa. WBH was applied under full anaesthesia, using a humidified radiant heat device (Enthermics Medical Systems RHS-7500) in combination with synchronous application of chemotherapy (ifosfamide and carboplatin). Four cycles of this combined treatment (one cycle per month) were given. Tumor oxygenation and temperature were continuously monitored by Licox catheters by means of one point measurement during each treatment (3.5 h). RESULTS: With a latency of 10 min, the increase of intratumoral temperature in the oral cavity was comparable to reference values in the esophagous. Maximum intratumoral temperature (oral cavity) was 41.8 degrees C (F). The average increase of tumor oxygenation was more than 100% in each individual cycle. Clinically, a partial tumor response was observed. CONCLUSIONS: During combined WBH and polychemotherapy, oxygenation is also significantly improved in a tumor in the head and neck area despite the fact that head and neck area remains outside the hyperthermia chamber during WBH. Intratumoral temperatures achieved are comparable to esophageal and rectal temperatures obtained during WBH.