Recurrent and novel fusions detected by targeted RNA sequencing as part of the diagnostic workflow of soft tissue and bone tumours.

The identification of gene fusions has become an integral part of soft tissue and bone tumour diagnosis. We investigated the added value of targeted RNA-based sequencing (targeted RNA-seq, Archer FusionPlex) to our current molecular diagnostic workflow of these tumours, which is based on fluorescence in situ hybridisation (FISH) for the detection of gene fusions using 25 probes. In a series of 131 diagnostic samples targeted RNA-seq identified a gene fusion, BCOR internal tandem duplication or ALK deletion in 47 cases (35.9%). For 74 cases, encompassing 137 FISH analyses, concordance between FISH and targeted RNA-seq was evaluated. A positive or negative FISH result was confirmed by targeted RNA-seq in 27 out of 49 (55.1%) and 81 out of 88 (92.0%) analyses, respectively. While negative concordance was high, targeted RNA-seq identified a canonical gene fusion in seven cases despite a negative FISH result. The 22 discordant FISH-positive analyses showed a lower percentage of rearrangement-positive nuclei (range 15-41%) compared to the concordant FISH-positive analyses (>41% of nuclei in 88.9% of cases). Six FISH analyses (in four cases) were finally considered false positive based on histological and targeted RNA-seq findings. For the EWSR1 FISH probe, we observed a gene-dependent disparity (p = 0.0020), with 8 out of 35 cases showing a discordance between FISH and targeted RNA-seq (22.9%). This study demonstrates an added value of targeted RNA-seq to our current diagnostic workflow of soft tissue and bone tumours in 19 out of 131 cases (14.5%), which we categorised as altered diagnosis (3 cases), added precision (6 cases), or augmented spectrum (10 cases). In the latter subgroup, four novel fusion transcripts were found for which the clinical relevance remains unclear: NAB2::NCOA2, YAP1::NUTM2B, HSPA8::BRAF, and PDE2A::PLAG1. Overall, targeted RNA-seq has proven extremely valuable in the diagnostic workflow of soft tissue and bone tumours.

Primary mediastinal large B-cell lymphoma is characterized by large-scale copy-neutral loss of heterozygosity.

Development of primary mediastinal B-cell lymphoma (PMBL) is driven by cumulative genomic aberrations. We discovered a unique copy-neutral loss of heterozygosity (CN-LOH) landscape of PMBL which distinguishes this tumor from other B-cell malignancies, including the biologically related diffuse large B-cell lymphoma. Using single nucleotide polymorphism array analysis we identified large-scale CN-LOH lesions in 91% (30/33) of diagnostic PMBLs and both investigated PMBL-derived cell lines. Altogether, the cohort showed 157 extra-large (25.3-248.4 Mb) CN-LOH lesions affecting up to 14 chromosomes per case (mean of 4.4) and resulting in a reduction of heterozygosity an average of 9.9% (range 1.3-51%) of the genome. Predominant involvement of terminal chromosomal segments suggests the implication of B-cell specific crossover events in the pathogenesis of PMBL. Notably, CN-LOH stretches non-randomly clustered on 6p (60%), 15 (37.2%), and 17q (40%), and frequently co-occurred with homozygous mutations in the MHC I (6p21), B2M (15q15), and GNA13 (17q23) genes, respectively, as shown by preliminary whole-exome/genome sequencing data. Altogether, our findings implicate CN-LOH as a novel and distinct mutational process contributing to the molecular pathogenesis of PMBL. The aberration acting as "second hit" in the Knudson hypothesis, ranks as the major mechanism converting to homozygosity the PMBL-related driver genes. Screening of the cohort of 199 B cell leukemia/lymphoma whole-genomes revealed significant differences in the CN-LOH landscape of PMBL and other B-cell malignancies, including the biologically related diffuse large B-cell lymphoma.

Targeted RNA sequencing for upfront analysis of actionable driver alterations in non-small cell lung cancer.

OBJECTIVES: Targeted RNA-based Next-Generation Sequencing (tRNA-seq) is increasingly being used in molecular diagnostics for gene fusion detection in non-small cell lung cancer (NSCLC). However, few data support its clinical application for the detection of single nucleotide variants (SNVs) and small insertions/deletions. In this study, we evaluated the performance of tRNA-seq using Archer FusionPlex for simultaneous detection of actionable gene fusions, splice variants, SNVs and indels in formalin-fixed, paraffin-embedded NSCLC tissue. MATERIALS AND METHODS: A total of 126 NSCLC samples, including 20 validation samples and 106 diagnostic cases, were analyzed by targeted DNA-based Next-Generation Sequencing (tDNA-seq) followed by tRNA-seq. RESULTS: All 28 SNVs and indels in the validation set, and 34 out of 35 mutations in the diagnostic set were identified by tRNA-seq. The only mutation undetected by tRNA-seq, ERBB2 p.(Ser310Tyr), was not included in the current Archer panel design. tRNA-seq revealed one additional BRAF p.(Val600Glu) mutation not found by tDNA-seq. SNVs and indels were correctly called by the vendor supplied software, except for ERBB2 duplication p.(Tyr772_A775dup) which was only detected by an additional in-house developed bio-informatics pipeline. Variant allelic frequency (VAF) values were generally higher at the expression level compared to the genomic level (range 6-96% for tRNA-seq versus 6-61% for tDNA-seq) and low VAF mutations in DNA (6-8% VAF) were all confirmed by tRNA-seq. Finally, tRNA-seq additionally identified a driver fusion or splice variant in 10 diagnostic NSCLC samples including one MET exon 14 skipping variant not detected by tDNA-seq. CONCLUSION: Our results demonstrate that tRNA-seq can be implemented in a diagnostic setting as an efficient strategy for simultaneous detection of actionable gene fusions, splice variants, SNVs and indels in NSCLC provided that adequate RNA-seq analysis tools are available, especially for the detection of indels. This approach allows upfront identification of currently recommended targetable molecular alterations in NSCLC samples.

Evaluation of quality of clinical management of neuroendocrine tumors.

Background: Neuroendocrine tumors (NETs) are a group of biologically and clinically heterogeneous neoplasms predominantly found in the gastrointestinal and bronchopulmonary tractus. Despite a rising incidence, implementation of evidence-based standardized care for this heterogenous group remains challenging. The European Neuroendocrine Tumor Society regularly reviews guidelines regarding diagnostic and treatment strategies for NETs. The aim of this study is to shed light on the care of patients with a NET in Belgian Limburg, to provide data as a basis for future studies and to check whether data and results are according to consensus guidelines and outcomes described in literature. Methods: Our study concerned a detailed observational data collection of two large Belgian hospitals (Jessa Hospital Hasselt and Hospital Oost-Limburg Genk) with special interest in patient profile, quality of pathology reports, use of diagnostic imaging, and overall survival. Data on 188 patients were assembled between January 2010 and December 2014 with follow-up until June 2016 (median follow-up: 33.6 months). Results: Fifty percent of patients were male. NETs were located mainly in the digestive tract (63.8%) and lung (20.2%). Appendiceal NETs were diagnosed at a significantly younger age than other tumors (41.3 vs. 64.0 years). Overall, a mean pathology report quality score of 3.0/5 was observed with the highest scores for small bowel NETs. Diagnostic and nuclear imaging was performed in 74.5% and 29.8% of cases, respectively. Seventy-four percent of the population survived until the end of the observation period with highest survival rates for appendiceal and small bowel NETs. Conclusion: Overall, epidemiological results were comparable with findings in the literature. Gastrointestinal NETs met most of the requirements of qualitative pathology reporting and diagnostic imaging as listed in the European Neuroendocrine Tumor Society consensus guidelines. However, consensus with regard to bronchopulmonary NETs is still scarce and remains an objective for future research. Moreover, discussing treatment strategies in specialized multidisciplinary tumor boards would facilitate regional care.

Salvage Lobectomy to Treat Necrotizing SARS-CoV-2 Pneumonia Complicated by a Bronchopleural Fistula.

We report a case of necrotizing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pneumonia complicated by a bronchopleural fistula and treated by decortication and salvage lobectomy. Owing to the unknown characteristics of the underlying SARS-CoV-2 infection, treatment of the abscess and bronchopleural fistula was delayed. This may have resulted in further deterioration of the patient, with ensuing multiple organ dysfunction. Complications of SARS-CoV-2 pneumonia, such as a bacterial abscess and a bronchopleural fistula, should be treated as if the patient were not infected with SARS-CoV-2.

Triple negative breast cancer: clinical characteristics in the different histological subtypes.

PURPOSE: To investigate the clinical behavior of triple negative breast cancer (TNC), including age distribution, occurrence of LN (lymph node) invasion and prognosis in different histological subtypes. METHODS: For this cohort study we used data on 476 patients with newly diagnosed TNC at the University Hospitals Leuven (Belgium) between 1999 and 2009. Of these, 395 received upfront surgery, 68 neoadjuvant chemotherapy and 21 had metastases at diagnosis. RESULTS: Apocrine and invasive lobular TNC occur more often in older patients compared to IDC-NOS. Of the primarily operated patients with TNC, 35.1% has pathological LN involvement. There were no significant differences in nodal invasion between different histological subtypes, but most subtypes contained few patients. In contrast to previous reports, 6/14 of apocrine TNC had LN involvement. Disease free survival (DFS) was different in different histological subtypes, but group sizes were insufficient to be able to draw firm conclusions. Within the histologically 'homogeneous' IDC-NOS group with primary surgery and outcome data (n = 300), DFS with 3.5 year median follow-up decreased with increasing age, but chemotherapy and radiotherapy were much less frequently given with increasing age. In multivariable analysis, lower age, presence of LN involvement, lack of administration of chemotherapy and radiotherapy were significant predictors of relapse. CONCLUSION: TNC is not a uniform disease. Different histological subtypes have different age distribution and behavior. The prognosis of the most common histological subgroup, IDC-NOS, is better in older patients, but this is counterbalanced by significantly decreased use of chemotherapy and radiotherapy.

Proteolytic processing of ErbB4 in breast cancer.

ErbB4 is a receptor tyrosine kinase that can signal by a mechanism involving proteolytic release of intracellular and extracellular receptor fragments. Proteolysis-dependent signaling of ErbB4 has been proposed to be enhanced in breast cancer, mainly based on immunohistochemical localization of intracellular epitopes in the nuclei. To more directly address the processing of ErbB4 in vivo, an ELISA was developed to quantify cleaved ErbB4 ectodomain from serum samples. Analysis of 238 breast cancer patients demonstrated elevated quantities of ErbB4 ectodomain in the serum (≥ 40 ng/mL) in 21% of the patients, as compared to 0% of 30 healthy controls (P = 0.002). Significantly, the elevated serum ectodomain concentration did not correlate with the presence of nuclear ErbB4 immunoreactivity in matched breast cancer tissue samples. However, elevated serum ectodomain concentration was associated with the premenopausal status at diagnosis (P = 0.04), and estradiol enhanced ErbB4 cleavage in vitro. A 3.4 Å X-ray crystal structure of a complex of ErbB4 ectodomain and the Fab fragment of anti-ErbB4 mAb 1479 localized the binding site of mAb 1479 on ErbB4 to a region on subdomain IV encompassing the residues necessary for ErbB4 cleavage. mAb 1479 also significantly blocked ErbB4 cleavage in breast cancer cell xenografts in vivo, and the inhibition of cleavage was associated with suppression of xenograft growth. These data indicate that ErbB4 processing is enhanced in breast cancer tissue in vivo, and that ErbB4 cleavage can be stimulated by estradiol and targeted with mAb 1479.

Relationship between age and axillary lymph node involvement in women with breast cancer.

PURPOSE: To study the relation between the presence of axillary lymph node (LN) involvement and age in breast cancer. PATIENTS AND METHODS: The breast cancer database of the University Hospitals Leuven contains complete data on 2,227 patients with early breast cancer consecutively treated between 2000 and 2005. A multivariate piecewise logistic regression model was used to analyze LN involvement in relation to age at diagnosis. A similar analysis was then performed on a large, independent, population-based database from the Eindhoven Cancer Registry to investigate whether the effects of the Leuven model could be replicated. RESULTS: We observed a piecewise effect of age. That is, women up to 70 years of age were less likely to have positive LNs with increasing age (odds ratio per 10-year increase, 0.87). In contrast, older women were more likely to have positive LNs with increasing age. However, for older women, the effect of age interacted with tumor size (P = .0044), suggesting that increasing age is associated with increased risk of LN involvement, mainly in small tumors. These findings were replicated in the Eindhoven Cancer Registry database. CONCLUSION: Axillary LN involvement varies with age at diagnosis; its probability decreases with increasing age up to the age of approximately 70 years, but increases again thereafter. However, this increase is mainly seen in smaller tumors and suggests a different behavior of small breast cancers in older adult patients. We hypothesize that decreased immune defense mechanisms, related with aging, may play a role in earlier invasion into LNs.

Triple-negative breast cancer.

PURPOSE OF REVIEW: Triple-negative breast cancer has been of great interest to oncologists because these cancers do not benefit from hormonal therapies or treatments targeted against human epidermal growth factor receptor 2 receptors. The only systemic therapy currently available is chemotherapy, and prognosis remains poor. We would like to describe recent pathological and genetic advancements and their possible implications on the development of new treatment strategies. RECENT FINDINGS: Increasing pathological and molecular knowledge has led to a better understanding of breast cancer in general. Triple-negative breast cancer, however, remains a heterogeneous subgroup with difficult-to-define subtypes. New targeted therapies are currently being developed and researched. SUMMARY: The recent advances in pathological and molecular knowledge have changed the landscape of breast cancer, with triple-negative breast cancer emerging as a pathological and clinical heterogeneous group. The main challenge lies in the development of new tailored treatment strategies for the various subgroups, with receptor kinase inhibition as the most promising new evolution to date. Further trials are needed to validate these new treatment options.

Granuloma possibly induced by palladium after ear piercing.

A case of sarcoidal-type allergic contact granuloma due to palladium is presented. The patient developed papulonodular lesions at the right earlobe after ear piercing, which after 3 to 4 years became more granulomatous and very resistant to treatment. Repeated intralesional injections with corticosteroids produced only a temporary regression of the lesions. Patch testing revealed a strong positive reaction to palladium (and nickel). Biopsy specimens taken from the persistent granulomatous lesion in the nodule at the earlobe, as well as from the site of the positive test reaction to palladium several weeks after patch testing, indicated epithelioid granulomas with some multinucleate histiocytes surrounded by a lymphocytic-histiocytic infiltrate. Similar cases (also with other metals) have been reported in the literature.

A gene signature of loss of oestrogen receptor (ER) function and oxidative stress links ER-positive breast tumours with an absent progesterone receptor and a poor prognosis.

Prognostic gene signatures like the wound and hypoxia signature differ by assumptions of cellular growth. Although gene signatures show little overlap, they also track within the group of luminal breast tumours those with a high proliferation and poor prognosis. Oxidative stress is another assumption of cellular growth. It affects several pathological conditions through its influence on the regulation of protein kinases and signal transduction pathways. A comprehensive set of 62 core genes from cultured oestrogen- and oestrogen receptor-deprived epithelial breast cancer cells is responsive to three forms of oxidative stress. Evidence is presented that oxidative stress involves the development of an aggressive subset of primary oestrogen receptor-positive breast tumours.

Aggressive angiomyxoma of the vulva: a case report and review of literature.

BACKGROUND: Aggressive angiomyxoma (AA) is a rare vulvovaginal mesenchymal neoplasm with a marked tendency to local recurrence but which usually does not metastasize. CASE REPORT: We describe a case of AA in the left labium majus pudendi in a 47-year-old woman who underwent incomplete surgical excision. Follow-up 2years later revealed no recurrence. CONCLUSION: In the past, most authors advocated wide excision even if genitourinary and digestive tract resection were necessary. These days, a less radical surgery is recommended, but the significance of hormonal treatment and/or radiation therapy is not clear yet. Further investigation is necessary.

Detection of head and neck squamous cell carcinoma with diffusion weighted MRI after (chemo)radiotherapy: correlation between radiologic and histopathologic findings.

PURPOSE: To investigate the value of diffusion weighted magnetic resonance imaging (DW-MRI) in differentiating persistent or recurrent head and neck squamous cell carcinoma (HNSCC) from nontumoral postradiotherapeutic alterations. METHODS AND MATERIALS: In 26 patients with suspicion of persistent or recurrent HNSCC, MRI of the head and neck was performed, including routine turbo spin-echo (TSE) sequences and an additional echo-planar DW-MRI sequence, using a large range of b-values (0-1000 s/mm(2)). Apparent diffusion coefficient (ADC) maps were calculated. In the suspect areas at the primary site and in the suspect lymph nodes, signal intensity was measured on the native b0 and b1000 images and ADC values were calculated for these tissues. The same was done for surrounding irradiated normal tissue. Imaging results were correlated to histopathology. RESULTS: Signal intensity on native b0 images was significantly lower for HNSCC than for nontumoral postradiotherapeutic tissue (p < 0.0001), resulting in a sensitivity of 66.2%, specificity of 60.8%, and accuracy of 62.4%. Signal intensity on native b1000 images was significantly higher for HNSCC than for nontumoral tissue (p < 0.0001), resulting in a sensitivity of 71.6%, specificity of 71.3%, and accuracy of 71.4%. ADC values were significantly lower for HNSCC than for nontumoral tissue (p < 0.0001), resulting in a sensitivity of 94.6%, specificity of 95.9%, and accuracy of 95.5%. When compared with computed tomography, TSE-MRI and fluorodeoxyglucose-positron emission tomography, DW-MRI yielded fewer false-positive results in persistent primary site abnormalities and in persistent adenopathies, and aided in the detection of subcentimetric nodal metastases. CONCLUSIONS: Diffusion weighted-MRI accurately differentiates persistent or recurrent HNSCC from nontumoral tissue changes after (chemo)radiotherapy.

Multifocal kaposiform haemangioendothelioma.

Kaposiform haemangioendothelioma (KHE) is a rare, locally aggressive vascular spindle cell proliferation, with resemblance to Kaposi's sarcoma. This tumour usually occurs in skin and retroperitoneum of infants and young children and is often complicated by the Kasabach-Merritt phenomenon (KMP). A 3-year-old boy presented with a right submandibular swelling due to lymphadenopathies, a violaceous skin lesion at the left commissure of the lips and an ill-defined lesion in the right thyroid lobe. There were some signs of KMP. Histological examination revealed a typical infiltrative multilobular spindle cell proliferation with slit-like vascular spaces in these three localisations. Immunohistochemical stains showed positivity for CD34 and CD31 and many alpha-smooth muscle actin-positive spindle cells around the vascular spaces. There was no Herpes virus type 8 expression. The presented case is unique in two ways. First, thyroid involvement of KHE has never been described in the literature until now. Secondly, and most remarkably, the multifocal presentation in three anatomically distinct and separated localisations is extremely unusual.

Epithelioid hemangioma of bone: a potentially metastasizing tumor?

The case of a 15-year-old boy with a severe swelling of the 2nd toe in the left foot and a large swelling of the left inguinofemoral region is described. Histologic examination of the foot lesion showed a typical epithelioid hemangioma of bone that extended into the soft tissues, with a secondary location in the groin, suggestive of lymph node involvement. Subsequently, imaging also revealed swelling of iliac and para-aortic lymph nodes, which probably indicates further lymph node spread.. Besides the fact that epithelioid hemangioma of the toe has not been reported yet, our case showed a non-continuous localization, most likely in draining lymph nodes. This finding suggests metastatic disease, an event that is extremely rare for epithelioid hemangioma. As such, epithelioid hemangioma might be comparable to another benign vascular tumor, the retiform hemangioendothelioma, which occasionally metastasizes to the lymph nodes without systemic spread.