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NTRK-rearranged spindle cell neoplasm represents an emerging entity included in the latest 5th edition of WHO classification of both soft tissue and female genital tumors. By immunohistochemistry, they are commonly positive for CD34, S100 protein, and CD30 and typically harbor fusions of kinase genes such as NTRK1/2/3, RET, and BRAF. In the gynecological tract, they typically affect the uterine cervix or uterine body. Most of the reported cases had fibrosarcoma-like morphology, occasionally showing perivascular and stromal hyalinization with only a few cases showing a less cellular spindle cell proliferation. Except for one case with RET fusion, all other gynecological cases harbored exclusively NTRK1/2/3 fusions. Besides kinase gene fusions, the analogous tumors in soft tissues may also harbor activating EGFR or BRAF point mutations, but no such case has been described in the uterus. Herein we are reporting two cases from the uterine cervix showing morphology and molecular features previously unreported at this anatomic site. The patients were 46 and 34 years old and clinically presented with unremarkable cervical polyps each measuring 8 mm in diameter. Histologically, both cases had a rounded polypoid outline and were composed of hypocellular proliferation of bland spindle cells lacking mitotic activity and growing in a fibrotic stroma which was punctuated by prominent small vessels with thick hyalinized walls. Immunohistochemically, both showed a diffuse expression of CD34, CD30, and S100 protein, whereas SOX10 was negative. Both cases harbored exon 20 EGFR mutation and did not reveal any fusions or significant copy number changes. The patient in case 1 was treated by hysterectomy with salpingectomy with no other residual tumor detected, and she was alive and well 27 months after the diagnosis. The patient in case 2 had no other known tumors at the time of diagnosis, but no follow-up is available. We believe the reported cases represent a hitherto unrecognized variant of "NTRK-rearranged spindle cell neoplasms" of the uterine cervix with novel EGFR mutations., (© 2024. The Author(s).)

Endometrial carcinomas (EC) of no special molecular profile (NSMP) represent the largest molecular category of EC, comprising a mixture of tumors with different histology and molecular profiles. These facts likely point to different tumor biology, clinical outcomes, and targeted therapy responses within this molecular category. The PIK3CA is currently the only targetable kinase oncoprotein directly implicated in EC carcinogenesis. Investigating a unique single-institution cohort, we attempted to stratify NSMP ECs based on the presence of the PIK3CA pathogenic mutation. Those cases were further analyzed for other well-established-associated oncogenic driver gene mutations. Histological and clinical variables were also correlated in each case. Altogether, 175 ECs were prospectively tested by a limited custom NGS panel containing ARID1A, BCOR, BRCA1, BRCA2, CTNNB1, KRAS, MLH1, MSH2, MSH6, NRAS, PIK3CA, PMS2, POLD1, POLE, PTEN,and TP53 genes. We identified 24 PIK3CA mutated cases in the group of 80 NSMP ECs, with another co-occurring mutation in at least one oncogenic driver gene (CTNNB1, PTEN, ARID1A, KRAS, BCOR, PMS2) in 19 cases. In conclusion, a limited NGS panel can effectively test EC tissue for specific pathogenetically relevant oncogene mutations. The NSMP EC category contains 30% of the PIK3CA mutated cases. Of those, 21% contain the PIK3CA mutation as a sole EC-associated oncogene mutation, while 79% harbor at least one more mutated gene. These findings may inform future healthcare planning and improve the effectiveness of EC patient selection for the PIK3CA-targeted therapy., (© 2024. The Author(s).)

Extreme ultraviolet (EUV) photon beam characterization techniques, Hartmann wavefront sensing and single shot ablation imprinting, were compared along the caustic of a tightly focused free-electron laser (FEL) beam at beamline FL24 of FLASH2, the Free-electron LASer in Hamburg at DESY. The transverse coherence of the EUV FEL was determined by a Young's double pinhole experiment and used in a back-propagation algorithm which includes partial coherence to calculate the beam intensity profiles along the caustic from the wavefront measurements. A very good agreement of the profile structure and size is observed for different wavelengths between the back-propagated profiles, an indirect technique, and ablation imprints. As a result, the Hartmann wavefront sensor including its software MrBeam is a very useful, single shot pulse resolved and fast tool for non-invasive determination of focal spot size and shape and also for beam profiles along the caustic.

Alterations in kinase genes such as NTRK1/2/3, RET, and BRAF underlie infantile fibrosarcoma (IFS), the emerging entity 'NTRK-rearranged spindle cell neoplasms' included in the latest WHO classification, and a growing set of tumors with overlapping clinical and pathological features. In this study, we conducted a comprehensive clinicopathological and molecular analysis of 22 cases of IFS and other kinase gene-altered spindle cell neoplasms affecting both pediatric and adult patients. Follow-up periods for 16 patients ranged in length from 10 to 130 months (mean 38 months). Six patients were treated with targeted therapy, achieving a partial or complete response in five cases. Overall, three cases recurred and one metastasized. Eight patients were free of disease, five were alive with disease, and two patients died. All cases showed previously reported morphological patterns. Based on the cellularity and level of atypia, cases were divided into three morphological grade groups. S100 protein and CD34 were at least focally positive in 12/22 and 14/22 cases, respectively. Novel PWWP2A::RET, NUMA1::RET, ITSN1::RAF1, and CAPZA2::MET fusions, which we report herein in mesenchymal tumors for the first time, were detected by RNA sequencing. Additionally, the first uterine case with BRAF and EGFR mutations and CD34 and S100 co-expression is described. DNA sequencing performed in 13 cases uncovered very rare additional genetic aberrations. The CNV profiles showed that high-grade tumors demonstrate a significantly higher percentage of copy number gains and losses across the genome compared with low- and intermediate-grade tumors. Unsupervised clustering of the tumors' methylation profiles revealed that in 8/9 cases, the methylation profiles clustered with the IFS methylation class, irrespective of their clinicopathological or molecular features. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland., (© 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.)

We report on experiments where solid-density Mg plasmas are created by heating with the focused output of the Linac Coherent Light Source x-ray free-electron laser. We study the K-shell emission from the helium- and lithium-like ions using Bragg crystal spectroscopy. Observation of the dielectronic satellites in lithium-like ions confirms that the M-shell electrons appear bound for these high charge states. An analysis of the intensity of these satellites indicates that when modeled with an atomic-kinetics code, the ionization potential depression model employed needs to produce depressions for these ions which lie between those predicted by the well known Stewart-Pyatt and Ecker-Kroll models. These results are largely consistent with recent density functional theory calculations.

Chordoma is a rare malignant tumor with notochordal differentiation, usually affecting the axial skeleton of young patients. We report a case of a high-grade epithelioid tumor involving the synovium and soft tissues of the knee in a 74-year-old male patient. The preliminary biopsy was inconclusive, but a diagnosis of metastatic clear-cell carcinoma of unknown origin was suggested. However, imaging studies did not reveal any primary lesions. The resection specimen consisted of nests and sheets of oval to polygonal cells with discernible cell borders, clear or lightly amphophilic cytoplasm, and round to oval nuclei with occasional well-visible eosinophilic nucleoli. Rare atypical mitoses, necrotic areas, and bizarre nuclei were noted. The biopsy and resection specimens underwent a wide molecular genetic analysis which included methylation profiling. The DKFZ sarcoma classifier assigned the methylation class chordoma (dedifferentiated) with a calibrated score of 0.96, and additionally, a loss of SMARCB1 locus was noted in the copy number variation plot. To verify these findings, T-brachyury and SMARCB1 immunostaining was performed afterward, showing diffuse nuclear positivity and complete loss in the tumor cells, respectively. To assess the prevalence of T-brachyury immunopositivity among SWI/SNF-deficient tumors and to evaluate its specificity for poorly differentiated chordoma, we analyzed a series of 23 SMARCB1- or SMARCA4-deficient tumors, all of which were negative. After incorporating all the available data, including the absence of any morphological features of conventional chordoma, the case was diagnosed as poorly differentiated chordoma. As illustrated herein, the utilization of methylation profiling in the diagnostic process of some carefully selected unclassifiable soft tissue neoplasms may lead to an increased detection rate of such extremely rare soft tissue tumors and enable their better characterization., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Branchioma is an uncommon benign neoplasm with an adult male predominance, typically occurring in the lower neck region. Different names have been used for this entity in the past (ectopic hamartomatous thymoma, branchial anlage mixed tumor, thymic anlage tumor, biphenotypic branchioma), but currently, the term branchioma has been widely accepted. Branchioma is composed of endodermal and mesodermal lineage derivatives, in particular epithelial islands, spindle cells, and mature adipose tissue without preexistent thymic tissue or evidence of thymic differentiation. Twenty-three branchiomas were evaluated morphologically. Eighteen cases with sufficient tissue were assessed by immunohistochemistry, next-generation sequencing (NGS) using the Illumina Oncology TS500 panel, and fluorescence in situ hybridization (FISH) using an RB1 dual-color probe. All cases showed a biphasic morphology of epithelial and spindle cells with intermingled fatty tissue. Carcinoma arising in branchioma was detected in three cases. The neoplastic cells showed strong AE1/3 immunolabeling (100%), while the spindle cells expressed CD34, p63, and SMA (100%); AR was detected in 40-100% of nuclei (mean, 47%) in 14 cases. Rb1 showed nuclear loss in ≥ 95% of neoplastic cells in 16 cases (89%), while two cases revealed retained expression in 10-20% of tumor cell nuclei. NGS revealed a variable spectrum of likely pathogenic variants (n = 5) or variants of unknown clinical significance (n = 6). Loss of Rb1 was detected by FISH in two cases. Recent developments support branchioma as a true neoplasm, most likely derived from the rudimental embryological structures of endoderm and mesoderm. Frequent Rb1 loss by immunohistochemistry and heterozygous deletion by FISH is a real pitfall and potential confusion with other Rb1-deficient head and neck neoplasms (i.e., spindle cell lipoma), especially in small biopsy specimens., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Methods of ablation imprints in solid targets are widely used to characterize focused X-ray laser beams due to a remarkable dynamic range and resolving power. A detailed description of intense beam profiles is especially important in high-energy-density physics aiming at nonlinear phenomena. Complex interaction experiments require an enormous number of imprints to be created under all desired conditions making the analysis demanding and requiring a huge amount of human work. Here, for the first time, we present ablation imprinting methods assisted by deep learning approaches. Employing a multi-layer convolutional neural network (U-Net) trained on thousands of manually annotated ablation imprints in poly(methyl methacrylate), we characterize a focused beam of beamline FL24/FLASH2 at the Free-electron laser in Hamburg. The performance of the neural network is subject to a thorough benchmark test and comparison with experienced human analysts. Methods presented in this Paper pave the way towards a virtual analyst automatically processing experimental data from start to end.

This article presents 2 cases of TFG::MET-rearranged mesenchymal tumor, an extremely rare molecular subset among an emerging group of mesenchymal neoplasms with kinase gene (NTRK, BRAF, RET and others) alterations. Both tumors were congenital, occurred in female patients and presented as huge masses on the trunk and thigh, measuring 18 and 20 cm in the largest dimension. Both cases showed identical areas with a distinctive triphasic morphology resembling fibrous hamartoma of infancy (FHI), consisting of haphazardly arranged ovoid to spindled cells traversed by variably cellular and hyalinized fascicles admixed with (most likely non-neoplastic) adipose tissue. In other areas, a high-grade infantile fibrosarcoma/malignant peripheral nerve sheath tumor-like (IFS/MPNST-like) morphology was present in both cases. While the first case co-expressed CD34 and S100 protein, the other case did not. When combined with the three previously reported MET-rearranged cases (of which two harbored TFG::MET fusion), 3/5 and 3/4 of MET-rearranged and TFG::MET fusion-associated tumors, respectively exhibited similar triphasic FHI-like low-grade morphology. This points toward the existence of a relatively distinct morphological subset among kinase-fusion-associated tumors which seems to be strongly associated with MET fusions. It seems some of these low-grade cases may transform into a high-grade variant with IFS/MPNST-like morphology as has been observed in other tumors with kinase gene fusions. While most cases seem to follow an indolent clinical course, the recognition of these tumors is clinically relevant as MET tyrosine kinase inhibitors might represent an effective treatment option for clinically aggressive or unresectable cases., (© 2022 Wiley Periodicals LLC.)

Extraskeletal myxoid chondrosarcoma (EMC) is a rare sarcoma of uncertain lineage. Insulinoma-associated protein 1 (INSM1) has recently been described as a highly specific and sensitive immunohistochemical marker for EMC. The goal of this study was to evaluate the diagnostic significance of INSM1 immunohistochemistry in EMC. Furthermore, correlations between molecular and morphological findings were performed. Sixteen of 17 EMC cases were stained with the INSM1 antibody. Tumors with at least 5% INSM1-positive cells and any staining intensity were considered positive. Molecular testing was successfully performed in 12/17 cases. The immunohistochemical analysis detected 13 INSM1-positive (81%) and 3 INSM1-negative tumors (19%). The extent of the staining was classified as 1+ in 7 cases (44%), 2+ in 2 cases (13%), 3+ in 2 cases (13%) and 4+ in 2 cases (13%). Intensity of immunostaining was weak in 5 cases (31%), moderate in 2 cases (13%) and strong in 6 cases (38%). Molecular assays revealed 8 EWSR1::NR4A3 positive tumors (67%), 2 TAF15::NR4A3 positive tumors (17%), 1 TCF12::NR4A3 positive tumor (8%) and 1 NR4A3 positive tumor (8%) in which no other gene alteration was identified. Two of them, namely TCF12 positive and one TAF15 positive tumors, were highly cellular and partially associated with pseudopapillary architecture. Our study found that moderate/strong expression of INSM1 in more than 25% of tumor cells was present in only 31% of cases. Thus, the diagnostic utility of INSM1 is rather low. Two morphologically unique cases of non-EWSR1 rearranged EMC with an extremely rare pseudopapillary growth pattern are also reported., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Epithelioid fibrous histiocytoma (EFH) is a distinctive cutaneous neoplasm with a relatively variable morphological appearance. Recently, it has been shown that this tumor is molecularly characterized by ALK gene fusions. We report three EFHs with unusual histological presentation represented by a prominent/predominant spindle cell proliferation arranged in a variably storiform/whirling architectural pattern with or without stromal sclerosis. One of the cases closely resembled cellular fibrous histiocytoma. All three cases were immunohistochemically ALK-positive and were analyzed for ALK gene rearrangements using a next-generation sequencing-based assay (FusionPlex Sarcoma Kit, ArcherDx). Three novel fusions, namely AP3D1::ALK, COL1A::ALK, and LRRFIP2::ALK, were detected and further confirmed by FISH in all 3 cases and RT-PCR in 1 case. All patients were elderly (62-63 years) and presented with a solitary polypoid lesion on the extremities. The awareness of these morphological variants is important since it entertains a wide and slightly different differential diagnosis than conventional EFH. We also presented evidence that a clear separation of EFH from BFH in all cases may not be as straightforward as previously thought. The consistent ALK immunoexpression and the continually expanding scale of ALK gene rearrangements provide a useful tool to distinguish EFH from its histologic mimics., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

We report 7 cases of an indolent, variably myxoid tumor of the vocal cords, characterized by overt cellular atypia with large cells containing intranuclear and intracytoplasmic vacuoles, delicate curvilinear vessels, and sparse inflammatory infiltrate. Six patients were male, aged 15 to 65 years, and 1 patient was a 54-year-old female. All tumors were located in the superficial portion of the vocal cord. One patient suffered a recurrence that was completely resected; all patients with available follow-up data currently have no evidence of disease. The tumors contained alternating areas with myxoid stroma and more compacted regions with tumor cells organized in short fascicles, interwoven with delicate curvilinear vasculature. Overt cellular atypia with large cells containing intranuclear and intracytoplasmic vacuoles or resembling ganglion cells was present in all cases but mitoses and necrosis were absent. ALK immunostaining was positive in all cases, while most tumors were negative for smooth muscle actin. Targeted RNA-sequencing revealed an identical TIMP3::ALK fusion with exon 1 of TIMP3 gene being fused with exon 12 of ALK gene in all analyzable cases. For various reasons discussed, it remains unclear whether this tumor represents a mere subtype of IMT or a separate entity. Nevertheless, it is a morphologically distinct and diagnostically challenging lesion that needs to be recognized by surgical pathologists in order to prevent overdiagnosis in this clinically very delicate area., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Myxoinflammatory fibroblastic sarcoma (MIFS) has been shown to harbor various recurrent molecular aberrations; most of which, however, seem to be present in only a minority of cases. In order to better characterize the molecular underpinnings of MIFS, fourteen cases were analyzed by targeted RNA-sequencing (RNA-seq), VGLL3 enumeration FISH probe, and BRAF break-apart and enumeration probes. Neither t(1;10)(p22;q24) nor BRAF gene amplifications were found. However, VGLL3 gene amplification was detected in 5 cases by FISH which corresponded with an increase in VGLL3 expression detected by RNA-seq. In 1 of these cases, RNA-seq additionally revealed a novel SND1::BRAF fusion. Two of the 9 cases lacking VGLL3 amplification harbored either a SEC23IP::VGLL3 or a TEAD1::MRTFB rearrangement by RNA-seq, both confirmed by RT-PCR and Sanger sequencing. The detected molecular aberrations have a potential to either activate the expression of genes regulated by the transcription factors of the TEAD family, which are involved in tumor initiation and progression, or switch on the MEK/ERK signaling cascade, which plays an important role in cell cycle progression. Our results broaden the molecular genetic spectrum of MIFS and point toward the importance of the VGLL3-TEAD interaction, as well as the deregulation of the MEK/ERK pathway in the pathogenesis of MIFS, and may represent a potential target for therapy of recurrent or advanced disease., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

A real-time and accurate characterization of the X-ray beam size is essential to enable a large variety of different experiments at free-electron laser facilities. Typically, ablative imprints are employed to determine shape and size of µm-focused X-ray beams. The high accuracy of this state-of-the-art method comes at the expense of the time required to perform an ex-situ image analysis. In contrast, diffraction at a curved grating with suitably varying period and orientation forms a magnified image of the X-ray beam, which can be recorded by a 2D pixelated detector providing beam size and pointing jitter in real time. In this manuscript, we compare results obtained with both techniques, address their advantages and limitations, and demonstrate their excellent agreement. We present an extensive characterization of the FEL beam focused to ≈1 µm by two Kirkpatrick-Baez (KB) mirrors, along with optical metrology slope profiles demonstrating their exceptionally high quality. This work provides a systematic and comprehensive study of the accuracy provided by curved gratings in real-time imaging of X-ray beams at a free-electron laser facility. It is applied here to soft X-rays and can be extended to the hard X-ray range. Furthermore, curved gratings, in combination with a suitable detector, can provide spatial properties of µm-focused X-ray beams at MHz repetition rate.

GLI1 fusions involving ACTB, MALAT1, PTCH1 and FOXO4 genes have been reported in a subset of malignant mesenchymal tumors with a characteristic nested epithelioid morphology and frequent S100 positivity. Typically, these multilobulated tumors consist of uniform epithelioid cells with bland nuclei and are organized into distinct nests and cords with conspicuously rich vasculature. We herein expand earlier findings by reporting a case of a 34-year-old female with an epithelioid mesenchymal tumor of the palate. The neoplastic cells stained positive for S100 protein and D2-40, whereas multiple other markers were negative. Genetic alterations were investigated by targeted RNA sequencing, and a PTCH1-GLI1 fusion was detected. Epithelioid mesenchymal tumors harboring a PTCH1-GLI1 fusion are vanishingly rare with only three cases reported so far. Due to the unique location in the mucosa of the soft palate adjacent to minor salivary glands, multilobulated growth, nested epithelioid morphology, focal clearing of the cytoplasm, and immunopositivity for S100 protein and D2-40, the differential diagnoses include primary salivary gland epithelial tumors, in particular myoepithelioma and myoepithelial carcinoma. Another differential diagnostic possibility is the ectomesenchymal chondromyxoid tumor. Useful diagnostic clues for tumors with a GLI1 rearrangement include a rich vascular network between the nests of neoplastic cells, tumor tissue bulging into vascular spaces, and absence of SOX10, GFAP and cytokeratin immunopositivity. Identifying areas with features of GLI1-rearranged tumors should trigger subsequent molecular confirmation. This is important for appropriate treatment measures as PTCH1-GLI1 positive mesenchymal epithelioid neoplasms have a propensity for locoregional lymph node and distant lung metastases., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

This article describes two cases of fatal complications of urolithiasis. The first case discusses the development of fatal urosepsis in a patient with a known mental health issue. The patient had self-introduced a thermometer into her bladder, which led to stone formation around the thermometer and consequently to fatal urosepsis. The second case deals with a motor vehicle accident in which a passenger with pre-existing hydronephrosis suffered a kidney injury leading to fatal bleeding into the surrounding adipose tissue. To provide a wider context, the paper also discusses the fatal complications and origin of urolithiasis., (© 2021. Springer Science+Business Media, LLC, part of Springer Nature.)

The knowledge of clinical features and, particularly, histopathological spectrum of EWSR1-PATZ1-rearranged spindle and round cell sarcomas (EPS) remains limited. For this reason, we report the largest clinicopathological study of EPS to date. Nine cases were collected, consisting of four males and five females ranging in age from 10 to 81 years (average: 49 years). Five tumors occurred in abdominal wall soft tissues, three in the thorax, and one in the back of the neck. Tumor sizes ranged from 2.5 to 18 cm (average 6.6 cm). Five patients had follow-up with an average of 38 months (range: 18-60 months). Two patients had no recurrence or metastasis 19 months after diagnosis. Four patients developed multifocal pleural or pulmonary metastasis and were treated variably by surgery, radiotherapy, and chemotherapy. The latter seemed to have little to no clinical benefit. One of the four patients was free of disease 60 months after diagnosis, two patients were alive with disease at 18 and 60 months, respectively. Morphologically, low, intermediate, and high-grade sarcomas composed of a variable mixture of spindled, ovoid, epithelioid, and round cells were seen. The architectural and stromal features also varied, resulting in a broad morphologic spectrum. Immunohistochemically, the following markers were most consistently expressed: S100-protein (7/9 cases), GFAP (7/8), MyoD1 (8/9), Pax-7 (4/5), desmin (7/9), and AE1/3 (4/9). By next-generation sequencing, all cases revealed EWSR1-PATZ1 gene fusion. In addition, 3/6 cases tested harbored CDKN2A deletion, while CDKN2B deletion and TP53 mutation were detected in one case each. Our findings confirm that EPS is a clinicopathologic entity, albeit with a broad morphologic spectrum. The uneventful outcome in some of our cases indicates that a subset of EPS might follow a more indolent clinical course than previously appreciated. Additional studies are needed to validate whether any morphological and/or molecular attributes have a prognostic impact.

Oncogenic gene fusions represent attractive targets for therapy of cancer. However, the frequency of actionable genomic rearrangements in colorectal cancer (CRC) is very low, and universal screening for these alterations seems to be impractical and costly. To address this problem, several large scale studies retrospectivelly showed that CRC with gene fusions are highly enriched in groups of tumors defined by MLH1 DNA mismatch repair protein deficiency (MLH1d), and hypermethylation of MLH1 promoter (MLH1ph), and/or the presence of microsatellite instability, and BRAF/KRAS wild-type status (BRAFwt/KRASwt). In this study, we used targeted next generation sequencing (NGS) to explore the occurence of potentially therapeutically targetable gene fusions in an unselected series of BRAFwt/KRASwt CRC cases that displayed MLH1d/MLH1ph. From the initially identified group of 173 MLH1d CRC cases, 141 cases (81.5%) displayed MLH1ph. BRAFwt/RASwt genotype was confirmed in 23 of 141 (~16%) of MLH1d/MLH1ph cases. Targeted NGS of these 23 cases identified oncogenic gene fusions in nine patients (39.1%; CI95: 20.5%-61.2%). Detected fusions involved NTRK (four cases), ALK (two cases), and BRAF genes (three cases). As a secondary outcome of NGS testing, we identified PIK3K-AKT-mTOR pathway alterations in two CRC cases, which displayed PIK3CA mutation. Altogether, 11 of 23 (~48%) MLH1d/MLH1ph/BRAFwt/RASwt tumors showed genetic alterations that could induce resistance to anti-EGFR therapy. Our study confirms that targeted NGS of MLH1d/MLH1ph and BRAFwt/RASwt CRCs could be a cost-effective strategy in detecting patients with potentially druggable oncogenic kinase fusions., (© 2020 Wiley Periodicals, Inc.)

Papillary thyroid carcinoma with desmoid-type fibromatosis or nodular fasciitis-like stroma is an extremely unusual and poorly understood subtype of papillary thyroid cancer. Although prior studies have demonstrated alterations in the Wnt/β-catenin signaling pathway in some of these tumors, controversy still exists regarding the nature of the stromal spindle component. We have studied seven cases of papillary thyroid carcinoma with prominent myofibroblastic stroma, including six men and one woman aged 20-65 years (mean age = 44). All cases displayed areas consistent with conventional papillary thyroid carcinoma embedded in abundant myofibroblastic-like stroma. The myofibroblastic stroma in six cases resembled desmoid-type fibromatosis and in one case it more closely resembled nodular fasciitis. By immunohistochemical staining, the stromal spindle component showed positivity for SMA and low MIB1 proliferation index in all cases, and there was at least patchy strong nuclear positivity for beta-catenin in six/seven cases. Stains for cytokeratin AE1/AE3 and PAX8 were positive in the epithelial elements but negative in the stromal component. Next-generation sequencing was performed on six of seven cases. CTNNB1 gene mutations were identified in six/seven cases. The epithelial component showed BRAF mutations in two cases and an NRAS mutation in one case. The case with fasciitis-like stroma was negative for beta-catenin by sequencing and immunostaining as well as negative for USP6 gene rearrangement. Our findings indicate that papillary thyroid carcinoma with prominent myofibroblastic stroma may represent more than one category of lesions.

Proper diagnostics of intense free-electron laser (FEL) X-ray pulses is indisputably important for experimental data analysis as well as for the protection of beamline optical elements. New challenges for beam diagnostic methods are introduced by modern FEL facilities capable of delivering powerful pulses at megahertz (MHz) repetition rates. In this paper, we report the first characterization of a defocused MHz 13.5-nm beam generated by the free-electron laser in Hamburg (FLASH) using the method of multi-pulse desorption imprints in poly(methyl methacrylate)(PMMA). The beam fluence profile is reconstructed in a novel and highly accurate way that takes into account the nonlinear response of material removal to total dose delivered by multiple pulses. The algorithm is applied to experimental data of single-shot ablation imprints and multi-shot desorption imprints at both low (10 Hz) and high (1 MHz) repetition rates. Reconstructed response functions show a great agreement with the theoretical desorption response function model.

Inflammatory leiomyosarcoma (ILMS) is a very rare soft tissue tumor that usually follows an indolent clinical course, but long-term follow-up studies are lacking. Recent publications primarily focused on its genetic profile characterized by a near haploid genome. One study also showed these tumors to have upregulation of genes known to be crucial for skeletal muscle differentiation. Nevertheless, immunohistochemical expression of skeletal muscle markers, as well as markers that would help to distinguish ILMS from a long list of relevant differential diagnostic entities, has not been extensively studied. Nine cases of ILMS were collected and stained by a broad IHC panel which, besides others, contained MyoD1, myogenin, and PAX-7. A subset of cases was also analyzed by 2 different NGS assays and by MDM2 fluorescence in situ hybridization. Five male and 4 female patients ranged in age from 25 to 54 years (mean, 36 years). The tumors showed a predilection for intramuscular sites of the lower limbs (n = 4) and back (n = 2), whereas the remaining 3 cases affected an unspecified skeletal muscle, lung, and omentum. Follow-up with an average length of 10.6 years (range 0.5-22) was available for 8 patients. The omental tumor spread locally within the abdominal cavity, but the patient has been free of disease 7 years after treatment. None of the 5 patients with somatic soft tissue tumors (and follow-up longer than 1.5 years) had either recurrence or metastasis. Immunohistochemical studies revealed a substantial expression of skeletal muscle markers in almost all cases. This phenotype coupled with a highly characteristic genotype and significantly more indolent clinical behavior as compared with conventional leiomyosarcoma of deep soft tissue offers a strong rationale to change the current nomenclature. Based on the clinicopathological features and gene expression profile, we propose the name low-grade inflammatory myogenic tumor.

We report on 51-year-old woman who presented with brown discharge and postcoital bleeding due to myoma nascens-like polypoid mass distending cervical canal. Histologically, the tumor consisted of high-grade spindle cell component with up to 15 mitotic figures per 10 HPF and also low-grade leiomyoma-like areas with focal myxoid change and so far undescribed cytoplasmic signet ring cell change. Immunohistochemically Desmin, actin, and h-caldesmon were negative. Conversely, BCOR positive expression was coupled with Cyclin D1 positivity and was antibody clone dependent. The molecular NGS and FISH study identified reciprocal fusion gene ZC3H7B-BCOR. In conclusion, these findings further support the idea of routine reflex molecular testing of uterine mesenchymal tumors with unusual clinical presentation or in case malignancy is suspected. Lastly, we suggest ZC3H7B-BCOR rearranged high-grade endometrial stromal sarcoma might be considered as a tumor suitable for BCL6-targeted treatment.

Contemporary health care is facing deep personal crisis appearing mainly in nurses. It particularly involves departments characterized by mentally and physically demanding workload especially departments of internal medicine, that are immediately endangered by the lack of the nursing staff. The importance and capability of motivation come to fore among other factors. Enough motivation could decrease the decline of nursing staff and lead to the increase of interest in work at departments of internal medicine. The article summarizes basic principles of motivation that is possible to employ.

Secretory carcinoma of the skin is a rare adnexal carcinoma, which is morphologically and immunohistochemically identical to secretory carcinoma of the breast and is associated with the presence of t (12;15) translocation, resulting in the ETV6-NTRK3 gene fusion. Nineteen cases of primary cutaneous secretory carcinoma have been previously published in the literature. In this study, we describe 6 new cases of secretory carcinoma of the skin. The study group consisted of 5 female patients and 1 male patient, ranging in age from 57 to 98 years (mean: 74.2, median: 74). Locations included the axilla (2), neck, eyelid, thigh, and nipple base, each one. Microscopically, all but 1 tumor were well circumscribed and nonencapsulated and exhibited characteristic abundant secretions within the microcystic and tubular spaces comprised by bland oval, round to cuboidal neoplastic cells. In addition, solid areas and focal pseudopapillae were seen, and, in 1 case, a focal mucinous component with small lakes of mucin containing small tumor nests or tubules of the neoplastic cells was present. The remaining neoplasm was mostly solid and papillary, with only few characteristic lumina containing secretions. Immunohistochemically, all cases expressed S-100 protein, mammaglobin, STAT5, GATA3, and NTRK. ETV6-NTRK3 gene fusion was detected in 5 cases, whereas, in the remaining tumor, a novel NFIX-PKN1 gene fusion was found.

Ruthenium is a perspective material to be used for XUV mirrors at free-electron laser facilities. Yet, it is still poorly studied in the context of ultrafast laser-matter interaction. In this work, we present single-shot damage studies of thin Ru films irradiated by femtosecond XUV free-electron laser pulses at FLASH. Ex-situ analysis of the damaged spots, performed by different types of microscopy, shows that the weakest detected damage is surface roughening. For higher fluences we observe ablation of Ru. Combined simulations using Monte-Carlo code XCASCADE(3D) and the two-temperature model reveal that the damage mechanism is photomechanical spallation, similar to the case of irradiating the target with optical lasers. The analogy with the optical damage studies enables us to explain the observed damage morphologies.

Electron-ion collisional dynamics is of fundamental importance in determining plasma transport properties, nonequilibrium plasma evolution, and electron damage in diffraction imaging applications using bright x-ray free-electron lasers (FELs). Here we describe the first experimental measurements of ultrafast electron impact collisional ionization dynamics using resonant core-hole spectroscopy in a solid-density magnesium plasma, created and diagnosed with the Linac Coherent Light Source x-ray FEL. By resonantly pumping the 1s→2p transition in highly charged ions within an optically thin plasma, we have measured how off-resonance charge states are populated via collisional processes on femtosecond time scales. We present a collisional cross section model that matches our results and demonstrates how the cross sections are enhanced by dense-plasma effects including continuum lowering. Nonlocal thermodynamic equilibrium collisional radiative simulations show excellent agreement with the experimental results and provide new insight on collisional ionization and three-body-recombination processes in the dense-plasma regime.

The durability of grazing- and normal-incidence optical coatings has been experimentally assessed under free-electron laser irradiation at various numbers of pulses up to 16 million shots and various fluence levels below 10% of the single-shot damage threshold. The experiment was performed at FLASH, the Free-electron LASer in Hamburg, using 13.5 nm extreme UV (EUV) radiation with 100 fs pulse duration. Polycrystalline ruthenium and amorphous carbon 50 nm thin films on silicon substrates were tested at total external reflection angles of 20° and 10° grazing incidence, respectively. Mo/Si periodical multilayer structures were tested in the Bragg reflection condition at 16° off-normal angle of incidence. The exposed areas were analysed post-mortem using differential contrast visible light microscopy, EUV reflectivity mapping and scanning X-ray photoelectron spectroscopy. The analysis revealed that Ru and Mo/Si coatings exposed to the highest dose and fluence level show a few per cent drop in their EUV reflectivity, which is explained by EUV-induced oxidation of the surface.

We present measurements of the spectrally resolved x rays emitted from solid-density magnesium targets of varying sub-μm thicknesses isochorically heated by an x-ray laser. The data exhibit a largely thickness-independent source function, allowing the extraction of a measure of the opacity to K-shell x rays within well-defined regimes of electron density and temperature, extremely close to local thermodynamic equilibrium conditions. The deduced opacities at the peak of the Kα transitions of the ions are consistent with those predicted by detailed atomic-kinetics calculations.

A nonlinear absorber in which the excited state absorption is larger than the ground state can undergo a process called reverse saturable absorption. It is a well-known phenomenon in laser physics in the optical regime, but is more difficult to generate in the x-ray regime, where fast nonradiative core electron transitions typically dominate the population kinetics during light matter interactions. Here, we report the first observation of decreasing x-ray transmission in a solid target pumped by intense x-ray free electron laser pulses. The measurement has been made below the K-absorption edge of aluminum, and the x-ray intensity ranges are 10^{16} -10^{17}  W/cm^{2}. It has been confirmed by collisional radiative population kinetic calculations, underscoring the fast spectral modulation of the x-ray pulses and charge states relevant to the absorption and transmission of x-ray photons. The processes shown through detailed simulations are consistent with reverse saturable absorption, which would be the first observation of this phenomena in the x-ray regime. These light matter interactions provide a unique opportunity to investigate optical transport properties in the extreme state of matters, as well as affording the potential to regulate ultrafast x-ray free-electron laser pulses.

The role played by heat accumulation in multi-shot damage of silicon was studied. Bulk silicon samples were exposed to intense XUV monochromatic radiation of a 13.5 nm wavelength in a series of 400 femtosecond pulses, repeated with a 1 MHz rate (pulse trains) at the FLASH facility in Hamburg. The observed surface morphological and structural modifications are formed as a result of sample surface melting. Modifications are threshold dependent on the mean fluence of the incident pulse train, with all threshold values in the range of approximately 36-40 mJ/cm 2 . Experimental data is supported by a theoretical model described by the heat diffusion equation. The threshold for reaching the melting temperature (45 mJ/cm 2 ) and liquid state (54 mJ/cm 2 ), estimated from this model, is in accordance with experimental values within measurement error. The model indicates a significant role of heat accumulation in surface modification processes.

The effect of a dense plasma environment on the energy levels of an embedded ion is usually described in terms of the lowering of its continuum level. For strongly coupled plasmas, the phenomenon is intimately related to the equation of state; hence, an accurate treatment is crucial for most astrophysical and inertial-fusion applications, where the case of plasma mixtures is of particular interest. Here we present an experiment showing that the standard density-dependent analytical models are inadequate to describe solid-density plasmas at the temperatures studied, where the reduction of the binding energies for a given species is unaffected by the different plasma environment (ion density) in either the element or compounds of that species, and can be accurately estimated by calculations only involving the energy levels of an isolated neutral atom. The results have implications for the standard approaches to the equation of state calculations.

The rate at which atoms and ions within a plasma are further ionized by collisions with the free electrons is a fundamental parameter that dictates the dynamics of plasma systems at intermediate and high densities. While collision rates are well known experimentally in a few dilute systems, similar measurements for nonideal plasmas at densities approaching or exceeding those of solids remain elusive. Here we describe a spectroscopic method to study collision rates in solid-density aluminium plasmas created and diagnosed using the Linac Coherent light Source free-electron X-ray laser, tuned to specific interaction pathways around the absorption edges of ionic charge states. We estimate the rate of collisional ionization in solid-density aluminium plasmas at temperatures ~30 eV to be several times higher than that predicted by standard semiempirical models.

High-intensity x-ray pulses from an x-ray free-electron laser are used to heat and probe a solid-density aluminum sample. The photon-energy-dependent transmission of the heating beam is studied through the use of a photodiode. Saturable absorption is observed, with the resulting transmission differing significantly from the cold case, in good agreement with atomic-kinetics simulations.

We present an extended theoretical background of so-called fluence scan (f-scan or F-scan) method, which is frequently being used for offline characterization of focused short-wavelength (EUV, soft X-ray, and hard X-ray) laser beams [J. Chalupský et al., Opt. Express 18, 27836 (2010)]. The method exploits ablative imprints in various solids to visualize iso-fluence beam contours at different fluence and/or clip levels. An f-scan curve (clip level as a function of the corresponding iso-fluence contour area) can be generated for a general non-Gaussian beam. As shown in this paper, fluence scan encompasses important information about energy distribution within the beam profile, which may play an essential role in laser-matter interaction research employing intense non-ideal beams. Here we for the first time discuss fundamental properties of the f-scan function and its inverse counterpart (if-scan). Furthermore, we extensively elucidate how it is related to the effective beam area, energy distribution, and to the so called Liu's dependence [J. M. Liu, Opt. Lett. 7, 196 (1982)]. A new method of the effective area evaluation based on weighted inverse f-scan fit is introduced and applied to real data obtained at the SCSS (SPring-8 Compact SASE Source) facility.

In situ focus characterization is demonstrated by working at an extreme ultraviolet (XUV) free-electron laser source using ablation technique. Design of the instrument reported here allows reaching a few micrometres resolution along with keeping the ultrahigh vacuum conditions and ensures high-contrast visibility of ablative imprints on optically transparent samples, e.g., PMMA. This enables on-line monitoring of the beam profile changes and thus makes possible in situ alignment of the XUV focusing optics. A good agreement between focal characterizations retrieved from in situ inspection of ablative imprints contours and from well-established accurate ex situ analysis with Nomarski microscope has been observed for a typical micro-focus experiment.

The x-ray intensities made available by x-ray free electron lasers (FEL) open up new x-ray matter interaction channels not accessible with previous sources. We report here on the resonant generation of Kα emission, that is to say the production of copious Kα radiation by tuning the x-ray FEL pulse to photon energies below that of the K edge of a solid aluminum sample. The sequential absorption of multiple photons in the same atom during the 80 fs pulse, with photons creating L-shell holes and then one resonantly exciting a K-shell electron into one of these holes, opens up a channel for the Kα production, as well as the absorption of further photons. We demonstrate rich spectra of such channels, and investigate the emission produced by tuning the FEL energy to the K-L transitions of those highly charged ions that have transition energies below the K edge of the cold material. The spectra are sensitive to x-ray intensity dependent opacity effects, with ions containing L-shell holes readily reabsorbing the Kα radiation.

We have used the Linac Coherent Light Source to generate solid-density aluminum plasmas at temperatures of up to 180 eV. By varying the photon energy of the x rays that both create and probe the plasma, and observing the K-α fluorescence, we can directly measure the position of the K edge of the highly charged ions within the system. The results are found to disagree with the predictions of the extensively used Stewart-Pyatt model, but are consistent with the earlier model of Ecker and Kröll, which predicts significantly greater depression of the ionization potential.

The interaction of free electron laser pulses with grating structure is investigated using 4.6±0.1 nm radiation at the FLASH facility in Hamburg. For fluences above 63.7±8.7 mJ/cm2, the interaction triggers a damage process starting at the edge of the grating structure as evidenced by optical and atomic force microscopy. Simulations based on solution of the Helmholtz equation demonstrate an enhancement of the electric field intensity distribution at the edge of the grating structure. A procedure is finally deduced to evaluate damage threshold.

Matter with a high energy density (>10(5) joules per cm(3)) is prevalent throughout the Universe, being present in all types of stars and towards the centre of the giant planets; it is also relevant for inertial confinement fusion. Its thermodynamic and transport properties are challenging to measure, requiring the creation of sufficiently long-lived samples at homogeneous temperatures and densities. With the advent of the Linac Coherent Light Source (LCLS) X-ray laser, high-intensity radiation (>10(17) watts per cm(2), previously the domain of optical lasers) can be produced at X-ray wavelengths. The interaction of single atoms with such intense X-rays has recently been investigated. An understanding of the contrasting case of intense X-ray interaction with dense systems is important from a fundamental viewpoint and for applications. Here we report the experimental creation of a solid-density plasma at temperatures in excess of 10(6) kelvin on inertial-confinement timescales using an X-ray free-electron laser. We discuss the pertinent physics of the intense X-ray-matter interactions, and illustrate the importance of electron-ion collisions. Detailed simulations of the interaction process conducted with a radiative-collisional code show good qualitative agreement with the experimental results. We obtain insights into the evolution of the charge state distribution of the system, the electron density and temperature, and the timescales of collisional processes. Our results should inform future high-intensity X-ray experiments involving dense samples, such as X-ray diffractive imaging of biological systems, material science investigations, and the study of matter in extreme conditions.

We present the x-ray optical design of the soft x-ray materials science instrument at the Linac Coherent Light Source, consisting of a varied line-spaced grating monochromator and Kirkpatrick-Baez refocusing optics. Results from the commissioning of the monochromator are shown. A resolving power of 3000 was achieved, which is within a factor of two of the design goal., (© 2011 American Institute of Physics)

We present a new technique for the characterization of non-Gaussian laser beams which cannot be described by an analytical formula. As a generalization of the beam spot area we apply and refine the definition of so called effective area (A(eff)) [1] in order to avoid using the full-width at half maximum (FWHM) parameter which is inappropriate for non-Gaussian beams. Furthermore, we demonstrate a practical utilization of our technique for a femtosecond soft X-ray free-electron laser. The ablative imprints in poly(methyl methacrylate) - PMMA and amorphous carbon (a-C) are used to characterize the spatial beam profile and to determine the effective area. Two procedures of the effective area determination are presented in this work. An F-scan method, newly developed in this paper, appears to be a good candidate for the spatial beam diagnostics applicable to lasers of various kinds.

It is well documented that the polysaccharide glucomannan (GM), an abundant constituent of the fungal cell wall, in the form of particulate induces strong activation of phagocytes, however, the effects of soluble GM are not known. Activation of phagocyte anti-microbial mechanisms is a crucial part of the innate host defense against invading pathogens. However, under uncontrolled inflammatory conditions they contribute to damage of surrounding tissues. Thus, to prevent these deleterious effects, the activation of phagocytes is a tightly regulated process. Therefore, in this study we analyzed the effect of soluble GM on some neutrophil functions such as reactive oxygen species production, degranulation, and receptor mobilization at the plasma membrane. Soluble GM at the tested concentrations did not stimulate oxidative burst of phagocytes directly but significantly potentiated oxidative burst in response to opsonized zymosan particles. GM induced significant phosphorylation of p47phox subunit of NADPH oxidase on Ser345. This priming effect of GM was accompanied by time and concentration dependent degranulation characterized by increased surface expression of receptors stored in neutrophil granules (CD10, CD11b, CD14, CD35, and CD66b). Degranulation was further confirmed by increase of elastase activity in media. Thus, it could be suggested that soluble GM induces priming of phagocytes connected with their degranulation, the increase of surface receptor expression, and potentiation of oxidative burst response to opsonized particles through the activation of NADPH oxidase.

We report the first observation of single-shot soft x-ray laser induced desorption occurring below the ablation threshold in a thin layer of poly (methyl methacrylate)--PMMA. Irradiated by the focused beam from the Free-electron LASer in Hamburg (FLASH) at 21.7 nm, the samples have been investigated by atomic-force microscope (AFM) enabling the visualization of mild surface modifications caused by the desorption. A model describing non-thermal desorption and ablation has been developed and used to analyze single-shot imprints in PMMA. An intermediate regime of materials removal has been found, confirming model predictions. We also report below-threshold multiple-shot desorption of PMMA induced by high-order harmonics (HOH) at 32 nm. Short-time exposure imprints provide sufficient information about transverse beam profile in HOH's tight focus whereas long-time exposed PMMA exhibits radiation-initiated surface ardening making the beam profile measurement infeasible.

We report on a single-shot micropatterning of an organic polymer achieved by ablation with demagnifying projection using a Ne-like Zn 21.2 nm soft x-ray laser. A nickel mesh with a period of 100 microm was approximately 10x demagnified and imprinted on poly(methyl methacrylate) via direct ablation. The quality of the ablated microstructure was found to be mainly dependent on the quality of the projected mask. This first demonstration (to our knowledge) of single-shot projection, single-step lithography illustrates the potential of soft x-ray lasers for the direct patterning of materials with a resolution scalable down to submicrometer domain.

A large-scale, double-stream gas puff target has been illuminated by sub-kJ, near-infrared (NIR) focused laser pulses at the PALS facility (Prague Asterix Laser System) to produce high-energy pulses of soft X rays from hot, dense plasma. The double-puff arrangement ensures high gas density and conversion efficiency from NIR to X rays approaching that typical for solid targets. In addition, its major advantage over solid targets is that it is free of debris and has substantially suppressed charged-particle emission. The X-ray emission characteristics of the source were determined for a range of gases that included krypton, xenon, N(2), CO and N(2)-CO. A demonstrated application of the xenon-based source is a single-shot damage induction to plasmid DNA. The yields of single-strand breaks (SSBs) and double-strand breaks (DSBs) were determined as a function of energy fluence adjusted by varying distance of sample from the source and thickness of aluminum filters.

A linear accelerator based source of coherent radiation, FLASH (Free-electron LASer in Hamburg) provides ultra-intense femtosecond radiation pulses at wavelengths from the extreme ultraviolet (XUV; lambda<100nm) to the soft X-ray (SXR; lambda<30nm) spectral regions. 25-fs pulses of 32-nm FLASH radiation were used to determine the ablation parameters of PMMA - poly (methyl methacrylate). Under these irradiation conditions the attenuation length and ablation threshold were found to be (56.9+/-7.5) nm and approximately 2 mJ*cm(-2), respectively. For a second wavelength of 21.7 nm, the PMMA ablation was utilized to image the transverse intensity distribution within the focused beam at mum resolution by a method developed here.

Consistent with the declaration of WHO "Health for all by the year 2000" and the FDI programme "Oral Health by the year 2000" the aim of our work was to become familiar with habits and knowledge on oral hygiene in 16-year-old adolescents. In 1988 we examined a total of 180 adolescents selected at random in one Prague secondary school and two centres for apprentices. For the assessment of habits regarding oral hygiene and knowledge on oral hygiene we used own questionnaire comprising 33 questions. The investigation provides evidence that it is essential to motivate patients more effectively to improve oral hygiene and to instruct during dental examinations on the importance of proper dental care. Dental surgeons should use every visit of the patient to explain oral hygiene and check adherence to it.

The DNA as isolated from duck fibroblasts transformed by a duck-adapted Prague strain of Rous sarcoma virus and used for transfection. Transformed recipient BLEF and DEF cultures exhibited considerable morphological variability. The virus designated daPR-RSV-C morphf was obtained from the culture with fusiform transformation and cloned. The virus retained the ability to induce fusiform transformation, even after 20 passages on chicken fibroblasts. There was a good correlation between focus forming activity of the virus and its tumorigenicity in chickens. The frequency of morphf mutation to another phenotype was less than 10(-3) in cloned virus. Foreign avian embryonic cells transformed by this virus clone had a similar morphological appearance as transformed chicken cells. The clone also retained two additional non-conditional markers - subgroup C specificity and the ability to replicate efficiently in duck cells ("duck adaptation"). Freshly obtained cloned virus was found not to contain a transformation-defective mutant. Such a mutant occurred in the second passage of the virus of DEF where the mutant was isolated. Inoculation of the td mutant into Brown Leghorn embryos gave rise to a sarcoma in one of the 36 examined chickens. However, no transforming virus was detected in the sarcoma. SDS-polyacrylamide gel electrophoresis showed that cloned daPR-RSV-C morphf contained only genomic RNA; its molecular weight 3.08 X 10(6) daltons corresponded to the molecular weight of a non-defective PR-RSV-C used as control.