TRIM28 inactivation in epithelial nephroblastoma is frequent and often associated with predisposing TRIM28 germline variants.

Wilms tumors (WTs) are histologically diverse childhood cancers with variable contributions of blastema, stroma, and epithelia. A variety of cancer genes operate in WTs, including the tripartite-motif-containing-28 gene (TRIM28). Case reports and small case series suggest that TRIM28 mutations are associated with epithelial morphology and WT predisposition. Here, we systematically investigated the prevalence of TRIM28 inactivation and predisposing mutations in a cohort of 126 WTs with >2/3 epithelial cells, spanning 20 years of biobanking in the German SIOP93-01/GPOH and SIOP2001/GPOH studies. Overall, 44.4% (56/126) cases exhibited loss of TRIM28 by immunohistochemical staining. Of these, 48 could be further analyzed molecularly, revealing TRIM28 sequence variants in each case - either homozygous (~2/3) or heterozygous with epigenetic silencing of the second allele (~1/3). The majority (80%) of the mutations resulted in premature stops and frameshifts. In addition, we detected missense mutations and small deletions predicted to destabilize the protein through interference with folding of key structural elements such as the zinc-binding clusters of the RING, B-box-2, and PHD domains or the central coiled-coil region. TRIM28-mutant tumors otherwise lacked WT-typical IGF2 alterations or driver events, except for rare TP53 progression events that occurred with expected frequency. Expression profiling identified TRIM28-mutant tumors as a homogeneous subset of epithelial WTs that mostly present with stage I disease. There was a high prevalence of perilobar nephrogenic rests, putative precursor lesions, that carried the same biallelic TRIM28 alterations in 7/7 cases tested. Importantly, 46% of the TRIM28 mutations were present in blood cells or normal kidney tissue, suggesting germline events or somatic mosaicism, partly supported by family history. Given the high prevalence of predisposing variants in TRIM28-driven WT, we suggest that immunohistochemical testing of TRIM28 be integrated into diagnostic practice as the management of WT in predisposed children differs from that with sporadic tumors. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

A familial disorder of altered DNA-methylation.

BACKGROUND: In a subset of imprinting disorders caused by epimutations, multiple imprinted loci are affected. Familial occurrence of multilocus imprinting disorders is rare. PURPOSE/OBJECTIVE: We have investigated the clinical and molecular features of a familial DNA-methylation disorder. METHODS: Tissues of affected individuals and blood samples of family members were investigated by conventional and molecular karyotyping. Sanger sequencing and RT-PCR of imprinting-associated genes (NLRP2, NLRP7, ZFP57, KHDC3L, DNMT1o), exome sequencing and locus-specific, array-based and genome-wide technologies to determine DNA-methylation were performed. RESULTS: In three offspring of a healthy couple, we observed prenatal onset of severe growth retardation and dysmorphism associated with altered DNA-methylation at paternally and maternally imprinted loci. Array-based analyses in various tissues of the offspring identified the DNA-methylation of 2.1% of the genes in the genome to be recurrently altered. Despite significant enrichment of imprinted genes (OR 9.49), altered DNA-methylation predominately (90.2%) affected genes not known to be imprinted. Sequencing of genes known to cause comparable conditions and exome sequencing in affected individuals and their ancestors did not unambiguously point to a causative gene. CONCLUSIONS: The family presented herein suggests the existence of a familial disorder of DNA-methylation affecting imprinted but also not imprinted gene loci potentially caused by a maternal effect mutation in a hitherto not identified gene.

ß1-Adrenoceptor mRNA level reveals distinctions between infantile hemangioma and vascular malformations.

BACKGROUND: Infantile hemangioma (IH) is the most frequent vascular tumor of early childhood. Recently, propranolol, a nonselective ß1- and ß2-adrenoceptor inhibitor, was introduced into the therapy of severe proliferating IH with excellent results. However, the underlying mechanism of action of propranolol is still unclear. METHODS: We performed immunohistochemistry for cluster of differentiation 31 (CD31), D2-40, glucose transporter-1 (GLUT-1), and Ki67 in order to characterize 21 vascular anomalies (nine IH, seven venous malformations (VMs), and five lymphatic malformations (LMs)). Furthermore, we analyzed the expression of ß1-, ß2-, and ß3-adrenoceptor mRNA in these specimens as well as in hemangioma-derived stem cells by quantitative real-time PCR (qPCR). RESULTS: We show that the expression of ß1-adrenoceptor mRNA is 10.7-fold higher in IH independent of the proliferative or regressive phase as well as 2.5-fold higher in hemangioma-derived stem cells as compared with ß2-adrenoceptor mRNA. In LM, the expression of ß2-adrenoceptor mRNA was ninefold higher than that of ß1-adrenoceptor mRNA. VM showed low expression levels of all ß-adrenoceptor mRNAs, and ß3-adrenoceptor mRNA was hardly detectable in any specimens examined. CONCLUSION: These results provide the first evidence of distinctions between IH and vascular malformations with regard to ß-adrenoceptor subtype mRNA levels.

Differential Diagnosis in Hypophysitis: First Report on a Spindle Cell Rhabdomyosarcoma of the Pituitary Gland.

Pituitary gland metastases are very rare. Most patients with pituitary gland metastases are asymptomatic; therefore, most cases of this disease are diagnosed during autopsies. Moreover, the four most common primary tumors that metastasize to the pituitary gland are breast, lung, thyroid, and renal carcinomas. We present a very rare case of pituitary metastasis of spindle cell rhabdomyosarcoma (RMS). Our patient presented with headache, visual disorder, panhypopituitarism, and diabetes insipidus. Due to tumor expansion, resection was not possible, so diagnosis was confirmed by biopsy, and chemotherapy and irradiation were administered. Our patient showed widespread spindle cell RMS, which harbors a mutation of myogenic differentiation 1 (MYOD1) and is associated with a poor prognosis. Even high-risk patients can show a remission after chemotherapy and irradiation. In the cases with indistinct lesions in the sella region, pituitary metastasis should always be considered.

UV-radiation and MC1R germline mutations are risk factors for the development of conventional and spitzoid melanomas in children and adolescents.

BACKGROUND: Genomic characterisation has led to an improved understanding of adult melanoma. However, the aetiology of melanoma in children is still unclear and identifying the correct diagnosis and therapeutic strategies remains challenging. METHODS: Exome sequencing of matched tumour-normal pairs from 26 paediatric patients was performed to study the mutational spectrum of melanomas. The cohort was grouped into different categories: spitzoid melanoma (SM), conventional melanoma (CM), and other melanomas (OT). FINDINGS: In all patients with CM (n = 10) germline variants associated with melanoma were found in low to moderate melanoma risk genes: in 8 patients MC1R variants, in 2 patients variants in MITF, PTEN and BRCA2. Somatic BRAF mutations were detected in 60% of CMs, homozygous deletions of CDKN2A in 20%, TERTp mutations in 30%. In the SM group (n = 12), 5 patients carried at least one MC1R variant; somatic BRAF mutations were detected in 8.3%, fusions in 25% of the cases. No SM showed a homozygous CDKN2A deletion nor a TERTp mutation. In 81.8% of the CM/SM cases the UV damage signatures SBS7 and/or DBS1 were detected. The patient with melanoma arising in giant congenital nevus (CNM) demonstrated the characteristic NRAS Q61K mutation. INTERPRETATION: UV-radiation and MC1R germline variants are risk factors in the development of conventional and spitzoid paediatric melanomas. Paediatric CMs share genomic similarities with adult CMs while the SMs differ genetically from the CM group. Consistent genetic characterization of all paediatric melanomas will potentially lead to better subtype differentiation, treatment, and prevention in the future. FUNDING: Found in Acknowledgement.

Impact of mammalian target of rapamycin inhibition on lymphoid homing and tolerogenic function of nanoparticle-labeled dendritic cells following allogeneic hematopoietic cell transplantation.

Dendritic cells (DC) play a major role in the pathogenesis of graft-vs-host disease (GvHD). Directed modification of surface molecules on DC that provide instructive signals for T cells may create a tolerogenic DC phenotype that affects GvHD severity. To investigate the impact of the mammalian target of rapamycin (mTOR) inhibitor rapamycin (RAPA) on in vivo migratory capacities, tolerogenic function, and B7 superfamily surface expression on DC following allogeneic hematopoietic cell transplantation (aHCT), we generated a platform for magnetic resonance imaging and bioluminescence imaging based cell trafficking studies. Luciferase transgenic DC were labeled with superparamagnetic iron oxide nanoparticles bound to a murine IgG Ab that allowed for Fc-gammaR-mediated endocytosis. Locally injected luc(+) DC could be tracked within their anatomical context by bioluminescence imaging and magnetic resonance imaging after aHCT, based on stable intracellular localization of superparamagnetic iron oxide-IgG complexes. RAPA preconditioned DC (DC-R) displayed reduced expression of MHC class II, B7-1 (CD80), and B7-2 (CD86) but not B7-H4 whose ligation of T cells has a profound inhibitory effect on their proliferation and cytokine secretion. DC-R of recipient genotype reduced GvHD severity that is compatible with their tolerogenic phenotype. CCR5, CCR7, and CD62L expression was not affected by mTOR inhibition, which allowed for DC-R in vivo trafficking to secondary lymphoid compartments where immunregulation is required. This study is the first to delineate the impact of RAPA on DC migration and tolerogenic function after aHCT. Modification of the DC phenotype by mTOR inhibition may have therapeutic potential in an attempt to reduce GvHD following aHCT.

Ischemic preconditioning improves stability of intestinal anastomoses in rats.

BACKGROUND: The aim of our study was to establish whether ischemic preconditioning (IPC) directly before performing a small bowel anastomosis has an effect on anastomotic stability and healing. MATERIAL AND METHODS: Forty male Wistar rats were randomized to five groups: control (CO, n = 8) with preparation of the superior mesenteric artery (SMA) but without IPC. IPC groups had different intervals of ischemia (occlusion of the SMA) and reperfusion: 10 min ischemia and 20 min reperfusion (IPC10/20, n = 7), 10 min ischemia and 30 min reperfusion (IPC10/30, n = 8), 15 min ischemia and 20 min reperfusion (IPC15/20, n = 8), and 15 min ischemia and 30 min reperfusion (IPC15/30, n = 9). On the fourth postoperative day, the animals were relaparotomized: bursting pressure, hydroxyproline concentration, and histological ischemia mucosal injury scale of the anastomosis were assessed. RESULTS: Four days after operation, the mean bursting pressure was 73 +/- 6 mmHg in the control group, whereas it was significantly higher in IPC10/20 (113 +/- 11 mmHg; p = 0.018), IPC10/30 (110 +/- 13 mmHg; p = 0.001), and IPC15/30 (124 +/- 9 mmHg; p = 0.003). IPC15/20 did not show a significant difference (63 +/- 2 mmHg; p = 0.4). We did not find a significant effect regarding hydroxyproline concentration, but IPC diminished mucosal injury. CONCLUSIONS: IPC directly before performing a small bowel anastomosis has a time-dependent beneficial effect on anastomotic stability, thus indicating a new clinical approach to improve the healing process of intestinal anastomosis.

Small intestinal submucosa for reinforcement of colonic anastomosis.

BACKGROUND: Different materials have been evaluated for anastomotic reinforcement to prevent gastrointestinal anastomotic leakage. In this experimental study, small intestinal submucosa (SIS) was tested as a sealing for stapled colonic anastomosis in a porcine model. The aims of this study were to determine the macroscopic and microscopic outcomes and to evaluate the safety and feasibility of applying SIS for anastomotic sealing. MATERIALS AND METHODS: Circular stapled anastomoses were performed in 18 pigs. Standard anastomosis in the control group (n = 8) was compared to an SIS-sealed anastomosis in the study group (n = 10). After 30 days, anastomotic segments were examined for macroscopic and microscopic regeneration and their resistance to mechanical stress. Furthermore, animal survival and clinical course were evaluated. RESULTS: None of the animals developed anastomotic leakage, intraabdominal abscess, or peritonitis. Shrinkage of SIS was evident in nine of ten animals. Encapsulation and displacement of the SIS patches were seen in two animals. Quantity of anastomotic granulation tissue and rate of complete mucosal coverage of anastomotic line were increased in SIS-sealed anastomoses without reaching significance. Moreover, no significant differences were found in the rate of survival of the animals, anastomotic stricture formation, intraabdominal adhesions, anastomotic bursting pressure, and microscopic healing parameters of the anastomosis between stapled colonic standard anastomosis and anastomosis protected by SIS. CONCLUSION: The results of this study indicate a safe use of SIS for anastomotic reinforcement in a porcine model. Adverse effects like strictures, increased adhesions, and anastomotic abscesses were absent. Promoting effects on colonic wound healing by SIS were microscopically evident. The results argue for a careful clinical evaluation in humans.

Differentiation of EBV-induced post-transplant Hodgkin lymphoma from Hodgkin-like post-transplant lymphoproliferative disease.

The development of lymphomas after SOT is a well-known complication of the immunosuppressive therapy necessary to prevent graft rejection. Epstein-Barr virus plays a central role in the pathogenesis of lymphomas because of its ability to transform infected cells. Differentiating PTLD from malignant lymphomas, especially HL can be challenging. We report on two patients, who developed EBV-associated lymphomas several years after SOT. A histological examination of lymph nodes led to a diagnosis of HL in both patients, who were started on chemotherapy according to current treatment protocols. A rapid and complete remission in one patient prompted us to analyze the expression pattern of EBV-latency genes. In this patient, the EBV expression profile revealed a latency type III suggesting the diagnosis of Hodgkin-like PTLD. The other patient required six courses of chemotherapy plus radiotherapy to reach a complete remission. In his tumor cells, a restricted EBV-latency type II pattern was found, suggesting a diagnosis of classical HL. These two cases demonstrate that in post-transplant lymphomas with histological features of HL, an analysis of the expression pattern of EBV proteins might aid in the differentiation between PTLD and HL.

Case report: value of gene expression profiling in the diagnosis of atypical neuroblastoma.

BACKGROUND: Nephroblastoma and neuroblastoma belong to the most common abdominal malignancies in childhood. Similarities in the initial presentation may provide difficulties in distinguishing between these two entities, especially if unusual variations to prevalent patterns of disease manifestation occur. Because of the risk of tumor rupture, European protocols do not require biopsy for diagnosis, which leads to misdiagnosis in some cases. CASE PRESENTATION: We report on a 4½-year-old girl with a renal tumor displaying radiological and laboratory characteristics supporting the diagnosis of nephroblastoma. Imaging studies showed tumor extension into the inferior vena cava and bilateral lung metastases while urine catecholamines and MIBG-scintigraphy were negative. Preoperative chemotherapy with vincristine, actinomycine D and adriamycin according to the SIOP2001/GPOH protocol for the treatment of nephroblastoma was initiated and followed by surgical tumor resection. Histopathology revealed an undifferentiated tumor with expression of neuronal markers, suggestive of neuroblastoma. MYCN amplification could not be detected. DNA-microarray analysis was performed using Affymetrix genechip human genome U133 plus 2.0 and artificial neural network analysis. Results were confirmed by multiplex RT-PCR. RESULTS: Principal component analysis using 84 genes showed that the patient sample was clearly clustering with neuroblastoma tumors. This was confirmed by hierarchical clustering of the multiplex RT-PCR data. The patient underwent treatment for high-risk neuroblastoma comprising chemotherapy including cisplatin, etoposide, vindesine, dacarbacine, ifosfamide, vincristine, adriamycine and autologous stem cell transplantation followed by maintenance therapy with 13-cis retinoic acid (GPOH NB2004 High Risk Trial Protocol) and is in complete long-term remission. CONCLUSION: The use of gene expression profiling in an individual patient strongly contributed to clarification in a diagnostic dilemma which finally led to a change of diagnosis from nephroblastoma to neuroblastoma. This case underlines the importance of gene-expression profiling in the correct diagnosis of childhood neoplasms with atypical presentation to ensure that adequate treatment regimens can be applied.

Reproducibility of Her2/neu scoring in gastric cancer and assessment of the 10% cut-off rule.

The application of Trastuzumab on gastric cancer patients is based on Her2/neu immunostaining. The testing method relies on visual estimation of both membranous staining intensity, and positive tumor ratio with respect to a 10% cutoff. We evaluated the effect of inter- and intraobserver variations of both factors on therapeutic decision, especially if the positive tumor ratio hovers around the 10% cutoff. Ten pathologists scored 12 Her2/neu immunohistologically stained whole sections of gastric cancer. Applying the common rules for Her2/neu testing for gastric cancer, they separately noted the strongest identifiable staining intensity and the corresponding positive tumor ratio. Scoring was done repeatedly using the microscope, plain virtual microscopy, and virtual microscopy with a manual outline drawing function. Agreements on the strongest identified staining intensities were moderate. Overall concordance correlation coefficients of positive tumor ratios ranged from 0.55 to 0.81. Reproducibility was not improved by virtual microscopy. Pathologists have a good ability to estimate ratios of clearly demarcated areas, but gradients in staining intensities hinder reproducible visual demarcation of positive tumor areas. When hovering around the 10% positive tumor ratio cutoff there is a risk of misinterpretation of the staining results. This could lead to a denial of Trastuzumab therapy. Assessment of Her2/neu expression should be carried out by experienced pathologists because they can more reproducibly rate membranous staining intensities. The low reproducibility of positive tumor ratio is inherent in the testing method and cannot be improved by virtual microscopy. Therefore, we propose to reconsider the 10% cut-off limit.

Graft-versus-host disease is enhanced by extracellular ATP activating P2X7R.

Danger signals released upon cell damage can cause excessive immune-mediated tissue destruction such as that found in acute graft-versus-host disease (GVHD), allograft rejection and systemic inflammatory response syndrome. Given that ATP is found in small concentrations in the extracellular space under physiological conditions, and its receptor P2X(7)R is expressed on several immune cell types, ATP could function as a danger signal when released from dying cells. We observed increased ATP concentrations in the peritoneal fluid after total body irradiation, and during the development of GVHD in mice and in humans. Stimulation of antigen-presenting cells (APCs) with ATP led to increased expression of CD80 and CD86 in vitro and in vivo and actuated a cascade of proinflammatory events, including signal transducer and activator of transcription-1 (STAT1) phosphorylation, interferon-γ (IFN-γ) production and donor T cell expansion, whereas regulatory T cell numbers were reduced. P2X(7)R expression increased when GVHD evolved, rendering APCs more responsive to the detrimental effects of ATP, thereby providing positive feedback signals. ATP neutralization, early P2X(7)R blockade or genetic deficiency of P2X(7)R during GVHD development improved survival without immune paralysis. These data have major implications for transplantation medicine, as pharmacological interference with danger signals that act via P2X(7)R could lead to the development of tolerance without the need for intensive immunosuppression.

Small intestinal submucosa as a bioscaffold for tissue regeneration in defects of the colonic wall.

BACKGROUND: Small intestinal submucosa (SIS) has proved considerable regenerative capacity for repair of bowel wall defects at different locations. This study assesses the effectiveness of SIS in the repair of defects at a gastrointestinal location with strong bacterial contamination. METHODS: Fourteen domestic pigs had a 4.5 x 1.5 cm full-thickness defect created on the wall of the descending colon. Repair was done by suturing an SIS patch to the defect. Grafts were harvested after 30, 60, and 90 days. Outcomes were evaluated on the basis of animal survival, clinical course, and macroscopic, histological, and immunohistochemical assessment. RESULTS: All animals survived the scheduled observation period. No patch failure and no postoperative leakage occurred. No luminal narrowing occurred at SIS-patched colon. Morphometric examination revealed contraction of the patched area of 77% after 30 days and more than 90% after 60 and 90 days. By 60 and 90 days, all animals showed mucosal regeneration at the margins of the graft. By 90 days, regeneration of smooth muscle cells was present at the original site of the muscularis mucosae. None of the reconstructed areas showed complete mucosal coverage or regeneration of a structured muscular layer. CONCLUSION: SIS can be used effectively for patch repair of colonic defects in a porcine model. Distinctive contraction of the reconstructed area and limited architectural regeneration of the bowel wall suggest limitation of morphologic regenerative capacities in large-bowel regeneration.

Lymphangiogenesis and its regulation in human neuroblastoma.

For the first time, we could detect lymph vessels in neuroblastoma (NB) by immunohistochemistry with the antibody D2_40. Furthermore, we demonstrate expression of the lymphangiogenic factors VEGF-C and VEGF-D and their receptors VEGFR-2 and VEGFR-3 in NB in vitro and in vivo by RT-PCR. However, addition of recombinant human VEGF-C or -D results in the absence of autocrine growth stimulus in NB cells. Treatment of NB cells with retinoic acid did not lead to a change in VEGF-C or VEGF-D mRNA expression. Incubation of the NB cells Lan-5 with 5-Aza-2'-deoxycytidine led to the up-regulation of VEGF-C mRNA expression, suggesting that the promotor of VEGF-C is methylated. Finally, VEGF-C mRNA expression could be effectively down-regulated by transfection with a specific siRNA in the NB cells Kelly. We conclude that lymphangiogenesis is involved in NB biology and that siRNA directed against VEGF-C may have a future role in anti-lymphangiogenic strategies in NB.