Diagnostic classification of childhood cancer using multiscale transcriptomics.

The causes of pediatric cancers' distinctiveness compared to adult-onset tumors of the same type are not completely clear and not fully explained by their genomes. In this study, we used an optimized multilevel RNA clustering approach to derive molecular definitions for most childhood cancers. Applying this method to 13,313 transcriptomes, we constructed a pediatric cancer atlas to explore age-associated changes. Tumor entities were sometimes unexpectedly grouped due to common lineages, drivers or stemness profiles. Some established entities were divided into subgroups that predicted outcome better than current diagnostic approaches. These definitions account for inter-tumoral and intra-tumoral heterogeneity and have the potential of enabling reproducible, quantifiable diagnostics. As a whole, childhood tumors had more transcriptional diversity than adult tumors, maintaining greater expression flexibility. To apply these insights, we designed an ensemble convolutional neural network classifier. We show that this tool was able to match or clarify the diagnosis for 85% of childhood tumors in a prospective cohort. If further validated, this framework could be extended to derive molecular definitions for all cancer types.

Ensuring no patient is lost along the way - a single-centre experience of the clinical genetics referral pathways for Lynch syndrome.

Lynch syndrome (LS), caused by heterozygous germline mutation in one of the key mismatch repair (MMR) genes, is the primary cause of inherited colorectal cancer (CRC). LS also increases susceptibility to several other cancers. It is estimated that just 5% of patients with LS are aware of their diagnosis. Therefore, in an attempt to increase the identification of cases within the UK population, the 2017 NICE guidelines recommend offering immunohistochemistry for MMR proteins or microsatellite instability (MSI) testing to all people with CRC when first diagnosed. Following identification of MMR deficiency, eligible patients should be assessed for underlying causes, including potential referral to the genetics service and/or germline LS testing (if appropriate). In our regional centre for CRC, we audited local pathways to identify what proportion of patients are being correctly referred, in line with national guidelines. Reflecting on these results, we highlight our practical concerns by identifying the pitfalls and issues faced with the recommended referral pathway. We also propose possible solutions to improve the efficacy of the system for both referrers and patients. Finally, we discuss the ongoing interventions that national bodies and regional centres are implementing to improve and further streamline this process.

Cutaneous Myoepithelial Neoplasms on Acral Sites Show Distinctive and Reproducible Histopathologic and Immunohistochemical Features.

Cutaneous myoepithelial neoplasms are a heterogenous group of neoplasms with mixed tumors typically affecting the head and myoepitheliomas showing a predilection for the extremities. Their malignant counterparts, myoepithelial carcinoma, and malignant mixed tumor are exceptionally rare in the skin, and the morphologic criteria for malignancy are only poorly defined. The aim of the present study was to characterize the clinicopathologic features of myoepithelial neoplasms presenting on acral skin. The clinical and histopathologic features of 11 tumors were recorded, and follow-up was obtained. Immunohistochemistry was performed for S100, SOX10, glial fibrillary acidic protein, keratins, epithelial membrane antigen, p63, p40, smooth muscle actin, desmin, and PLAG1. The tumors mainly affected the feet of adults (range: 26 to 78 y; median: 47 y) with a predilection for the great toe and a male predominance of 1.8:1. Most tumors (91%) displayed a lobular architecture composed of solid and nested growth of epithelioid cells with plasmacytoid features in a myxoid or angiomatous stroma. Scattered cytologic atypia and rare duct differentiation were frequently noted. Three tumors with confluent cytologic atypia, infiltrative growth, and lymphovascular invasion were classified as malignant. By immunohistochemistry, the tumors were positive for S100, SOX10, keratins AE1/AE3, CK5/6 and CK7, and PLAG1. Local recurrence and bilateral pulmonary metastasis were observed in a patient presenting with a histopathologically benign-appearing tumor. Two patients with malignant tumors experienced local recurrences, and 1 developed metastasis to soft tissue, lung, and mediastinal lymph nodes. All patients are currently alive, all but 1 with no evidence of disease after a median follow-up interval of 96 months (range: 2 to 360 mo). In conclusion, acral myoepithelial neoplasms show distinctive and reproducible histopathologic and immunohistochemical features. They are best regarded as a distinctive subset of mixed tumors with features reminiscent of their salivary gland counterparts. While most tumors pursue a benign disease course, histopathologic features appear to be a poor indicator of prognosis.

Blood and immune development in human fetal bone marrow and Down syndrome.

Haematopoiesis in the bone marrow (BM) maintains blood and immune cell production throughout postnatal life. Haematopoiesis first emerges in human BM at 11-12 weeks after conception1,2, yet almost nothing is known about how fetal BM (FBM) evolves to meet the highly specialized needs of the fetus and newborn. Here we detail the development of FBM, including stroma, using multi-omic assessment of mRNA and multiplexed protein epitope expression. We find that the full blood and immune cell repertoire is established in FBM in a short time window of 6-7 weeks early in the second trimester. FBM promotes rapid and extensive diversification of myeloid cells, with granulocytes, eosinophils and dendritic cell subsets emerging for the first time. The substantial expansion of B lymphocytes in FBM contrasts with fetal liver at the same gestational age. Haematopoietic progenitors from fetal liver, FBM and cord blood exhibit transcriptional and functional differences that contribute to tissue-specific identity and cellular diversification. Endothelial cell types form distinct vascular structures that we show are regionally compartmentalized within FBM. Finally, we reveal selective disruption of B lymphocyte, erythroid and myeloid development owing to a cell-intrinsic differentiation bias as well as extrinsic regulation through an altered microenvironment in Down syndrome (trisomy 21).

Assessment of Apparent Diffusion Coefficients and SUVs as Predicators of Histological Differentiation in Anal Squamous Cell Carcinoma.

AIM: The study aims to assess minimal apparent diffusion coefficient (ADCmin) and SUVmax as predictors of histological differentiation in patients with anal squamous cell carcinoma (ASCC) and to determine cutoff values for each histopathological tumor grade. PATIENTS AND METHODS: A retrospective study of 41 ASCC patients (14 males, 27 females; mean age, 65 ± 13 years) staged with FDG PET/CT and MRI (mean scan time interval, 21 ± 11 days). SUVmax and ADCmin values were measured and compared with histopathological tumor grading obtained from biopsy. RESULTS: The mean size and tumor volume were 3 ± 2 cm and 16.5 ± 27.3 cm3, respectively. The mean ADCmin values for well-, moderately, and poorly differentiated ASCC were 935 ± 179, 896 ± 123, and 637 ± 114, respectively. The mean SUVmax for well-, moderately, and poorly differentiated ASCC were 6.9 ± 1.8, 11.5 ± 4.1, and 13.4 ± 2.6, respectively. The difference in mean ADCmin values between poorly and moderately/well-differentiated tumors was statistically significant, whereas this was not significant between moderately and well-differentiated tumors. Differences in SUVmax values were statistically significant between poorly/moderately and well-differentiated tumors, whereas there was no statistical significance between poorly and moderately differentiated tumors. By combining the 2 modalities using cutoff values of 675 × 10-6 mm2·s-1 for ADCmin and 8.5 for SUVmax, it was possible to differentiate the tumor categories with a sensitivity, specificity, positive predictive value, and negative predictive value, respectively, of 84.6%, 96.4%, 91.7%, and 93.1% for well-differentiated ASCC, 76.5%, 87.5%, 81.3%, and 84% for moderately, and 90.9%, 89.3%, 76.9%, and 96.2% for poorly differentiated ASCC, respectively. CONCLUSIONS: ADCmin and SUVmax values correlated with the degree of differentiation in ASCC and can be used as predictors of tumor grading and aggressiveness. Combined ADCmin and SUVmax cutoff values can therefore be used for early patient risk stratification and treatment decision making.

Membrane-bound estrogen receptor alpha initiated signaling is dynamin dependent in breast cancer cells.

BACKGROUND: Although membrane-associated estrogen receptors (mERs) have been known to play important role in steroid-induced signal transmission, we still know little about their function in the estrogen-induced proliferation of breast cancer cells. METHODS: In our current work we tried to separate membrane-initiated estrogen receptor signaling from the overall estrogenic effect in MCF-7 breast carcinoma cells. Re-analyzing expression data from multiple microarray experiments, we selected a set of key regulatory genes involved in proliferation regulation and estrogen signaling to monitor estrogen-induced transcription changes. We then compared these expression changes after 17ß-estradiol and a membrane receptor selective estrogen-BSA treatment using quantitative real-time PCR. In order to follow receptor trafficking we used light and electron microscopy. RESULTS: Our quantitative real-time PCR results confirmed that the selective membrane receptor agonist, estrogen-BSA induces similarly pronounced expression changes regarding these genes as 17ß-estradiol. Morphological study revealed that the membrane-bound form of classical estrogen receptor alpha is internalized after ligand binding via dynamin-dependent, caveola-mediated endocytosis. Inhibition of this internalization with dynamin inhibitor, dynasore practically abolished the regulatory effect of E2-BSA, suggesting that interaction and internalization with the scaffold protein is necessary for effective signaling. CONCLUSIONS: The physiological role of plasma membrane estrogen receptor alpha is intensively studied, yet there are still several aspects of it to be resolved. The dynamin-dependent, ligand-mediated internalization of mERs seems to play an important role in estrogen signaling. Our results may serve as another example of how membrane initiated estrogen signaling and nuclear receptor initiated signaling overlap and form an intertwined system.

Primary alveolar rhabdomyosarcoma of the bone: two cases and review of the literature.

BACKGROUND: Rhabdomyosarcoma (RMS) is a malignant tumor of mesenchymal origin and comprises the largest category of soft-tissue sarcomas both in children and adolescents. From a pediatric oncology point of view, RMS has traditionally been classified into alveolar (ARMS) and embryonal (ERMS) subtypes. The anatomical localization of the tumor may vary, but commonly involve the head/neck regions, male and female urogenital tract or the trunk and extremities. CASE PRESENTATION: Here, we report two challenging cases involving 17- and 9-years-olds males where diffuse and multiplex bone lesions suggested either a hematological disease or a primary bone tumor (mesenchymal chondrosarcoma). Biopsies, proved a massive infiltration of the bone marrow cavity with rhabdomyosarcoma. In both cases, the ARMS subtype was confirmed using FOXO1 break-apart probes (FISH). Radiological examination could not identify primary soft tissue component in any localization at the time of diagnosis in either cases. CONCLUSIONS: Primary alveolar rhabdomyosarcoma of the bone as a subtype of ARMS, seems to be a distinct clinico-pathological entity with challenging diagnostic difficulties and different, yet better, biological behavior in comparison to soft tissue ARMS. However, it is difficult to be characterized or predict its prognosis and long-term survival as only sporadic cases (four) were reported so far.

Autophagy may contribute to the recovery of rat mesothelium following acute inflammation in vivo.

Following Freund's adjuvant-induced acute inflammation, the regeneration of rat mesothelium is accompanied by the reduction of cell organelles. The aim of the present study is to test whether autophagy may play a role in the recovery process of mesothelial cells by eliminating accumulated cell organelles and also to investigate the presence of potential inducers and molecular transmitters of the process. Control and treated (from day 2 to day 11; D2-D11) mesothelial cells (n = 16 samples/group) obtained from male rats were isolated and phenotypically characterized. Morphological studies included light and electron microscopy. Biochemical studies performed on tissue samples as well as isolated cells were used to evaluate the dynamics of autophagy and also to detect the expression levels of TNF-α, LC3B, estrogen receptors (ER-α and GPR30) and Erk1/2. Gene expression was measured by individual Taqman assays on quantitative RT-PCR. Protein expression study was performed by Western blotting and immunolabeling. Estradiol concentration was measured both in peritoneal fluid and plasma samples in control and treated animals (n = 3-10 animals per group). Our conventional electron microscopic and morphometric results showed a progressive autophagosome formation with a peak by the termination of inflammation (D5). Subsequently, autophagolysosome formation dominated between D6 and D8 with a concomitant expression of LC3B proved by immunoblotting. We further observed the reduction of cell compartments by D11 parallel with the morphological restitution of mesothelium. Estradiol showed a sustained level in the peritoneal fluid but not in plasma samples between D3 and D11 compared to levels obtained from untreated animals. The mRNA expression of TNF-α was increased between D2 and D11 compared to control. Western blot analysis showed a constitutive expression of GPR30, while ER-α could not be detected between D6 and D11. Erk1/2 was activated by phosphorylation with a peak at D6. Considering our present in vivo results, we hypothesize that the facilitated autophagy might play an important role in the removal of cytoplasmic organelles during the recovery of mesothelium, while our results also suggest that the detected peritoneal estradiol as well as TNF-α may contribute to this process.

The subcellular compartmentalization of TGFß-RII and the dynamics of endosomal formation during the signaling events: An in vivo study on rat mesothelial cells.

We previously showed that intraperitoneal administration of Freund's adjuvant treatment resulted in acute peritonitis and TGF-ß was found to be one of the main organizers of the subsequent EMT in mesothelial cells. In the present study, we investigated whether TGF-ß signaling molecules are present in mesothelial cells and how their compartmentalization pattern changes with the dynamics of inflammatory events in vivo. In addition, we tried to evaluate the turnover of endosomal compartments concomitant with the internalization of signaling molecules and examine whether caveola-mediated internalization might play a role in the termination of TGF-ß signaling. Using immunocytochemical approach, we could detect TßRII in EEA1 positive compartments and as the inflammation progressed, at D3, the receptor appeared in caveolin-1 positive intracellular structures as well. The latter event was accompanied by the appearance of negative regulatory protein, Smad7 in caveolae. We also found EEA1 and caveolin-1 double positive vesicular structures that were corresponded to forming MVBs affirmed by our immuno-electron microscopical results. Fine structural, morphometric and immunoblot analysis proved that Cd63 positive multivesicular body (MVB) formation was significantly increased by D3 and the IP results confirmed that TßRII as well as caveolin-1 were strongly associated with these endosomal compartments at this time. In contrast, by the termination of inflammation, by D5, caveolin-1 was found to be associated with late endosomal marker, Rab7 and entirely degraded from the system. Despite the limitations of an in vivo system, our results provide both morphological and biochemical data about the endosomal compartments involved in the internalization of TßRII upon inflammatory stimuli. Furthermore, our study implies the possible role of caveola-mediated endocytosis in the attenuation of TGF-ß signaling and highlight the significance of endosomal compartments via which caveolae might meet the classical endocytic pathway under in vivo inflammatory conditions.

Estrogen receptor alpha is expressed in mesenteric mesothelial cells and is internalized in caveolae upon Freund's adjuvant treatment.

Transformation of epithelial cells into connective tissue cells (epithelial-mesenchymal transition, EMT) is a complex mechanism involved in tumor metastasis, and in normal embryogenesis, while type II EMT is mainly associated with inflammatory events and tissue regenaration. In this study we examined type II EMT at the ultrastructural and molecular level during the inflammatory process induced by Freund's adjuvant treatment in rat mesenteric mesothelial cells. We found that upon the inflammatory stimulus mesothelial cells lost contact with the basal lamina and with each other, and were transformed into spindle-shaped cells. These morphological changes were accompanied by release of interleukins IL-1alpha, -1beta and IL-6 and by secretion of transforming growth factor beta (TGF-ß) into the peritoneal cavity. Mesothelial cells also expressed estrogen receptor alpha (ER-α) as shown by immunolabeling at the light and electron microscopical levels, as well as by quantitative RT-PCR. The mRNA level of ER-α showed an inverse correlation with the secretion of TGF-ß. At the cellular and subcellular levels ER-α was colocalized with the coat protein caveolin-1 and was found in the plasma membrane of mesothelial cells, in caveolae close to multivesicular bodies (MVBs) or in the membrane of these organelles, suggesting that ER-α is internalized via caveola-mediated endocytosis during inflammation. We found asymmetric, thickened, electron dense areas on the limiting membrane of MVBs (MVB plaques) indicating that these sites may serve as platforms for collecting and organizing regulatory proteins. Our morphological observations and biochemical data can contribute to form a potential model whereby ER-α and its caveola-mediated endocytosis might play role in TGF-ß induced type II EMT in vivo.

Epithelial-to-mesenchymal transition induced by Freund's adjuvant treatment in rat mesothelial cells: a morphological and immunocytochemical study.

Intraperitoneal injection of Freund's adjuvant induces acute peritonitis. By the time of the Freund's adjuvant treatment the flat, simple squamous epithelial cells became rounded, cuboidal shaped, many of them have lost their connection with the neighbouring cells and detached from the basement membrane. The macrophage markers' (ED1, OX43 and CD68) expression also increased in the mesothelial cells and more mesothelin and anti-ED1 double-labelled cells were found freely present close to the surface. The cytokeratin expression of the mesothelial cells has gradually decreased. At the 5th day of the inflammation practically there was no cytokeratin labelling present in the mesothelial cells and the mesothelin expression has significantly decreased. Parallel to this mesothelial cells started to express vimentin, a characteristic mesenchymal intermediate filament protein indicating that they gradually lost their epithelial character and gained mesenchymal phenotype. These results strongly suggest that under the effect of Freund's adjuvant treatment (inflammation) mesothelial cells can undergo epithelial-to-mesenchymal transition and differentiate into phagocytotic (macrophage-like) cells. Studying the caveolae/caveolin-1 on the plasma membrane of mesothelial cells we found that the Freund's adjuvant treatment has changed the cellular distribution of caveolin-1: as the inflammation progressed strong caveolin-1 labelling was found inside of the cytoplasm (in perinuclear localization) indicating that inflammation induced the caveolae internalization. These results indicate that caveolae/caveolin-1 might play important regulatory role in signal transduction leading to trasdifferentiation.

Mesothelial cells can detach from the mesentery and differentiate into macrophage-like cells.

Peritoneal cell suspension is composed of heterogeneous cell population. Macrophages are the most numerous cells among them. They can originate from different sources and can be resident, exudate and elicited. When we used Freund's adjuvant to elicit peritoneal macrophages, cells having large amount of caveolae on their plasma membrane appeared in the peritoneal wash. The number of these caveolae-rich cells increased by the time of the Freund's adjuvant treatment. Although their morphology was different form from the common macrophages, they were labelled with pan-macrophage antibodies. As the origin of these cells is unknown in this work, we tried to find out where they can originate from. Our interest turned towards the mesothelial cells. We found that the adjuvant treatment resulted in significant morphological changes in these cells and stimulate them to leave the surface of the mesentery. By the time of the adjuvant treatment, the macrophage markers expression increased in the mesothelial cells and more cells were found to detach from the mesentery. These results strongly suggest that under special stimuli mesothelial cells can leave the mesentery and differentiate into phagocytotic (macrophage-like) cells. These data raises the idea that mesothelial cells might not entirely differentiated and represent a multipotential cell lineage. To study whether this is the case we used anti-nestin antibody, which is a specific marker for multifunctional, multi-lineage progenitor cells. Mesothelial cells showed strong labelling with this antibody indicating that these cells really represent a 'young', not entirely differentiated cell population.