A Gradual Transition Toward Anaplasia in Wilms Tumor Through Tolerance to Genetic Damage.

Patients with Wilms tumor (WT) in general have excellent survival, but the prognosis of patients belonging to the subgroup of WT with diffuse anaplasia (DA) is poor due to frequent resistance to chemotherapy. We hypothesized that DA WT cells might undergo changes, such as acquiring a persistent tolerance to DNA damage and copy number aberrations (CNAs), which could eventually lead to their resistance to chemotherapy treatment. Tissue sections from chemotherapy-treated DA WTs (n = 12) were compared with chemotherapy-treated nonanaplastic WTs (n = 15) in a tissue microarray system, enabling analysis of 769 tumor regions. All regions were scored for anaplastic features and immunohistochemistry was used to quantify p53 expression, proliferation index (Ki67), and DNA double-strand breaks (γH2AX). CNAs were assessed by array-based genotyping and TP53 mutations using targeted sequencing. Proliferation index and the frequency of DNA double-strand breaks (γH2AX dot expression) increased with higher anaplasia scores. Almost all (95.6%) areas with full-scale anaplasia had TP53 mutations or loss of heterozygosity, along with an increased amount of CNAs. Interestingly, areas with wild-type TP53 with loss of heterozygosity and only one feature of anaplasia (anaplasia score 1) also had significantly higher proliferation indices, more DNA double-strand breaks, and more CNAs than regions without any anaplastic features (score 0); such areas may be preanaplastic cell populations under selective pressure for TP53 mutations. In conclusion, we suggest that chemoresistance of DA WTs may be partly explained by a high proliferative capability of anaplastic cells, which also have a high burden of double-stranded DNA breaks and CNAs, and that there is a gradual emergence of anaplasia in WT.

Inactivation of RB1, CDKN2A, and TP53 have distinct effects on genomic stability at side-by-side comparison in karyotypically normal cells.

Chromosomal instability is a common feature in malignant tumors. Previous studies have indicated that inactivation of the classical tumor suppressor genes RB1, CDKN2A, and TP53 may contribute to chromosomal aberrations in cancer by disrupting different aspects of the cell cycle and DNA damage checkpoint machinery. We performed a side-by-side comparison of how inactivation of each of these genes affected chromosomal stability in vitro. Using CRISPR-Cas9 technology, RB1, CDKN2A, and TP53 were independently knocked out in karyotypically normal immortalized cells, after which these cells were followed over time. Bulk RNA sequencing revealed a distinct phenotype with upregulation of pathways related to cell cycle control and proliferation in all three knockouts. Surprisingly, the RB1 and CDKN2A knocked out cell lines did not harbor more copy number aberrations than wild-type cells, despite culturing for months. The TP53-knocked out cells, in contrast, showed a massive amount of copy number alterations and saltatory evolution through whole genome duplication. This side-by-side comparison indicated that the effects on chromosomal stability from inactivation of RB1 and CDKN2A are negligible compared to inactivation of TP53, under the same conditions in a nonstressful environment, even though partly overlapping regulatory pathways are affected. Our data suggest that loss of RB1 and CDKN2A alone is not enough to trigger surviving detectable aneuploid clones while inactivation of TP53 on its own caused massive CIN leading to saltatory clonal evolution in vitro and clonal selection.

Tracing the evolution of aneuploid cancers by multiregional sequencing with CRUST.

Clonal deconvolution of mutational landscapes is crucial to understand the evolutionary dynamics of cancer. Two limiting factors for clonal deconvolution that have remained unresolved are variation in purity and chromosomal copy number across different samples of the same tumor. We developed a semi-supervised algorithm that tracks variant calls through multi-sample spatiotemporal tumor data. While normalizing allele frequencies based on purity, it also adjusts for copy number changes at clonal deconvolution. Absent à priori copy number data, it renders in silico copy number estimations from bulk sequences. Using published and simulated tumor sequences, we reliably segregated clonal/subclonal variants even at a low sequencing depth (~50×). Given at least one pure tumor sample (>70% purity), we could normalize and deconvolve paired samples down to a purity of 40%. This renders a reliable clonal reconstruction well adapted to multi-regionally sampled solid tumors, which are often aneuploid and contaminated by non-cancer cells.

Mosaicism in health and disease - clones picking up speed.

Post-zygotic variation refers to genetic changes that arise in the soma of an individual and that are not usually inherited by the next generation. Although there is a paucity of research on such variation, emerging studies show that it is common: individuals are complex mosaics of genetically distinct cells, to such an extent that no two somatic cells are likely to have the exact same genome. Although most types of mutation can be involved in post-zygotic variation, structural genetic variants are likely to leave the largest genomic footprint. Somatic variation has diverse physiological roles and pathological consequences, particularly when acquired variants influence the clonal trajectories of the affected cells. Post-zygotic variation is an important confounder in medical genetic testing and a promising avenue for research: future studies could involve analyses of sorted and single cells from multiple tissue types to fully explore its potential.

Tumor biology, biomarkers, and liquid biopsy in pediatric renal tumors.

The expansion of knowledge regarding driver mutations for Wilms tumor (WT) and malignant rhabdoid tumor of the kidney (MRT) and various translocations for other pediatric renal tumors opens up new possibilities for diagnosis and treatment. In addition, there are growing data surrounding prognostic factors that can be used to stratify WT treatment to improve outcomes. Here, we review the molecular landscape of WT and other pediatric renal tumors as well as WT prognostic factors. We also review incorporation of circulating tumor DNA/liquid biopsies to leverage this molecular landscape, with potential use in the future for distinguishing renal tumors at the time of diagnosis and elucidating intratumor heterogeneity, which is not well evaluated with standard biopsies. Incorporation of liquid biopsies will require longitudinal collection of multiple biospecimens. Further preclinical research, identification and validation of biomarkers, molecular studies, and data sharing among investigators are crucial to inform therapeutic strategies that improve patient outcomes.

Systematic orientation of fresh rectal suction biopsies improves histopathological diagnostics in hirschsprung's disease - a method description and preliminary report.

BACKGROUND: Optimizing rectal suction biopsy (RSB) diagnostics in Hirschsprung's disease (HD) may shorten diagnostic time and prevent need for repeated biopsies. AIM: To explore whether systematic orientation of fresh RSB specimens increased biopsy quality, diagnostic times, diagnostic efficacy, and histopathologic workload, and to explore these outcome measures for aganglionic specimens. MATERIALS/METHODS: This was an observational case-control study conducted at a national referral center for HD on data collected from the local HD-diagnostic register. From 2019 each fresh RSB was oriented by the collector in a notch in a foam cushion, placed in a separate cassette, and sent in formalin for pathological analysis. Outcome measures of oriented RSB samples collected 2019-2021 were compared to those of non-oriented RSB samples collected 2015-2018. Staining/immunohistochemistry consisted of hematoxylin eosin, S-100 and calretinin. RESULTS: 78 children with 81 RSBs and 242 biopsy analyzes were included. The frequency of high-quality RSB specimens was higher in oriented: 40% (42/106) versus non-oriented 25% (34/136) (p = 0.018), the diagnostic turnaround time was shorter: 2 days (1-5) versus 3 days (2-8) (p = 0.015), and the number of additional sectioning/leveling/re-orientation per biopsy was lower: 7 (3-26) versus 16 (7-72) (p = 0.011). Specifically for aganglionic specimens, the frequency of high-quality biopsies was generally higher in oriented than in non-oriented RSB specimens: 47% (28/59) versus 14% (7/50) (p < 0.001); the diagnostic efficacy was higher 95% (19/20) versus 60% (9/15) (p = 0.027) and the diagnostic turnaround time shorter: 2 days (2-3) versus 3 days (2-8) (p = 0.036). CONCLUSIONS: Systematic orientation of fresh RSB specimens improves HD diagnostics. Improvement was consistent in aganglionic specimens.

Amplification of ERBB2 (HER2) in embryonal rhabdomyosarcoma: A potential treatment target in rare cases?

The ERBB2 gene encodes a receptor tyrosine kinase also known as HER2. The gene is amplified and overexpressed in one-fifth of breast carcinomas; patients with such tumors benefit from targeted treatment with trastuzumab or other drugs blocking the receptor. In addition, ERBB2 has been shown to be amplified and/or overexpressed in a variety of other malignancies. Notably, both alveolar and embryonal rhabdomyosarcoma (RMS), especially in children, often show increased expression of ERBB2. Although high-level amplification of the gene has not been described in RMS, its frequent expression at the cell surface of RMS cells has been exploited for chimeric antigen receptor T-cell (CAR T)-based treatment strategies. We here describe two cases of pediatric, fusion-negative embryonal RMS with high-level amplification of the ERBB2 gene. One patient is currently treated with conventional chemotherapy for a recently detected standard risk RMS, whereas the other patient died from metastatic disease. Both tumors displayed focal amplicons (210 and 274 Kb, respectively) in chromosome band 17q12, with proximal and distal borders corresponding to those typically seen in breast cancer. In both tumors, the ERBB2 amplicon correlated with high expression at the RNA and protein levels. Thus, breast cancer-like ERBB2 amplification is a very rare, but recurrent feature of pediatric RMS, and should be exploited as an alternative treatment target.

SIX1 as a Novel Immunohistochemical Marker in the Differential Diagnosis of Rhabdomyosarcoma.

Background: Differential diagnosis of rhabdomyosarcoma (RMS) is challenging. Sineoculis homeobox homolog 1 (SIX1) is an oncogene involved in skeletal muscle differentiation. We compared protein expression patterns of SIX1 in RMS and its most common differential diagnoses. Methods: SIX1 immunohistochemistry in 36 RMS and in 33 tumors from seven differential diagnostic subtypes were evaluated. The fraction of SIX1 positive tumor cells was scored by three independent observers. Results: A majority (75%) of the evaluated RMS expressed SIX1 in at least 50% of tumor cells and all except one RMS had more than 25% positive tumor cells. Neuroblastoma had less than 1% SIX1 positive tumor cells. Gonadoblastoma, malignant rhabdoid tumor, and Ewing sarcoma had 10% or less positive tumor cells. Pleuropulmonary blastoma exhibited 26-50% positive tumor cells and synovial sarcoma >50% positive cells. Conclusion: SIX1 immunohistochemistry is positive in most RMS, and occasionally in some tumors within the differential diagnoses of RMS.

Delineating the intra-patient heterogeneity of molecular alterations in treatment-naïve colorectal cancer with peritoneal carcinomatosis.

In a non-negligible number of patients with metastatic colorectal cancer (mCRC), the peritoneum is the predominant site of dissemination. Cure can be achieved by cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC), but this procedure is associated with long-term morbidity and high relapse rates. Thus, there is a pressing need for improved therapeutic strategies and complementary biomarkers. The present study explored the molecular heterogeneity in mCRC with peritoneal carcinomatosis (PC), and the potential clinical implications thereof. Multi-region immunohistochemical profiling and deep targeted DNA-sequencing was performed on chemotherapy-naïve tumours from seven patients with synchronous colorectal PC who underwent CRS and HIPEC. In total, 88 samples (5-19 per patient) were analysed, representing primary tumour, lymph node metastases, tumour deposits, PC and liver metastases. Expression of special AT-rich sequence-binding protein 2 (SATB2), a marker of colorectal lineage, was lacking in the majority of cases, and a conspicuous intra-patient heterogeneity was denoted for expression of the proposed prognostic and predictive biomarker RNA-binding motif protein 3 (RBM3). Loss of mismatch repair proteins MLH1 and PSM2, observed in one case, was concordant with microsatellite instability and the highest tumour mutational burden. When present in a patient, mutations in key CRC driver genes, i.e., KRAS, APC and TP53, were homogenously distributed across all samples, while less common mutations were more heterogenous. On the same note, copy number variations showed intra-patient as well inter-patient heterogeneity. In two out of seven cases, hierarchical clustering revealed that samples from the PC and lymph node metastases were more similar to each other than to the primary tumour. In summary, these findings should encourage additional studies addressing the potential distinctiveness of mCRC with PC, which might pave the way for improved personalized treatment of these patients.

Clinical-pathological features in placentas of pregnancies with SARS-CoV-2 infection and adverse outcome: case series with and without congenital transmission.

OBJECTIVE: To correlate clinical outcomes to pathology in SARS-CoV-2 infected placentas in stillborn and live-born infants presenting with fetal distress. DESIGN: Retrospective, observational. SETTING: Nationwide. POPULATION: Five stillborn and nine live-born infants from 13 pregnant women infected with SARS-CoV-2 seeking care at seven different maternity units in Sweden. METHODS: Clinical outcomes and placental pathology were studied in 14 cases (one twin pregnancy) of maternal SARS-CoV-2 infection with impaired fetal outcome. Outcomes were correlated to placental pathology in order to investigate the impact of virus-related pathology on the villous capillary endothelium, trophoblast and other cells. MAIN OUTCOME MEASURES: Maternal and fetal clinical outcomes and placental pathology in stillborn and live-born infants. RESULTS: Reduced fetal movements were reported (77%) and time from onset of maternal COVID-19 symptoms to signs of fetal distress among live-born infants was 6 (3-12) days and to diagnosis of stillbirth 11 (2-25) days. Two of the live-born infants died during the postnatal period. Signs of fetal distress led to emergency caesarean section in all live-born infants with umbilical cord blood gases and low Apgar scores confirming intrauterine hypoxia. Five stillborn and one live-born neonate had confirmed congenital transmission. Massive perivillous fibrinoid deposition, intervillositis and trophoblast necrosis were associated with SARS-CoV-2 placental infection and congenital transmission. CONCLUSIONS: SARS-CoV-2 can cause rapid placental dysfunction with subsequent acute fetal hypoxia leading to intrauterine fetal compromise. Associated placental pathology included massive perivillous fibrinoid deposition, intervillositis and trophoblast degeneration.

Histopathological dimensions differ between aganglionic and ganglionic bowel wall in children with Hirschsprung's disease.

BACKGROUND: In the validation of new imaging technology for children with Hirschsprung's disease (HSCR), basic anatomical parameters of the bowel wall must be established specifically for this patient group. AIM: To explore differences in histoanatomical layers of bowel wall, comparing ganglionic and aganglionic bowel walls, and to examine if the bowel wall thickness is linked to patient weight. METHODS: This was an observational study of bowel specimens from children weighing 0-10 kg, operated on consecutively during 2018-2020. Ganglionic and aganglionic bowel walls were measured in digitalized microscopy images from 10 sites per trans-sectional specimen and compared regarding the thickness of their histoanatomical layers. RESULTS: Bowel walls were measured in 21 children. Full bowel wall thickness did not differ between aganglionic and ganglionic bowel (2.20 vs 2.04; p = 0.802) while weight at surgery correlated positively with both ganglionic and aganglionic bowel wall thickness (r = 0.688 and 0.849, respectively), and age at surgery with ganglionic bowel wall thickness (r = 0.517). In aganglionic segments, the muscularis externa layer was thicker compared to that in ganglionosis (0.45 vs 0.31 mm, p = 0.012) whereas the muscularis interna was thinner (0.45 vs 0.62 mm, p < 0.001). A diagnostic index was identified whereby a lower ratio of muscularis interna/externa thickness followed by a thinner muscularis interna differed between aganglionic and ganglionic bowel in all specimens. CONCLUSION: Thicknesses of the bowel wall's muscle layers differ between aganglionic and ganglionic bowel walls in children with HSCR. These findings support a diagnostic index that could be validated for transfer to instant diagnostic imaging techniques. LEVEL OF EVIDENCE: Diagnostic: 3.

Differential Activation of Immune Effector Processes in Mature Compared to Immature Sacrococcygeal Teratomas.

Objective: This study aims to characterize the molecular signatures of sacrococcygeal teratomas (SCTs). Methods: Three SCTs were analyzed with whole genome genotyping. RNA sequencing of 10 SCTs dominated by mature, immature and neuroglial elements was analyzed. Expression in SCT-samples with different elements were compared to each other and to a reference group of malignant pediatric tumors. Macrophages, T- and B-lymphocytes were detected by immunohistochemistry. Results: No chromosomal imbalances were detected. SCTs showed overexpression of genes involved in neurosignaling, DNA-binding molecules and pathways of early germ cells. Genes associated with immune effector processes were overexpressed in mature compared to immature SCTs, and immune cell infiltration was found predominantly around mature epithelial elements. Conclusion: The broad repertoire of histological elements in SCTs reflects differences in transcriptional regulation rather than differences in gene copy numbers. A paucity of immune response in immature SCTs may be a factor contributing to their uninhibited growth.

A dynamic mutational landscape associated with an inter-regionally diverse immune response in malignant rhabdoid tumour.

Malignant rhabdoid tumour (MRT) is a childhood neoplasm of high malignancy characterised by biallelic mutation and/or loss of the epigenetic master regulator SMARCB1, accompanied by no or few other oncogenic drivers. In spite of their generally low mutational burden, an intratumoural T-cell response has been reported in a subset of MRTs, indicating that immune checkpoint inhibition may be considered a viable therapy option for some patients. We assess here the evolution over time and space of predicted neoantigens and indicators of immune checkpoint status in two MRT patients who progressed under treatment. Both patients showed an accumulation of novel clonal and subclonal mutations, including predicted neoantigens, in metastases compared to their inferred ancestral clones in the primary tumours. The first patient had peritoneal metastases from an MRT of the liver. Clonal deconvolution revealed polyclonal seeding from the primary tumour to a single metastatic site, followed by a local subclonal burst of mutations. The second patient had a renal MRT with multiple pulmonary metastases, each of which could be traced back to a single genetically unique founder cell, with formation of novel subclones in two metastases. Both patients showed a regionally heterogeneous landscape of predicted neoantigens and of tumour-infiltrating lymphocytes expressing CD8 and PD1. In both patients, some tumour regions fulfilled established criteria for PD-L1 positivity (> 1% of tumour cells), while others did not. This suggests that even in a tumour type like MRT, with a single driver mutation, there can be heterogeneity in neoantigen repertoire, immune response, and biomarkers for checkpoint blockade among sampled locations. This must be taken into account when assessing progressed MRT patients for checkpoint inhibition therapy. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Chemotherapy, host response and molecular dynamics in periampullary cancer: the CHAMP study.

BACKGROUND: Pancreatic cancer is a devastating disease with a dismal prognosis. Despite profound medical advances in systemic therapies for other types of aggressive tumours during recent years, a diagnosis of pancreatic cancer is still often synonymous with a fatal outcome. The term periampullary cancer includes pancreatic cancer and applies to the group of tumours found in proximity to the ampulla of Vater. Molecular events and immune response in the host during chemotherapy remain largely unexplored in this group of tumours. Therefore, the "Chemotherapy, Host Response and Molecular Dynamics in Periampullary Cancer (CHAMP)" study aims to monitor these processes to gain new insight into this perplexing disease. METHODS: The CHAMP study is a prospective, single-arm observational study. All patients diagnosed with pancreatic or other periampullary adenocarcinoma undergoing adjuvant or palliative chemotherapy treatment in the Department of Oncology, Skåne University Hospital, are invited to participate. Clinical and pathological data will be compiled at study entry. A single tissue microarray (TMA) block is constructed for each patient with a resected tumour and blood samples are drawn before, during and after chemotherapy in order to sample peripheral blood mononuclear cells (PBMC), cytokines and circulating tumour DNA (ctDNA). Next generation sequencing will be performed on tumour tissue and ctDNA to detect changes in the clonal landscape over space and time. DISCUSSION: Despite the recent emergence of some promising biomarkers for periampullary cancer, there has been a lack of success in clinical implementation. Cancer cells continuously adapt and become resistant to treatment during chemotherapy. To be able to keep pace with and hopefully overtake this rapid evolution we must, with the help of new diagnostic tools, be ready to adapt and alter treatment accordingly. It seems to us that the only way forward is to gain a better understanding of the dynamics of the disease during treatment. With insights gained from the CHAMP study we hope to find answers to key questions in this largely unexplored territory. TRIAL REGISTRATION: This study has been registered 30th October 2018 at clinicaltrials.gov as NCT03724994.

The Iroquois homeobox proteins IRX3 and IRX5 have distinct roles in Wilms tumour development and human nephrogenesis.

Wilms tumour is a paediatric malignancy with features of halted kidney development. Here, we demonstrate that the Iroquois homeobox genes IRX3 and IRX5 are essential for mammalian nephrogenesis and govern the differentiation of Wilms tumour. Knock-out Irx3- /Irx5- mice showed a strongly reduced embryonic nephron formation. In human foetal kidney and Wilms tumour, IRX5 expression was already activated in early proliferative blastema, whereas IRX3 protein levels peaked at tubular differentiation. Accordingly, an orthotopic xenograft mouse model of Wilms tumour showed that IRX3-/- cells formed bulky renal tumours dominated by immature mesenchyme and active canonical WNT/ß-catenin-signalling. In contrast, IRX5-/- cells displayed activation of Hippo and non-canonical WNT-signalling and generated small tumours with abundant tubulogenesis. Our findings suggest that promotion of IRX3 signalling or inhibition of IRX5 signalling could be a route towards differentiation therapy for Wilms tumour, in which WNT5A is a candidate molecule for enforced tubular maturation. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Clonal evolution through genetic bottlenecks and telomere attrition: Potential threats to in vitro data reproducibility.

Tissue cultures of immortalized human cells, also known as established cell lines, are broadly accessible and cost-efficient tools for biomedical research. We here review potential genetic sources of systematic error in cell line experiments due to clonal evolution in vitro. In particular, the authors highlight alterations in telomere function over prolonged culture and population bottlenecks, respectively, as two commonly overlooked phenomena that can result in significant alterations in cell line genotypes over just one or a few passages in vitro. These alterations may include changes in mutation status of oncogenes and large scale chromosomal imbalances. We introduce a simple list of factors to be avoided in order to reduce the risk of data misinterpretation due to clonal evolution, including unacknowledged in vitro selection pressures, prolonged culture per se, harsh population size reductions, experiments at early phases after establishment, and the employment of cell lines not sufficiently analyzed by high resolution genetic techniques.

Changes in the Prevalence of Embryologic Remnants in Umbilical Cord With Gestational Age.

The aim of this study was to examine the prevalence of embryologic remnants in umbilical cords of different gestational ages. Sections from 392 umbilical cords were examined using light microscopy. Of these, 52% contained at least 1 remnant, most commonly of the allantoic duct type. Although there was a significant decrease in vitelline duct remnants over increasing gestational age, from 11% at weeks 11-25 to 1.6% at weeks 36-42 (P = .009; χ2 test), the allantoic duct remnants remained constant in prevalence irrespective of gestational age.

Convergent evolution of 11p allelic loss in multifocal Wilms tumors arising in WT1 mutation carriers.

Wilms tumors in patients with constitutional WT1 mutations are examples of Knudson's tumor suppressor paradigm, with somatic inactivation of the second allele occurring through 11p loss of heterozygosity. The time point of this second hit has remained unknown. We analyzed seven Wilms tumors from two patients with constitutional WT1 mutations by whole exome sequencing and genomic array. All tumors exhibited wild type WT1 loss through uniparental isodisomy. Each tumor had a unique genomic breakpoint in 11p, typically accompanied by a private activating mutation of CTNNB1. Hence, convergent evolution rather than field carcinogenesis underlies multifocal tumors in WT1 mutation carriers.

BCOR internal tandem duplication and YWHAE-NUTM2B/E fusion are mutually exclusive events in clear cell sarcoma of the kidney.

Clear cell sarcoma of the kidney (CCSK) is the second most common pediatric renal tumor. Two recurrent genetic aberrations have been described in CCSK. One is a fusion of YWHAE and NUTM2B/E, the other is an internal tandem duplication (ITD) in the BCOR gene. Here it is shown that YWHAE-NUTM2B/E fusion and the BCOR ITD are mutually exclusive events and activated different downstream signaling systems. This has important diagnostic implications and opens up for further mechanistic studies of CCSK pathogenesis.