Spatially resolved clonal copy number alterations in benign and malignant tissue.

Defining the transition from benign to malignant tissue is fundamental to improving early diagnosis of cancer1. Here we use a systematic approach to study spatial genome integrity in situ and describe previously unidentified clonal relationships. We used spatially resolved transcriptomics2 to infer spatial copy number variations in >120,000 regions across multiple organs, in benign and malignant tissues. We demonstrate that genome-wide copy number variation reveals distinct clonal patterns within tumours and in nearby benign tissue using an organ-wide approach focused on the prostate. Our results suggest a model for how genomic instability arises in histologically benign tissue that may represent early events in cancer evolution. We highlight the power of capturing the molecular and spatial continuums in a tissue context and challenge the rationale for treatment paradigms, including focal therapy.

The Swedish childhood tumor biobank: systematic collection and molecular characterization of all pediatric CNS and other solid tumors in Sweden.

The Swedish Childhood Tumor Biobank (BTB) is a nonprofit national infrastructure for collecting tissue samples and genomic data from pediatric patients diagnosed with central nervous system (CNS) and other solid tumors. The BTB is built on a multidisciplinary network established to provide the scientific community with standardized biospecimens and genomic data, thereby improving knowledge of the biology, treatment and outcome of childhood tumors. As of 2022, over 1100 fresh-frozen tumor samples are available for researchers. We present the workflow of the BTB from sample collection and processing to the generation of genomic data and services offered. To determine the research and clinical utility of the data, we performed bioinformatics analyses on next-generation sequencing (NGS) data obtained from a subset of 82 brain tumors and patient blood-derived DNA combined with methylation profiling to enhance the diagnostic accuracy and identified germline and somatic alterations with potential biological or clinical significance. The BTB procedures for collection, processing, sequencing, and bioinformatics deliver high-quality data. We observed that the findings could impact patient management by confirming or clarifying the diagnosis in 79 of the 82 tumors and detecting known or likely driver mutations in 68 of 79 patients. In addition to revealing known mutations in a broad spectrum of genes implicated in pediatric cancer, we discovered numerous alterations that may represent novel driver events and specific tumor entities. In summary, these examples reveal the power of NGS to identify a wide number of actionable gene alterations. Making the power of NGS available in healthcare is a challenging task requiring the integration of the work of clinical specialists and cancer biologists; this approach requires a dedicated infrastructure, as exemplified here by the BTB.

Multidisciplinary management of clival chordomas; long-term clinical outcome in a single-institution consecutive series.

OBJECTIVE: Chordomas of the skull base have high recurrence rates even after radical resection and adjuvant radiotherapy. We evaluate the long-term clinical outcome using multidisciplinary management in the treatment of clival chordomas. METHODS: Between 1984 and 2015, 22 patients diagnosed with an intracranial chordoma were treated at the Karolinska University Hospital, Stockholm, Sweden. Sixteen of 22 were treated with Gamma Knife radiosurgery (GKRS) for tumour residual or progression during the disease course. Seven of 22 received adjuvant fractionated radiotherapy and 5 of these also received proton beam radiotherapy. RESULTS: Fifteen of 22 (68%) patients were alive at follow-up after a median of 80 months (range 22-370 months) from the time of diagnosis. Six were considered disease free after >10-year follow-up. The median tumour volume at the time of GKRS was 4.7 cm3, range 0.8-24.3 cm3. Median prescription dose was 16 Gy, range 12-20 Gy to the 40-50% isodose curve. Five patients received a second treatment with GKRS while one received three treatments. After GKRS patients were followed with serial imaging for a median of 34 months (range 6-180 months). Four of 16 patients treated with GKRS were in need of a salvage microsurgical procedure compared to 5/7 treated with conventional or proton therapy. CONCLUSION: After surgery, 7/22 patients received conventional and/or photon therapy, while 15/22 were treated with GKRS for tumour residual or followed with serial imaging with GKRS as needed upon tumour progression. With this multidisciplinary management, 5- and 10-year survivals of 82% and 50% were achieved, respectively.

Simultaneous Ultra-Sensitive Detection of Structural and Single Nucleotide Variants Using Multiplex Droplet Digital PCR in Liquid Biopsies from Children with Medulloblastoma.

Medulloblastoma is a malignant embryonal tumor of the central nervous system (CNS) that mainly affects infants and children. Prognosis is highly variable, and molecular biomarkers for measurable residual disease (MRD) detection are lacking. Analysis of cell-free DNA (cfDNA) in cerebrospinal fluid (CSF) using broad genomic approaches, such as low-coverage whole-genome sequencing, has shown promising prognostic value. However, more sensitive methods are needed for MRD analysis. Here, we show the technical feasibility of capturing medulloblastoma-associated structural variants and point mutations simultaneously in cfDNA using multiplexed droplet digital PCR (ddPCR). Assay sensitivity was assessed with a dilution series of tumor in normal genomic DNA, and the limit of detection was below 100 pg of input DNA for all assays. False positive rates were zero for structural variant assays. Liquid biopsies (CSF and plasma, n = 47) were analyzed from 12 children with medulloblastoma, all with negative CSF cytology. MRD was detected in 75% (9/12) of patients overall. In CSF samples taken before or within 21 days of surgery, MRD was detected in 88% (7/8) of patients with localized disease and in one patient with the metastasized disease. Our results suggest that this approach could expand the utility of ddPCR and complement broader analyses of cfDNA for MRD detection.

Spatially resolved transcriptomic profiling of degraded and challenging fresh frozen samples.

Spatially resolved transcriptomics has enabled precise genome-wide mRNA expression profiling within tissue sections. The performance of methods targeting the polyA tails of mRNA relies on the availability of specimens with high RNA quality. Moreover, the high cost of currently available spatial resolved transcriptomics assays requires a careful sample screening process to increase the chance of obtaining high-quality data. Indeed, the upfront analysis of RNA quality can show considerable variability due to sample handling, storage, and/or intrinsic factors. We present RNA-Rescue Spatial Transcriptomics (RRST), a workflow designed to improve mRNA recovery from fresh frozen specimens with moderate to low RNA quality. First, we provide a benchmark of RRST against the standard Visium spatial gene expression protocol on high RNA quality samples represented by mouse brain and prostate cancer samples. Then, we test the RRST protocol on tissue sections collected from five challenging tissue types, including human lung, colon, small intestine, pediatric brain tumor, and mouse bone/cartilage. In total, we analyze 52 tissue sections and demonstrate that RRST is a versatile, powerful, and reproducible protocol for fresh frozen specimens of different qualities and origins.

The spatial RNA integrity number assay for in situ evaluation of transcriptome quality.

The RNA integrity number (RIN) is a frequently used quality metric to assess the completeness of rRNA, as a proxy for the corresponding mRNA in a tissue. Current methods operate at bulk resolution and provide a single average estimate for the whole sample. Spatial transcriptomics technologies have emerged and shown their value by placing gene expression into a tissue context, resulting in transcriptional information from all tissue regions. Thus, the ability to estimate RNA quality in situ has become of utmost importance to overcome the limitation with a bulk rRNA measurement. Here we show a new tool, the spatial RNA integrity number (sRIN) assay, to assess the rRNA completeness in a tissue wide manner at cellular resolution. We demonstrate the use of sRIN to identify spatial variation in tissue quality prior to more comprehensive spatial transcriptomics workflows.

A challenging case of concurrent multiple sclerosis and anaplastic astrocytoma.

BACKGROUND: Cases of gliomas coexisting with multiple sclerosis (MS) have been described over the past few decades. However, due to the complex clinical and radiological traits inherent to both entities, this concurrent phenomenon remains difficult to diagnose. Much has been debated about whether this coexistence is incidental or mirrors a poorly understood neoplastic phenomenon engaging glial cells in the regions of demyelination. CASE DESCRIPTION: We present the case of a 41-year-old patient diagnosed with a left-sided frontal contrast enhancing lesion initially assessed as a tumefactive MS. Despite systemic treatment, the patient gradually developed signs of mass effect, which led to decompressive surgery. The initial microscopic evaluation demonstrated the presence of MS and oligodendroglioma; the postoperative evolution proved complex due to a series of MS-relapses and tumor recurrence. An ulterior revaluation of the samples for the purpose of this report showed an MS-concurrent anaplastic astrocytoma. We describe all relevant clinical aspects of this case and review the medical literature for possible causal mechanisms. CONCLUSION: Although cases of concurrent glioma and MS remain rare, we present a case illustrating this phenomenon and explore a number of theories behind a potential causal relationship.

Pituitary carcinomas: Rare and challenging.

BACKGROUND: Pituitary carcinomas (PCs) are defined as adenohypophyseal tumors with metastatic activity within and outside the boundaries of the central nervous system (CNS). The condition is rare and therefore seldom reported; most lesions are hormone producing and have a tendency for complex evolution. As such, the management of PCs remains difficult. We present an illustrative case of PC with a brief review of the recent medical literature. CASE DESCRIPTION: A 58-year-old patient was diagnosed with prolactinoma in 2005. The ensuing biochemical and radiological evolution proved contentious; local tumor control was never fully achieved despite multimodal management including pharmacological treatment, repeated resections, and radiotherapy. In late 2017, the patient developed metastatic lesions within the confinements of the CNS requiring further surgical interventions, high-dose radiation, and systemic treatment. CONCLUSION: As it was the case in our patient, PCs require tailored, multimodal treatments according to the degree of infiltration, site of invasion, and hormone status. Further studies are necessary to understand the mechanisms promoting "extra-sellar" activity, particularly at distant sites; the identification of biomarkers exposing the risk of PC remains a crucial aspect of diagnostics, prevention and future customized therapies.

Gamma knife radiosurgery in the management of endolymphatic sac tumors.

BACKGROUND: Although widely regarded as rare epithelial tumors with a low grade of malignancy, endolymphatic sac tumors (ELST) often lead to disabling petrous bone destruction and significantly impairing symptoms at the time of primary diagnosis and/or recurrence. ELST is not uncommon in von Hippel Lindau (VHL) patients. Although open surgery is regarded as the best treatment option, recurrence remains a challenge, particularly when gross tumor resection (GTR) is deemed unachievable due to topographic conditions. Tumor recurrence successfully treated with fractionated radiotherapy and radiosurgery have been reported in selected cases. We present the case of a patient with recurrent ELST treated with salvage gamma knife radiosurgery (GKRS) adding a review of current literature. CASE DESCRIPTION: A 65-year-old patient underwent GKRS of an unresectable, recurrent ELST. Tumor volumetric analysis showed almost 15% increase in tumor volume in the 4 months between the pre-GKRS magnetic resonance imaging (MRI) and the stereotactic MRI (s-MRI) at treatment. Follow-up MRI at 12 and 20 months showed significant decrease in local tumor volume, decreased contrast enhancement and no perifocal edema. The patient's general and neurological status remains stable to the present day. CONCLUSION: In the present case, GKRS was effective in the management of a recurrent ELST over the course of 20 months. Because of ELSTs recurrence potential, long-term follow up is required. The present case as well as previous reports might suggest a possible salvage/adjunctive role of radiosurgery in the management of ELST. Further studies are deemed necessary.

Clinical response of the novel activating ALK-I1171T mutation in neuroblastoma to the ALK inhibitor ceritinib.

Tumors with anaplastic lymphoma kinase (ALK) fusion rearrangements, including non-small-cell lung cancer and anaplastic large cell lymphoma, are highly sensitive to ALK tyrosine kinase inhibitors (TKIs), underscoring the notion that such cancers are addicted to ALK activity. Although mutations in ALK are heavily implicated in childhood neuroblastoma, response to the ALK TKI crizotinib has been disappointing. Embryonal tumors in patients with DNA repair defects such as Fanconi anemia (FA) often have a poor prognosis, because of lack of therapeutic options. Here we report a child with underlying FA and ALK mutant high-risk neuroblastoma responding strongly to precision therapy with the ALK TKI ceritinib. Conventional chemotherapy treatment caused severe, life-threatening toxicity. Genomic analysis of the initial biopsy identified germline FANCA mutations as well as a novel ALK-I1171T variant. ALK-I1171T generates a potent gain-of-function mutant, as measured in PC12 cell neurite outgrowth and NIH3T3 transformation. Pharmacological inhibition profiling of ALK-I1171T in response to various ALK TKIs identified an 11-fold improved inhibition of ALK-I1171T with ceritinib when compared with crizotinib. Immunoaffinity-coupled LC-MS/MS phosphoproteomics analysis indicated a decrease in ALK signaling in response to ceritinib. Ceritinib was therefore selected for treatment in this child. Monotherapy with ceritinib was well tolerated and resulted in normalized catecholamine markers and tumor shrinkage. After 7.5 mo treatment, the residual primary tumor shrunk, was surgically removed, and exhibited hallmarks of differentiation together with reduced Ki67 levels. Clinical follow-up after 21 mo treatment revealed complete clinical remission including all metastatic sites. Therefore, ceritinib presents a viable therapeutic option for ALK-positive neuroblastoma.

Salvage gamma knife radiosurgery in the management of dysembryoplastic neuroepithelial tumors: Long-term outcome in a single-institution case series.

BACKGROUND: Dysembryoplastic neuroepithelial tumors (DNT/DNET) are rare epileptogenic tumors. Microsurgery remains the best treatment option, although case reports exist on the use of gamma knife radiosurgery (GKRS) in selected cases. We investigated the long-term outcome of GKRS-treated DNTs at our institution in the context of current diagnostic and treatment options. CASE DESCRIPTIONS: We conducted a retrospective review of three consecutive adult patients (≥18 years) treated with salvage GKRS between 2002 and 2010 at Karolinska University Hospital, Stockholm, Sweden. The case series was supplemented by a review of current literature. A 20-year-old male underwent subtotal resection (STR) in 1997 and 2002 of DNT resulting in temporary control of intractable epilepsy despite antiepileptic drug treatment (AED). Long-term seizure control was obtained after GKRS of two separate residual DNT components along the surgical margin (2005 and 2010). A 27-year-old male undergoing gross total resection of the contrast-enhancing portion of a DNT (1999) resulted in temporary control of intractable epilepsy despite AEDs; lasting clinical control of seizures was achieved in 2002 after GKRS of a small, recurrent DNT component. A 28-year-old male underwent STR of DNT (1994 and 2004) resulting in temporary control of intractable epilepsy. Lasting seizure control was gained after GKRS of a residual tumor (2005). CONCLUSION: GKRS as performed in our series was effective in terms of tumor and seizure control. No adverse radiation effects were recorded. Prospective studies are warranted to establish the role of GKRS in the treatment of DNTs.

Subclinical inflammation with increased neutrophil activity in healthy twin siblings reflect environmental influence in the pathogenesis of inflammatory bowel disease.

BACKGROUND: The mechanisms behind increased fecal calprotectin (FC) in healthy relatives of patients with inflammatory bowel disease (IBD) are unknown. Our aims were to explore if there is a subclinical inflammation with increased neutrophil activity in healthy twin siblings in discordant twin pairs with IBD and to assess the influence of genetics in this context. METHODS: Nuclear factor kappa B (NF-κB) and neutrophil activity, based on myeloperoxidase (MPO) and FC, were analyzed in healthy twin siblings in discordant twin pairs with IBD and compared with healthy controls. NF-κB and MPO were assessed by immunohistochemistry and FC by enzyme-linked immunosorbent assay. RESULTS: In total, 33 of 34 healthy twin siblings were histologically normal. Increased NF-κB was more often observed in healthy twin siblings in discordant twin pairs with Crohn's disease (13/18 [73%]) and with ulcerative colitis (12/16 [75%]) than in healthy controls (8/45 [18%]). MPO was more often increased in healthy twin siblings in discordant pairs with Crohn's disease (12/18 [67%]) than in healthy controls (11/45 [24%]) and FC more often in healthy twin siblings in discordant pairs with ulcerative colitis (14/21 [67%]) than in healthy controls (6/31 [19%]). Interestingly, the observed differences remained when healthy monozygotic and dizygotic twin siblings were analyzed separately. CONCLUSIONS: We observed increased NF-κB, MPO, and FC in healthy twins in both monozygotic and dizygotic discordant pairs with IBD. These novel findings speak for an ongoing subclinical inflammation with increased neutrophil activity in healthy first-degree relatives.