Treatment Monitoring of a Patient with Synchronous Metastatic Angiosarcoma and Breast Cancer Using ctDNA.

Angiosarcoma is a rare and aggressive type of soft-tissue sarcoma with high propensity to metastasize. For patients with metastatic angiosarcoma, prognosis is dismal and treatment options are limited. To improve the outcomes, identifying patients with poor treatment response at an earlier stage is imperative, enabling alternative therapy. Consequently, there is a need for improved methods and biomarkers for treatment monitoring. Quantification of circulating tumor-DNA (ctDNA) is a promising approach for patient-specific monitoring of treatment response. In this case report, we demonstrate that quantification of ctDNA using SiMSen-Seq was successfully utilized to monitor a patient with metastatic angiosarcoma. By quantifying ctDNA levels using 25 patient-specific mutations in blood plasma throughout surgery and palliative chemotherapy, we predicted the outcome and monitored the clinical response to treatment. This was accomplished despite the additional complexity of the patient having a synchronous breast cancer. The levels of ctDNA showed a superior correlation to the clinical outcome compared with the radiological evaluations. Our data propose a promising approach for personalized biomarker analysis to monitor treatment in angiosarcomas, with potential applicability to other cancers and for patients with synchronous malignancies.

Pulmonary Adenocarcinoma In Situ and Minimally Invasive Adenocarcinomas in European Patients Have Less KRAS and More EGFR Mutations Compared to Advanced Adenocarcinomas.

Pulmonary adenocarcinoma (ADC) is a very diverse disease, both genetically and histologically, which displays extensive intratumor heterogeneity with numerous acquired mutations. ADC is the most common type of lung cancer and is believed to arise from adenocarcinoma in situ (AIS) which then progresses to minimally invasive adenocarcinoma (MIA). In patients of European ethnicity, we analyzed genetic mutations in AIS (n = 10) and MIA (n = 18) and compared the number of genetic mutations with advanced ADC (n = 2419). Using next-generation sequencing, the number of different mutations detected in both AIS (87.5%) and MIA (94.5%) were higher (p < 0.001) than in advanced ADC (53.7%). In contrast to the high number of mutations in Kirsten rat sarcoma virus gene (KRAS) in advanced ADC (34.6%), there was only one case of AIS with KRAS G12C mutation (3.5%; p < 0.001) and no cases of MIA with KRAS mutation (p < 0.001). In contrast to the modest prevalence of epidermal growth factor receptor (EGFR) mutations in advanced ADC (15.0%), the fraction of EGFR mutant cases was higher in both in AIS (22.2%) and MIA (59.5%; p < 0.001). The EGFR exon 19 deletion mutation was more common in both MIA (50%; n = 6/12) and ADC (41%; n = 149/363), whereas p.L858R was more prevalent in AIS (75%; n = 3/4). In contrast to pulmonary advanced ADC, KRAS driver mutations are less common, whereas mutations in EGFR are more common, in detectable AIS and MIA.

Tissue specificity of oncogenic BRAF targeted to lung and thyroid through a shared lineage factor.

Cells of origin in cancer determine tumor phenotypes, but whether lineage-defining transcription factors might influence tissue specificity of tumorigenesis among organs with similar developmental traits are unknown. We demonstrate here that tumor development and progression markedly differ in lung and thyroid targeted by Braf mutation in Nkx2.1CreERT2 mice heterozygous for Nkx2-1. In absence of tamoxifen, non-induced Nkx2.1CreERT2;BrafCA/+ mutants developed multiple full-blown lung adenocarcinomas with a latency of 1-3 months whereas thyroid tumors were rare and constrained, although minute BrafCA activation documented by variant allele sequencing was similar in both tissues. Induced oncogene activation accelerated neoplastic growth only in the lungs. By contrast, NKX2-1+ progenitor cells were equally responsive to constitutive expression of mutant Braf during lung and thyroid development. Both lung and thyroid cells transiently downregulated NKX2-1 in early tumor stages. These results indicate that BRAFV600E-induced tumorigenesis obey organ-specific traits that might be differentially modified by a shared lineage factor.

Low-grade intestinal inflammation two decades after pelvic radiotherapy.

BACKGROUND: Radiotherapy is effective in the treatment of cancer but also causes damage to non-cancerous tissue. Pelvic radiotherapy may produce chronic and debilitating bowel symptoms, yet the underlying pathophysiology is still undefined. Most notably, although pelvic radiotherapy causes an acute intestinal inflammation there is no consensus on whether the late-phase pathophysiology contains an inflammatory component or not. To address this knowledge gap, we examined the potential presence of a chronic inflammation in mucosal biopsies from irradiated pelvic cancer survivors. METHODS: We biopsied 24 cancer survivors two to 20 years after pelvic radiotherapy, and four non-irradiated controls. Using tandem mass tag (TMT) mass spectrometry and mRNA sequencing (mRNA-seq), we charted proteomic and transcriptomic profiles of the mucosal tissue previously exposed to a high or a low/no dose of radiation. Changes in the immune cell populations were determined with flow cytometry. The integrity of the protective mucus layers were determined by permeability analysis and 16S rRNA bacterial detection. FINDINGS: 942 proteins were differentially expressed in mucosa previously exposed to a high radiation dose compared to a low radiation dose. The data suggested a chronic low-grade inflammation with neutrophil activity, which was confirmed by mRNA-seq and flow cytometry and further supported by findings of a weakened mucus barrier with bacterial infiltration. INTERPRETATION: Our results challenge the idea that pelvic radiotherapy causes an acute intestinal inflammation that either heals or turns fibrotic without progression to chronic inflammation. This provides a rationale for exploring novel strategies to mitigate chronic bowel symptoms in pelvic cancer survivors. FUNDING: This study was supported by the King Gustav V Jubilee Clinic Cancer Foundation (CB), The Adlerbertska Research Foundation (CB), The Swedish Cancer Society (GS), The Swedish State under the ALF agreement (GS and CB), Mary von Sydow's foundation (MA and VP).

Diagnostic Yield From a Nationwide Implementation of Precision Medicine for all Children With Cancer.

PURPOSE: Several studies have indicated that broad genomic characterization of childhood cancer provides diagnostically and/or therapeutically relevant information in selected high-risk cases. However, the extent to which such characterization offers clinically actionable data in a prospective broadly inclusive setting remains largely unexplored. METHODS: We implemented prospective whole-genome sequencing (WGS) of tumor and germline, complemented by whole-transcriptome sequencing (RNA-Seq) for all children diagnosed with a primary or relapsed solid malignancy in Sweden. Multidisciplinary molecular tumor boards were set up to integrate genomic data in the clinical decision process along with a medicolegal framework enabling secondary use of sequencing data for research purposes. RESULTS: During the study's first 14 months, 118 solid tumors from 117 patients were subjected to WGS, with complementary RNA-Seq for fusion gene detection in 52 tumors. There was no significant geographic bias in patient enrollment, and the included tumor types reflected the annual national incidence of pediatric solid tumor types. Of the 112 tumors with somatic mutations, 106 (95%) exhibited alterations with a clear clinical correlation. In 46 of 118 tumors (39%), sequencing only corroborated histopathological diagnoses, while in 59 cases (50%), it contributed to additional subclassification or detection of prognostic markers. Potential treatment targets were found in 31 patients (26%), most commonly ALK mutations/fusions (n = 4), RAS/RAF/MEK/ERK pathway mutations (n = 14), FGFR1 mutations/fusions (n = 5), IDH1 mutations (n = 2), and NTRK2 gene fusions (n = 2). In one patient, the tumor diagnosis was revised based on sequencing. Clinically relevant germline variants were detected in 8 of 94 patients (8.5%). CONCLUSION: Up-front, large-scale genomic characterization of pediatric solid malignancies provides diagnostically valuable data in the majority of patients also in a largely unselected cohort.

Novel TPR::ROS1 Fusion Gene Activates MAPK, PI3K and JAK/STAT Signaling in an Infant-type Pediatric Glioma.

BACKGROUND/AIM: Although fusion genes involving the proto-oncogene receptor tyrosine kinase ROS1 are rare in pediatric glioma, targeted therapies with small inhibitors are increasingly being approved for histology-agnostic fusion-positive solid tumors. PATIENT AND METHODS: Here, we present a 16-month-old boy, with a brain tumor in the third ventricle. The patient underwent complete resection but relapsed two years after diagnosis and underwent a second operation. The tumor was initially classified as a low-grade glioma (WHO grade 2); however, methylation profiling suggested the newly WHO-recognized type: infant-type hemispheric glioma. To further refine the molecular background, and search for druggable targets, whole genome (WGS) and whole transcriptome (RNA-Seq) sequencing was performed. RESULTS: Concomitant WGS and RNA-Seq analysis revealed several segmental gains and losses resulting in complex structural rearrangements and fusion genes. Among the top-candidates was a novel TPR::ROS1 fusion, for which only the 3' end of ROS1 was expressed in tumor tissue, indicating that wild type ROS1 is not normally expressed in the tissue of origin. Functional analysis by Western blot on protein lysates from transiently transfected HEK293 cells showed the TPR::ROS1 fusion gene to activate the MAPK-, PI3K- and JAK/STAT- pathways through increased phosphorylation of ERK, AKT, STAT and S6. The downstream pathway activation was also confirmed by immunohistochemistry on tumor tissue slides from the patient. CONCLUSION: We have mapped the activated oncogenic pathways of a novel ROS1-fusion gene and broadened the knowledge of the newly recognized infant-type glioma subtype. The finding facilitates suitable targeted therapies for the patient in case of relapse.

KRAS Mutations Impact Clinical Outcome in Metastatic Non-Small Cell Lung Cancer.

There is an urgent need to identify new predictive biomarkers for treatment response to both platinum doublet chemotherapy (PT) and immune checkpoint blockade (ICB). Here, we evaluated whether treatment outcome could be affected by KRAS mutational status in patients with metastatic (Stage IV) non-small cell lung cancer (NSCLC). All consecutive patients molecularly assessed and diagnosed between 2016−2018 with Stage IV NSCLC in the region of West Sweden were included in this multi-center retrospective study. The primary study outcome was overall survival (OS). Out of 580 Stage IV NSCLC patients, 35.5% harbored an activating mutation in the KRAS gene (KRASMUT). Compared to KRAS wild-type (KRASWT), KRASMUT was a negative factor for OS (p = 0.014). On multivariate analysis, KRASMUT persisted as a negative factor for OS (HR 1.478, 95% CI 1.207−1.709, p < 0.001). When treated with first-line platinum doublet (n = 195), KRASMUT was a negative factor for survival (p = 0.018), with median OS of 9 months vs. KRASWT at 11 months. On multivariate analysis, KRASMUT persisted as a negative factor for OS (HR 1.564, 95% CI 1.124−2.177, p = 0.008). KRASMUT patients with high PD-L1 expression (PD-L1high) had better OS than PD-L1highKRASWT patients (p = 0.036). In response to first-line ICB, KRASMUT patients had a significantly (p = 0.006) better outcome than KRASWT patients, with a median OS of 23 vs. 6 months. On multivariable Cox analysis, KRASMUT status was an independent prognostic factor for better OS (HR 0.349, 95% CI 0.148−0.822, p = 0.016). kRAS mutations are associated with better response to treatment with immune checkpoint blockade and worse response to platinum doublet chemotherapy as well as shorter general OS in Stage IV NSCLC.

Endoscopic ultrasound-guided side-fenestrated needle biopsy sampling is sensitive for pancreatic neuroendocrine tumors but inadequate for tumor grading: a prospective study.

Accurate pretreatment grading of pancreatic neuroendocrine tumors (PanNETs) is important to guide patient management. We aimed to evaluate endoscopic ultrasound-guided fine needle biopsy sampling (EUS-FNB) for the preoperative diagnosis and grading of PanNETs. In a tertiary-center setting, patients with suspected PanNETs were prospectively subjected to 22-gauge, reverse-bevel EUS-FNB. The EUS-FNB samples (Ki-67EUS) and corresponding surgical specimens (Ki-67SURG) were analyzed with Ki-67 indexing and thereafter tumor grading, (GRADEEUS) and (GRADESURG) respectively. In total 52 PanNET-patients [median age: 66 years; females: 25/52; surgical resection 22/52 (42%)] were included. EUS-FNB was diagnostic in 44/52 (85%). In 42 available FNB-slides, the median neoplastic cell count was 1034 (IQR: 504-3667) with 32/42 (76%), 22/42 (52%), and 14/42 (33%) cases exceeding 500, 1000, and 2000 neoplastic cells respectively. Ki-67SURG was significantly higher compared to Ki-67EUS with a moderate correlation comparing Ki-67EUS and Ki-67SURG (Pearson r = 0.60, r2 = 0.36, p = 0.011). The GRADEEUS had a weak level of agreement (κ = 0.08) compared with GRADESURG. Only 2/12 (17%) G2-tumors were correctly graded in EUS-FNB-samples. EUS-guided fine needle biopsy sampling is sensitive for preoperative diagnosis of PanNET but biopsy quality is relatively poor. Therefore, the approach seems suboptimal for pretreatment grading of PanNET.

Different HSP90 Inhibitors Exert Divergent Effect on Myxoid Liposarcoma In Vitro and In Vivo.

The therapeutic options for patients with relapsed or metastatic myxoid liposarcoma (MLS) remain scarce and there is currently no targeted therapy available. Inhibition of the HSP90 family of chaperones has been suggested as a possible therapeutic option for patients with MLS. However, the clinical effect of different HSP90 inhibitors vary considerably and no comparative study in MLS has been performed. Here, we evaluated the effects of the HSP90 inhibitors 17-DMAG, AUY922 and STA-9090 on MLS cell lines and in an MLS patient-derived xenograft (PDX) model. Albeit all drugs inhibited in vitro growth of MLS cell lines, the in vivo responses were discrepant. Whereas 17-DMAG inhibited tumor growth, AUY922 surprisingly led to increased tumor growth and a more aggressive morphological phenotype. In vitro, 17-DMAG and STA-9090 reduced the activity of the MAPK and PI3K/AKT signaling pathways, whereas AUY922 led to a compensatory upregulation of downstream ERK. Furthermore, all three tested HSP90 inhibitors displayed a synergistic combination effect with trabectidin, but not with doxorubicin. In conclusion, our results indicate that different HSP90 inhibitors, albeit having the same target, can vary significantly in downstream effects and treatment outcomes. These results should be considered before proceeding into clinical trials against MLS or other malignancies.

FET fusion oncoproteins interact with BRD4 and SWI/SNF chromatin remodelling complex subtypes in sarcoma.

FET fusion oncoproteins containing one of the FET (FUS, EWSR1, TAF15) family proteins juxtaposed to alternative transcription-factor partners are characteristic of more than 20 types of sarcoma and leukaemia. FET oncoproteins bind to the SWI/SNF chromatin remodelling complex, which exists in three subtypes: cBAF, PBAF and GBAF/ncBAF. We used comprehensive biochemical analysis to characterize the interactions between FET oncoproteins, SWI/SNF complexes and the transcriptional coactivator BRD4. Here, we report that FET oncoproteins bind all three main SWI/SNF subtypes cBAF, PBAF and GBAF, and that FET oncoproteins interact indirectly with BRD4 via their shared interaction partner SWI/SNF. Furthermore, chromatin immunoprecipitation sequencing and proteomic analysis showed that FET oncoproteins, SWI/SNF components and BRD4 co-localize on chromatin and interact with mediator and RNA Polymerase II. Our results provide a possible molecular mechanism for the FET-fusion-induced oncogenic transcriptional profiles and may lead to novel therapies targeting aberrant SWI/SNF complexes and/or BRD4 in FET-fusion-caused malignancies.

Overlapping morphological, immunohistochemical and genetic features of superficial CD34-positive fibroblastic tumor and PRDM10-rearranged soft tissue tumor.

Superficial CD34-positive fibroblastic tumor (SCD34FT) is a recently recognized soft tissue tumor that is considered to be of borderline malignancy. The pathogenesis of this tumor remains incompletely understood, but it has been suggested that SCD34FT overlaps with tumors showing fusions involving the PRDM10 gene. Previous analyses of PRDM10-rearranged tumors have demonstrated that they have a distinct gene expression profile, resulting in high expression of CADM3 (also known as SynCam3), which can be detected immunohistochemically. Here, we investigated a series (n = 43) of SCD34FT or PRDM10-rearranged tumors and potential mimics (n = 226) with regard to morphological, genetic, and immunohistochemical features. The results show that SCD34FT and PRDM10-rearranged tumor are morphologically indistinguishable; 41 of 43 tumors of both entities are CADM3-positive. Hence, we suggest that they constitute a single entity, preferably referred to as SCD34FT. Expression of CADM3 was only rarely seen in other soft tissue tumors, except in tumors with Schwann cell differentiation. Thus, IHC for CADM3, in combination with the characteristic morphological features, is a valuable adjunct in the diagnosis of SCD34FT.

Tissue architecture delineates field cancerization in BRAFV600E-induced tumor development.

Cancer cells hijack developmental growth mechanisms but whether tissue morphogenesis and architecture modify tumorigenesis is unknown. Here, we characterized a new mouse model of sporadic thyroid carcinogenesis based on inducible expression of BRAF carrying a Val600 Glu (V600E) point mutation (BRAFV600E) from the thyroglobulin promoter (TgCreERT2). Spontaneous activation of this Braf-mutant allele due to leaky activity of the Cre recombinase revealed that intrinsic properties of thyroid follicles determined BRAF-mutant cell fate. Papillary thyroid carcinomas developed multicentrically within a normal microenvironment. Each tumor originated from a single follicle that provided a confined space for growth of a distinct tumor phenotype. Lineage tracing revealed oligoclonal tumor development in infancy and early selection of BRAFV600E kinase inhibitor-resistant clones. Somatic mutations were few, non-recurrent and limited to advanced tumors. Female mice developed larger tumors than males, reproducing the gender difference of human thyroid cancer. These data indicate that BRAFV600E-induced tumorigenesis is spatiotemporally regulated depending on the maturity and heterogeneity of follicles. Moreover, thyroid tissue organization seems to determine whether a BRAF-mutant lineage becomes a cancerized lineage. The TgCreERT2;BrafCA/+ sporadic thyroid cancer mouse model provides a new tool to evaluate drug therapy at different stages of tumor evolution.

Discordance of PIK3CA and TP53 mutations between breast cancer brain metastases and matched primary tumors.

There is limited knowledge of the biology of breast cancer (BC) brain metastasis (BM). We primarily aimed to determine the mutations in BCBM and to compare the mutational pattern with the matched primary breast cancer (BC). Secondary aims were to determine mutations in each subgroup (Luminal A-/B-like, HER2+ and TNBC) of BCBM, and to determine survival according to specific mutations. We investigated 57 BCBMs, including 46 cases with matched primary tumors (PT) by targeted Next Generation Sequencing (NGS) using the Cancer Hotspot Panel v2 (ThermoFisher Scientific) covering 207 targeted regions in 50 cancer related genes. Subtype according to immunohistochemistry was re-evaluated. NGS results fulfilling sequencing quality criteria were obtained from 52 BM and 41 PT, out of which 37 were matched pairs. Pathogenic mutations were detected in 66% of PTs (27/41), and 62% of BMs (32/52). TP53 mutations were most frequent; 49% (20/41) of PTs and 48% (25/52) in BMs, followed by PIK3CA mutations; 22% (9/42) in PTs and 25% (13/52) in BMs. Mutations in CDH1, EGFR, HRAS, RB1 CDKN2A and PTEN were detected in single pairs or single samples. Mutational pattern was discordant in 24% of matched pairs. We show a discordance of PIK3CA and TP53 mutations of roughly 25% indicating the need to develop methods to assess mutational status in brain metastasis where analysis of cell-free DNA from cerebrospinal fluid (CSF) has shown promising results.

Monitoring Circulating Tumor DNA During Surgical Treatment in Patients with Gastrointestinal Stromal Tumors.

The majority of patients diagnosed with advanced gastrointestinal stromal tumors (GISTs) are successfully treated with a combination of surgery and tyrosine kinase inhibitors (TKIs). However, it remains challenging to monitor treatment efficacy and identify relapse early. Here, we utilized a sequencing strategy based on molecular barcodes and developed a GIST-specific panel to monitor tumor-specific and TKI resistance mutations in cell-free DNA and applied the approach to patients undergoing surgical treatment. Thirty-two patients with GISTs were included, and 161 blood plasma samples were collected and analyzed at routine visits before and after surgery and at the beginning, during, and after surgery. Patients were included regardless of their risk category. Our GIST-specific sequencing approach allowed detection of tumor-specific mutations and TKI resistance mutations with mutant allele frequency < 0.1%. Circulating tumor DNA (ctDNA) was detected in at least one timepoint in nine of 32 patients, ranging from 0.04% to 93% in mutant allele frequency. High-risk patients were more often ctDNA positive than other risk groups (P < 0.05). Patients with detectable ctDNA also displayed higher tumor cell proliferation rates (P < 0.01) and larger tumor sizes (P < 0.01). All patients who were ctDNA positive during surgery became negative after surgery. Finally, in two patients who progressed on TKI treatment, we detected multiple resistance mutations. Our data show that ctDNA may become a clinically useful biomarker in monitoring treatment efficacy in patients with high-risk GISTs and can assist in treatment decision making.

Proteomic analysis in diffuse large B-cell lymphoma identifies dysregulated tumor microenvironment proteins in non-GCB/ABC subtype patients.

The complexity of the activated B-cell like (ABC) diffuse large B-cell lymphoma (DLBCL) subtype is probably not only explained by genetic alterations and methods to measure global protein expression could bring new knowledge regarding the pathophysiology. We used quantitative proteomics to analyze the global protein expression of formalin-fixed paraffin-embedded (FFPE) tumor tissues from 202 DLBCL patients. We identified 6430 proteins and 498 were significantly regulated between the germinal center B-cell like (GCB) and non-GCB groups. A number of proteins previously not described to be upregulated in non-GCB or ABC DLBCL was found, e.g. CD64, CD85A, guanylate-binding protein 1 (GBP1), interferon-induced proteins with tetratricopeptide repeat (IFIT)2, and mixed lineage kinase domain-like protein (MLKL) and immunohistochemical staining showed higher expression of GBP1 and MLKL. A cluster analysis revealed that the most prominent cluster contained proteins involved in the tumor microenvironment and regulation of the immune system. Our data suggest that the therapeutic focus should be expanded toward the tumor microenvironment in non-GCB/ABC subtype patients.

[Molecular pathology - the key to precision oncology]. / Molekylär patologi ­ nyckeln till målinriktad cancerbehandling.

Rapidly expanding knowledge of the molecular landscape of cancers has resulted in the implementation of an increasing number of specific therapies targeted at tumors with specific molecular aberrations. In response to this development, new tools for predictive testing for molecular targets need to be implemented in routine health care. To achieve robust future molecular diagnostic pathology, and equal opportunity for patients to qualify for targeted therapy, the national working group for Solid Tumors in the initiative Genomic Medicine Sweden (GMS) aims to implement regional and national platforms for comprehensive genomic tumor profiling and linked analysis pipelines. Novel IT-infrastrucutures and recruitment of bioinformaticians and molecular biologists to hospital labotatories are paramount. The infrastructure will allow wider inclusion into clinical trials and supplement the national cancer registries with molecular ¼real world data« for research and evaluation of implemented cancer therapies and diagnostic procedures.

Discovery of a rare GKAP1-NTRK2 fusion in a pediatric low-grade glioma, leading to targeted treatment with TRK-inhibitor larotrectinib.

Here we report a case of an 11-year-old girl with an inoperable tumor in the optic chiasm/hypothalamus, who experienced several tumor progressions despite three lines of chemotherapy treatment. Routine clinical examination classified the tumor as a BRAF-negative pilocytic astrocytoma. Copy-number variation profiling of fresh frozen tumor material identified two duplications in 9q21.32-33 leading to breakpoints within the GKAP1 and NTRK2 genes. RT-PCR Sanger sequencing revealed a GKAP1-NTRK2 exon 10-16 in-frame fusion, generating a putative fusion protein of 658 amino acids with a retained tyrosine kinase (TK) domain. Functional analysis by transient transfection of HEK293 cells showed the GKAP1-NTRK2 fusion protein to be activated through phosphorylation of the TK domain (Tyr705). Subsequently, downstream mediators of the MAPK- and PI3K-signaling pathways were upregulated in GKAP1-NTRK2 cells compared to NTRK2 wild-type; phosphorylated (p)ERK (3.6-fold), pAKT (1.8- fold), and pS6 ribosomal protein (1.4-fold). Following these findings, the patient was enrolled in a clinical trial and treated with the specific TRK-inhibitor larotrectinib, resulting in the arrest of tumor growth. The patient's condition is currently stable and the quality of life has improved significantly. Our findings highlight the value of comprehensive clinical molecular screening of BRAF-negative pediatric low-grade gliomas, to reveal rare fusions serving as targets for precision therapy.

Asynchrony of Apical Polarization, Luminogenesis, and Functional Differentiation in the Developing Thyroid Gland.

Follicular thyroid tissue originates from progenitors derived from a midline endodermal primordium. Current understanding infers that folliculogenesis in the embryonic thyroid designates the latest morphogenetic event taking place after the final anatomical shape and position of the gland is established. However, this concept does not consider the fact that the thyroid isthmus develops chronologically before the lobes and also contains all progenitors required for lobulation. To elucidate whether cells committed to a thyroid fate might be triggered to differentiate asynchronously related to maturation and developmental stage, mouse embryonic thyroid tissues from E12.5-17.5 were subjected to immunofluorescent labeling of biomarkers (progenitors: NKX2-1; differentiation: thyroglobulin/TG); folliculogenesis: E-cadherin/CDH1; luminogenesis: mucin 1/MUC1; apical polarity: pericentrin/PCNT; basement membrane: laminin; growth: Ki67), quantitative RT-PCR analysis (Nkx2.1, Tg, Muc1) and transmission electron microscopy. Tg expression was detectable as early as E12.5 and gradually increased >1000-fold until E17.5. Muc1 and Nkx2.1 transcript levels increased in the same time interval. Prior to lobulation (E12.5-13.5), MUC1 and TG distinguished pre-follicular from progenitor cells in the developing isthmus characterized by intense cell proliferation. Luminogenesis comprised redistribution of MUC1+ vesicles or vacuoles, transiently associated with PCNT, to the apical cytoplasm and the subsequent formation of MUC1+ nascent lumens. Apical polarization of pre-follicular cells and lumen initiation involved submembraneous vesicular traffic, reorganization of adherens junctions and ciliogenesis. MUC1 did not co-localize with TG until a lumen with a MUC1+ apical membrane was established. MUC1 delineated the lumen of all newly formed follicles encountered in the developing lobes at E15.5-17.5. Folliculogenesis started before establishment of a complete follicular basal lamina. These observations indicate that embryonic thyroid differentiation is an asynchronous process consistent with the idea that progenitors attaining a stationary position in the connecting isthmus portion undergo apical polarization and generate follicles already at a primordial stage of thyroid development, i.e. foregoing growth of the lobes. Although the thyroid isthmus eventually comprises minute amounts of the total thyroid volume and contributes little to the overall hormone production, it is of principal interest that local cues related to the residence status of cells - independently of a prevailing high multiplication rate - govern the thyroid differentiation program.

TBLR1 and CREBBP as potential novel prognostic immunohistochemical biomarkers in diffuse large B-cell lymphoma.

Recent studies have identified prognostic mutational clusters for diffuse large B-cell lymphoma (DLBCL) patients, both within and outside the original cell-of-origin (COO) classification. For many of these mutations, there is limited information regarding the corresponding protein expression. With the aim to determine the relationship of protein expression and intensity to COO and prognosis, we used digital image analysis to quantitate immunohistochemical staining of CREBBP, IRF8, EZH2, and TBLR1 in 209 DLBCL patients. We found that patients with strong nuclear expression of TBLR1 had inferior progression-free survival (PFS) and overall survival (OS) in univariable analysis and inferior PFS in multivariable analysis. Patients with higher proportion of intermediate to strong nuclear CREBBP expression had a worse PFS and OS in univariable analysis. CREBBP was expressed with stronger intensity in non-GCB patients and the prognostic impact was restricted to this subgroup. These findings suggest that high nuclear protein expression of TBLR1 and CREBBP is negatively associated with prognosis in DLBCL.

Circulating cell-free tumor DNA analysis in pediatric cancers.

Analysis of circulating cell-free tumor DNA (ctDNA) has shown promising results within several clinical applications, including cancer detection, mutation profiling, treatment monitoring and early detection of relapse. Here, we discuss the potential and limitations of ctDNA analysis in pediatric cancer detection, therapy decision making and research. Biological properties associated to ctDNA are highlighted and related to technical constraints in downstream analyses. The effects of ctDNA release and clearance dynamics are illustrated and we argue that reporting ctDNA as a fraction of mutated compared to normal wild-type DNA may be problematic from a biological point of view. We have summarized experimental details, data and conclusions from 50 pediatric ctDNA studies. We discuss the genomic landscape of several pediatric entities and how their specific mutation profiles affects ctDNA analysis, often requiring custom-made technical solutions. Finally, we outline future aspects of ctDNA analysis and what is needed to fully implement it into clinical routine in pediatric oncology.