Identification and characterization of the genetic alterations underlying neuroendocrine tumors of the small intestine

Neuroendocrine tumors of the small intestine (SI-NETs) have experienced a dramatic increase in incidence over the last three decades. Although defined by a small proliferative index (mostly G1 and G2 tumors), the tumors give frequently and early rise to metastases, which often exceed the size of the primary tumor and kill the patients in the end. SI-NETs are genetically poorly characterized, the frequent loss of one chromosome 18 (Chr18) being the exception. Therefore, this doctoral thesis focused on this lesion in order to investigate potential tumor suppressors located on this chromosome (SMAD2, SMAD4, Elongin A3, CABLES, PMAIP1, and DCC). SMAD2 and SMAD4 showed retained expression in the 14 SI-NET samples investigated (12 with loss of Chr18), leaving only haploinsuffiency as possible mechanism in tumor development and progression. Elongin A3 and CABLES mRNAs were differentially expressed between the 1xChr18 and 2xChr18 cohort, suggesting that the loss of Chr18 has an impact on mRNA level. However, western blot analysis of 21 SI-NETs revealed preserved protein expression of Elongin A3 and CABLES. Interestingly, CABLES western blot depicted – in addition to the normal doublet-isoform – an additional isoform at ~55 kDa in the tumor samples, which was not present in the HEK293 control. Among alternative splicing, aberrant splicing of this protein is known in tumors, which could lead to the loss of the CDK-binding domain of CABLES, resulting in enhanced cell growth and tumor formation due to faster progression through the cell cycle [1]. PMAIP1 was not expressed in eight samples investigated. Since a 100% loss of a tumor suppressor is rare, the hypothesis that the lack of PMAIP1 is a normal feature of normal neuroendocrine enterochromaffin cells is favored, rather than the loss being a characteristic of neuroendocrine tumor cells. Remarkably, the tumor suppressor protein DCC showed total loss or, at least, clearly reduced expression in nearly 30% (6/21) of the tumor samples. Abridged DCC function can result in reduced apoptosis, giving rise to tumor growth and dissemination. Alternative splicing and mutations in the intronic region of DCC [2] render this gene even more interesting. Further investigations of our lab will focus on the transcriptome and proteome of SI-NETs, and thereby on the differential expression of gene transcripts and proteins between tumors with and without loss of Chr18; hoping to shed light on the role of DCC (and CABLES), which we found to be altered in SI-NETs. In 2013, Banck et al. published the genomic landscape of SI-NETs with amplifications of the PI3K/AKT/mTOR pathway being the most frequent aberration [3]. Subsequently, we analyzed six genes (PDGFRα, PDGFRβ, PIK3CD, AKT1, AKT2, mTOR) involved in this pathway by FISH; revealing advanced, metastatic tumors as well as more invasive ¬¬tumors to harbor significantly more copy number (CN) alterations than tumors of early stage without metastases and less invasive tumors (UICC and T stage comparison). However, no association with protein expression or activation could be identified. One possible explanation for the discrepancy between gene and protein expression might be that epigenetic events play a role in the transcriptional control of amplified genes, thereby preventing protein overexpression. Since especially the more aggressive tumors (defined by UICC stage IIIB and IV, as well as tumor stage 3 and 4) are lacking effective treatment, the inhibition of the PI3K/AKT/mTOR pathway could be a useful new tool in the therapy of SI-NETs. Therefore, a similar trial to the RADIANT-4 study [4] with the inclusion of functional gastrointestinal neuroendocrine tumors should enlighten the possible effect of everolimus or another inhibitor of the PI3K/AKT/mTOR pathway on the tumor progression

Is Upregulation of Aquaporin 4-M1 Isoform Responsible for the Loss of Typical Orthogonal Arrays of Particles in Astrocytomas?

The astrocytic endfoot membranes of the healthy blood-brain barrier—contacting the capillary—are covered with a large number of the water channel aquaporin 4 (AQP4). They form orthogonal arrays of particles (OAPs), which consist of AQP4 isoform M1 and M23. Under pathologic conditions, AQP4 is distributed over the whole cell and no or only small OAPs are found. From cell culture experiments, it is known that cells transfected only with AQP4-M1 do not form OAPs or only small ones. We hypothesized that in astrocytomas the situation may be comparable to the in vitro experiments expecting an upregulation of AQP4-M1. Quantitative Real-time PCR (qRT-PCR) of different graded astrocytomas revealed an upregulation of both isoforms AQP4 M1 and M23 in all astrocytomas investigated. In freeze fracture replicas of low-grade malignancy astrocytomas, more OAPs than in high-grade malignancy astrocytomas were found. In vitro, cultured glioma cells did not express AQP4, whereas healthy astrocytes revealed a slight upregulation of both isoforms and only a few OAPs in freeze fracture analysis. Taken together, we found a correlation between the decrease of OAPs and increasing grade of malignancy of astrocytomas but this was not consistent with an upregulation of AQP4-M1 in relation to AQP4 M23

Mapping Spatial Genetic Landscapes in Tissue Sections through Microscale Integration of Sampling Methodology into Genomic Workflows

In cancer research, genomic profiles are often extracted from homogenized macrodissections of tissues, with the histological context lost and a large fraction of material underutilized. Pertinently, the spatial genomic landscape provides critical complementary information in deciphering disease heterogeneity and progression. Microscale sampling methods such as microdissection to obtain such information are often destructive to a sizeable fraction of the biopsy sample, thus showing limited multiplexability and adaptability to different assays. A modular microfluidic technology is here implemented to recover cells at the microscale from tumor tissue sections, with minimal disruption of unsampled areas and tailored to interface with genome profiling workflows, which is directed here toward evaluating intratumoral genomic heterogeneity. The integrated workflow-GeneScape-is used to evaluate heterogeneity in a metastatic mammary carcinoma, showing distinct single nucleotide variants and copy number variations in different tumor tissue regions, suggesting the polyclonal origin of the metastasis as well as development driven by multiple location-specific drivers

Spatial Genomics: Mapping Spatial Genetic Landscapes in Tissue Sections through Microscale Integration of Sampling Methodology into Genomic Workflows (Small 23/2021)

The GeneScape workflow, presented in article number 2007901 by Govind V. Kaigala and co-workers, uses a modular multipurpose microfluidic probe to facilitate evaluation of spatial genomic heterogeneity in tumors. Cancer cells essentially contain the same genomic blueprint but accumulate distinct corruptions at different spatial and temporal coordinates. Since these cells reside in several interacting ecosystems, it becomes very critical to study the spatial mutational landscape to evaluate and quantify tumor progression

Mapping Spatial Genetic Landscapes in Tissue Sections through Microscale Integration of Sampling Methodology into Genomic Workflows

In cancer research, genomic profiles are often extracted from homogenized macrodissections of tissues, with the histological context lost and a large fraction of material underutilized. Pertinently, the spatial genomic landscape provides critical complementary information in deciphering disease heterogeneity and progression. Microscale sampling methods such as microdissection to obtain such information are often destructive to a sizeable fraction of the biopsy sample, thus showing limited multiplexability and adaptability to different assays. A modular microfluidic technology is here implemented to recover cells at the microscale from tumor tissue sections, with minimal disruption of unsampled areas and tailored to interface with genome profiling workflows, which is directed here toward evaluating intratumoral genomic heterogeneity. The integrated workflow-GeneScape-is used to evaluate heterogeneity in a metastatic mammary carcinoma, showing distinct single nucleotide variants and copy number variations in different tumor tissue regions, suggesting the polyclonal origin of the metastasis as well as development driven by multiple location-specific drivers

Additional file 1: of Frequency of BRAF V600E mutations in 969 central nervous system neoplasms

List of all analyzed tumor samples with the corresponding histopathological diagnosis and age atdiagnosis. (XLS 129 kb

Loss of chromosome 18 in neuroendocrine tumors of the small intestine

$\textit {Background/Aims:}$ Neuroendocrine tumors of the small intestine (SI-NETs) exhibit an increasing incidence and high mortality rate. Until now, no fundamental molecular event has been linked to the tumorigenesis and progression of these tumors. Only the loss of chromosome 18 (Chr18) has been shown in up to two thirds of SI-NETs, whereby the significance of this alteration is still not understood. We therefore performed the first comprehensive study to identify Chr18-related events at the genetic, epigenetic and gene/protein expression levels. $\textit {Methods:}$ We did expression analysis of all seven putative Chr18-related tumor suppressors by quantitative real-time PCR (qRT-PCR), Western blot and immunohistochemistry. Next-generation exome sequencing and SNP array analysis were performed with five SI-NETs with (partial) loss of Chr18. Finally, we analyzed all microRNAs (miRNAs) located on Chr18 by qRT-PCR, comparing Chr18+/– and Chr18+/+ SI-NETs. $\textit {Results:}$ Only DCC (deleted in colorectal cancer) revealed loss of/greatly reduced expression in 6/21 cases (29%). No relevant loss of SMAD2, SMAD4, elongin A3 and CABLES was detected. PMAIP1 and maspin were absent at the protein level. Next-generation sequencing did not reveal relevant recurrent somatic mutations on Chr18 either in an exploratory cohort of five SI-NETs, or in a validation cohort (n = 30). SNP array analysis showed no additional losses. The quantitative analysis of all 27 Chr18-related miRNAs revealed no difference in expression between Chr18+/– and Chr18+/+ SI-NETs. $\textit {Conclusion:}$ DCC seems to be the only Chr18-related tumor suppressor affected by the monoallelic loss of Chr18 resulting in a loss of DCC protein expression in one third of SI-NETs. No additional genetic or epigenetic alterations were present on Chr18