Andreas Muth

Adrenal lesion frequency: A prospective, cross-sectional CT study in a defined region, including systematic re-evaluation

Background: Incidentally detected adrenal lesions have become a growing clinical problem. Purpose: To prospectively estimate and validate the prevalence of incidentally detected adrenal lesions (adrenal incidentaloma) in patients with or without malignant disease undergoing CT. Material and Methods: During 18 months all adult patients with incidentally discovered adrenal lesions detected at CT were prospectively reported from the radiology departments of all hospitals in Western Sweden (1.66 million inhabitants). Frequencies of adrenal lesions initially reported at CT and at a systematic re-evaluation were compared. The interobserver variation in blindly assessing adrenal lesions was also analyzed. Results: Adrenal lesions were reported and verified in 339 patients (193 females; mean age 69 years, range 30–94 years). Mean lesion size was 25.8 mm (range 8–94 mm). The mean frequency of originally reported adrenal lesions was 0.9% (range 0–2.4% between hospitals). The systematic re-evaluation of 3801 randomly selected cases showed a mean frequency of 4.5% (range 1.8–7.1% between hospitals). The re-evaluation revealed 177 cases with adrenal lesions, 30% of these were submitted by the local radiologist in accordance with the study design, 23% were described in the local radiology report but not submitted to the study center, while 47% were neither locally reported nor submitted. Conclusion: Adrenal lesions are under-reported in clinical practice. Prevalence figures for adrenal incidentalomas should therefore be interpreted with caution, especially in multi-center settings.

Cohort study of patients with adrenal lesions discovered incidentally

This prospective cohort study investigated the incidence, clinical features and natural history of incidentally discovered adrenal mass lesions (adrenal incidentaloma, AI) in an unselected population undergoing radiological examination.

Systematic review of surgery and outcomes in patients with primary aldosteronism

Primary aldosteronism (PA) is the most common cause of secondary hypertension. The main aims of this paper were to review outcome after surgical versus medical treatment of PA and partial versus total adrenalectomy in patients with PA.

Can quantification of VMAT and SSTR expression be helpful for planning radionuclide therapy of malignant pheochromocytomas

Abstract:  Tumor‐specific uptake of the radio‐iodinated norepinephrine analogue meta‐iodobenzylguanidine (MIBG) or uptake of radiolabeled somatostatin analogues via somatostatin receptors (SSTRs) are possibilities to diagnose and treat malignant pheochromocytomas/paragangliomas (PCs/PGs) . The aims of this study were to investigate the quantitative expression of vesicular monoamine transporters (VMAT 1, 2) and all five SSTRs in malignant pheochromocytoma/paraganglioma (PC/PG) to evaluate the possibilities for tumor‐specific radionuclide therapy. High scintigraphic 123I‐MIBG uptake was found in two malignant PGs with high VMAT expression (500–730 copies of VMAT 1, 1,500–1,700 copies of VMAT 2 per 1,000 β‐actin), while no 123I‐MIBG uptake was found in the malignant PG with low VMAT expression (330 copies of VMAT 1, 350 copies of VMAT 2 per 1,000 β‐actin). The two patients with high VMAT expression and high 123I‐MIBG uptake were treated with 131I‐MIBG (2–3 × 8 GBq). In vitro, the VMAT antagonist, reserpine, and the membrane pump inhibitor, clomipramine, inhibited the uptake of 123I‐MIBG into tumor cells equally well (48% and 53% reduction respectively, P < 0.001). SSTR2 was the most abundant receptor subtype, but in the two malignant PGs its expression was only 110–260 copies/1,000 β‐actin. The transporters at the cell membrane and in the vesicular membrane both appear to be of importance for the uptake of 123I‐MIBG into malignant PC/PG. Quantitative determination of VMAT expression may be helpful in selecting patients suitable for radionuclide therapy with 131I‐MIBG. The present data indicate that SSTR‐mediated radionuclide therapy will not be effective treatment of malignant PC/PG.

Malignant pheochromocytoma in a population-based study: survival and clinical results.

Abstract:  One hundred fifty‐four consecutive patients with pheochromocytoma (PC, n= 137) or paraganglioma (PG, n= 17) were treated at our unit. Twenty patients had MEN 2, 15 VRD, and 1 VHL tumors. Twelve had malignant tumors and were classified according to mode of presentation: (1) Distant metastases (n= 4); three underwent surgical debulking (with chemotherapy in one); and three had 131I‐MIBG therapy. Within 4 years two patients died of tumor progression. (2) Locally advanced disease (n= 4), all resected for cure. (3) Malignancy disclosed during follow‐up after adrenalectomy with “benign” histopathology (n= 4). All patients in groups 2 and 3 developed recurrence 9 (1–17) years after primary surgery; four underwent resection, one remains tumor‐free. The others were treated chronically with phenoxybenzamine, combined with 131I‐MIBG in one. These eight patients were observed 20 (5–35) years after primary surgery and 11 (1–19) years after recurrence. This series is population‐based and may better reflect the natural history of malignant PC/PG than the series from national referral centers. Active surgical treatment and phenoxybenzamine resulted in low tumor‐related mortality in groups 2 and 3; five patients died 8–30 years after diagnosis, four of PC/PG (three from group 2 and one from group 3 ) and one of other causes. We propose tumor uptake studies (MIBG‐ and octreotide scintigraphy) in patients with nonresectable metastases; to select individual radionuclide therapy data on the expression of CA‐transporters/somatostatin receptors may be helpful. To diagnose PC/PG early, screening of adrenal incidentalomas has been suggested. In a regional population‐based prospective study, 503 incidentalomas were reported during 18 months, but only one patient with PG was identified.

Acquired hypermethylation of the P16INK4A promoter in abdominal paraganglioma: relation to adverse tumor phenotype and predisposing mutation

Recurrent alterations in promoter methylation of tumor suppressor genes (TSGs) and LINE1 (L1RE1) repeat elements were previously reported in pheochromocytoma and abdominal paraganglioma. This study was undertaken to explore CpG methylation abnormalities in an extended tumor panel and assess possible relationships between metastatic disease and mutation status. CpG methylation was quantified by bisulfite pyrosequencing for selected TSG promoters and LINE1 repeats. Methylation indices above normal reference were observed for DCR2 (TNFRSF10D), CDH1, P16 (CDKN2A), RARB, and RASSF1A. Z-scores for overall TSG, and individual TSG methylation levels, but not LINE1, were significantly correlated with metastatic disease, paraganglioma, disease predisposition, or outcome. Most strikingly, P16 hypermethylation was strongly associated with SDHB mutation as opposed to RET/MEN2, VHL/VHL, or NF1-related disease. Parallel analyses of constitutional, tumor, and metastasis DNA implicate an order of events where constitutional SDHB mutations are followed by TSG hypermethylation and 1p loss in primary tumors, later transferred to metastatic tissue. In the combined material, P16 hypermethylation was prevalent in SDHB-mutated samples and was associated with short disease-related survival. The findings verify the previously reported importance of P16 and other TSG hypermethylation in an independent tumor series. Furthermore, a constitutional SDHB mutation is proposed to predispose for an epigenetic tumor phenotype occurring before the emanation of clinically recognized malignancy.

Malignant pheochromocytomas/paragangliomas harbor mutations in transport and cell adhesion genes.

One out of ten patients with pheochromocytoma (PCC) and paraganglioma (PGL) develop malignant disease. Today there are no reliable pathological methods to predict malignancy at the time of diagnosis. Tumors harboring mutations in the succinate dehydrogenase subunit B (SDHB) gene often metastasize but the sequential genetic events resulting in malignant progression are not fully understood. The aim of this study was to identify somatic mutations that contribute to the malignant transformation of PCC/PGL. We performed pair‐wise (tumor‐normal) whole‐exome sequencing to analyze the somatic mutational landscape in five malignant and four benign primary PCC/sympathetic PGL (sPGL), including two biological replicates from each specimen. In total, 225 unique somatic mutations were identified in 215 genes, with an average mutation rate of 0.54 mutations/megabase. Malignant tumors had a significantly higher number of mutations compared to benign tumors (p < 0.001). Three novel genes were identified as recurrently mutated; MYCN, MYO5B and VCL, and mutations in these genes were exclusively found in malignant sPGL tumors. Mutations in the MYO5B gene could be verified in two publicly available data sets. A gene ontology analysis of mutated genes showed enrichment of cellular functions related to cytoskeletal protein binding, myosin complex and motor activity, many of which had functions in Rab and Rac/Rho GTPase pathways. In conclusion, we have identified recurrent mutations in genes related to intracellular transport and cell adhesion, and we have confirmed MYO5B to be recurrently mutated in PCC/PGL cases with malignant potential. Our study suggests that deregulated Rab and Rac/Rho pathways may be important in PCC/PGL tumorigenesis.

Mutational Signature and Transcriptomic Classification Analyses as the Decisive Diagnostic Tools for a Cancer of Unknown Primary

Purpose Cancer of unknown primary is a group of metastatic tumors in which the standard diagnostic workup fails to identify the site of origin of the tumor. The potential impact of precision oncology on this group of patients is large, because actionable driver mutations and a correct diagnosis could provide treatment options otherwise not available for patients with these fatal cancers. This study investigated if comprehensive genomic analyses could provide information on the origin of the tumor. Patients and Methods Here we describe a patient whose tumor was misdiagnosed at least three times. Next-generation sequencing, a patient-derived xenograft mouse model, and bioinformatics were used to identify an actionable mutation, predict resistance development to the targeted therapy, and correctly diagnose the origin of the tumor. Transcriptomic classification was benchmarked using The Cancer Genome Atlas (TCGA). Results Despite the lack of a known primary tumor site and the absence of diagnostic immunohistochemical markers, the origin of the patient’s tumor was established using the novel bioinformatic workflow. This included a mutational signature analysis of the sequenced metastases and comparison of their transcriptomic profiles to a pan-cancer panel of tumors from TCGA. We further discuss the strengths and limitations of the latter approaches in the context of three potentially incorrectly diagnosed TCGA lung tumors. Conclusion Comprehensive genomic analyses can provide information on the origin of tumors in patients with cancer of unknown primary.

Abstract A11: Exome sequencing of FFPE material: An evaluation

Background: The current project aims to explore the possibility to use formalin fixed paraffin embedded (FFPE) samples for exome sequencing, which would open up a large collection of tissue samples for molecular studies. Usage of formalin is known to degrade and modify the DNA both during treatment and storage resulting in problems in downstream molecular analyses. The aim of the present study is to evaluate exome sequencing of FFPE samples by comparing data from FFPE normal tissue to corresponding snap frozen (SF) blood controls with focus on data output such as amount of data removed due to low sequencing quality, amount of data mapped to the genome, GC-content, and duplicate levels. Methods: In the current study we used the western Swedish biobank of neural tumors archived at the Sahlgrenska Hospital, Gothenburg. We performed exome sequencing of FFPE normal adrenal tissue samples and corresponding SF blood samples from five patients diagnosed with pheochromocytoma/paraganglioma. Two of the patients had known heterozygous germline mutations and were included to determine if these mutations could be found in both sample types. Libraries for SF samples were prepared according to protocol using Agilent Technologies SureSelect Human All Exon 50 Mb library prep kit. FFPE samples were prepared with slight modifications in the protocol, with additional PCR cycles used to increase amplification. 75 bp paired end reads were generated on Illumina HiScan SQ according to manufacturer9s protocol (v.3). Results: Between 92-99% of the raw data were kept after removal of data due to low sequencing quality independent of sample type. For the SF samples 99-100% of the trimmed data mapped to the genome, the number for FFPE samples were slightly lower ranging from 87-98%. The GC content in the exome enriched DNA from FFPE and SF tissue were similar, ranging between 45-51%. Looking at the mapped data we found higher levels of duplicates in the FFPE samples compared to the SF samples, 19-78% vs. 5-15%. For one of the two patients with a known germline mutation the variant was detected at approximately the same frequency in both FFPE and SF sample (39% in FFPE vs. 44% in SF). For the other patient the variant were found at higher frequency in the FFPE sample compared to SF sample (80% vs. 50%). Conclusions: We saw no difference in amount data removed due to low sequencing quality between the five FFPE and SF samples evaluated in this study. Also, we saw only slightly lower mapping frequencies for the FFPE samples. The GC-content for both FFPE and SF samples were within the range of what9s expected for exome sequencing data for libraries prepared with this kit and sequenced on Illumina HiScan SQ platform. Due to the additional PCR cycles used for FFPE samples we saw higher duplicate levels in these samples, suggesting that deeper sequencing is necessary for FFPE samples to get the same amount of unique reads as for the SF samples. A probable explanation to the higher allele frequency of the variant observed in the FFPE sample from one of the patients with a known germline mutation is the presence of contaminant tumor cells in the normal tissue sample, resulting in a shifted allele frequency. To conclude, SF samples are to prefer for exome sequencing. However, if SF material is not available FFPE tissue is a good alternative to increase the sample cohort when studying rare and complex diseases. Citation Format: Annica Wilzen, Heidi Ottesen, Anna Larsson, Bo Wangberg, Andreas Muth, Ola Nilsson, Frida Abel. Exome sequencing of FFPE material: An evaluation. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A11.