Glucagonoma With Necrolytic Migratory Erythema: Metabolic Profile and Detection of Biallelic Inactivation of DAXX Gene

Glucagonoma With Necrolytic Migratory Erythema: Metabolic Profile and Detection of Biallelic... Abstract Context Necrolytic migratory erythema (NME) occurs in approximately 70% of patients with glucagonoma syndrome. Excessive stimulation of metabolic pathways by hyperglucagonemia, which leads to hypoaminoacidemia, contributes to NME pathogenesis. However, the molecular pathogenesis of glucagonoma and relationships between metabolic abnormalities and clinical symptoms remain unclear. Patient A 53-year-old woman was referred to our hospital with a generalized rash and weight loss. NME was diagnosed by histopathological examination of skin biopsy tissue. Laboratory tests revealed diabetes, hyperglucagonemia, marked insulin resistance, severe hypoaminoacidemia, ketosis, and anemia. Enhanced computed tomography scans detected a 29-mm pancreatic hypervascular tumor, which was eventually diagnosed as glucagonoma. Preoperative treatment with octreotide long-acting release reduced the glucagon level, improved the amino acid profile, and produced NME remission. Surgical tumor excision normalized the metabolic status and led to remission of symptoms, including NME. Interventions Whole-exome sequencing (WES) and subsequent targeted capture sequencing, followed by Sanger sequencing and pyrosequencing, identified biallelic alteration of death-domain associated protein (DAXX) with a combination of loss of heterozygosity and frameshift mutations (c.553_554del:p.R185fs and c.1884dupC:p.C629fs) in the glucagonoma. Consistently, immunohistochemistry confirmed near-absence of DAXX staining in the tumor cells. Tumor expression of glucagon and somatostatin receptor subtype 2 and 3 messenger RNA was markedly upregulated. Conclusions This is a report of glucagonoma with biallelic inactivation of DAXX determined by WES. The tumor manifested as glucagonoma syndrome with generalized NME. This case showed the relationship between hypoaminoacidemia and NME status. Further investigations are required to elucidate the underlying mechanisms of NME onset and glucagonoma tumorigenesis. Glucagonoma is a rare pancreatic neuroendocrine tumor (PanNET) originating from islets of Langerhans α-cells. Glucagonoma occurs in 3% to 7% of patients with PanNETs, and 8% to 10% of glucagonomas are associated with multiple endocrine neoplasia type 1 (1–4). Glucagonoma syndrome includes weight loss, cheilosis or stomatitis, diarrhea, necrolytic migratory erythema (NME), and diabetes mellitus. NME occurs in 50% to 70% of patients with the syndrome (1, 3, 5). It has been attributed to hypoaminoacidemia and zinc or essential fatty acid deficiency (6); however, details of NME pathogenesis are still unclear. Recent genome-wide analysis identified death-domain associated protein (DAXX) and alpha thalassemia/mental retardation syndrome X-linked (ATRX) as candidate pathogenetic genes for PanNET (7, 8), but the genetics of glucagonoma remain unknown. We describe metabolic and NME changes in a woman with glucagonoma during her clinical course. Genetic analyses including whole-exome sequencing (WES) and subsequent targeted capture sequencing identified somatic mutations at two DAXX sites along with chromosomal loss in broad genomic regions associated with the tumor. Case Description A 53-year-old woman presented to our hospital with anorexia, weight loss, and generalized rash. The rash began on the scalp and demonstrated irregular erythema with erosions and crusts (Fig. 1A). It waxed and waned, without obvious triggers. Lesion biopsy showed parakeratosis, necrosis, and separation of the upper epidermis with vacuolization of keratinocytes (Supplemental Fig. 1), consistent with NME. Figure 1. View largeDownload slide Clinical presentation of glucagonoma syndrome. (A, B) Skin lesions of NME. (A) At patient’s initial visit to our hospital, she had severe erythema with erosions and crusts involving mainly the back, hips, and genital area. (B) At 3 months postoperatively, the NME was completely resolved. (C-E) Imaging studies. (C) Contrast-enhanced CT scan showing a 29 × 25 × 19 mm hypervascular pancreatic tumor (red arrowheads). (D) Gadolinium-enhanced MRI of the tumor demonstrating high signal intensity on T1-weighted imaging (red arrowheads). (E) 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography/CT showing mild 18F-FDG uptake in the tumor lesion (maximum standardized uptake value, 2.32). (F, G) Pathological findings of the glucagonoma. (F) Gross appearance of the tumor, which was 30 × 30 mm and contained hemorrhage and necrosis. Scale bar = 1 cm. (G) Hematoxylin and eosin staining showing tumor cells forming a rosette arrangement. (H-K) Immunostaining of the tumor for glucagon (H, in brown), insulin (I, in brown), Ki-67 (J, in brown), and SSTR2 (K, in brown). (L) Chronologic changes in amino acids during the clinical course. Glucogenic, ketogenic, and both amino acids are shown in red, green, and purple characters, respectively. Numbers 1 and 2 represent fold-induction determined by the lower limit of the reference range for each amino acid. On admission, all amino acids (glucogenic and ketogenic) except aspartic acid and glutamic acid were remarkably low (blue line). After Oct-LAR treatment, both essential and nonessential amino acids increased, with some reaching the lower limit of the reference range (yellow line). After surgical treatment, almost all amino acids levels normalized immediately (red line). Amino acids in the pyruvic acid pathway changed more than those in the acetoacetate pathway. Figure 1. View largeDownload slide Clinical presentation of glucagonoma syndrome. (A, B) Skin lesions of NME. (A) At patient’s initial visit to our hospital, she had severe erythema with erosions and crusts involving mainly the back, hips, and genital area. (B) At 3 months postoperatively, the NME was completely resolved. (C-E) Imaging studies. (C) Contrast-enhanced CT scan showing a 29 × 25 × 19 mm hypervascular pancreatic tumor (red arrowheads). (D) Gadolinium-enhanced MRI of the tumor demonstrating high signal intensity on T1-weighted imaging (red arrowheads). (E) 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography/CT showing mild 18F-FDG uptake in the tumor lesion (maximum standardized uptake value, 2.32). (F, G) Pathological findings of the glucagonoma. (F) Gross appearance of the tumor, which was 30 × 30 mm and contained hemorrhage and necrosis. Scale bar = 1 cm. (G) Hematoxylin and eosin staining showing tumor cells forming a rosette arrangement. (H-K) Immunostaining of the tumor for glucagon (H, in brown), insulin (I, in brown), Ki-67 (J, in brown), and SSTR2 (K, in brown). (L) Chronologic changes in amino acids during the clinical course. Glucogenic, ketogenic, and both amino acids are shown in red, green, and purple characters, respectively. Numbers 1 and 2 represent fold-induction determined by the lower limit of the reference range for each amino acid. On admission, all amino acids (glucogenic and ketogenic) except aspartic acid and glutamic acid were remarkably low (blue line). After Oct-LAR treatment, both essential and nonessential amino acids increased, with some reaching the lower limit of the reference range (yellow line). After surgical treatment, almost all amino acids levels normalized immediately (red line). Amino acids in the pyruvic acid pathway changed more than those in the acetoacetate pathway. Laboratory tests revealed normochromic normocytic anemia, hypoalbuminemia, and hyperglucagonemia (hemoglobin, 10.4 g/dL; mean corpuscular volume, 86.3 fL; albumin, 3.5 g/dL; plasma glucagon, 402 pg/mL) (Table 1; Supplemental Table 1). Severe hypoaminoacidemia along with marked ketosis suggested a hypermetabolic state (total amino acids, 820.2 nmol/mL; total ketone body, 1263 μmol/L; 3-hydroxybutyric acid, 986 μmol/L) (Table 1; Supplemental Table 1). Glycated hemoglobin was 6.5%, and a 75-g oral glucose tolerance test revealed diabetes with marked hyperinsulinemia, possibly because of hepatic insulin resistance (Table 1). Plasma zinc and other pancreatic hormone levels were normal (Supplemental Table 1). Table 1. NME and Metabolic Profile Changes During the Clinical Course On Admission After Oct-LAR After Surgery NME Waxing and Waning; Generalized Remission; No New Lesions Complete Remission Reference Range Metabolic profile  Glucagon (pg/mL) 402 228 167 7-174  Total ketone body (μmol/L) 1263 184 106 ≧130  Total amino acids (nmol/mL) 820.2 1337.1 2787.5 2068.2–3510.3  EAAs (nmol/mL) 333.0 509.1 792.8 660.0–1222.3  NEAAs (nmol/mL) 469.2 828.0 1994.7 1381.6–2379.4  α-Linolenic acid (μg/mL) 23.3 ND 21.1 11.5–45.8  Linoleic acid (μg/mL) 865.5 ND 1094.5 708.1–1286.0  Arachidonic acid (μg/mL) 160.2 194.5 245.2 135.7–335.3 On Admission After Oct-LAR After Surgery NME Waxing and Waning; Generalized Remission; No New Lesions Complete Remission Reference Range Metabolic profile  Glucagon (pg/mL) 402 228 167 7-174  Total ketone body (μmol/L) 1263 184 106 ≧130  Total amino acids (nmol/mL) 820.2 1337.1 2787.5 2068.2–3510.3  EAAs (nmol/mL) 333.0 509.1 792.8 660.0–1222.3  NEAAs (nmol/mL) 469.2 828.0 1994.7 1381.6–2379.4  α-Linolenic acid (μg/mL) 23.3 ND 21.1 11.5–45.8  Linoleic acid (μg/mL) 865.5 ND 1094.5 708.1–1286.0  Arachidonic acid (μg/mL) 160.2 194.5 245.2 135.7–335.3 On Admission, Time (min) After Surgery, Time (min) 75-g OGTT 0 15 30 60 90 120 0 15 30 60 90 120 Plasma glucose (mmol/L) 6.6 9.3 10.3 10.4 10.5 12.8 4.8 6.2 8.1 8.5 7.3 6.8 IRI (μIU/mL) 12 129 124 120 159 362 3 29 35 48 73 50 On Admission, Time (min) After Surgery, Time (min) 75-g OGTT 0 15 30 60 90 120 0 15 30 60 90 120 Plasma glucose (mmol/L) 6.6 9.3 10.3 10.4 10.5 12.8 4.8 6.2 8.1 8.5 7.3 6.8 IRI (μIU/mL) 12 129 124 120 159 362 3 29 35 48 73 50 Abbreviations: EAA, essential amino acid; IRI, immunoreactive insulin; ND, no data; NEAA, nonessential amino acid; OGTT, oral glucose tolerance test. View Large Table 1. NME and Metabolic Profile Changes During the Clinical Course On Admission After Oct-LAR After Surgery NME Waxing and Waning; Generalized Remission; No New Lesions Complete Remission Reference Range Metabolic profile  Glucagon (pg/mL) 402 228 167 7-174  Total ketone body (μmol/L) 1263 184 106 ≧130  Total amino acids (nmol/mL) 820.2 1337.1 2787.5 2068.2–3510.3  EAAs (nmol/mL) 333.0 509.1 792.8 660.0–1222.3  NEAAs (nmol/mL) 469.2 828.0 1994.7 1381.6–2379.4  α-Linolenic acid (μg/mL) 23.3 ND 21.1 11.5–45.8  Linoleic acid (μg/mL) 865.5 ND 1094.5 708.1–1286.0  Arachidonic acid (μg/mL) 160.2 194.5 245.2 135.7–335.3 On Admission After Oct-LAR After Surgery NME Waxing and Waning; Generalized Remission; No New Lesions Complete Remission Reference Range Metabolic profile  Glucagon (pg/mL) 402 228 167 7-174  Total ketone body (μmol/L) 1263 184 106 ≧130  Total amino acids (nmol/mL) 820.2 1337.1 2787.5 2068.2–3510.3  EAAs (nmol/mL) 333.0 509.1 792.8 660.0–1222.3  NEAAs (nmol/mL) 469.2 828.0 1994.7 1381.6–2379.4  α-Linolenic acid (μg/mL) 23.3 ND 21.1 11.5–45.8  Linoleic acid (μg/mL) 865.5 ND 1094.5 708.1–1286.0  Arachidonic acid (μg/mL) 160.2 194.5 245.2 135.7–335.3 On Admission, Time (min) After Surgery, Time (min) 75-g OGTT 0 15 30 60 90 120 0 15 30 60 90 120 Plasma glucose (mmol/L) 6.6 9.3 10.3 10.4 10.5 12.8 4.8 6.2 8.1 8.5 7.3 6.8 IRI (μIU/mL) 12 129 124 120 159 362 3 29 35 48 73 50 On Admission, Time (min) After Surgery, Time (min) 75-g OGTT 0 15 30 60 90 120 0 15 30 60 90 120 Plasma glucose (mmol/L) 6.6 9.3 10.3 10.4 10.5 12.8 4.8 6.2 8.1 8.5 7.3 6.8 IRI (μIU/mL) 12 129 124 120 159 362 3 29 35 48 73 50 Abbreviations: EAA, essential amino acid; IRI, immunoreactive insulin; ND, no data; NEAA, nonessential amino acid; OGTT, oral glucose tolerance test. View Large Enhanced CT and MRI scans revealed a 29-mm pancreatic tumor (Fig. 1C and1D). Positron emission tomography/CT showed mild uptake in the tumor and normal uptake elsewhere (Fig. 1E). The patient was diagnosed with glucagonoma, and preoperative treatment with 20 mg octreotide long-acting release (Oct-LAR) was begun because she had severe malnutrition and NME; octreotide treatment has been reported to improve NME symptoms in glucagonoma (2, 9–12). Indeed, this treatment improved her glucagon levels, hypoaminoacidemia, and ketosis, and produced NME remission within a month (Table 1). She underwent excision of the central pancreatic lesion. The tumor was 30 × 30 mm and exhibited typical NET pathological findings (Fig. 1F and 1G; Supplemental Fig. 2A). Immunohistochemistry was positive for glucagon (Fig. 1H; Supplemental Fig. 2B), synaptophysin, and chromogranin A, and negligible for insulin (Fig. 1I; Supplemental Fig. 2C). Ki-67 labeling index was approximately 3% (Fig. 1J). The tumor was diagnosed histologically as grade 2 (G2) PanNET (World Health Organization 2010 criteria). Postoperatively, hypoaminoacidemia, ketosis, and diabetes parameters, especially severe insulin resistance, returned to near normal almost immediately (Table 1; Supplemental Table 2). NME resolved completely within one month after surgery (Fig. 1B; Table 1). Materials and Methods The Committee on Ethics in Human Research of Chiba University approved this study. The patient provided written informed consent for publication. Tumor and attached nontumor tissues were carefully dissected from formalin-fixed, paraffin-embedded (FFPE) tissue samples (Supplemental Fig. 3A). Genomic DNA and total RNA were extracted from these tissues using QIAamp DNA FFPE Tissue Kit (Qiagen) and RNeasy FFPE Kit (Qiagen). Genomic DNA was extracted from peripheral blood using MagneSil Blood Genomic, Max Yield System (Promega). WES, targeted capture sequencing, Sanger sequencing, and pyrosequencing are described in the Supplemental Material and Methods. We performed reverse-transcription quantitative PCR as previously described (13, 14). All mRNA expression values were normalized to β-actin. Primers and experimental conditions are described in Supplemental Material and Methods. Detailed information about immunohistochemistry and confocal microscopy analysis is also described in the Supplemental Materials and Methods. Genetic and Gene Expression Analyses To assess the pathophysiology of our patient’s glucagonoma, we performed genetic and gene expression analyses using genomic DNA from peripheral blood and genomic DNA and total RNA from the tumor and attached nontumor tissue. We analyzed the WES data as described in the Supplemental Materials and Methods for somatic mutations and chromosomal aberrations using our in-house pipeline, as previously reported (15). WES analyses based on paired tumor-normal DNA samples identified 31 genes with single-nucleotide variants and/or insertions-deletions as potential candidate genes of somatic mutations in this glucagonoma; a number of altered genes were reasonably distributed by their variant allele frequencies (VAFs) (Supplemental Table 3; Supplemental Fig. 3B). There were two distinct DAXX alterations: a c.553_554del:p.R185fs in exon3 and a c.1884dupC:p.C629fs in exon6 (Fig. 2A), which was located at a colon cancer mutation site reported in the Catalogue of Somatic Mutations in Cancer database (mutation ID: COSM1443783) (Supplemental Materials and Methods). Given that a DAXX gene alteration has been suggested to function as a putative driver gene for PanNET (8), the validation of these mutations by targeted capture sequencing was subsequently performed. We confirmed the presence of these somatic mutations at the DAXX gene locus (Fig. 2A). In this context, it has been shown that copy number changes in PanNET are classified into four groups based on arm length copy number patterns; one of them, recurrent pattern of whole chromosomal loss, was significantly enriched in G2 PanNET (8). Therefore, we next investigated copy number variants according to our in-house pipeline using WES data (Supplemental Materials and Methods) and detected chromosomal loss patterns over broad regions, including chromosomes 1, 2, 3, 6, 8, 10, 11, 15, 16, and 22 that have been previously found in G2 PanNET (Fig. 2B) (8). Notably, chromosomal loss, which appears to be related to a loss of heterozygosity (LOH), was observed at 6p21.32 containing the entire DAXX locus (Fig. 2B, vertical line). These results, together with our findings showing VAF of 0.367 and 0.24 for c.553_554del:p.R185fs and c.1884dupC:p.C629fs, respectively (Fig. 2A), which both result in a truncated form of the DAXX gene product (Fig. 2C), suggest that chromosomal loss, including at the DAXX gene locus, and subsequent truncated mutations cause biallelic inactivation and loss of DAXX function in this case of glucagonoma and may play a pathogenic role in this disease. Because biallelic inactivation of DAXX was detected in approximately 20% of PanNET (8), we decided to confirm a putative second hit mutation of the DAXX locus, in addition to LOH determined by copy number analysis, using Sanger sequencing and pyrosequencing of paired tumor-normal tissue DNA samples. Expectedly, we detected the same DAXX somatic mutations in tumor, but not in nontumor, tissue (Fig. 2D and 2E; Supplemental Fig. 3C and 3D). To assess the DAXX expression at the protein level in tumor cells, we performed immunohistochemistry and confocal microscopy analyses with single and/or double staining for DAXX and glucagon. Consistent with its genetic status, immunohistochemistry revealed no or little DAXX protein expression in tumor cells, particularly in glucagon-positive tumor cells (Fig. 2F; Supplemental Fig. 4C). In contrast, in nontumor tissues, DAXX-positive cells were clearly observed as the major cell population, with high expression in islet cells (Supplemental Fig. 4A, 4B, and 4D). In addition, confocal microscopy analysis confirmed DAXX protein inactivation in glucagon-positive tumor cells at the single-cell level (Fig. 2G), suggesting that DAXX biallelic inactivation is present in this tumor. Figure 2. View largeDownload slide Whole-exome and targeted capture sequencing identification of biallelic inactivation of DAXX gene in the glucagonoma. (A) DAXX mutations revealed by WES and subsequent TCS. Sequencing data of the DAXX gene locus was aligned using the Integrative Genomics Viewer, which identified a frameshift deletion in exon3 (left) and a frameshift insertion in exon6 (right). Sequence depth, variant number, and VAF in WES and TCS are shown at bottom. (B) CN alterations in the patient’s tumor. Total CN is shown in the at top (blue). Hetero-SNPs and allelic ratio are shown in the center (dark green) and bottom (red and green). Chromosomal loss was found in chromosomes 1, 2, 3, 5, 6, 8, 9, 10, 11, 15, 16, 18, 21, 22, and X as indicated by the dark blue line. LOH at 6p21.32 containing the DAXX gene locus is indicated by the vertical line. (C) Domain structure of human DAXX protein. Two distinct gene alterations causing a truncate form of DAXX gene product are shown (arrowheads). (D, E) DAXX mutations were confirmed by Sanger sequencing (D, left: exon3, c.553_554del:p.R185fs; E, right: exon6, c.1884dupC:p.C629fs). (F) Immunostaining for DAXX in the tumor (brown). A magnified view is shown with representative images of DAXX-negative tumor cells and a DAXX-positive nontumor cell (inset). (G) Confocal laser scanning microscopy analysis for DAXX (red), glucagon (green), and DAPI (nuclear staining in blue). (H) Reverse-transcription quantitative PCR analysis showing high mRNA expression of glucagon, SSTR2, and SSTR3 in the tumor part (T), compared with nontumor tissue (N). By contrast, insulin and DAXX mRNA expression was lower in the tumor. Data represent mean ± SEM. CN, copy number; DAPI, 4′,6-diamidino-2-phenylindole; SNP, single nucleotide polymorphism; TCS, targeted capture sequencing. Figure 2. View largeDownload slide Whole-exome and targeted capture sequencing identification of biallelic inactivation of DAXX gene in the glucagonoma. (A) DAXX mutations revealed by WES and subsequent TCS. Sequencing data of the DAXX gene locus was aligned using the Integrative Genomics Viewer, which identified a frameshift deletion in exon3 (left) and a frameshift insertion in exon6 (right). Sequence depth, variant number, and VAF in WES and TCS are shown at bottom. (B) CN alterations in the patient’s tumor. Total CN is shown in the at top (blue). Hetero-SNPs and allelic ratio are shown in the center (dark green) and bottom (red and green). Chromosomal loss was found in chromosomes 1, 2, 3, 5, 6, 8, 9, 10, 11, 15, 16, 18, 21, 22, and X as indicated by the dark blue line. LOH at 6p21.32 containing the DAXX gene locus is indicated by the vertical line. (C) Domain structure of human DAXX protein. Two distinct gene alterations causing a truncate form of DAXX gene product are shown (arrowheads). (D, E) DAXX mutations were confirmed by Sanger sequencing (D, left: exon3, c.553_554del:p.R185fs; E, right: exon6, c.1884dupC:p.C629fs). (F) Immunostaining for DAXX in the tumor (brown). A magnified view is shown with representative images of DAXX-negative tumor cells and a DAXX-positive nontumor cell (inset). (G) Confocal laser scanning microscopy analysis for DAXX (red), glucagon (green), and DAPI (nuclear staining in blue). (H) Reverse-transcription quantitative PCR analysis showing high mRNA expression of glucagon, SSTR2, and SSTR3 in the tumor part (T), compared with nontumor tissue (N). By contrast, insulin and DAXX mRNA expression was lower in the tumor. Data represent mean ± SEM. CN, copy number; DAPI, 4′,6-diamidino-2-phenylindole; SNP, single nucleotide polymorphism; TCS, targeted capture sequencing. We also compared gene expression using reverse-transcription quantitative PCR in tumor and nontumor tissue. Accordingly, glucagon mRNA expression was drastically upregulated in the tumor, whereas insulin mRNA was downregulated (Fig. 2H). Consistent with the effectiveness of Oct-LAR in our patient, both mRNA and protein expression of somatostatin receptor subtypes SSTR2 and SSTR3 were higher in tumor than in nontumor tissues (Fig. 1K; Fig. 2H; Supplemental Fig. 2D and 2E); this reflects previous reports of elevated SSTR2 expression in pancreatic tumors (16). In accordance with our notion, the expression of DAXX mRNA was markedly downregulated in the tumor (Fig. 2H). Discussion In our patient, glucagonoma syndrome presented as diabetes with severe insulin resistance, hypoaminoacidemia, and ketosis. Glucagon directly and indirectly regulates various enzymes, stimulating glycogenolysis and gluconeogenesis and inhibiting glycogenesis and glycolysis (17). Hepatic insulin resistance pivotally influences impaired glucose tolerance in glucagonoma, which likely contributed to the marked insulin resistance in our patient. Because glucagon also regulates amino acid uptake in the liver, severe hypoaminoacidemia mediated by excessive amino acid catabolism occurs frequently with glucagonoma. Although glucagon normally functions in ketogenesis primarily through free fatty acid β-oxidation, catabolism of ketogenic amino acids could promote ketone bodies formation in a pathological context such as glucagonoma. Indeed, hypolipidemia is uncommon in glucagonoma, as in our case. Intriguingly, we found that Oct-LAR treatment produced NME remission in parallel with a large improvement in hypoaminoacidemia and only minor changes in lipids. This suggests that hypoaminoacidemia was a more important contributor to NME than essential fatty acid deficiency. Furthermore, amino acids involved in the pyruvic acid pathway were altered to a greater extent than those in the acetoacetate pathway after treatment (Fig. 1L). This suggests that glucagon-signaling effects on glucogenic amino acid catabolism occur via the liver and may have resulted in NME in this patient. Several somatic mutations and chromosomal aberrations were previously identified in PanNET, including alterations of MENIN, CDKN1B, mTOR signaling pathway, and DAXX/ATRX pathway genes (7, 8, 18). Loss of DAXX/ATRX is thought to be involved in disease pathophysiology and progression through telomerase-independent methods of telomere stabilization: alternative lengthening of telomeres and chromosomal instability (8, 19). DAXX/ATRX mutations were associated with large tumor size, late tumor stage, and poor prognosis in G2 subgroup PanNET (8, 19); thus, biallelic inactivation of ATRX and/or DAXX and LOH may be related to malignant alteration rather than tumorigenesis. Biallelic DAXX inactivation through a combination of LOH and frameshift mutations in our patient suggests that close follow-up will be necessary because malignancy cannot be excluded in terms of the genetic background of this tumor. The DAXX mutation site is located in the histone-binding domain (Fig. 2C) (20), which is functionally linked to formation of the ATRX-DAXX histone chaperone complex that is implicated in gene repression and telomere chromatin structure. Because this complex mediates histone 3.3 deposition, histone-3 lysine-9 trimethylation levels, and DNA methyltransferase 1 recruitment followed by alternative lengthening of telomere activation and/or chromosomal instability, the detected DAXX mutation may result in the loss of epigenetic regulation and thereby contribute to pathogenesis of the glucagonoma. Conclusion In our patient with glucagonoma, NME, and diabetes resolved with treatment. Hypoaminoacidemia was closely related to NME status. This article reports on biallelic DAXX inactivation in glucagonoma. Abbreviations: Abbreviations: ATRX alpha thalassemia/mental retardation syndrome X-linked DAXX death-domain associated protein G2 grade 2 LOH loss of heterozygosity NME necrolytic migratory erythema Oct-LAR octreotide long-acting release PanNET pancreatic neuroendocrine tumor VAF variant allele frequency WES whole-exome sequencing Acknowledgments We thank Erika Sugawara and Noriko Yamanaka for technical support. Financial Support: This work was supported by Grants-in Aid for the Foundation for Growth Science; Advanced Research and Development Programs for Medical Innovation, Scientific Research (B) 17H04037 (T.T.) and (C) 17K09875 (S.S.); Young Scientists (B) 17K16160 (I.S.); the Takeda Science Foundation (T.T.); Foundation for Growth Science in Japan (T.T.); SENSHINE Medical Research Foundation (T.T.); and the Cooperative Research Project Program of the Medical Institute of Bioregulation, Kyushu University (T.T.). 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Endocrine Society
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Copyright © 2018 Endocrine Society
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0021-972X
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1945-7197
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10.1210/jc.2017-02646
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Abstract

Abstract Context Necrolytic migratory erythema (NME) occurs in approximately 70% of patients with glucagonoma syndrome. Excessive stimulation of metabolic pathways by hyperglucagonemia, which leads to hypoaminoacidemia, contributes to NME pathogenesis. However, the molecular pathogenesis of glucagonoma and relationships between metabolic abnormalities and clinical symptoms remain unclear. Patient A 53-year-old woman was referred to our hospital with a generalized rash and weight loss. NME was diagnosed by histopathological examination of skin biopsy tissue. Laboratory tests revealed diabetes, hyperglucagonemia, marked insulin resistance, severe hypoaminoacidemia, ketosis, and anemia. Enhanced computed tomography scans detected a 29-mm pancreatic hypervascular tumor, which was eventually diagnosed as glucagonoma. Preoperative treatment with octreotide long-acting release reduced the glucagon level, improved the amino acid profile, and produced NME remission. Surgical tumor excision normalized the metabolic status and led to remission of symptoms, including NME. Interventions Whole-exome sequencing (WES) and subsequent targeted capture sequencing, followed by Sanger sequencing and pyrosequencing, identified biallelic alteration of death-domain associated protein (DAXX) with a combination of loss of heterozygosity and frameshift mutations (c.553_554del:p.R185fs and c.1884dupC:p.C629fs) in the glucagonoma. Consistently, immunohistochemistry confirmed near-absence of DAXX staining in the tumor cells. Tumor expression of glucagon and somatostatin receptor subtype 2 and 3 messenger RNA was markedly upregulated. Conclusions This is a report of glucagonoma with biallelic inactivation of DAXX determined by WES. The tumor manifested as glucagonoma syndrome with generalized NME. This case showed the relationship between hypoaminoacidemia and NME status. Further investigations are required to elucidate the underlying mechanisms of NME onset and glucagonoma tumorigenesis. Glucagonoma is a rare pancreatic neuroendocrine tumor (PanNET) originating from islets of Langerhans α-cells. Glucagonoma occurs in 3% to 7% of patients with PanNETs, and 8% to 10% of glucagonomas are associated with multiple endocrine neoplasia type 1 (1–4). Glucagonoma syndrome includes weight loss, cheilosis or stomatitis, diarrhea, necrolytic migratory erythema (NME), and diabetes mellitus. NME occurs in 50% to 70% of patients with the syndrome (1, 3, 5). It has been attributed to hypoaminoacidemia and zinc or essential fatty acid deficiency (6); however, details of NME pathogenesis are still unclear. Recent genome-wide analysis identified death-domain associated protein (DAXX) and alpha thalassemia/mental retardation syndrome X-linked (ATRX) as candidate pathogenetic genes for PanNET (7, 8), but the genetics of glucagonoma remain unknown. We describe metabolic and NME changes in a woman with glucagonoma during her clinical course. Genetic analyses including whole-exome sequencing (WES) and subsequent targeted capture sequencing identified somatic mutations at two DAXX sites along with chromosomal loss in broad genomic regions associated with the tumor. Case Description A 53-year-old woman presented to our hospital with anorexia, weight loss, and generalized rash. The rash began on the scalp and demonstrated irregular erythema with erosions and crusts (Fig. 1A). It waxed and waned, without obvious triggers. Lesion biopsy showed parakeratosis, necrosis, and separation of the upper epidermis with vacuolization of keratinocytes (Supplemental Fig. 1), consistent with NME. Figure 1. View largeDownload slide Clinical presentation of glucagonoma syndrome. (A, B) Skin lesions of NME. (A) At patient’s initial visit to our hospital, she had severe erythema with erosions and crusts involving mainly the back, hips, and genital area. (B) At 3 months postoperatively, the NME was completely resolved. (C-E) Imaging studies. (C) Contrast-enhanced CT scan showing a 29 × 25 × 19 mm hypervascular pancreatic tumor (red arrowheads). (D) Gadolinium-enhanced MRI of the tumor demonstrating high signal intensity on T1-weighted imaging (red arrowheads). (E) 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography/CT showing mild 18F-FDG uptake in the tumor lesion (maximum standardized uptake value, 2.32). (F, G) Pathological findings of the glucagonoma. (F) Gross appearance of the tumor, which was 30 × 30 mm and contained hemorrhage and necrosis. Scale bar = 1 cm. (G) Hematoxylin and eosin staining showing tumor cells forming a rosette arrangement. (H-K) Immunostaining of the tumor for glucagon (H, in brown), insulin (I, in brown), Ki-67 (J, in brown), and SSTR2 (K, in brown). (L) Chronologic changes in amino acids during the clinical course. Glucogenic, ketogenic, and both amino acids are shown in red, green, and purple characters, respectively. Numbers 1 and 2 represent fold-induction determined by the lower limit of the reference range for each amino acid. On admission, all amino acids (glucogenic and ketogenic) except aspartic acid and glutamic acid were remarkably low (blue line). After Oct-LAR treatment, both essential and nonessential amino acids increased, with some reaching the lower limit of the reference range (yellow line). After surgical treatment, almost all amino acids levels normalized immediately (red line). Amino acids in the pyruvic acid pathway changed more than those in the acetoacetate pathway. Figure 1. View largeDownload slide Clinical presentation of glucagonoma syndrome. (A, B) Skin lesions of NME. (A) At patient’s initial visit to our hospital, she had severe erythema with erosions and crusts involving mainly the back, hips, and genital area. (B) At 3 months postoperatively, the NME was completely resolved. (C-E) Imaging studies. (C) Contrast-enhanced CT scan showing a 29 × 25 × 19 mm hypervascular pancreatic tumor (red arrowheads). (D) Gadolinium-enhanced MRI of the tumor demonstrating high signal intensity on T1-weighted imaging (red arrowheads). (E) 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography/CT showing mild 18F-FDG uptake in the tumor lesion (maximum standardized uptake value, 2.32). (F, G) Pathological findings of the glucagonoma. (F) Gross appearance of the tumor, which was 30 × 30 mm and contained hemorrhage and necrosis. Scale bar = 1 cm. (G) Hematoxylin and eosin staining showing tumor cells forming a rosette arrangement. (H-K) Immunostaining of the tumor for glucagon (H, in brown), insulin (I, in brown), Ki-67 (J, in brown), and SSTR2 (K, in brown). (L) Chronologic changes in amino acids during the clinical course. Glucogenic, ketogenic, and both amino acids are shown in red, green, and purple characters, respectively. Numbers 1 and 2 represent fold-induction determined by the lower limit of the reference range for each amino acid. On admission, all amino acids (glucogenic and ketogenic) except aspartic acid and glutamic acid were remarkably low (blue line). After Oct-LAR treatment, both essential and nonessential amino acids increased, with some reaching the lower limit of the reference range (yellow line). After surgical treatment, almost all amino acids levels normalized immediately (red line). Amino acids in the pyruvic acid pathway changed more than those in the acetoacetate pathway. Laboratory tests revealed normochromic normocytic anemia, hypoalbuminemia, and hyperglucagonemia (hemoglobin, 10.4 g/dL; mean corpuscular volume, 86.3 fL; albumin, 3.5 g/dL; plasma glucagon, 402 pg/mL) (Table 1; Supplemental Table 1). Severe hypoaminoacidemia along with marked ketosis suggested a hypermetabolic state (total amino acids, 820.2 nmol/mL; total ketone body, 1263 μmol/L; 3-hydroxybutyric acid, 986 μmol/L) (Table 1; Supplemental Table 1). Glycated hemoglobin was 6.5%, and a 75-g oral glucose tolerance test revealed diabetes with marked hyperinsulinemia, possibly because of hepatic insulin resistance (Table 1). Plasma zinc and other pancreatic hormone levels were normal (Supplemental Table 1). Table 1. NME and Metabolic Profile Changes During the Clinical Course On Admission After Oct-LAR After Surgery NME Waxing and Waning; Generalized Remission; No New Lesions Complete Remission Reference Range Metabolic profile  Glucagon (pg/mL) 402 228 167 7-174  Total ketone body (μmol/L) 1263 184 106 ≧130  Total amino acids (nmol/mL) 820.2 1337.1 2787.5 2068.2–3510.3  EAAs (nmol/mL) 333.0 509.1 792.8 660.0–1222.3  NEAAs (nmol/mL) 469.2 828.0 1994.7 1381.6–2379.4  α-Linolenic acid (μg/mL) 23.3 ND 21.1 11.5–45.8  Linoleic acid (μg/mL) 865.5 ND 1094.5 708.1–1286.0  Arachidonic acid (μg/mL) 160.2 194.5 245.2 135.7–335.3 On Admission After Oct-LAR After Surgery NME Waxing and Waning; Generalized Remission; No New Lesions Complete Remission Reference Range Metabolic profile  Glucagon (pg/mL) 402 228 167 7-174  Total ketone body (μmol/L) 1263 184 106 ≧130  Total amino acids (nmol/mL) 820.2 1337.1 2787.5 2068.2–3510.3  EAAs (nmol/mL) 333.0 509.1 792.8 660.0–1222.3  NEAAs (nmol/mL) 469.2 828.0 1994.7 1381.6–2379.4  α-Linolenic acid (μg/mL) 23.3 ND 21.1 11.5–45.8  Linoleic acid (μg/mL) 865.5 ND 1094.5 708.1–1286.0  Arachidonic acid (μg/mL) 160.2 194.5 245.2 135.7–335.3 On Admission, Time (min) After Surgery, Time (min) 75-g OGTT 0 15 30 60 90 120 0 15 30 60 90 120 Plasma glucose (mmol/L) 6.6 9.3 10.3 10.4 10.5 12.8 4.8 6.2 8.1 8.5 7.3 6.8 IRI (μIU/mL) 12 129 124 120 159 362 3 29 35 48 73 50 On Admission, Time (min) After Surgery, Time (min) 75-g OGTT 0 15 30 60 90 120 0 15 30 60 90 120 Plasma glucose (mmol/L) 6.6 9.3 10.3 10.4 10.5 12.8 4.8 6.2 8.1 8.5 7.3 6.8 IRI (μIU/mL) 12 129 124 120 159 362 3 29 35 48 73 50 Abbreviations: EAA, essential amino acid; IRI, immunoreactive insulin; ND, no data; NEAA, nonessential amino acid; OGTT, oral glucose tolerance test. View Large Table 1. NME and Metabolic Profile Changes During the Clinical Course On Admission After Oct-LAR After Surgery NME Waxing and Waning; Generalized Remission; No New Lesions Complete Remission Reference Range Metabolic profile  Glucagon (pg/mL) 402 228 167 7-174  Total ketone body (μmol/L) 1263 184 106 ≧130  Total amino acids (nmol/mL) 820.2 1337.1 2787.5 2068.2–3510.3  EAAs (nmol/mL) 333.0 509.1 792.8 660.0–1222.3  NEAAs (nmol/mL) 469.2 828.0 1994.7 1381.6–2379.4  α-Linolenic acid (μg/mL) 23.3 ND 21.1 11.5–45.8  Linoleic acid (μg/mL) 865.5 ND 1094.5 708.1–1286.0  Arachidonic acid (μg/mL) 160.2 194.5 245.2 135.7–335.3 On Admission After Oct-LAR After Surgery NME Waxing and Waning; Generalized Remission; No New Lesions Complete Remission Reference Range Metabolic profile  Glucagon (pg/mL) 402 228 167 7-174  Total ketone body (μmol/L) 1263 184 106 ≧130  Total amino acids (nmol/mL) 820.2 1337.1 2787.5 2068.2–3510.3  EAAs (nmol/mL) 333.0 509.1 792.8 660.0–1222.3  NEAAs (nmol/mL) 469.2 828.0 1994.7 1381.6–2379.4  α-Linolenic acid (μg/mL) 23.3 ND 21.1 11.5–45.8  Linoleic acid (μg/mL) 865.5 ND 1094.5 708.1–1286.0  Arachidonic acid (μg/mL) 160.2 194.5 245.2 135.7–335.3 On Admission, Time (min) After Surgery, Time (min) 75-g OGTT 0 15 30 60 90 120 0 15 30 60 90 120 Plasma glucose (mmol/L) 6.6 9.3 10.3 10.4 10.5 12.8 4.8 6.2 8.1 8.5 7.3 6.8 IRI (μIU/mL) 12 129 124 120 159 362 3 29 35 48 73 50 On Admission, Time (min) After Surgery, Time (min) 75-g OGTT 0 15 30 60 90 120 0 15 30 60 90 120 Plasma glucose (mmol/L) 6.6 9.3 10.3 10.4 10.5 12.8 4.8 6.2 8.1 8.5 7.3 6.8 IRI (μIU/mL) 12 129 124 120 159 362 3 29 35 48 73 50 Abbreviations: EAA, essential amino acid; IRI, immunoreactive insulin; ND, no data; NEAA, nonessential amino acid; OGTT, oral glucose tolerance test. View Large Enhanced CT and MRI scans revealed a 29-mm pancreatic tumor (Fig. 1C and1D). Positron emission tomography/CT showed mild uptake in the tumor and normal uptake elsewhere (Fig. 1E). The patient was diagnosed with glucagonoma, and preoperative treatment with 20 mg octreotide long-acting release (Oct-LAR) was begun because she had severe malnutrition and NME; octreotide treatment has been reported to improve NME symptoms in glucagonoma (2, 9–12). Indeed, this treatment improved her glucagon levels, hypoaminoacidemia, and ketosis, and produced NME remission within a month (Table 1). She underwent excision of the central pancreatic lesion. The tumor was 30 × 30 mm and exhibited typical NET pathological findings (Fig. 1F and 1G; Supplemental Fig. 2A). Immunohistochemistry was positive for glucagon (Fig. 1H; Supplemental Fig. 2B), synaptophysin, and chromogranin A, and negligible for insulin (Fig. 1I; Supplemental Fig. 2C). Ki-67 labeling index was approximately 3% (Fig. 1J). The tumor was diagnosed histologically as grade 2 (G2) PanNET (World Health Organization 2010 criteria). Postoperatively, hypoaminoacidemia, ketosis, and diabetes parameters, especially severe insulin resistance, returned to near normal almost immediately (Table 1; Supplemental Table 2). NME resolved completely within one month after surgery (Fig. 1B; Table 1). Materials and Methods The Committee on Ethics in Human Research of Chiba University approved this study. The patient provided written informed consent for publication. Tumor and attached nontumor tissues were carefully dissected from formalin-fixed, paraffin-embedded (FFPE) tissue samples (Supplemental Fig. 3A). Genomic DNA and total RNA were extracted from these tissues using QIAamp DNA FFPE Tissue Kit (Qiagen) and RNeasy FFPE Kit (Qiagen). Genomic DNA was extracted from peripheral blood using MagneSil Blood Genomic, Max Yield System (Promega). WES, targeted capture sequencing, Sanger sequencing, and pyrosequencing are described in the Supplemental Material and Methods. We performed reverse-transcription quantitative PCR as previously described (13, 14). All mRNA expression values were normalized to β-actin. Primers and experimental conditions are described in Supplemental Material and Methods. Detailed information about immunohistochemistry and confocal microscopy analysis is also described in the Supplemental Materials and Methods. Genetic and Gene Expression Analyses To assess the pathophysiology of our patient’s glucagonoma, we performed genetic and gene expression analyses using genomic DNA from peripheral blood and genomic DNA and total RNA from the tumor and attached nontumor tissue. We analyzed the WES data as described in the Supplemental Materials and Methods for somatic mutations and chromosomal aberrations using our in-house pipeline, as previously reported (15). WES analyses based on paired tumor-normal DNA samples identified 31 genes with single-nucleotide variants and/or insertions-deletions as potential candidate genes of somatic mutations in this glucagonoma; a number of altered genes were reasonably distributed by their variant allele frequencies (VAFs) (Supplemental Table 3; Supplemental Fig. 3B). There were two distinct DAXX alterations: a c.553_554del:p.R185fs in exon3 and a c.1884dupC:p.C629fs in exon6 (Fig. 2A), which was located at a colon cancer mutation site reported in the Catalogue of Somatic Mutations in Cancer database (mutation ID: COSM1443783) (Supplemental Materials and Methods). Given that a DAXX gene alteration has been suggested to function as a putative driver gene for PanNET (8), the validation of these mutations by targeted capture sequencing was subsequently performed. We confirmed the presence of these somatic mutations at the DAXX gene locus (Fig. 2A). In this context, it has been shown that copy number changes in PanNET are classified into four groups based on arm length copy number patterns; one of them, recurrent pattern of whole chromosomal loss, was significantly enriched in G2 PanNET (8). Therefore, we next investigated copy number variants according to our in-house pipeline using WES data (Supplemental Materials and Methods) and detected chromosomal loss patterns over broad regions, including chromosomes 1, 2, 3, 6, 8, 10, 11, 15, 16, and 22 that have been previously found in G2 PanNET (Fig. 2B) (8). Notably, chromosomal loss, which appears to be related to a loss of heterozygosity (LOH), was observed at 6p21.32 containing the entire DAXX locus (Fig. 2B, vertical line). These results, together with our findings showing VAF of 0.367 and 0.24 for c.553_554del:p.R185fs and c.1884dupC:p.C629fs, respectively (Fig. 2A), which both result in a truncated form of the DAXX gene product (Fig. 2C), suggest that chromosomal loss, including at the DAXX gene locus, and subsequent truncated mutations cause biallelic inactivation and loss of DAXX function in this case of glucagonoma and may play a pathogenic role in this disease. Because biallelic inactivation of DAXX was detected in approximately 20% of PanNET (8), we decided to confirm a putative second hit mutation of the DAXX locus, in addition to LOH determined by copy number analysis, using Sanger sequencing and pyrosequencing of paired tumor-normal tissue DNA samples. Expectedly, we detected the same DAXX somatic mutations in tumor, but not in nontumor, tissue (Fig. 2D and 2E; Supplemental Fig. 3C and 3D). To assess the DAXX expression at the protein level in tumor cells, we performed immunohistochemistry and confocal microscopy analyses with single and/or double staining for DAXX and glucagon. Consistent with its genetic status, immunohistochemistry revealed no or little DAXX protein expression in tumor cells, particularly in glucagon-positive tumor cells (Fig. 2F; Supplemental Fig. 4C). In contrast, in nontumor tissues, DAXX-positive cells were clearly observed as the major cell population, with high expression in islet cells (Supplemental Fig. 4A, 4B, and 4D). In addition, confocal microscopy analysis confirmed DAXX protein inactivation in glucagon-positive tumor cells at the single-cell level (Fig. 2G), suggesting that DAXX biallelic inactivation is present in this tumor. Figure 2. View largeDownload slide Whole-exome and targeted capture sequencing identification of biallelic inactivation of DAXX gene in the glucagonoma. (A) DAXX mutations revealed by WES and subsequent TCS. Sequencing data of the DAXX gene locus was aligned using the Integrative Genomics Viewer, which identified a frameshift deletion in exon3 (left) and a frameshift insertion in exon6 (right). Sequence depth, variant number, and VAF in WES and TCS are shown at bottom. (B) CN alterations in the patient’s tumor. Total CN is shown in the at top (blue). Hetero-SNPs and allelic ratio are shown in the center (dark green) and bottom (red and green). Chromosomal loss was found in chromosomes 1, 2, 3, 5, 6, 8, 9, 10, 11, 15, 16, 18, 21, 22, and X as indicated by the dark blue line. LOH at 6p21.32 containing the DAXX gene locus is indicated by the vertical line. (C) Domain structure of human DAXX protein. Two distinct gene alterations causing a truncate form of DAXX gene product are shown (arrowheads). (D, E) DAXX mutations were confirmed by Sanger sequencing (D, left: exon3, c.553_554del:p.R185fs; E, right: exon6, c.1884dupC:p.C629fs). (F) Immunostaining for DAXX in the tumor (brown). A magnified view is shown with representative images of DAXX-negative tumor cells and a DAXX-positive nontumor cell (inset). (G) Confocal laser scanning microscopy analysis for DAXX (red), glucagon (green), and DAPI (nuclear staining in blue). (H) Reverse-transcription quantitative PCR analysis showing high mRNA expression of glucagon, SSTR2, and SSTR3 in the tumor part (T), compared with nontumor tissue (N). By contrast, insulin and DAXX mRNA expression was lower in the tumor. Data represent mean ± SEM. CN, copy number; DAPI, 4′,6-diamidino-2-phenylindole; SNP, single nucleotide polymorphism; TCS, targeted capture sequencing. Figure 2. View largeDownload slide Whole-exome and targeted capture sequencing identification of biallelic inactivation of DAXX gene in the glucagonoma. (A) DAXX mutations revealed by WES and subsequent TCS. Sequencing data of the DAXX gene locus was aligned using the Integrative Genomics Viewer, which identified a frameshift deletion in exon3 (left) and a frameshift insertion in exon6 (right). Sequence depth, variant number, and VAF in WES and TCS are shown at bottom. (B) CN alterations in the patient’s tumor. Total CN is shown in the at top (blue). Hetero-SNPs and allelic ratio are shown in the center (dark green) and bottom (red and green). Chromosomal loss was found in chromosomes 1, 2, 3, 5, 6, 8, 9, 10, 11, 15, 16, 18, 21, 22, and X as indicated by the dark blue line. LOH at 6p21.32 containing the DAXX gene locus is indicated by the vertical line. (C) Domain structure of human DAXX protein. Two distinct gene alterations causing a truncate form of DAXX gene product are shown (arrowheads). (D, E) DAXX mutations were confirmed by Sanger sequencing (D, left: exon3, c.553_554del:p.R185fs; E, right: exon6, c.1884dupC:p.C629fs). (F) Immunostaining for DAXX in the tumor (brown). A magnified view is shown with representative images of DAXX-negative tumor cells and a DAXX-positive nontumor cell (inset). (G) Confocal laser scanning microscopy analysis for DAXX (red), glucagon (green), and DAPI (nuclear staining in blue). (H) Reverse-transcription quantitative PCR analysis showing high mRNA expression of glucagon, SSTR2, and SSTR3 in the tumor part (T), compared with nontumor tissue (N). By contrast, insulin and DAXX mRNA expression was lower in the tumor. Data represent mean ± SEM. CN, copy number; DAPI, 4′,6-diamidino-2-phenylindole; SNP, single nucleotide polymorphism; TCS, targeted capture sequencing. We also compared gene expression using reverse-transcription quantitative PCR in tumor and nontumor tissue. Accordingly, glucagon mRNA expression was drastically upregulated in the tumor, whereas insulin mRNA was downregulated (Fig. 2H). Consistent with the effectiveness of Oct-LAR in our patient, both mRNA and protein expression of somatostatin receptor subtypes SSTR2 and SSTR3 were higher in tumor than in nontumor tissues (Fig. 1K; Fig. 2H; Supplemental Fig. 2D and 2E); this reflects previous reports of elevated SSTR2 expression in pancreatic tumors (16). In accordance with our notion, the expression of DAXX mRNA was markedly downregulated in the tumor (Fig. 2H). Discussion In our patient, glucagonoma syndrome presented as diabetes with severe insulin resistance, hypoaminoacidemia, and ketosis. Glucagon directly and indirectly regulates various enzymes, stimulating glycogenolysis and gluconeogenesis and inhibiting glycogenesis and glycolysis (17). Hepatic insulin resistance pivotally influences impaired glucose tolerance in glucagonoma, which likely contributed to the marked insulin resistance in our patient. Because glucagon also regulates amino acid uptake in the liver, severe hypoaminoacidemia mediated by excessive amino acid catabolism occurs frequently with glucagonoma. Although glucagon normally functions in ketogenesis primarily through free fatty acid β-oxidation, catabolism of ketogenic amino acids could promote ketone bodies formation in a pathological context such as glucagonoma. Indeed, hypolipidemia is uncommon in glucagonoma, as in our case. Intriguingly, we found that Oct-LAR treatment produced NME remission in parallel with a large improvement in hypoaminoacidemia and only minor changes in lipids. This suggests that hypoaminoacidemia was a more important contributor to NME than essential fatty acid deficiency. Furthermore, amino acids involved in the pyruvic acid pathway were altered to a greater extent than those in the acetoacetate pathway after treatment (Fig. 1L). This suggests that glucagon-signaling effects on glucogenic amino acid catabolism occur via the liver and may have resulted in NME in this patient. Several somatic mutations and chromosomal aberrations were previously identified in PanNET, including alterations of MENIN, CDKN1B, mTOR signaling pathway, and DAXX/ATRX pathway genes (7, 8, 18). Loss of DAXX/ATRX is thought to be involved in disease pathophysiology and progression through telomerase-independent methods of telomere stabilization: alternative lengthening of telomeres and chromosomal instability (8, 19). DAXX/ATRX mutations were associated with large tumor size, late tumor stage, and poor prognosis in G2 subgroup PanNET (8, 19); thus, biallelic inactivation of ATRX and/or DAXX and LOH may be related to malignant alteration rather than tumorigenesis. Biallelic DAXX inactivation through a combination of LOH and frameshift mutations in our patient suggests that close follow-up will be necessary because malignancy cannot be excluded in terms of the genetic background of this tumor. The DAXX mutation site is located in the histone-binding domain (Fig. 2C) (20), which is functionally linked to formation of the ATRX-DAXX histone chaperone complex that is implicated in gene repression and telomere chromatin structure. Because this complex mediates histone 3.3 deposition, histone-3 lysine-9 trimethylation levels, and DNA methyltransferase 1 recruitment followed by alternative lengthening of telomere activation and/or chromosomal instability, the detected DAXX mutation may result in the loss of epigenetic regulation and thereby contribute to pathogenesis of the glucagonoma. Conclusion In our patient with glucagonoma, NME, and diabetes resolved with treatment. Hypoaminoacidemia was closely related to NME status. This article reports on biallelic DAXX inactivation in glucagonoma. Abbreviations: Abbreviations: ATRX alpha thalassemia/mental retardation syndrome X-linked DAXX death-domain associated protein G2 grade 2 LOH loss of heterozygosity NME necrolytic migratory erythema Oct-LAR octreotide long-acting release PanNET pancreatic neuroendocrine tumor VAF variant allele frequency WES whole-exome sequencing Acknowledgments We thank Erika Sugawara and Noriko Yamanaka for technical support. Financial Support: This work was supported by Grants-in Aid for the Foundation for Growth Science; Advanced Research and Development Programs for Medical Innovation, Scientific Research (B) 17H04037 (T.T.) and (C) 17K09875 (S.S.); Young Scientists (B) 17K16160 (I.S.); the Takeda Science Foundation (T.T.); Foundation for Growth Science in Japan (T.T.); SENSHINE Medical Research Foundation (T.T.); and the Cooperative Research Project Program of the Medical Institute of Bioregulation, Kyushu University (T.T.). 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Journal of Clinical Endocrinology and MetabolismOxford University Press

Published: Apr 23, 2018

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