Endocr Pathol DOI 10.1007/s12022-016-9415-x
Hypoglycemic Syndrome without Hyperinsulinemia. A Diagnostic Challenge Belén Pérez-Pevida 1 & Miguel Ángel Idoate 2 & Sara Fernández-Landázuri 3 & Nerea Varo 3 & Javier Escalada 1
# Springer Science+Business Media New York 2016
Abstract The most common cause of organic fasting hypoglycemia in adults is the presence of an insulin-producing pancreatic adenoma, but when high insulin levels are not found, the differential diagnosis is challenging. Misdiagnosis can lead to an unnecessary pancreatectomy. Insulin concentrations may be low in some cases despite a clinical history suggestive of insulinoma. In these cases, a proinsulinoma should be suspected, although the rarity of this condition requires an extensive workup before reaching a final diagnosis. We describe an unusual case of a 38-year-old man with a severe hypoglycemic syndrome due to a proinsulin-secreting pancreatic adenoma. Insulin was measured by the specific assay and suppressed under the lower detection limit during fasting hypoglycemia. Serum proinsulin and C-peptide levels were abnormally elevated, and further tests revealed an islet cell tumor. The tumor was surgically removed, relieving the fasting hypoglycemia. Histopathological study showed a conventional well-differentiated neuroendocrine tumor with high immunoreactivity against proinsulin and with lesser intensity against insulin. Interestingly, GS-9A8 antibody clone used for immunostaining proinsulin did not cross-react with human insulin or C-peptide, providing an unbiased picture of proinsulin secretion. The resolution of symptoms, the fall of
* Belén Pérez-Pevida [email protected]; [email protected]
1
Department of Endocrinology and Nutrition, Clínica Universidad de Navarra, Avenida Pio XII 36, 31008 Pamplona, Spain
2
Department of Pathology, Clínica Universidad de Navarra, Pamplona, Spain
3
Department of Biochemistry, Clínica Universidad de Navarra, Pamplona, Spain
proinsulin concentrations after tumor removal and the histopathology study confirmed the diagnosis of proinsulinoma. Keywords Proinsulinoma . Hypoglycemic syndrome . Proinsulin-producing pancreatic adenoma . Neuroendocrine tumors . Monoclonal antibody GS-9A8 against proinsulin Insulinomas are the most common cause of organic fasting hypoglycemia in adults and constitute approximately 20– 30 % of pancreatic neuroendocrine tumors [1, 2]. The classical clinical syndrome is due to low plasma glucose levels secondary to insulin hypersecretion. Hypoglycemia due to enhanced proinsulin secretion is infrequently reported. Clinical diagnosis is relatively complicated, and in certain cases, the difficulty is even greater, such as when low insulin concentrations occur despite a clinical history suggestive of insulinoma. In these cases, a proinsulinoma should be suspected, but the rarity of this neuroendocrine tumor requires an extensive workup before reaching a final diagnosis [2, 3].
Case Report A 38-year-old man was admitted for evaluation due to episodes of dizziness, together with loss of balance and lack of attention, for over a period of 4 years. He had been diagnosed with complex partial epileptic seizures by the neurology department, having initiated pharmacologic treatment with oxcarbazepine. These episodes had continued, with concomitant fasting glucose levels ranging from 2.2 to 3.3 mmol/L (40–60 mg/dL). The aforementioned symptoms appeared several hours after meals and disappeared after food intake, being more intense when doing exercise. As a result, he was studied by several endocrinologists
Endocr Pathol
without obtaining a definitive diagnosis. In October 2012, he was referred to our hospital due to persisting symptoms. The patient brought analytical studies that had been previously performed, showing the following results: glucose 2.53–3.79 mmol/L (46–69 mg/dL) [reference value (RV), 4.4–6.05 mmol/L (80–110 mg/dL)]; value suggestive for insulinoma (VSI), 1.5 ng/mL)], chromogranin A 146 ng/mL (RV, 0–100 ng/mL), and intact human proinsulin 29.9 pmol/L (RV, 0–6 pmol/L; VSI, >5 pmol/L). Growth hormone, IGF-1, parathyroid hormone, and calcitonin levels were within normal range. He had never had a fasting test. A previous abdominal computed tomography (CT) scan was normal. Weight had remained stable over the past 4 years, although in the last 6 months, he experienced a slight increase because of decreased physical activity. At the time of our medical consultation, there was nothing remarkable in the physical examination. A study consisting of continuous subcutaneous glucose monitoring was carried out over a week (CGMS) (iPro™2; Medtronic, Northridge, CA), observing an average glucose level below 4.12 mmol/L (75 mg/dL) and a minimum blood glucose average level below 4.12 mml/L (40 mg/dL). After this, the patient underwent a 72-h prolonged fasting test in hospital regime. Average capillary glucose levels were 64.5 mg/dL (81-38), blood glucose levels were 55 mg/dL (67-33), and basal insulin levels were below 2.0 μU/mL throughout the test. Ketone bodies were 0.03 mmol/L, and serum β-hydroxybutyrate was 0.02 mmol/L. At the beginning and the end of the test, C-peptide levels and intact human proinsulin were 1.01 and 1.37 ng/mL and 33 and 57.4 pMol/L, respectively. The fasting test was interrupted after 5 h due to plasma glucose of 2.09 mmol/L (38 mg/dL) and hypoglycemic symptoms. Plasma glucose was analyzed in a Cobas® 8000 (Roche) by the hexokinase method. Insulin and C-peptide were measured by a solid-phase enzyme-labeled chemiluminescent immunometric assay in the Immulite® 2000 analyzer (Siemens) using monoclonal antibodies (MAb). Proinsulin was measured by enzyme immunoassay, sulfonylurea by high-performance liquid chromatography, and anti-insulin antibodies by radioimmunoassay. During the hypoglycemic period, blood insulin levels were constantly undetectable. Heterophile antibodies and anti-insulin antibodies were negative (4.0 %) as well as plasma levels of metformin, sulfonylureas (glibenclamide, gliclazide, glimepiride, glipizide, gliquidone, glisentide, and chlorpropamide), pioglitazone, sitagliptin, vildagliptin, repaglinide, and nateglinide by liquid chromatography/mass spectrometry (LC-MS/MS). The low plasma insulin concentration during the hypoglycemic episodes and the elevated proinsulin levels suggested
a proinsulinoma or a predominantly proinsulin-secreting adenoma. Afterwards, the patient underwent an abdominal magnetic resonance which did not show any mass suggestive of a neuroendocrine tumor (Fig. 1a, b). Subsequently, an upper eco-endoscopy was conducted, which revealed a highly vascularized small nodule in the pancreatic head, consistent with a neuroendocrine tumor (Fig. 1c, d). Based on the clinical, biochemical, and imaging studies, the patient was referred to the Department of General Surgery and a tumor-enucleating surgery was performed on October 25, 2012. A nodular lesion of 1 cm in maximum diameter with irregular outer surface and yellowish gray coloration was described in the macroscopic description of the surgical piece. Representative samples of the tumor were obtained, fixed in formalin, and stained with H&E (Fig. 2). Immunohistochemistry was performed against chromogranin A, (clone DAK-A3, dilution, 1:200, DAKO), synaptophysin (clone DAK-SYNAP, dilution 1:200, DAKO), insulin (polyclonal, dilution, DAKO), proinsulin, and Ki-67 (clone MIB-1, dilution, DAKO). An antigen recovery by TrisCl/EDTA, at pH 9.0, and during 5 min at 98 °C, was carried out on every antibody. In the case of proinsulin, a MAb against proinsulin (clone GS-9A8, Developmental Studies Hybridoma Bank, University of Iowa) was used. PT link (DAKO) at pH 6, during 12 min and at 98 °C, was carried out. Endogenous peroxidase was blocked for 20 min. The slides were incubated with the primary antibody at 1:50 dilution, overnight, at room temperature. An amplification method using EnVision was used. Diaminobenzidine was applied as a chromogen. The slides were counterstained with hematoxylin. (Fig. 2a). A pathological study showed a pancreatic well-differentiated neuroendocrine tumor, as can be observed by the staining against both chromogranin A and synaptophysin. Interestingly, the immunostaining against proinsulin showed an intense and diffuse cell tumor expression with a granular cytoplasmic distribution (Fig. 2b). The insulin staining was moderate and diffuse. Tumor showed less than 1 % of proliferation index against Ki-67 and no mitotic figures. Three lymph nodes were also studied, showing no significant microscopic abnormalities. As a result, the tumor was diagnosed as a well-differentiated proinsulinoma and staged as pT1, N0, and G1, according to international TNM pathological staging recommendations for neuroendocrine tumors. Margins and lymph nodes studied were free of tumor. Postoperative period was uneventful. During hospitalization, we initiated treatment with octreotide (3 doses/day), which was maintained until the following checkup. Postsurgical hyperglycemia with daily requirements of 60 units of insulin was observed in the first 24 h, and afterwards, during the rest of the hospital stay, the maximum observed capillary blood glucose level was 10.39 mmol/L (189 mg/dL), and the patient did not require intravenous glucose delivery or subcutaneous insulin
Endocr Pathol
Fig. 1 Imaging studies. Axial T1-weighted magnetic resonance image, through the upper abdomen, in arterial (a) and portal venous (b) phase imaging, reveals a pancreas of normal size and morphology, without observing focal lesions. The second row shows images of EUS. In the pancreatic head, toward its medial edge, there is a discretely hypoechoic ovoid nodule with a hypoechoic halo, slightly encapsulated, measuring
12 × 8 × 9 mm in diameter (c), which exhibits high peripheral reticule vascularization (d). The lesion is consistent with a neuroendocrine tumor. The rest of the pancreas shows no abnormalities. The main pancreatic duct in its cephalic level is virtually undetectable and appears to be very close to the lesion. Other anomalies with this technique are not visualized
administration. The patient was discharged in stable condition on the 10th postoperative day. The patient came back for a checkup 2 months after surgery. During that period, he had not suffered any hypoglycemic
episodes, providing complete glycemic profiles made with his glucometer in which normal glucose values were confirmed. Six weeks after tumor removal, fasting glucose levels (5.22 mmol/L (95 mg/dL)), C-peptide (0.26 nmol/L
A Fig. 2 Histopathological analysis of the surgical piece. a A welldifferentiated neuroendocrine tumor with clearly delimited borders is observed. A uniformity of tumor cells is observed with a rather
B conventional aspect (H&E ×200). b Immunohistochemical staining. An intense expression of proinsulin is observed in tumor cells with a granular cytoplasmic distribution (DAB, ×200)
Endocr Pathol
(0.79 ng/mL)), proinsulin (1.4 pmol/L; 4.0 pg/mL), and insulin 2.4 μU/mL concentrations were all within normal range. Medical consultations continued, with the latest one taking place in July 2014. The patient is asymptomatic without evidence of recurrence.
Discussion The clinical history of this patient was highly suggestive of an organic hypoglycemia due to an insulinoma, although the analytical data showed suppressed insulin levels, thereby hampering a definitive diagnosis despite being evaluated by several endocrinologists. As a result, it was necessary to make a careful differential diagnosis in order to reach the correct definitive diagnosis. Patients suspected of having an insulinoma should undergo a monitored 72-h fasting test. Development of signs and symptoms of hypoglycemia, a low plasma glucose level, and relief of symptoms with glucose administration are called Whipple’s triad [1]. According to Per de Herder et al. [4], insulinoma has six strict diagnostic criteria, including a documented blood glucose of 2.47 mmol/L (45 mg/dL) or below, concomitant serum insulin levels of 36 μU/L or above [≥36 pmol/L; ≥3 μU/L by immunochemiluminometric assay (ICMA)], plasma/serum C-peptide levels of 200 pmol/L (0.6 ng/mL) or above, serum proinsulin levels of 5 pmol/L or above, serum B-hydroxybutyrate levels of 2.7 mmol/L or below, and the absence of sulfonylurea in the plasma or urine [5]. Although the patient’s clinical and pathological diagnosis justified the presence of a pancreatic neuroendocrine tumor, surprisingly, the plasma insulin levels were undetectable. This could be primarily explained by the presence of anti-insulin antibodies; however, they were negative in the patient’s blood. Moreover, to avoid possible interferences or the effect of extremely high concentrations of insulin, several dilutions of the sample were performed, but plasma insulin levels remained undetectable. Finally, other possible explanation could be an existing cross-reaction with proinsulin by the insulin assay used (Immulite® 2000 analyzer (Siemens)). Proinsulin is synthesized as a single chain, 110 amino acid (aa) preproprecursor that contains a 24-aa signal sequence and an 86-aa proinsulin. Following removal of the signal peptide, the proinsulin peptide undergoes further proteolysis to generate mature insulin, a 51-aa disulfide-linked dimer that consists of a 30-aa B chain (aa 25–54) bound to a 21-aa A chain (aa 90–110). The 34-aa intervening peptide (aa 55–89) that connects the B and A chains is termed the C-peptide. In the Immulite® insulin assay, the antibodies employed have epitopes on the A and B chains. This is the reason why there could be some cross-reactivities in the insulin assay to proinsulin since the A and B chains are
present in both molecules. This is a fact of all immunoassays for insulin. However, such low levels of insulin could hardly be explained just by this supposed cross-reaction. We then considered the diagnostic possibility of the existence of a proinsulinoma due to the presence of that high proinsulin levels with undetectable insulin [6]. However, proinsulin-only secreting tumors are very rare, while pancreatic endocrine tumors usually produce and secrete both insulin and proinsulin [7]. Therefore, pathologic confirmation was paramount. From a histopathological perspective, the tumor was a pancreatic, well-differentiated neuroendocrine tumor whose phenotype is similar to other pancreatic neuroendocrine neoplasias. However, the immunohistochemistry should be performed carefully and the MAb chosen the indicated because it has been described that the immunocytochemical application of a MAb against the Arg-Arg region at the junction of the C-peptide and the insulin p-chain of the human proinsulin molecule on rat pancreatic tissue has resulted in a positive immunogold labeling over the insulin as well as the glucagon-seating cells [8]. We chose the MAb GS-9A8 against proinsulin because it has been shown to be a IgG1-specific antibody which is linked to the B-C junction of proinsulin [9]. It is known that this MAb stains only intact, non-processed proinsulin found in the immature secretory granules of islet beta cells. Furthermore, it has been shown that this antibody does not cross-react with human insulin or C-peptide, yet there was no information about it’s possible cross-reaction with glucagon. This could be of interest for other purposes. The immunohistochemistry against proinsulin revealed an intense cytoplasmic expression, and only moderate staining against insulin was observed. The immunoreactivity observed against insulin in the tumor should be interpreted as the result of the immunoreaction of the polyclonal antibody against a portion of amino acid shared between the insulin and proinsulin molecules. Thus, this intense immunoreaction against proinsulin in the tumor cells correlated with the high proinsulin level detected in the peripheral blood. Consequently, the tumor was diagnosed as a proinsulinoma and not as an insulinoma. Pathologists must recognize the possibility of obtaining a false-positive finding with a polyclonal antibody against insulin. If not, proinsulinomas could be misdiagnosed. A conservative approach by enucleation of the tumor resulted in a favorable prognosis, without exposing the patient to a larger pancreatic resection and the risk of developing diabetes. Resolution of symptoms and normal proinsulin concentrations after removal of the tumor, together with the histological analysis, confirmed the diagnosis of proinsulinoma. Nearly two and a half years after diagnosis and treatment, the patient has not presented symptoms compatible with hypoglycemia and no analytical data indicate tumor recurrence. In conclusion, given the existence of a hypoglycemic syndrome, the first suspected diagnosis must remain insulinoma.
Endocr Pathol
Yet, when insulin levels do not accompany the diagnosis, other options must be considered, including proinsulinoma. Diagnosis requires the systematic measurement of proinsulin levels during the 72-h fasting test. Moreover, it is important to emphasize the need to use specific antibodies against proinsulin for correct characterization of the tumor.
3.
4. 5.
Compliance with Ethical Standards Conflict Interest The authors declare that they have no competing interests.
6.
7.
References 1.
2.
Goldin SB, Aston J and Wahi MM. Sporadically occurring functional pancreatic endocrine tumors: review of recent literature. Curr Opin Oncol 20: 1: 25-33, 2008. Grant CS. Insulinoma. Best Pract Res Clin Gastroenterol 19: 5: 783798, 2005.
8.
9.
Fadini GP, Maran A, Valerio A, Meduri F, Pelizzo M, Miotto D, Lanza C, Altavilla G and Avogaro A. Hypoglycemic syndrome in a patient with proinsulin-only secreting pancreatic adenoma (proinsulinoma). Case Rep Med 2011: 930904, 2011. de Herder WW. Biochemistry of neuroendocrine tumours. Best Pract Res Clin Endocrinol Metab 21: 1: 33-41, 2007. Vinik AI, Woltering EA, Warner RR, Caplin M, O'Dorisio TM, Wiseman GA, Coppola D, Go VL and North American Neuroendocrine Tumor Society (NANETS). NANETS consensus guidelines for the diagnosis of neuroendocrine tumor. Pancreas 39: 6: 713-734, 2010. Guettier JM, Lungu A, Goodling A, Cochran C and Gorden P. The role of proinsulin and insulin in the diagnosis of insulinoma: a critical evaluation of the Endocrine Society clinical practice guideline. J Clin Endocrinol Metab 98: 12: 4752-4758, 2013. Creutzfeldt W, Creutzfeldt C, Frerichs H, Track NS and Arnold R. Histochemistry, ultrastructure and hormone content of human insulinomas. Horm Metab Res Suppl 6: 7-18, 1976. Bendayan M. Possibilities of false immunocytochemical results generated by the use of monoclonal antibodies: the example of the antiproinsulin antibody. J Histochem Cytochem 43:9:881-886, 1995. Madsen OD, Cohen RM, Fitch FW, Rubenstein AH and Steiner DF. The production and characterization of monoclonal antibodies specific for human proinsulin using a sensitive microdot assay procedure. Endocrinology 113: 6: 2135-2144, 1983.
Hypoglycemic Syndrome without Hyperinsulinemia. A Diagnostic Challenge Belén Pérez-Pevida 1 & Miguel Ángel Idoate 2 & Sara Fernández-Landázuri 3 & Nerea Varo 3 & Javier Escalada 1
# Springer Science+Business Media New York 2016
Abstract The most common cause of organic fasting hypoglycemia in adults is the presence of an insulin-producing pancreatic adenoma, but when high insulin levels are not found, the differential diagnosis is challenging. Misdiagnosis can lead to an unnecessary pancreatectomy. Insulin concentrations may be low in some cases despite a clinical history suggestive of insulinoma. In these cases, a proinsulinoma should be suspected, although the rarity of this condition requires an extensive workup before reaching a final diagnosis. We describe an unusual case of a 38-year-old man with a severe hypoglycemic syndrome due to a proinsulin-secreting pancreatic adenoma. Insulin was measured by the specific assay and suppressed under the lower detection limit during fasting hypoglycemia. Serum proinsulin and C-peptide levels were abnormally elevated, and further tests revealed an islet cell tumor. The tumor was surgically removed, relieving the fasting hypoglycemia. Histopathological study showed a conventional well-differentiated neuroendocrine tumor with high immunoreactivity against proinsulin and with lesser intensity against insulin. Interestingly, GS-9A8 antibody clone used for immunostaining proinsulin did not cross-react with human insulin or C-peptide, providing an unbiased picture of proinsulin secretion. The resolution of symptoms, the fall of
* Belén Pérez-Pevida [email protected]; [email protected]
1
Department of Endocrinology and Nutrition, Clínica Universidad de Navarra, Avenida Pio XII 36, 31008 Pamplona, Spain
2
Department of Pathology, Clínica Universidad de Navarra, Pamplona, Spain
3
Department of Biochemistry, Clínica Universidad de Navarra, Pamplona, Spain
proinsulin concentrations after tumor removal and the histopathology study confirmed the diagnosis of proinsulinoma. Keywords Proinsulinoma . Hypoglycemic syndrome . Proinsulin-producing pancreatic adenoma . Neuroendocrine tumors . Monoclonal antibody GS-9A8 against proinsulin Insulinomas are the most common cause of organic fasting hypoglycemia in adults and constitute approximately 20– 30 % of pancreatic neuroendocrine tumors [1, 2]. The classical clinical syndrome is due to low plasma glucose levels secondary to insulin hypersecretion. Hypoglycemia due to enhanced proinsulin secretion is infrequently reported. Clinical diagnosis is relatively complicated, and in certain cases, the difficulty is even greater, such as when low insulin concentrations occur despite a clinical history suggestive of insulinoma. In these cases, a proinsulinoma should be suspected, but the rarity of this neuroendocrine tumor requires an extensive workup before reaching a final diagnosis [2, 3].
Case Report A 38-year-old man was admitted for evaluation due to episodes of dizziness, together with loss of balance and lack of attention, for over a period of 4 years. He had been diagnosed with complex partial epileptic seizures by the neurology department, having initiated pharmacologic treatment with oxcarbazepine. These episodes had continued, with concomitant fasting glucose levels ranging from 2.2 to 3.3 mmol/L (40–60 mg/dL). The aforementioned symptoms appeared several hours after meals and disappeared after food intake, being more intense when doing exercise. As a result, he was studied by several endocrinologists
Endocr Pathol
without obtaining a definitive diagnosis. In October 2012, he was referred to our hospital due to persisting symptoms. The patient brought analytical studies that had been previously performed, showing the following results: glucose 2.53–3.79 mmol/L (46–69 mg/dL) [reference value (RV), 4.4–6.05 mmol/L (80–110 mg/dL)]; value suggestive for insulinoma (VSI), 1.5 ng/mL)], chromogranin A 146 ng/mL (RV, 0–100 ng/mL), and intact human proinsulin 29.9 pmol/L (RV, 0–6 pmol/L; VSI, >5 pmol/L). Growth hormone, IGF-1, parathyroid hormone, and calcitonin levels were within normal range. He had never had a fasting test. A previous abdominal computed tomography (CT) scan was normal. Weight had remained stable over the past 4 years, although in the last 6 months, he experienced a slight increase because of decreased physical activity. At the time of our medical consultation, there was nothing remarkable in the physical examination. A study consisting of continuous subcutaneous glucose monitoring was carried out over a week (CGMS) (iPro™2; Medtronic, Northridge, CA), observing an average glucose level below 4.12 mmol/L (75 mg/dL) and a minimum blood glucose average level below 4.12 mml/L (40 mg/dL). After this, the patient underwent a 72-h prolonged fasting test in hospital regime. Average capillary glucose levels were 64.5 mg/dL (81-38), blood glucose levels were 55 mg/dL (67-33), and basal insulin levels were below 2.0 μU/mL throughout the test. Ketone bodies were 0.03 mmol/L, and serum β-hydroxybutyrate was 0.02 mmol/L. At the beginning and the end of the test, C-peptide levels and intact human proinsulin were 1.01 and 1.37 ng/mL and 33 and 57.4 pMol/L, respectively. The fasting test was interrupted after 5 h due to plasma glucose of 2.09 mmol/L (38 mg/dL) and hypoglycemic symptoms. Plasma glucose was analyzed in a Cobas® 8000 (Roche) by the hexokinase method. Insulin and C-peptide were measured by a solid-phase enzyme-labeled chemiluminescent immunometric assay in the Immulite® 2000 analyzer (Siemens) using monoclonal antibodies (MAb). Proinsulin was measured by enzyme immunoassay, sulfonylurea by high-performance liquid chromatography, and anti-insulin antibodies by radioimmunoassay. During the hypoglycemic period, blood insulin levels were constantly undetectable. Heterophile antibodies and anti-insulin antibodies were negative (4.0 %) as well as plasma levels of metformin, sulfonylureas (glibenclamide, gliclazide, glimepiride, glipizide, gliquidone, glisentide, and chlorpropamide), pioglitazone, sitagliptin, vildagliptin, repaglinide, and nateglinide by liquid chromatography/mass spectrometry (LC-MS/MS). The low plasma insulin concentration during the hypoglycemic episodes and the elevated proinsulin levels suggested
a proinsulinoma or a predominantly proinsulin-secreting adenoma. Afterwards, the patient underwent an abdominal magnetic resonance which did not show any mass suggestive of a neuroendocrine tumor (Fig. 1a, b). Subsequently, an upper eco-endoscopy was conducted, which revealed a highly vascularized small nodule in the pancreatic head, consistent with a neuroendocrine tumor (Fig. 1c, d). Based on the clinical, biochemical, and imaging studies, the patient was referred to the Department of General Surgery and a tumor-enucleating surgery was performed on October 25, 2012. A nodular lesion of 1 cm in maximum diameter with irregular outer surface and yellowish gray coloration was described in the macroscopic description of the surgical piece. Representative samples of the tumor were obtained, fixed in formalin, and stained with H&E (Fig. 2). Immunohistochemistry was performed against chromogranin A, (clone DAK-A3, dilution, 1:200, DAKO), synaptophysin (clone DAK-SYNAP, dilution 1:200, DAKO), insulin (polyclonal, dilution, DAKO), proinsulin, and Ki-67 (clone MIB-1, dilution, DAKO). An antigen recovery by TrisCl/EDTA, at pH 9.0, and during 5 min at 98 °C, was carried out on every antibody. In the case of proinsulin, a MAb against proinsulin (clone GS-9A8, Developmental Studies Hybridoma Bank, University of Iowa) was used. PT link (DAKO) at pH 6, during 12 min and at 98 °C, was carried out. Endogenous peroxidase was blocked for 20 min. The slides were incubated with the primary antibody at 1:50 dilution, overnight, at room temperature. An amplification method using EnVision was used. Diaminobenzidine was applied as a chromogen. The slides were counterstained with hematoxylin. (Fig. 2a). A pathological study showed a pancreatic well-differentiated neuroendocrine tumor, as can be observed by the staining against both chromogranin A and synaptophysin. Interestingly, the immunostaining against proinsulin showed an intense and diffuse cell tumor expression with a granular cytoplasmic distribution (Fig. 2b). The insulin staining was moderate and diffuse. Tumor showed less than 1 % of proliferation index against Ki-67 and no mitotic figures. Three lymph nodes were also studied, showing no significant microscopic abnormalities. As a result, the tumor was diagnosed as a well-differentiated proinsulinoma and staged as pT1, N0, and G1, according to international TNM pathological staging recommendations for neuroendocrine tumors. Margins and lymph nodes studied were free of tumor. Postoperative period was uneventful. During hospitalization, we initiated treatment with octreotide (3 doses/day), which was maintained until the following checkup. Postsurgical hyperglycemia with daily requirements of 60 units of insulin was observed in the first 24 h, and afterwards, during the rest of the hospital stay, the maximum observed capillary blood glucose level was 10.39 mmol/L (189 mg/dL), and the patient did not require intravenous glucose delivery or subcutaneous insulin
Endocr Pathol
Fig. 1 Imaging studies. Axial T1-weighted magnetic resonance image, through the upper abdomen, in arterial (a) and portal venous (b) phase imaging, reveals a pancreas of normal size and morphology, without observing focal lesions. The second row shows images of EUS. In the pancreatic head, toward its medial edge, there is a discretely hypoechoic ovoid nodule with a hypoechoic halo, slightly encapsulated, measuring
12 × 8 × 9 mm in diameter (c), which exhibits high peripheral reticule vascularization (d). The lesion is consistent with a neuroendocrine tumor. The rest of the pancreas shows no abnormalities. The main pancreatic duct in its cephalic level is virtually undetectable and appears to be very close to the lesion. Other anomalies with this technique are not visualized
administration. The patient was discharged in stable condition on the 10th postoperative day. The patient came back for a checkup 2 months after surgery. During that period, he had not suffered any hypoglycemic
episodes, providing complete glycemic profiles made with his glucometer in which normal glucose values were confirmed. Six weeks after tumor removal, fasting glucose levels (5.22 mmol/L (95 mg/dL)), C-peptide (0.26 nmol/L
A Fig. 2 Histopathological analysis of the surgical piece. a A welldifferentiated neuroendocrine tumor with clearly delimited borders is observed. A uniformity of tumor cells is observed with a rather
B conventional aspect (H&E ×200). b Immunohistochemical staining. An intense expression of proinsulin is observed in tumor cells with a granular cytoplasmic distribution (DAB, ×200)
Endocr Pathol
(0.79 ng/mL)), proinsulin (1.4 pmol/L; 4.0 pg/mL), and insulin 2.4 μU/mL concentrations were all within normal range. Medical consultations continued, with the latest one taking place in July 2014. The patient is asymptomatic without evidence of recurrence.
Discussion The clinical history of this patient was highly suggestive of an organic hypoglycemia due to an insulinoma, although the analytical data showed suppressed insulin levels, thereby hampering a definitive diagnosis despite being evaluated by several endocrinologists. As a result, it was necessary to make a careful differential diagnosis in order to reach the correct definitive diagnosis. Patients suspected of having an insulinoma should undergo a monitored 72-h fasting test. Development of signs and symptoms of hypoglycemia, a low plasma glucose level, and relief of symptoms with glucose administration are called Whipple’s triad [1]. According to Per de Herder et al. [4], insulinoma has six strict diagnostic criteria, including a documented blood glucose of 2.47 mmol/L (45 mg/dL) or below, concomitant serum insulin levels of 36 μU/L or above [≥36 pmol/L; ≥3 μU/L by immunochemiluminometric assay (ICMA)], plasma/serum C-peptide levels of 200 pmol/L (0.6 ng/mL) or above, serum proinsulin levels of 5 pmol/L or above, serum B-hydroxybutyrate levels of 2.7 mmol/L or below, and the absence of sulfonylurea in the plasma or urine [5]. Although the patient’s clinical and pathological diagnosis justified the presence of a pancreatic neuroendocrine tumor, surprisingly, the plasma insulin levels were undetectable. This could be primarily explained by the presence of anti-insulin antibodies; however, they were negative in the patient’s blood. Moreover, to avoid possible interferences or the effect of extremely high concentrations of insulin, several dilutions of the sample were performed, but plasma insulin levels remained undetectable. Finally, other possible explanation could be an existing cross-reaction with proinsulin by the insulin assay used (Immulite® 2000 analyzer (Siemens)). Proinsulin is synthesized as a single chain, 110 amino acid (aa) preproprecursor that contains a 24-aa signal sequence and an 86-aa proinsulin. Following removal of the signal peptide, the proinsulin peptide undergoes further proteolysis to generate mature insulin, a 51-aa disulfide-linked dimer that consists of a 30-aa B chain (aa 25–54) bound to a 21-aa A chain (aa 90–110). The 34-aa intervening peptide (aa 55–89) that connects the B and A chains is termed the C-peptide. In the Immulite® insulin assay, the antibodies employed have epitopes on the A and B chains. This is the reason why there could be some cross-reactivities in the insulin assay to proinsulin since the A and B chains are
present in both molecules. This is a fact of all immunoassays for insulin. However, such low levels of insulin could hardly be explained just by this supposed cross-reaction. We then considered the diagnostic possibility of the existence of a proinsulinoma due to the presence of that high proinsulin levels with undetectable insulin [6]. However, proinsulin-only secreting tumors are very rare, while pancreatic endocrine tumors usually produce and secrete both insulin and proinsulin [7]. Therefore, pathologic confirmation was paramount. From a histopathological perspective, the tumor was a pancreatic, well-differentiated neuroendocrine tumor whose phenotype is similar to other pancreatic neuroendocrine neoplasias. However, the immunohistochemistry should be performed carefully and the MAb chosen the indicated because it has been described that the immunocytochemical application of a MAb against the Arg-Arg region at the junction of the C-peptide and the insulin p-chain of the human proinsulin molecule on rat pancreatic tissue has resulted in a positive immunogold labeling over the insulin as well as the glucagon-seating cells [8]. We chose the MAb GS-9A8 against proinsulin because it has been shown to be a IgG1-specific antibody which is linked to the B-C junction of proinsulin [9]. It is known that this MAb stains only intact, non-processed proinsulin found in the immature secretory granules of islet beta cells. Furthermore, it has been shown that this antibody does not cross-react with human insulin or C-peptide, yet there was no information about it’s possible cross-reaction with glucagon. This could be of interest for other purposes. The immunohistochemistry against proinsulin revealed an intense cytoplasmic expression, and only moderate staining against insulin was observed. The immunoreactivity observed against insulin in the tumor should be interpreted as the result of the immunoreaction of the polyclonal antibody against a portion of amino acid shared between the insulin and proinsulin molecules. Thus, this intense immunoreaction against proinsulin in the tumor cells correlated with the high proinsulin level detected in the peripheral blood. Consequently, the tumor was diagnosed as a proinsulinoma and not as an insulinoma. Pathologists must recognize the possibility of obtaining a false-positive finding with a polyclonal antibody against insulin. If not, proinsulinomas could be misdiagnosed. A conservative approach by enucleation of the tumor resulted in a favorable prognosis, without exposing the patient to a larger pancreatic resection and the risk of developing diabetes. Resolution of symptoms and normal proinsulin concentrations after removal of the tumor, together with the histological analysis, confirmed the diagnosis of proinsulinoma. Nearly two and a half years after diagnosis and treatment, the patient has not presented symptoms compatible with hypoglycemia and no analytical data indicate tumor recurrence. In conclusion, given the existence of a hypoglycemic syndrome, the first suspected diagnosis must remain insulinoma.
Endocr Pathol
Yet, when insulin levels do not accompany the diagnosis, other options must be considered, including proinsulinoma. Diagnosis requires the systematic measurement of proinsulin levels during the 72-h fasting test. Moreover, it is important to emphasize the need to use specific antibodies against proinsulin for correct characterization of the tumor.
3.
4. 5.
Compliance with Ethical Standards Conflict Interest The authors declare that they have no competing interests.
6.
7.
References 1.
2.
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