- Email: [email protected]
Morphologic analysis of focal and diffuse forms of congenital hyperinsulinism
Seminars in Pediatric Surgery (2011) 20, 3-12
Morphologic analysis of focal and diffuse forms of congenital hyperinsulinism Jacques Rahier, MD, PhD, Yves Guiot, PhD, Christine Sempoux, MD, PhD From the Department of Pathology, Cliniques Universitaires Saint-Luc, Université catholique de Louvain, Tour Rosalind Franklin, Brussels, Belgium. KEYWORDS Hypoglycemia; Histology; Islets of Langerhans; Nesidioblastosis; Congenital hyperinsulinism
Congenital hyperinsulinism is clinically characterized by an inappropriate insulin secretion resulting in recurrent severe hypoglycemia. Nesidioblastosis, the proliferation of islet cells budding off from ducts, has been considered for years as the histologic lesion responsible for the syndrome. In our morphologic studies, we demonstrate that nesidioblastosis is not specific of the disease, which is actually not a single entity. Indeed, we recognize the existence of 2 different forms—a diffuse form and a focal form—and demonstrate that they can be differentiated by morphologic criteria, even on frozen sections during surgery. This histologic distinction directs the therapeutic approach because the patients experiencing the focal form of the syndrome can be completely cured by a very limited pancreatectomy. Molecular findings confirmed the reliability of this histologic distinction, showing a specific background for each form. © 2011 Elsevier Inc. All rights reserved.
Hypoglycemia is not a rare event in neonates and infants and has various causes.1 It can be the result of a lack of hepatic production of glucose, mostly related to congenital metabolic diseases, or the consequence of hormonal imbalance linked to pituitary, adrenal, or thyroid deficiency. Hypoglycemia is also observed in small-for-date neonates, after salicylate consumption or in Reye syndrome. However, recurrent severe hypoglycemia may also be related to inappropriately elevated plasma insulin levels, which is the topic of this review. The syndrome of persistent hyperinsulinemic hypoglycemia of infancy (PHHI) was recognized more than 50 years ago by McQuarrie.2 The estimated incidence of PHHI in the general population is 1/30,000 to 1/50,000 but it increases to 1/2500 in communities with substantial in-
Address reprint requests and correspondence: Jacques Rahier, MD, PhD, Department of Pathology, Cliniques Universitaires Saint-Luc, Université catholique de Louvain, Tour Rosalind Franklin, 10 Avenue Hippocrate, 1200, Brussels, Belgium. E-mail address: [email protected].
1055-8586/$ -see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1053/j.sempedsurg.2010.10.010
breeding. Despite the number of studies devoted to this subject, the pathogenicity of the disease remained unsolved for several decades.3,4 Fortunately, major progress in genetic and molecular biology has brought enlightenment in most cases.5-13 The age at onset is quite variable: the vast majority of cases are revealed early in life, a few hours or days after birth, but occasionally, symptoms can take as long as 5 or 6 months to become apparent. Most infants with an early onset of symptoms have a heavy birth weight. The severity of the disease is quite variable but tends to be greater in the neonatal form, many patients remaining unresponsive to diazoxide.14 In those infants who remain nonresponsive to medical treatment, persistent hyperinsulinic hypoglycemia can cause great neurological damage.5,15 For many years, surgery was the only therapeutic option. The extent of surgical resection has long been a matter of debate, with some surgeons recommending a 95% pancreatic resection to avoid recurrence of the disease. However, subsequent diabetes is a major side effect of resection.16-18
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Seminars in Pediatric Surgery, Vol 20, No 1, February 2011
Figure 1 Nesidioblastosis B cells budding off from ducts in a case of diffuse PHHI. (Color version of figure is available online.)
The 2 first cases we encountered in the 1980s were of particular interest, with very curious clinical progress: both were of neonatal onset, had a high birth weight and were insensitive to diazoxide.19 Both pancreases were apparently macroscopically normal, with no evident lesion at microscopy, but the first one was not cured despite a near-total pancreatectomy (in 2 steps) and the second case was cured by a hemipancreatectomy. At that time, people used to consider that “nesidioblastosis” was the underlying pathologic condition responsible for the disease. Nesidioblastosis was defined by G. Laidlaw20 as a diffuse and disseminated proliferation of islet cells budding off from ducts (Figure 1) and the PHHI syndrome was also called nesidioblastosis. Judging by the strange
Figure 2
evolution of these cases, we were at that time already convinced that PHHI was not a single entity. By conducting further investigations in the patient cured by partial pancreatectomy, we realized that, although there was no visible lesion either macroscopically or after conventional Hemalun Eosine staining, immunohistochemical analysis with the use of an antibody directed against insulin revealed a small focal endocrine “tumor-like” lesion (Figure 2), the removal of which was sufficient and necessary for the patient to be cured.19 However, despite careful analysis, such a focal lesion was never found in the other patient who remained sick despite near-total pancreatectomy. Investigations of the pathogenicity of the nonfocal, also called the diffuse type of PHHI, were the next step: first we wondered whether “nesidioblastosis” really existed at all and whether that process could be responsible for hyperinsulinism. Morphologic analysis clearly showed that nesidioblastosis, as defined previously, was present not only in symptomatic but also in normoglycemic control patients of a similar age (Figure 3): it could thus not be considered pathognomonic of this syndrome. Quantitative double immunohistochemical techniques with simultaneously anti-insulin and Ki-67 antibodies enabled Sempoux et al21 to demonstrate that there was no proliferation in these small groups of B cells in PHHI (Figure 4). Furthermore, when the number of B cells was measured by the point counting method, we observed that there was no significant difference in the numbers of pancreatic B cells between normoglycemic infants and patients with the diffuse type of PHHI (Figure 5). We therefore suggested that the nonfocal or diffuse type of PHHI was related to a B-cell functional abnormality.19
Small focal adenomatous hyperplasia not recognized on hematoxylin and eosin staining. (Color version of figure is available online.)
Rahier et al
Morphologic Analysis of Focal and Diffuse Forms of CHI
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Figure 5 The amount of B cells is similar in normoglycemic controls and in infants with diffuse PHHI.
further concluded that nesidioblastosis does exist, but that it is not specific to PHHI and does not correspond to permanent B cell proliferation.
Subclassification and characteristics of the different PHHI types Figure 3 Nesidioblastosis in cases of normoglycemic infant (A) or infant with diffuse PHHI (B). (Color version of figure is available online.)
We were therefore able to conclude at that time in the early eighties that different types of PHHI indeed do exist: one is secondary to a focal tumor-like lesion and can be cured by partial resection, the other appears to be a diffuse lesion that cannot be cured by partial pancreatectomy. It was
Figure 4
PHHI does not actually correspond to a single entity.5-13,19 Many different forms have now been reported on the basis of specific genetic defects. According to the type of genetic abnormality, the histologic feature may be different and the morphologic pattern may help to differentiate the various entities. On a practical or surgical point of view, one of the most important distinction remains that of focal versus diffuse character of the lesion because a focal lesion may be cured by a resection limited to the abnormal focus.
Nesidioblastosis: not a permanent proliferation of B cells. (Color version of figure is available online.)
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Figure 6 Focal adenomatous hyperplasia is made by the confluence of hyperplastic but normally structured islets with a peripheral rim of non-B cells. (Color version of figure is available online.)
Focal type of PHHI The focal type is a focal adenomatous hyperplasia,19,22 characterized by the presence of a small endocrine lesion in the pancreas, histologically corresponding to the confluence of hyperplastic but apparently normally structured islets, with a large core of B cells and a peripheral rim of endocrine non-B cells (Figure 6). Within the focal lesion, the B cells are hyperactive, with an enlarged cytoplasm and a large Golgi region full of proinsulin but relatively few insulin granules and little insulin labeling because the lesion hypersecretes and does not store insulin. The lesion may be very small and its limits are sometimes irregular. Outside the focus the islets are small, their B cells are resting with a high storage of insulin and a weak production of proinsulin (Figure 7). Their cytoplasm is shrunken and their nuclei are small.23,24 The focal adenomatous hyperplasia is different from adenoma: adenoma do not seem to be formed by the confluence of true islets and we have never observed adenomas in neonates or infants. Focal adenomatous hyperplasia is related to an loss of heterozygosity of 11p15 on the maternal allele and a sulfonylurea receptor (SUR)
mutation on the paternal one.25-28 This can be easily demonstrated because p57 is located in this region and is thus not expressed in cases with 11p15 loss of heterozygosity (Figure 8).29,30 Focal adenomatous hyperplasia differs from adenoma or insulinoma both morphologically and by molecular biology.30 Selective resection restricted to the lesion cures the patient provided the resection is complete and the lesion solitary.
Diffuse type of PHHI In the diffuse type of PHHI the pancreas appears normal both macro- and microscopically, but even near-total pancreatectomy can be insufficient to cure the patient.19,22 Careful analysis of the morphology of the islets of Langerhans uncovers the presence of large B cells with abnormally large nuclei as observed in hyperactive endocrine cells (Figure 9). Immunostaining with a specific antibody to proinsulin reveals the labeling of a large Golgi apparatus reflecting a very high activity of proinsulin synthesis. However, there is only very weak labeling with insulin antibody, traducing low insulin storage because of a major nonregulated insulin release. This
Figure 7 (A) Islets from outside a focal lesion. (B) Cells have reduced cytoplasm and small nuclei and low proinsulin production. (Color version of figure is available online.)
Rahier et al
Morphologic Analysis of Focal and Diffuse Forms of CHI
Figure 8
Figure 9 line.)
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p57 within the focal lesion (A) or outside (B). (Color version of figure is available online.)
Islets from diffuse type of PHHI (A and C) and from normoglycemic control (B). (Color version of figure is available on-
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Figure 10 Normal insulin content (A), proinsulin production (B), and SUR 1 expression (C) in a case of GLUD overactivity. (Color version of figure is available online.)
particular aspect contrasts very much with that of islets located outside the lesion in cases of focal adenomatous hyperplasia. It is now well demonstrated that these diffuse lesions are secondary to a mutation of one of the genes of the KATP-dependent channel.31,32 The KATP-dependent channels are composed of 2 subunits: the sulfonylurea receptor, ABCC8 (previously SUR 1), and a member of the inwardly rectifying potassium channels, KCNJ11 (previously Kir 6.2). The permanent closure of these channels caused by their absence or inefficiency increases intracellular K⫹ and depolarizes the B-cell membrane. This leads to the opening of the voltage-dependent Ca2⫹ channels, which in turn, activates the exocytic machinery for insulin secretion. Only in cases of SUR mutation leading to a total lack of SUR synthesis, the negativity of SUR immunocytochemistry is useful for diagnosis. The diffuse type of PHHI used to represent 60% of all operated cases before the distinction of the various types of PHHI was made. This percentage is nowadays greater because the tendency is to be conservative as much as possible in the diffuse form.
Other forms of PHHI The 2 types we described in the previous chapter are both related to a pathology of the sulfonylurea receptor (the K⫹ channel) and represent the more frequent types of PHHI in the material we analyzed (nearly 90%). Other causes exist but their frequency is much lower in the material we analyzed.
in the regulatory domain of the GLUD1 gene with resulting impaired sensitivity of the enzyme to the inhibition by guanosine triphosphate.13,33 Insulin secretion, in infants with this abnormality, is triggered by the consumption of highprotein diets. These infants therefore present a severe hypoglycemia after an oral leucine load, known in the early PHHI literature as leucine sensitive hypoglycemia. The 2 cases we were able to analyze were macroscopically unremarkable. Microscopically, a few B-cell nuclei showed a moderate increase in size, but the cytoplasm remained unchanged. At immunohistochemistry, insulin staining was not lowered as in the classical diffuse form, proinsulin production was high and SUR1 expression was normal (Figure 10).
Glucokinase overactivity In 1998, Glaser et al34 reported a family with autosomal dominant familial hyperinsulinism suggesting a defect in the glucose threshold at which insulin secretion is switched off. Other cases were reported by the group of CuestaMunoz and that Stanley.35-37 It is a very rare cause of PHHI. In the 2 cases we had the opportunity to analyze, the pancreas was reported as macroscopically normal. At microscopy, a few rare B-cell nuclei were slightly increased in size, and immunohistochemistry staining for insulin and proinsulin was normal. The only difference with the pancreas of age-matched controls was a major increase in the islet size (Figure 11). This peculiarity was not reported in the cases analyzed in by researchers in Philadelphia.36
Glutamate deshydrogenase hyperactivity Increased insulin secretion can also be consecutive to overactivity of glutamate deshydrogenase. This is the case of patients with autosomal dominant hyperinsulinism-hyperammonemia syndrome. It results from genomic mutations
Atypical forms A few cases have been classified as atypical because they did not fit the described categories. Symptoms of most of these patients do not appear at birth but later, at 5 or 6
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Morphologic Analysis of Focal and Diffuse Forms of CHI
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Figure 11 Normoglycemic control (A) and a case with glucokinase overactivity (B). The size of the islets is higher in this type of PHHI than in control patients. (Color version of figure is available online.)
months, and are less prominent than in the usual diffuse form. These infants are often improved by diazoxide therapy, suggesting that KATP channels are not involved in this
Figure 12
pathology. Some islets show signs of hyperactivity but others appear normal. The genetic abnormalities underlying this type of PHHI are so far unknown.38
Mean radius of 50 selected B-cell nuclei measured outside a focal lesion or in cases devoid of focal lesion.
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Figure 13 B cells from inside (A) or outside (B) a focal lesion and in diffuse type of PHHI in frozen sections; the B cells abnormalities (C) are easily recognized with toluidine blue staining. (Color version of figure is available online.)
The challenge The distinction between the focal and diffuse types of PHHI is very important because it has a major impact on the therapeutic management of the patient. The differential diagnosis long relied on morphologic analysis, and because the focal lesion might be very small, multiple sections were required to ascertain that the lesion was actually diffuse rather than focal. Furthermore, the diagnosis always arrived after the pancreas had been removed, sometimes after total pancreatectomy had been performed for a very small lesion susceptible to be cured by a small resection restricted to the focal lesion. We therefore decided to reanalyze our series of pancreases removed for PHHI with to analyze whether differences existed between islets of the diffuse types and islets outside a focal lesion. It was then observed that in diffuse PHHI the islets consisted of hyperactive B cells while those outside a focal lesion were resting islets. The existence of objective differences between resting and hyperactive B cells was first evaluated on paraffin retrospective material by measuring the size of the B-cell nuclei and the nuclear B-cell crowding (which is the main number of B cell nuclei per 100 squared micrometers). Figure 12 shows major and highly significant differences for both parameters between B cells from diffuse and focal types of PHHI.23 In a second step, a prospective study was performed. Frozen sections were made during surgery on small specimens (3-4 mm in large axis) obtained from the head, isthmus, corpus and tail of the pancreas, with the objective of assessing the presence of resting islets with shrunken B-cell cytoplasm or that of islets with hyperactive B cells with abnormally large cytoplasm and B cell nuclei (Figure 13). This technique enabled us to make the right distinction in most cases between focal versus diffuse types of PHHI and
in all cases with neonatal onset, large birth weight, and resistance to diazoxide.39,40 Before the arrival of selective venous samplings and later, of the 18-fluorodopa positron emission tomography scan, it was the only way to distinguish focal from diffuse disease before pancreatectomy. Nowadays, intraoperative frozen sections remain very useful to ascertain the adequacy of the diagnosis, recognize the atypical or bizarre forms and most of all to check during surgery whether the resection margins are free from lesion.
Difficulties in management Some cases may be very difficult to manage by the pathologist during surgery. Cases with a pathology related to
Figure 14 The limits of a focal lesion may be very irregular. (Color version of figure is available online.)
Rahier et al
Morphologic Analysis of Focal and Diffuse Forms of CHI
Figure 15 The focal lesions are sometimes very small and difficult to share for the various analyses. (Color version of figure is available online.)
KATP channels are usually easy to analyze. Sometimes the frequency of B-cell occurrence with large nuclei is not as high as usual and the recognition of a lesion as a diffuse type may not be so evident. On fortunately rare occasions, hyperinsulinism results from bifocal lesions that are only revealed after surgery, because the infant was not cured by an apparently complete resection of the lesion. More frequently, the focal lesion is small, not actually recognizable at the naked eye, but the limits of the abnormal focus is markedly irregular (Figure 14), making it difficult to ascertain whether resection is complete. One case of a focal lesion involving more than 90% of the pancreas has also been observed, the absence of p57 labeling and its persistence in the healthy part of the pancreas enabled the confirmation of the diagnosis of focal PHHI in this particular case. In general, atypical cases, such as glucokinase or glutamate dehydrogenase activation are always more difficult to diagnose on per surgical frozen sections than hypoglycemias secondary to K channelopathy. Last but not least, the pathologist also has to share the material and that is not easy, because the lesion, when focal, may be very small, and physiological studies (electrophysiology or insulin secretion studies) require that the tissue given to physiologists must not only be well characterized but also still living (Figure 15).
References 1. Haymond MW. Hypoglycemia in infants and children. Endocrinol Metab Clin North Am 1989;18:211-52. 2. McQuarrie I. Idiopathic spontaneously occurring hypoglycemia in infants: clinical significance of problem and treatment. Am J Dis Child 1954;87:399-428. 3. Aynsley-Green A. Nesidioblastosis of the pancreas in infancy. Dev Med Child Neurol 1981;23:372-9. 4. Landau H, Perlman M, Meyer S, et al. Persistent neonatal hypoglycemia due to hyperinsulinism: medical aspects. Pediatrics 1982;70: 440-6.
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5. de Lonlay-Debeney P, Fournet J-C, Touati G, et al. Heterogeneity of persistent hyperinsulinaemic hypoglycemia. A series of 175 cases. Eur J Pediatr 2002;161:37-48. 6. Aguilar-Bryan L, Bryan J, Nakazaki M. Of mice and men: K(ATP) channels and insulin secretion. Recent Prog Horm Res 2001;56:47-68. 7. Aguilar-Bryan L, Nichols C, Wechsler S, et al. Cloning of the beta cell high-affinity sulfonylurea receptor: a regulator of insulin secretion. Science 1995;268:423-6. 8. Glaser B, Chiu KC, Anker R, et al. Familial hyperinsulinism maps to chromosome 11p14-15.1, 30 cM centromeric to the insulin gene. Nat Genet 1994;7:185-8. 9. Glaser B, Thornton P, Otonkoski T, et al. Genetics of neonatal hyperinsulinism. Arch Disord Child Fetal Neonat 2000;82:F79-86. 10. Meissner T, Brune W, Mayatepek E. Persistent hyperinsulinemic hypoglycemia of infancy: therapy, clinical outcome and mutational analysis. Eur J Pediatr 1997;156:754-7. 11. Otonkoski T, ämmälä C, Huopio H, et al. A point mutation inactivating the sulfonylurea receptor causes the severe form of persistent hyperinsulinemic hypoglycemia of infancy in Finland. Diabetes 1999;48: 408-15. 12. Nestorowicz A, Wilson BA, Schoor KP, et al. Mutations in the sulfonylurea receptor gene are associated with familial hyperinsulinism in Ashkenazi Jews. Hum Mol Genet 1996;5:1813-22. 13. Stanley C, Lieu Y, Hsu B, et al. Hyperinsulinism and hyperammonemia in infants with regulatory mutations of the glutamate dehydrogenase gene. N Engl J Med 1998;338:1352-7. 14. de Lonlay-Debeney P, Fournet JC, Martin D, et al. Hypoglycémie hyperinsulinémique persistante du nouveau-né et du nourrisson. Arch Pediatr 1998;5:1347-52. 15. Aynsley-Green A, Polak JM, Bloom SR, et al. Nesidioblastosis of the pancreas: definition of the syndrome and the management of the severe neonatal hyperinsulinemic hypoglycemia. Arch Dis Child 1981;56: 496-508. 16. Shilyansky J, Fisher S, Cutz E, et al. Is 95% pancreatectomy the procedure of choice for treatment of persistent hyperinsulinemic hypoglycaemia of the neonate? J Pediatr Surg 1997;32:342-6. 17. Lovvorn HN, Nance ML, Ferry RJ, et al. Congenital hyperinsulinism and the surgeon: lessons learned over 35 years. J Pediatr Surg 1999; 34:786-92. 18. Cade A, Walters M, Puntis JW, et al. Pancreatic exocrine and endocrine function after pancreatectomy for persistent hyperinsulinaemic hypoglycaemia of infancy. Arch Dis Child 1998;79:435-9. 19. Rahier J, Fält K, Müntefering H, et al. The basic structural lesion of persistent neonatal hypoglycaemia with hyperinsulinism: deficiency of pancreatic D cells or hyperactivity of B-cells? Diabetologia 1984;26: 282-9. 20. Laidlaw GF. Nesidioblastoma, the islet tumor of the pancreas. Am J Pathol 1938;14:125-34. 21. Sempoux C, Guiot Y, Dubois D, et al. Pancreatic B-cell proliferation in persistent hyperinsulinemic hypoglycemia of infancy : an immunohistochemical study of 18 cases. Mod Pathol 1998;11:444-9. 22. Goossens A, Gepts W, Saudubray JM, et al. Diffuse and focal nesidioblastosis. A clinicopathological study of 24 patients with persistent neonatal hyperinsulinemic hypoglycemia. Am J Surg Pathol 1989;13: 766-75. 23. Sempoux C, Guiot Y, Lefebre A, et al. Neonatal hyperinsulinemic hypoglycemia: heterogeneity of the syndrome and keys for differential diagnosis. J Clin Endocrinol Metab 1998;83:1455-61. 24. Sempoux C, Guiot Y, Rahier J. The focal form of persistent hyperinsulinemic hypoglycemia of infancy. Diabetes 2001;50:S182-183. 25. de Lonlay P, Fournet JC, Rahier J, et al. Somatic deletion of the imprinted 11p15 region in sporadic persistent hyperinsulinemic hypoglycemia of infancy is specific of focal adenomatous hyperplasia and endorses partial pancreatectomy. J Clin Invest 1997;100:802-7. 26. Verkarre V, Fournet JC, de Lonlay P, et al. Paternal mutation of the sulfonylurea receptor (SUR1) gene and maternal loss of 11p15 imprinted genes lead to persistent hyperinsulinism in focal adenomatous hyperplasia. J Clin Invest 1998;102:1286-91.
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27. Ryan F, Devaney D, Joyce C, et al. Hyperinsulinism: molecular aetiology of focal disease. Arch Dis Child 1998;79:445-7. 28. Fournet JC, Mayaud C, de Lonlay P, et al. Unbalanced expression of 11p15 imprinted genes in focal form of congenital hyperinsulinism: association with a reduction to homozygosity of a mutation in ABCC8 or KCNJ11. Am J Pathol 2001;158:2177-84. 29. Kassem SA, Ariel I, Thornton PS, et al. p57KIP2 expression in normal islet cells and in hyperinsulinism of infancy. Diabetes 2001;50:2763-9. 30. Sempoux C, Guiot Y, Dahan K, et al. The focal form of persistent hyperinsulinemic hypoglycemia of infancy: morphological and molecular studies show structural and functional differences with insulinoma. Diabetes 2003;52:784-94. 31. Thomas P, Ye Y, Lightner E. Mutation of the pancreatic islet inward rectifier Kir 6.2 also leads to familial persistent hyperinsulinemic hypoglycemia of infancy. Hum Mol Genet 1996;5:1809-12. 32. Thomas PM, Cote GJ, Wohllk N, et al. Mutations in the sulfonylurea receptor gene in familial persistent hyperinsulinemic hypoglycemia of infancy. Science 1995;268:426-9. 33. Stanley CA, Fang J, Kutyna K, et al. Molecular basis and characterization of the hyperinsulinism/hyperammonemia syndrome. Predominance of mutations in exons 11 and 12 of the glutamate dehydrogenase Gene. Diabetes 2000;49:667-73.
34. Glaser B, Kesavan P, Heyman M, et al. Familial hyperinsulinism caused by an activating glucokinase mutation. N Engl J Med 1998; 338:226-30. 35. Cuesta-Muñoz AL, Huopio H, Otonkoski T, et al. Severe persistent hyperinsulinemic hypoglycemia due to a de novo glucokinase mutation. Diabetes 2004;53:2164-8. 36. Sayed S, Langdon DR, Odili S, et al. Extremes of clinical and enzymatic phenotypes in children with hyperinsulinism caused by glucokinase activating mutations. Diabetes 2009;58:1419-27. 37. Kassem S, Heyman M, Glaser B, et al. Large islets, beta-cell proliferation, and a glucokinase mutation. N Engl J Med 2010;362: 1348-50. 38. Sempoux C, Guiot Y, Cosgrove K, et al. A new morphological form of persistent hyperinsulinaemic hypoglycaemia of infancy: correlation with the clinical and phydiological data. Horm Res 2002;58: 44. 39. Rahier J, Sempoux C, Fournet JC, et al. Partial or near-total pancreatectomy for persistent neonatal hyperinsulinemic hypoglycemia: the pathologist’s role. Histopathology 1998;32:15-9. 40. Suchi M, Thornton PS, Adzick NS, et al. Congenital hyperinsulinism: intraoperative biopsy interpretation can direct the extent of pancreatectomy. Am J Surg Pathol 2004;28:1326-35.
Morphologic analysis of focal and diffuse forms of congenital hyperinsulinism Jacques Rahier, MD, PhD, Yves Guiot, PhD, Christine Sempoux, MD, PhD From the Department of Pathology, Cliniques Universitaires Saint-Luc, Université catholique de Louvain, Tour Rosalind Franklin, Brussels, Belgium. KEYWORDS Hypoglycemia; Histology; Islets of Langerhans; Nesidioblastosis; Congenital hyperinsulinism
Congenital hyperinsulinism is clinically characterized by an inappropriate insulin secretion resulting in recurrent severe hypoglycemia. Nesidioblastosis, the proliferation of islet cells budding off from ducts, has been considered for years as the histologic lesion responsible for the syndrome. In our morphologic studies, we demonstrate that nesidioblastosis is not specific of the disease, which is actually not a single entity. Indeed, we recognize the existence of 2 different forms—a diffuse form and a focal form—and demonstrate that they can be differentiated by morphologic criteria, even on frozen sections during surgery. This histologic distinction directs the therapeutic approach because the patients experiencing the focal form of the syndrome can be completely cured by a very limited pancreatectomy. Molecular findings confirmed the reliability of this histologic distinction, showing a specific background for each form. © 2011 Elsevier Inc. All rights reserved.
Hypoglycemia is not a rare event in neonates and infants and has various causes.1 It can be the result of a lack of hepatic production of glucose, mostly related to congenital metabolic diseases, or the consequence of hormonal imbalance linked to pituitary, adrenal, or thyroid deficiency. Hypoglycemia is also observed in small-for-date neonates, after salicylate consumption or in Reye syndrome. However, recurrent severe hypoglycemia may also be related to inappropriately elevated plasma insulin levels, which is the topic of this review. The syndrome of persistent hyperinsulinemic hypoglycemia of infancy (PHHI) was recognized more than 50 years ago by McQuarrie.2 The estimated incidence of PHHI in the general population is 1/30,000 to 1/50,000 but it increases to 1/2500 in communities with substantial in-
Address reprint requests and correspondence: Jacques Rahier, MD, PhD, Department of Pathology, Cliniques Universitaires Saint-Luc, Université catholique de Louvain, Tour Rosalind Franklin, 10 Avenue Hippocrate, 1200, Brussels, Belgium. E-mail address: [email protected].
1055-8586/$ -see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1053/j.sempedsurg.2010.10.010
breeding. Despite the number of studies devoted to this subject, the pathogenicity of the disease remained unsolved for several decades.3,4 Fortunately, major progress in genetic and molecular biology has brought enlightenment in most cases.5-13 The age at onset is quite variable: the vast majority of cases are revealed early in life, a few hours or days after birth, but occasionally, symptoms can take as long as 5 or 6 months to become apparent. Most infants with an early onset of symptoms have a heavy birth weight. The severity of the disease is quite variable but tends to be greater in the neonatal form, many patients remaining unresponsive to diazoxide.14 In those infants who remain nonresponsive to medical treatment, persistent hyperinsulinic hypoglycemia can cause great neurological damage.5,15 For many years, surgery was the only therapeutic option. The extent of surgical resection has long been a matter of debate, with some surgeons recommending a 95% pancreatic resection to avoid recurrence of the disease. However, subsequent diabetes is a major side effect of resection.16-18
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Seminars in Pediatric Surgery, Vol 20, No 1, February 2011
Figure 1 Nesidioblastosis B cells budding off from ducts in a case of diffuse PHHI. (Color version of figure is available online.)
The 2 first cases we encountered in the 1980s were of particular interest, with very curious clinical progress: both were of neonatal onset, had a high birth weight and were insensitive to diazoxide.19 Both pancreases were apparently macroscopically normal, with no evident lesion at microscopy, but the first one was not cured despite a near-total pancreatectomy (in 2 steps) and the second case was cured by a hemipancreatectomy. At that time, people used to consider that “nesidioblastosis” was the underlying pathologic condition responsible for the disease. Nesidioblastosis was defined by G. Laidlaw20 as a diffuse and disseminated proliferation of islet cells budding off from ducts (Figure 1) and the PHHI syndrome was also called nesidioblastosis. Judging by the strange
Figure 2
evolution of these cases, we were at that time already convinced that PHHI was not a single entity. By conducting further investigations in the patient cured by partial pancreatectomy, we realized that, although there was no visible lesion either macroscopically or after conventional Hemalun Eosine staining, immunohistochemical analysis with the use of an antibody directed against insulin revealed a small focal endocrine “tumor-like” lesion (Figure 2), the removal of which was sufficient and necessary for the patient to be cured.19 However, despite careful analysis, such a focal lesion was never found in the other patient who remained sick despite near-total pancreatectomy. Investigations of the pathogenicity of the nonfocal, also called the diffuse type of PHHI, were the next step: first we wondered whether “nesidioblastosis” really existed at all and whether that process could be responsible for hyperinsulinism. Morphologic analysis clearly showed that nesidioblastosis, as defined previously, was present not only in symptomatic but also in normoglycemic control patients of a similar age (Figure 3): it could thus not be considered pathognomonic of this syndrome. Quantitative double immunohistochemical techniques with simultaneously anti-insulin and Ki-67 antibodies enabled Sempoux et al21 to demonstrate that there was no proliferation in these small groups of B cells in PHHI (Figure 4). Furthermore, when the number of B cells was measured by the point counting method, we observed that there was no significant difference in the numbers of pancreatic B cells between normoglycemic infants and patients with the diffuse type of PHHI (Figure 5). We therefore suggested that the nonfocal or diffuse type of PHHI was related to a B-cell functional abnormality.19
Small focal adenomatous hyperplasia not recognized on hematoxylin and eosin staining. (Color version of figure is available online.)
Rahier et al
Morphologic Analysis of Focal and Diffuse Forms of CHI
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Figure 5 The amount of B cells is similar in normoglycemic controls and in infants with diffuse PHHI.
further concluded that nesidioblastosis does exist, but that it is not specific to PHHI and does not correspond to permanent B cell proliferation.
Subclassification and characteristics of the different PHHI types Figure 3 Nesidioblastosis in cases of normoglycemic infant (A) or infant with diffuse PHHI (B). (Color version of figure is available online.)
We were therefore able to conclude at that time in the early eighties that different types of PHHI indeed do exist: one is secondary to a focal tumor-like lesion and can be cured by partial resection, the other appears to be a diffuse lesion that cannot be cured by partial pancreatectomy. It was
Figure 4
PHHI does not actually correspond to a single entity.5-13,19 Many different forms have now been reported on the basis of specific genetic defects. According to the type of genetic abnormality, the histologic feature may be different and the morphologic pattern may help to differentiate the various entities. On a practical or surgical point of view, one of the most important distinction remains that of focal versus diffuse character of the lesion because a focal lesion may be cured by a resection limited to the abnormal focus.
Nesidioblastosis: not a permanent proliferation of B cells. (Color version of figure is available online.)
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Figure 6 Focal adenomatous hyperplasia is made by the confluence of hyperplastic but normally structured islets with a peripheral rim of non-B cells. (Color version of figure is available online.)
Focal type of PHHI The focal type is a focal adenomatous hyperplasia,19,22 characterized by the presence of a small endocrine lesion in the pancreas, histologically corresponding to the confluence of hyperplastic but apparently normally structured islets, with a large core of B cells and a peripheral rim of endocrine non-B cells (Figure 6). Within the focal lesion, the B cells are hyperactive, with an enlarged cytoplasm and a large Golgi region full of proinsulin but relatively few insulin granules and little insulin labeling because the lesion hypersecretes and does not store insulin. The lesion may be very small and its limits are sometimes irregular. Outside the focus the islets are small, their B cells are resting with a high storage of insulin and a weak production of proinsulin (Figure 7). Their cytoplasm is shrunken and their nuclei are small.23,24 The focal adenomatous hyperplasia is different from adenoma: adenoma do not seem to be formed by the confluence of true islets and we have never observed adenomas in neonates or infants. Focal adenomatous hyperplasia is related to an loss of heterozygosity of 11p15 on the maternal allele and a sulfonylurea receptor (SUR)
mutation on the paternal one.25-28 This can be easily demonstrated because p57 is located in this region and is thus not expressed in cases with 11p15 loss of heterozygosity (Figure 8).29,30 Focal adenomatous hyperplasia differs from adenoma or insulinoma both morphologically and by molecular biology.30 Selective resection restricted to the lesion cures the patient provided the resection is complete and the lesion solitary.
Diffuse type of PHHI In the diffuse type of PHHI the pancreas appears normal both macro- and microscopically, but even near-total pancreatectomy can be insufficient to cure the patient.19,22 Careful analysis of the morphology of the islets of Langerhans uncovers the presence of large B cells with abnormally large nuclei as observed in hyperactive endocrine cells (Figure 9). Immunostaining with a specific antibody to proinsulin reveals the labeling of a large Golgi apparatus reflecting a very high activity of proinsulin synthesis. However, there is only very weak labeling with insulin antibody, traducing low insulin storage because of a major nonregulated insulin release. This
Figure 7 (A) Islets from outside a focal lesion. (B) Cells have reduced cytoplasm and small nuclei and low proinsulin production. (Color version of figure is available online.)
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Morphologic Analysis of Focal and Diffuse Forms of CHI
Figure 8
Figure 9 line.)
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p57 within the focal lesion (A) or outside (B). (Color version of figure is available online.)
Islets from diffuse type of PHHI (A and C) and from normoglycemic control (B). (Color version of figure is available on-
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Figure 10 Normal insulin content (A), proinsulin production (B), and SUR 1 expression (C) in a case of GLUD overactivity. (Color version of figure is available online.)
particular aspect contrasts very much with that of islets located outside the lesion in cases of focal adenomatous hyperplasia. It is now well demonstrated that these diffuse lesions are secondary to a mutation of one of the genes of the KATP-dependent channel.31,32 The KATP-dependent channels are composed of 2 subunits: the sulfonylurea receptor, ABCC8 (previously SUR 1), and a member of the inwardly rectifying potassium channels, KCNJ11 (previously Kir 6.2). The permanent closure of these channels caused by their absence or inefficiency increases intracellular K⫹ and depolarizes the B-cell membrane. This leads to the opening of the voltage-dependent Ca2⫹ channels, which in turn, activates the exocytic machinery for insulin secretion. Only in cases of SUR mutation leading to a total lack of SUR synthesis, the negativity of SUR immunocytochemistry is useful for diagnosis. The diffuse type of PHHI used to represent 60% of all operated cases before the distinction of the various types of PHHI was made. This percentage is nowadays greater because the tendency is to be conservative as much as possible in the diffuse form.
Other forms of PHHI The 2 types we described in the previous chapter are both related to a pathology of the sulfonylurea receptor (the K⫹ channel) and represent the more frequent types of PHHI in the material we analyzed (nearly 90%). Other causes exist but their frequency is much lower in the material we analyzed.
in the regulatory domain of the GLUD1 gene with resulting impaired sensitivity of the enzyme to the inhibition by guanosine triphosphate.13,33 Insulin secretion, in infants with this abnormality, is triggered by the consumption of highprotein diets. These infants therefore present a severe hypoglycemia after an oral leucine load, known in the early PHHI literature as leucine sensitive hypoglycemia. The 2 cases we were able to analyze were macroscopically unremarkable. Microscopically, a few B-cell nuclei showed a moderate increase in size, but the cytoplasm remained unchanged. At immunohistochemistry, insulin staining was not lowered as in the classical diffuse form, proinsulin production was high and SUR1 expression was normal (Figure 10).
Glucokinase overactivity In 1998, Glaser et al34 reported a family with autosomal dominant familial hyperinsulinism suggesting a defect in the glucose threshold at which insulin secretion is switched off. Other cases were reported by the group of CuestaMunoz and that Stanley.35-37 It is a very rare cause of PHHI. In the 2 cases we had the opportunity to analyze, the pancreas was reported as macroscopically normal. At microscopy, a few rare B-cell nuclei were slightly increased in size, and immunohistochemistry staining for insulin and proinsulin was normal. The only difference with the pancreas of age-matched controls was a major increase in the islet size (Figure 11). This peculiarity was not reported in the cases analyzed in by researchers in Philadelphia.36
Glutamate deshydrogenase hyperactivity Increased insulin secretion can also be consecutive to overactivity of glutamate deshydrogenase. This is the case of patients with autosomal dominant hyperinsulinism-hyperammonemia syndrome. It results from genomic mutations
Atypical forms A few cases have been classified as atypical because they did not fit the described categories. Symptoms of most of these patients do not appear at birth but later, at 5 or 6
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Morphologic Analysis of Focal and Diffuse Forms of CHI
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Figure 11 Normoglycemic control (A) and a case with glucokinase overactivity (B). The size of the islets is higher in this type of PHHI than in control patients. (Color version of figure is available online.)
months, and are less prominent than in the usual diffuse form. These infants are often improved by diazoxide therapy, suggesting that KATP channels are not involved in this
Figure 12
pathology. Some islets show signs of hyperactivity but others appear normal. The genetic abnormalities underlying this type of PHHI are so far unknown.38
Mean radius of 50 selected B-cell nuclei measured outside a focal lesion or in cases devoid of focal lesion.
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Figure 13 B cells from inside (A) or outside (B) a focal lesion and in diffuse type of PHHI in frozen sections; the B cells abnormalities (C) are easily recognized with toluidine blue staining. (Color version of figure is available online.)
The challenge The distinction between the focal and diffuse types of PHHI is very important because it has a major impact on the therapeutic management of the patient. The differential diagnosis long relied on morphologic analysis, and because the focal lesion might be very small, multiple sections were required to ascertain that the lesion was actually diffuse rather than focal. Furthermore, the diagnosis always arrived after the pancreas had been removed, sometimes after total pancreatectomy had been performed for a very small lesion susceptible to be cured by a small resection restricted to the focal lesion. We therefore decided to reanalyze our series of pancreases removed for PHHI with to analyze whether differences existed between islets of the diffuse types and islets outside a focal lesion. It was then observed that in diffuse PHHI the islets consisted of hyperactive B cells while those outside a focal lesion were resting islets. The existence of objective differences between resting and hyperactive B cells was first evaluated on paraffin retrospective material by measuring the size of the B-cell nuclei and the nuclear B-cell crowding (which is the main number of B cell nuclei per 100 squared micrometers). Figure 12 shows major and highly significant differences for both parameters between B cells from diffuse and focal types of PHHI.23 In a second step, a prospective study was performed. Frozen sections were made during surgery on small specimens (3-4 mm in large axis) obtained from the head, isthmus, corpus and tail of the pancreas, with the objective of assessing the presence of resting islets with shrunken B-cell cytoplasm or that of islets with hyperactive B cells with abnormally large cytoplasm and B cell nuclei (Figure 13). This technique enabled us to make the right distinction in most cases between focal versus diffuse types of PHHI and
in all cases with neonatal onset, large birth weight, and resistance to diazoxide.39,40 Before the arrival of selective venous samplings and later, of the 18-fluorodopa positron emission tomography scan, it was the only way to distinguish focal from diffuse disease before pancreatectomy. Nowadays, intraoperative frozen sections remain very useful to ascertain the adequacy of the diagnosis, recognize the atypical or bizarre forms and most of all to check during surgery whether the resection margins are free from lesion.
Difficulties in management Some cases may be very difficult to manage by the pathologist during surgery. Cases with a pathology related to
Figure 14 The limits of a focal lesion may be very irregular. (Color version of figure is available online.)
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Morphologic Analysis of Focal and Diffuse Forms of CHI
Figure 15 The focal lesions are sometimes very small and difficult to share for the various analyses. (Color version of figure is available online.)
KATP channels are usually easy to analyze. Sometimes the frequency of B-cell occurrence with large nuclei is not as high as usual and the recognition of a lesion as a diffuse type may not be so evident. On fortunately rare occasions, hyperinsulinism results from bifocal lesions that are only revealed after surgery, because the infant was not cured by an apparently complete resection of the lesion. More frequently, the focal lesion is small, not actually recognizable at the naked eye, but the limits of the abnormal focus is markedly irregular (Figure 14), making it difficult to ascertain whether resection is complete. One case of a focal lesion involving more than 90% of the pancreas has also been observed, the absence of p57 labeling and its persistence in the healthy part of the pancreas enabled the confirmation of the diagnosis of focal PHHI in this particular case. In general, atypical cases, such as glucokinase or glutamate dehydrogenase activation are always more difficult to diagnose on per surgical frozen sections than hypoglycemias secondary to K channelopathy. Last but not least, the pathologist also has to share the material and that is not easy, because the lesion, when focal, may be very small, and physiological studies (electrophysiology or insulin secretion studies) require that the tissue given to physiologists must not only be well characterized but also still living (Figure 15).
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34. Glaser B, Kesavan P, Heyman M, et al. Familial hyperinsulinism caused by an activating glucokinase mutation. N Engl J Med 1998; 338:226-30. 35. Cuesta-Muñoz AL, Huopio H, Otonkoski T, et al. Severe persistent hyperinsulinemic hypoglycemia due to a de novo glucokinase mutation. Diabetes 2004;53:2164-8. 36. Sayed S, Langdon DR, Odili S, et al. Extremes of clinical and enzymatic phenotypes in children with hyperinsulinism caused by glucokinase activating mutations. Diabetes 2009;58:1419-27. 37. Kassem S, Heyman M, Glaser B, et al. Large islets, beta-cell proliferation, and a glucokinase mutation. N Engl J Med 2010;362: 1348-50. 38. Sempoux C, Guiot Y, Cosgrove K, et al. A new morphological form of persistent hyperinsulinaemic hypoglycaemia of infancy: correlation with the clinical and phydiological data. Horm Res 2002;58: 44. 39. Rahier J, Sempoux C, Fournet JC, et al. Partial or near-total pancreatectomy for persistent neonatal hyperinsulinemic hypoglycemia: the pathologist’s role. Histopathology 1998;32:15-9. 40. Suchi M, Thornton PS, Adzick NS, et al. Congenital hyperinsulinism: intraoperative biopsy interpretation can direct the extent of pancreatectomy. Am J Surg Pathol 2004;28:1326-35.