Hypophysitis Secondary to Cytotoxic T-Lymphocyte–Associated Protein 4 Blockade
2016; Elsevier BV; Volume: 186; Issue: 12 Linguagem: Inglês
10.1016/j.ajpath.2016.08.020
ISSN1525-2191
AutoresPatrizio Caturegli, Giulia Di Dalmazi, Martina Lombardi, Federica Grosso, H. Benjamin Larman, Tatianna Larman, G Taverna, Mirco Cosottini, Isabella Lupi,
Tópico(s)Cancer Immunotherapy and Biomarkers
ResumoHypophysitis that develops in cancer patients treated with monoclonal antibodies blocking cytotoxic T-lymphocyte–associated protein 4 (CTLA-4; an inhibitory molecule classically expressed on T cells) is now reported at an incidence of approximately 10%. Its pathogenesis is unknown, in part because no pathologic examination of the pituitary gland has been reported to date. We analyzed at autopsy the pituitary glands of six cancer patients treated with CTLA-4 blockade, one with clinical and pathologic evidence of hypophysitis, one with mild lymphocytic infiltration in the pituitary gland but no clinical signs of hypophysitis, and four with normal pituitary structure and function. CTLA-4 antigen was expressed by pituitary endocrine cells in all patients but at different levels. The highest levels were found in the patient who had clinical and pathologic evidence of severe hypophysitis. This high pituitary CTLA-4 expression was associated with T-cell infiltration and IgG-dependent complement fixation and phagocytosis, immune reactions that induced an extensive destruction of the adenohypophyseal architecture. Pituitary CTLA-4 expression was confirmed in a validation group of 37 surgical pituitary adenomas and 11 normal pituitary glands. The study suggests that administration of CTLA-4 blocking antibodies to patients who express high levels of CTLA-4 antigen in the pituitary can cause an aggressive (necrotizing) form of hypophysitis through type IV (T-cell dependent) and type II (IgG dependent) immune mechanisms. Hypophysitis that develops in cancer patients treated with monoclonal antibodies blocking cytotoxic T-lymphocyte–associated protein 4 (CTLA-4; an inhibitory molecule classically expressed on T cells) is now reported at an incidence of approximately 10%. Its pathogenesis is unknown, in part because no pathologic examination of the pituitary gland has been reported to date. We analyzed at autopsy the pituitary glands of six cancer patients treated with CTLA-4 blockade, one with clinical and pathologic evidence of hypophysitis, one with mild lymphocytic infiltration in the pituitary gland but no clinical signs of hypophysitis, and four with normal pituitary structure and function. CTLA-4 antigen was expressed by pituitary endocrine cells in all patients but at different levels. The highest levels were found in the patient who had clinical and pathologic evidence of severe hypophysitis. This high pituitary CTLA-4 expression was associated with T-cell infiltration and IgG-dependent complement fixation and phagocytosis, immune reactions that induced an extensive destruction of the adenohypophyseal architecture. Pituitary CTLA-4 expression was confirmed in a validation group of 37 surgical pituitary adenomas and 11 normal pituitary glands. The study suggests that administration of CTLA-4 blocking antibodies to patients who express high levels of CTLA-4 antigen in the pituitary can cause an aggressive (necrotizing) form of hypophysitis through type IV (T-cell dependent) and type II (IgG dependent) immune mechanisms. Hypophysitis is a chronic inflammation of the pituitary gland of idiopathic (primary) or known (secondary) etiology.1Leporati P. Landek-Salgado M.A. Lupi I. Chiovato L. Caturegli P. IgG4-related hypophysitis: a new addition to the hypophysitis spectrum.J Clin Endocrinol Metab. 2011; 96: 1971-1980Crossref PubMed Scopus (196) Google Scholar Primary hypophysitis is rare but significant because it enters in the differential diagnosis of other, more common, non–hormone-secreting pituitary masses, such as pituitary adenomas. It typically presents with signs and symptoms of sellar compression and/or various degrees of hypopituitarism. If unrecognized, it can also cause death because of irreversible adrenal insufficiency. Primary hypophysitis encompasses a spectrum of pathologic lesions,2Caturegli P. Iwama S. From Japan with love: another tessera in the hypophysitis mosaic.J Clin Endocrinol Metab. 2013; 98: 1865-1868Crossref PubMed Scopus (34) Google Scholar, 3Caturegli P. Newschaffer C. Olivi A. Pomper M.G. Burger P.C. Rose N.R. Autoimmune hypophysitis.Endocr Rev. 2005; 26: 599-614Crossref PubMed Scopus (511) Google Scholar ranging from the most common lymphocytic and granulomatous variants to the more recently described xanthomatous,4Deodhare S.S. Bilbao J.M. Kovacs K. Horvath E. Nomikos P. Buchfelder M. Reschke K. Lehnert H. Xanthomatous hypophysitis: a novel entity of obscure etiology.Endocr Pathol. 1999; 10: 237-241Crossref PubMed Scopus (38) Google Scholar IgG4 plasmacytic,5van der Vliet H.J. Perenboom R.M. Multiple pseudotumors in IgG4-associated multifocal systemic fibrosis.Ann Intern Med. 2004; 141: 896-897Crossref PubMed Scopus (119) Google Scholar and necrotizing6Ahmed S.R. Aiello D.P. Page R. Hopper K. Towfighi J. Santen R.J. Necrotizing infundibulo-hypophysitis: a unique syndrome of diabetes insipidus and hypopituitarism.J Clin Endocrinol Metab. 1993; 76: 1499-1504Crossref PubMed Scopus (97) Google Scholar variants. A total of 1005 patients with primary hypophysitis have been described in publications from 1917 to June 2016 (Table 1), diagnosed by surgical pathology [631 (63%)], clinical and imaging criteria [331 (33%)], or autopsy [43 (4%)].Table 1Key Features of Primary Hypophysitis and Hypophysitis Secondary to CTLA-4 BlockadeFeaturesPrimary hypophysitisHypophysitis secondary to CTLA-4 blockadeP valueNo. of patients described in publications∗Meeting abstracts are not included.1005127 + this caseNo. of articles featuring these patients59845Publication time span, years (range)100 (1917 to 2016)14 (2003 to 2016)Diagnosis established by Surgical pathology6310 Autopsy431 (this case) Clinical and imaging criteria331127F:M, n (ratio)718:287 (2.5:1)28:100 (1:4)<0.001Mean age at onset, years41 ± 1659 ± 13<0.001Time after the initiating event, means ± SDUnknown, likely years10 ± 5 weeks after first antibody injectionSymptoms at presentation†Data are given as % (number/total). Headache47 (397/852)60 (70/117) Low cortisol35 (288/824)72 (82/113)0.002 Polydipsia and polyuria35 (297/845)0.9 (1/116)<0.001 Visual disturbances31 (264/861)3 (4/117)<0.001 Low sex steroids20 (168/834)15 (17/112)<0.001 Low thyroxine16 (132/824)20 (22/112)Endocrine abnormalities at diagnosis†Data are given as % (number/total). Secondary hypocortisolism60 (412/682)91 (85/93) Secondary hypothyroidism52 (363/701)84 (80/95)<0.001 Secondary hypogonadism55 (345/624)83 (65/78)<0.001 Central diabetes insipidus39 (320/813)1 (1/75)<0.001 Increased PRL37 (236/630)9 (5/53)<0.001 Decreased GH38 (184/481)43 (13/31)<0.001MRI findings†Data are given as % (number/total). Abnormal98 (632/646)77 (68/88)<0.001 Normal2 (13/646)23 (20/88)Pathologic variants†Data are given as % (number/total). Lymphocytic68 (461/674)0 Granulomatous20 (133/674)0 IgG4 plasmacytic4 (27/674)0 Mixed forms4 (26/674)0 Xanthomatous3 (23/674)0 Necrotizing0.6 (4/674)0.8 (1/128): this casePathogenesisAutoimmuneType II and IV hypersensitivityInitiating pituitary autoantigen(s)UnknownPituitary CTLA-4Systemic high-dose glucocorticoidsOften efficaciousConsidered efficaciousOutcomeVariable: from complete recovery to deathPituitary function rarely recoversF, female; M, male; CTLA-4, cytotoxic T-lymphocyte–associated protein 4; GH, growth hormone; MRI, magnetic resonance imaging; PRL, prolactin.∗ Meeting abstracts are not included.† Data are given as % (number/total). Open table in a new tab F, female; M, male; CTLA-4, cytotoxic T-lymphocyte–associated protein 4; GH, growth hormone; MRI, magnetic resonance imaging; PRL, prolactin. Hypophysitis secondary to the administration of monoclonal antibodies directed against cytotoxic T-lymphocyte–associated protein 4 (CTLA-4), a molecule classically expressed on T cells, was first reported in 20037Phan G.Q. Yang J.C. Sherry R.M. Hwu P. Topalian S.L. Schwartzentruber D.J. Restifo N.P. Haworth L.R. Seipp C.A. Freezer L.J. Morton K.E. Mavroukakis S.A. Duray P.H. Steinberg S.M. Allison J.P. Davis T.A. Rosenberg S.A. Cancer regression and autoimmunity induced by cytotoxic T lymphocyte-associated antigen 4 blockade in patients with metastatic melanoma.Proc Natl Acad Sci U S A. 2003; 100: 8372-8377Crossref PubMed Scopus (1377) Google Scholar and first reviewed in 2009.8Gutenberg A. Landek-Salgado M.A. Tzou S.C. Lupi I. Geis A. Kimura H. Caturegli P. Autoimmune hypophysitis: expanding the differential diagnosis to CTLA-4 blockade.Expert Rev Endocrinol Metab. 2009; 4: 681-698Crossref Scopus (15) Google Scholar This form of hypophysitis is now observed in approximately 10%9Albarel F. Gaudy C. Castinetti F. Carre T. Morange I. Conte-Devolx B. Grob J.J. Brue T. Long-term follow-up of ipilimumab-induced hypophysitis, a common adverse event of the anti-CTLA-4 antibody in melanoma.Eur J Endocrinol. 2014; 172: 195-204Crossref Scopus (186) Google Scholar, 10Faje A.T. Sullivan R. Lawrence D. Tritos N.A. Fadden R. Klibanski A. Nachtigall L. Ipilimumab-induced hypophysitis: a detailed longitudinal analysis in a large cohort of patients with metastatic melanoma.J Clin Endocrinol Metab. 2014; 99: 4078-4085Crossref PubMed Scopus (291) Google Scholar, 11Min L. Hodi F.S. Giobbie-Hurder A. Ott P.A. Luke J.J. Donahue H. Davis M. Carroll R.S. Kaiser U.B. Systemic high dose corticosteroid treatment does not improve the outcome of ipilimumab-related hypophysitis: a retrospective cohort study.Clin Cancer Res. 2014; 21: 749-755Crossref Scopus (178) Google Scholar of cancer patients treated with ipilimumab (an IgG1 produced by Bristol-Myers Squibb, New York, NY). It occurs less frequently in patients receiving tremelimumab (an IgG2 monoclonal antibody against CTLA-4 produced by Pfizer, New York, NY), and rarely in those treated with other immune checkpoint inhibitors, such as antibodies against PD-1,12Topalian S.L. Hodi F.S. Brahmer J.R. Gettinger S.N. Smith D.C. McDermott D.F. Powderly J.D. Carvajal R.D. Sosman J.A. Atkins M.B. Leming P.D. Spigel D.R. Antonia S.J. Horn L. Drake C.G. Pardoll D.M. Chen L. Sharfman W.H. Anders R.A. Taube J.M. McMiller T.L. Xu H. Korman A.J. Jure-Kunkel M. Agrawal S. McDonald D. Kollia G.D. Gupta A. Wigginton J.M. Sznol M. Safety, activity, and immune correlates of anti-PD-1 antibody in cancer.N Engl J Med. 2012; 366: 2443-2454Crossref PubMed Scopus (9274) Google Scholar or PD-L1.13Brahmer J.R. Tykodi S.S. Chow L.Q. Hwu W.J. Topalian S.L. Hwu P. Drake C.G. Camacho L.H. Kauh J. Odunsi K. Pitot H.C. Hamid O. Bhatia S. Martins R. Eaton K. Chen S. Salay T.M. Alaparthy S. Grosso J.F. Korman A.J. Parker S.M. Agrawal S. Goldberg S.M. Pardoll D.M. Gupta A. Wigginton J.M. Safety and activity of anti-PD-L1 antibody in patients with advanced cancer.N Engl J Med. 2012; 366: 2455-2465Crossref PubMed Scopus (5831) Google Scholar Overall, however, hypophysitis is the most common endocrine adverse event associated with immune checkpoint inhibitors.14Abdel-Rahman O. ElHalawani H. Fouad M. Risk of endocrine complications in cancer patients treated with immune check point inhibitors: a meta-analysis.Future Oncol. 2016; 12: 413-425Crossref PubMed Scopus (98) Google Scholar, 15Bertrand A. Kostine M. Barnetche T. Truchetet M.E. Schaeverbeke T. Immune related adverse events associated with anti-CTLA-4 antibodies: systematic review and meta-analysis.BMC Med. 2015; 13: 211Crossref PubMed Scopus (471) Google Scholar In addition, hypophysitis is the most costly adverse event in hospitalized patients with metastatic melanoma, adding an average expense per hospitalization of €10,265 in Spain, €5316 in France, $9735 in Canada, $7231 in Australia,16Wehler E. Zhao Z. Pinar Bilir S. Munakata J. Barber B. Economic burden of toxicities associated with treating metastatic melanoma in eight countries.Eur J Health Econ. 2015; ([Epub ahead of print] doi:10.1007/s10198-015-0757-y)Google Scholar and $8490 in the United States.17Barzey V. Atkins M.B. Garrison L.P. Asukai Y. Kotapati S. Penrod J.R. Ipilimumab in 2nd line treatment of patients with advanced melanoma: a cost-effectiveness analysis.J Med Econ. 2013; 16: 202-212Crossref PubMed Scopus (41) Google Scholar Since the original report,7Phan G.Q. Yang J.C. Sherry R.M. Hwu P. Topalian S.L. Schwartzentruber D.J. Restifo N.P. Haworth L.R. Seipp C.A. Freezer L.J. Morton K.E. Mavroukakis S.A. Duray P.H. Steinberg S.M. Allison J.P. Davis T.A. Rosenberg S.A. Cancer regression and autoimmunity induced by cytotoxic T lymphocyte-associated antigen 4 blockade in patients with metastatic melanoma.Proc Natl Acad Sci U S A. 2003; 100: 8372-8377Crossref PubMed Scopus (1377) Google Scholar 127 patients have been described in publications as individual case reports or case series (Table 1). A similar number of patients have appeared as counts, without specific information about their clinical characteristics, in studies of ipilimumab or tremelimumab, as recently reviewed by Bertrand et al15Bertrand A. Kostine M. Barnetche T. Truchetet M.E. Schaeverbeke T. Immune related adverse events associated with anti-CTLA-4 antibodies: systematic review and meta-analysis.BMC Med. 2015; 13: 211Crossref PubMed Scopus (471) Google Scholar in a meta-analysis of 22 clinical trials. The pathogenesis of hypophysitis secondary to CTLA-4 blockade remains undetermined, a knowledge gap that often leads to increased morbidity and therapy interruptions. Part of the gap relates to the absence of pathologic information, because none of the published patients underwent pituitary biopsy or autopsy. In this study, we report the first pathologically proven cases of hypophysitis secondary to CTLA-4 and provide mechanistic insights into the pathogenesis of this emerging condition. The index case, designated herein as autopsy case 1, was provided by the Saints Anthony and Biagio, and Cesare Arrigo Hospital (Alessandria, Italy), in a 79-year-old woman with a history of environmental asbestos exposure. The patient was diagnosed with unresectable pleural mesothelioma in October 2013 and treated with conventional chemotherapy but without significant response. In October 2014, she began treatment with the CTLA-4 blocking antibody tremelimumab (10 mg/kg every 4 weeks). After the third i.v. injection (early December 2014), she developed severe diarrhea, profound fatigue, and vomiting. No signs or symptoms of pituitary mass expansion (headache and/or visual disturbance) or diabetes insipidus (polyuria and polydipsia) were present. Laboratory test results revealed severe hyponatremia (112 mEq/L; reference range, 135 to 150 mEq/L), mild neutrophilic leukocytosis (11,300 cells per microliter with 83% neutrophils), central adrenal insufficiency [low morning cortisol: 3.1 μg/dL (reference range, 5 to 25 μg/dL); and inappropriately normal adrenocorticotropic hormone: 12.9 pg/mL (reference range, 5 to 46 pg/mL)], and central hypothyroidism [low free triiodothyronine: 1.38 pg/mL (reference range, 2.3 to 4.2 pg/mL) and low thyroid-stimulating hormone: 0.023 mU/L (reference range, 0.55 to 4.78 mU/L)]. Cranial magnetic resonance imaging showed a slightly enlarged pituitary gland (Supplemental Figure S1A), with a height of 6.08 mm (reference range for sex and age is 4.87 ± 0.95 mm18Tsunoda A. Okuda O. Sato K. MR height of the pituitary gland as a function of age and sex: especially physiological hypertrophy in adolescence and in climacterium.AJNR Am J Neuroradiol. 1997; 18: 551-554PubMed Google Scholar). On T1-weighted images after contrast, the anterior pituitary contained scattered areas of hypointensity consistent with necrosis (Supplemental Figure S1B). Diagnoses of hypophysitis and colitis were made on clinical and imaging grounds. The patient began glucocorticoids (hydrocortisone, 100 mg four times per day; and methylprednisolone, 250 mg/day), thyroid hormones (levothyroxine, 75 μg/day), and saline infusions in mid-December. After an initial improvement, she died in January 2015 because of acute respiratory failure. An autopsy was performed. We then queried the autopsy database of The Johns Hopkins Hospital Pathology Department (searchable electronically from June 1985 to present by authorized personnel) for additional cases and found, as of June 30, 2016, five metastatic melanoma patients who had received ipilimumab, all at a dose of 3 mg/kg and all without clinical signs and symptoms of hypophysitis. Autopsy case 2: A 67-year-old man underwent amputation of the third and fourth left toes and sentinel lymph node biopsy in February 2014 for a diagnosis of B-rapidly accelerated fibrosarcoma (BRAF) wild-type melanoma, followed by inguinal, femoral, and pelvic lymph node dissection. Twenty months later, chest and abdominal computed tomographic scans showed enlarged lymph nodes and pulmonary nodules suggestive of metastatic disease, as well as peritoneal carcinomatosis. He began treatment with the anti–CTLA-4 antibody ipilimumab and the anti–PD-1 antibody nivolumab in October 2015. During follow-up, laboratory studies revealed primary hypothyroidism [decreased free thyroxine: 0.7 ng/dL (reference range, 0.8 to 1.8 ng/dL); and increased thyroid-stimulating hormone: 16.8 UI/L (reference range, 0.5 to 4.5 UI/L)], severe anemia, and coagulopathy. He received treatment with levothyroxine, anticoagulant, and palliative care but died 2 months later because of widely metastatic disease. Autopsy case 3: A 30-year-old man underwent resection of a left heel tumor in February 2014 that yielded a diagnosis of lentiginous, BRAF-V600 mutant melanoma. Chest and abdominal computed tomographic scans showed multiple pulmonary nodules, accompanied by mediastinal and hilar lymphadenopathy. Lymph node dissection of the left groin revealed metastatic melanoma. The patient began treatment with the BRAF inhibitor dabrafenib. Repeat imaging in September 2014 showed regression of the lung nodules and pelvic lymph nodes, but appearance of renal and subcutaneous metastases. He then began treatment with ipilimumab, which was discontinued after the first dose because of the appearance of autoimmune hepatitis. In November 2014, the patient received the anti-PD1 monoclonal antibody pembrolizumab but developed colitis, requiring the administration of glucocorticoids. He then developed brain metastases and died in March 2015 because of widely metastatic disease. Autopsy case 4: A 59-year-old man underwent excision of an ulcerating lesion on the left jaw in June 2010. Pathology showed BRAF wild type and cKit L567P mutant melanoma. The patient began therapy with recombinant IL-2 and remained stable until January 2013 when new pulmonary and adrenal nodules were found. He then received one full treatment cycle with ipilimumab (four doses at 3-week intervals) with minimal response. Pembrolizumab was then administered in the winter of 2013, but new metastases to the rib and omentum were found in July 2014. The patient then underwent treatment with recombinant IL-15 but gradually deteriorated because of the appearance of new metastases. He began a new cycle of ipilimumab in December 2014 but died the following month because of aggressive disease. Autopsy case 5: A 65-year-old man underwent excision of a mass on his left arm that was shown to be BRAF wild-type melanoma in December 2009. Two years later, the lesion recurred and metastasized to the left axillary lymph nodes. The patient received one full cycle of ipilimumab (from September to November 2011) with stabilization of disease. The following year, the arm lesion recurred and ipilimumab was re-established. In February 2013, a chest computed tomographic scan revealed bilateral pleural effusion and soft tissue involvement. He received the anti–PD-1 antibody nivolumab, initially with partial control of the metastases. The patient then developed renal and heart failure, and died in November 2013 after an acute myocardial infarction. Autopsy case 6: A 58-year-old man underwent resection of a right flank lesion and dissection of right axilla lymph nodes in January 2013, yielding a diagnosis of metastatic melanoma. In February 2014, because of the presence of multiple pulmonary metastases, he began treatment with ipilimumab and nivolumab, obtaining a partial response that eventually prompted the resection of two residual lung metastases in October 2015. Two months later, however, he developed multiple brain metastases and discontinued the immune checkpoint inhibitors. Despite additional treatments, the disease progressed and caused the patient's demise on May 12, 2016. To validate the CTLA-4 immunostainings performed on the six autopsy cases described above, we queried the surgical pathology paraffin block database of The Johns Hopkins Department of Pathology (searchable electronically from March 1984 to present) for pituitary adenomas. Of the total 3274 pituitary surgeries performed in this 33-year interval, 2186 were for pituitary adenomas. We randomly selected 37 specimens, composed of 13 prolactin-secreting, 9 corticotropin-secreting, 8 growth hormone–secreting, and 7 nonsecreting adenomas. We also included 11 normal pituitary specimens from autopsy (n = 7) or surgical pathology (n = 4) sources. All pituitary specimens were deidentified and analyzed blindly (P.C.) under institutional review board protocol number 04-07-12-05e. Pituitary sections were first stained with hematoxylin and eosin and Masson's trichrome to assess morphology and presence of fibrosis. Pituitary sections were then used in immunohistochemistry to detect markers for anterior pituitary hormones, immune cells, immunoglobulins, complement, and CTLA-4. Antibodies to the six anterior pituitary hormones (growth hormone, prolactin, adrenocorticotropic hormone, thyroid-stimulating hormone, luteinizing hormone, and follicle-stimulating hormone) were used to characterize the type and frequency of adenohypophyseal cells. Immunostainings were performed on the BenchMark Ultra automated stainer (Ventana Medical System, Tucson, AZ) using routine clinical diagnostic protocols. Antibodies to CD3 (all T cells), CD4 (helper T cells), CD8 (cytotoxic T cells), CD20 (all B cells), Foxp3 (regulatory T cells), CD138 (plasma cells), and CD68 (macrophages) were used to characterize the immune infiltrate, using automated stainer and routine protocols. To determine whether the injected CTLA-4 blocking antibodies had bound in vivo to the patient's own pituitary gland, we stained the pituitary gland with an antibody against human IgG2 (05-3500; Invitrogen, Carlsbad, CA) or human IgG1 (5218-9850; Serotec, Raleigh, NC). We also stained pituitary sections for C4d (BR-RC4d; ALPCO, Salem, NH), a complement component that binds covalently to surfaces where complement activation is initiated. These experiments were performed manually, with 10-minute antigen retrieval and 30-minute incubations. A mouse monoclonal antibody directed against human CTLA-4 (clone 14D3; eBioscience, San Diego, CA) was optimized on the spleen to achieve an optimal signal/noise ratio, which corresponded to a concentration of 2.5 μg/mL. The protocol was then added to the automated stainer and used to analyze our collection of autopsy pituitary sections. We used the pituitary gland from autopsy case 6 (which, as discussed in Results, showed pathologic evidence of mild hypophysitis and retained great part of the normal pituitary architecture) to extend our original report of CTLA-4 expression in a subset of prolactin- and thyrotropin-secreting cells.19Iwama S. De Remigis A. Callahan M.K. Slovin S.F. Wolchok J.D. Caturegli P. Pituitary expression of ctla-4 mediates hypophysitis secondary to administration of ctla-4 blocking antibody.Sci Transl Med. 2014; 6: 230ra45Crossref PubMed Scopus (427) Google Scholar A portion of this gland was frozen in optimal cutting temperature compound, cut (5 μm thick), and double stained for CTLA-4 plus one of the antibodies directed against prolactin, thyroid-stimulating hormone, follicle-stimulating hormone, luteinizing hormone, adrenocorticotropic hormone, or growth hormone (information regarding these antibodies can be found in the study by Iwama et al19Iwama S. De Remigis A. Callahan M.K. Slovin S.F. Wolchok J.D. Caturegli P. Pituitary expression of ctla-4 mediates hypophysitis secondary to administration of ctla-4 blocking antibody.Sci Transl Med. 2014; 6: 230ra45Crossref PubMed Scopus (427) Google Scholar). Sections were stained as described by Pollock et al.20Pollock A.J. Seibert T.S. Salvatori C. Caturegli P. Allen D.B. Pituitary antibodies in an adolescent with secondary adrenal insufficiency and Turner syndrome.Hormone Res Paediatr. 2016; ([Epub ahead of print] doi:10.1159/000446912)Google Scholar Then, cells were counted at 40× to calculate the total number of endocrine cells and, of them, the number of cells positive for the hormone, CTLA-4, or both. Pituitary CTLA-4 expression was scored on a continuous scale from 0 (no cell stained) to 300 (all cells stained strongly), using the semiquantitative system we previously published.21Zheng G. Chaux A. Sharma R. Netto G. Caturegli P. LMP2, a novel immunohistochemical marker to distinguish renal oncocytoma from the eosinophilic variant of chromophobe renal cell carcinoma.Exp Mol Pathol. 2013; 94: 29-32Crossref Scopus (10) Google Scholar The score was modeled by multiple linear regression using three predictors: diagnostic category (coded as 0 for normal pituitary and 1 for autopsy pituitaries of patients treated with CTLA-4 blocking antibodies), sex, and age. Differences in the proportions of patients with a particular clinical feature when comparing primary to secondary hypophysitis were assessed by Fisher's exact test. Statistical analyses were performed using Stata software release 14 (Stata, College Station, TX). The most striking pathologic feature of autopsy case 1 was the near complete destruction of her anterior pituitary gland, because of extensive necrosis (in approximately 30% of the parenchyma) (Figure 1A) and fibrosis (Figure 1B). Only a few acini of endocrine cells remained in two isolated areas of the gland (Figure 1A), which were heavily infiltrated with hematopoietic mononuclear cells (Figure 1, C and D). The few remaining endocrine cells were mainly of the acidophil class (Figure 1E) and growth hormone producing (Figure 1F). Prolactin- or adrenocorticotropic hormone–producing cells were rare, and gonadotrophs and thyrotrophs absent (data not shown). The posterior pituitary was normal (data not shown), consistent with the absence of central diabetes insipidus in our patients, and the rarity of diabetes insipidus in the published cases [1 (0.9%) of 116] (Table 1). The histologic appearance of the other autopsy cases, who also had received CTLA-4 blockade for the treatment of their melanoma but had not developed clinical or radiological signs of hypophysitis, was that of a normal pituitary gland for cases 2 through 5 (data not shown), and that of mild lymphocytic hypophysitis for case 6 (as presented below). Type IV hypersensitivity reactions, commonly seen in autoimmune diseases, are mediated by T lymphocytes that cause tissue damage by production of cytokines, activation of B lymphocytes and phagocytes, and direct granzyme/perforin-mediated cytotoxicity.22Uzzaman A. Cho S.H. Classification of hypersensitivity reactions.Allergy Asthma Proc. 2012; 33: S96-S99Crossref PubMed Scopus (90) Google Scholar The pituitary gland of autopsy case 1 was markedly infiltrated with lymphocytes, scattered throughout the parenchyma (Figure 2A) or occasionally aggregating to form ectopic lymphoid follicles, a hallmark of autoimmune pathology. Lymphocytes were mainly composed of CD4-positive T cells (Figure 2B) and CD20-positive B cells (Figure 2C). They made intimate contact with the endocrine cells (Figure 2D) and in some cases penetrated their cytosol (Figure 2D), a phenomenon known as emperipolesis, also a feature of autoimmune pathology. The autopsy of case 6, who had no clinical or radiological signs of hypophysitis, showed pathologic evidence of mild lymphocytic hypophysitis. This consisted of a fine, extravascular presence of lymphocytes scattered throughout the pituitary parenchyma, at times clustered in groups of three to five cells (Supplemental Figure S2A) and associated with loss of the normal endocrine cells (Supplemental Figure S2A). The infiltrating cells were mainly CD3-positive T cells (Supplemental Figure S2B). Type II hypersensitivity reactions are mediated by IgG antibodies that bind to antigens expressed on target cells.22Uzzaman A. Cho S.H. Classification of hypersensitivity reactions.Allergy Asthma Proc. 2012; 33: S96-S99Crossref PubMed Scopus (90) Google Scholar This binding causes tissue damage by promoting phagocytosis of the target via macrophages and activating the complement cascade. The pituitary gland of the index case was heavily infiltrated with CD68-positive macrophages, which represented the most abundant hematopoietic cell type (Figure 3A). Macrophages permeated throughout the entire parenchyma and featured a prominent, vacuolated, foamy cytoplasm (Figure 3A). They contained fragments of endocrine cells inside their cytoplasm. For example, remnants of prolactin-secreting cells were observed inside the phagocytic macrophages (Figure 3B). Macrophages did not fuse to form multinucleated giant cells or assume the epithelioid appearance typically seen in granulomas. Staining with an anti-IgG2 antibody revealed in autopsy case 1 the presence of IgG2 on some of the remaining pituitary cells (Figure 3C), suggesting that the administered tremelimumab (an IgG2 antibody) had bound to its target (CTLA-4) on the pituitary cells. No significant pituitary cell staining was found for IgG1 (Figure 3C) or IgG2 in the other autopsy pituitary glands. The complement factor C4d was found to be deposited onto the pituitary cells (Figure 3D) and vessels (Figure 3D) of the index case. No C4d staining was seen in the other pituitary glands (F
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