Histological characterization of liver involvement in systemic mastocytosis

Systemic mastocytosis (SM) is characterized by the accumulation of atypical mast cells (MCs) in organs. Liver histology of SM has been marginally described and accurate histological classification is critical, given the consequences of aggressive SM diagnosis. We aimed to describe the histological features associated with liver SM using updated tools.


| INTRODUC TI ON
Mastocytosis corresponds to a heterogeneous spectrum of diseases characterized by the accumulation of neoplastic mast cells (MCs) in various organs. 1 The most commonly affected organs are the skin, the bone marrow (BM), and the gastrointestinal (GI) tract, whereas the liver is less commonly involved.The World Health Organization (WHO)'s mastocytosis classification distinguishes between cutaneous mastocytosis (CM), systemic mastocytosis (SM), and MC sarcoma. 2,3Systemic mastocytosis is defined by the involvement of an organ other than the skin and can be classified as indolent (ISM), smouldering (SSM), aggressive (ASM), associated with other haematological neoplasms (SM-AHN), or MC leukaemia.The last three entities compose the advanced SM (Adv-SM) group.The diagnosis of SM subtypes is based on the degree of organ infiltration (B-findings) and dysfunction (C-findings).In the WHO classification, the so-called "Bfindings" include a high MC burden, myeloproliferation, signs of dysplasia in a non-MC lineage (without meeting the criteria for haematological neoplasms), and organomegaly (including splenomegaly, hepatomegaly, and adenopathy) without organ impairment (when at least 2 B-findings and no C-findings are found, the final diagnosis is SSM).The so-called "C-findings" correspond to organ dysfunction; for the liver, they include hepatomegaly on palpation with the impairment of liver function, ascites, and/or portal hypertension (when at least one C-finding is found, the final diagnosis is ASM).
][10][11][12] Indeed, most histological studies were published before the introduction of the WHO classification and featured small numbers of patients (i.e.no more than 10 centrally reviewed patients with Adv-SM).A series reported by Mican et al. described the histopathological features of 10 Adv-SM (including 8 cases of SM-AHN), relative to those observed in 17 patients with ISM or CM.These authors found common MC infiltration, which was more severe in advanced diseases.In addition, they observed correlations between MC infiltration, the blood alkaline phosphatase (ALP) level, and liver fibrosis.However, they did not use immunohistochemistry to accurately study the infiltration by immune cells (including MCs) or MC CD25/CD30 expression and did not address the histological-clinical correlations with hepatic C-findings.
Results: A total of 28 patients were included: 6 had indolent SM, 9 had aggressive SM, and 13 had SM with an associated hematologic neoplasm.Twenty-five (89%) patients presented hepatomegaly, and 19 (68%) had portal hypertension.The LB frequently showed slight sinusoid dilatation (82%).Fibrosis was observed in 3/6 indolent SM and in almost all advanced SM cases (21/22), but none of them showed cirrhosis.A high MC burden (>50 MCs/high-power field) was correlated with elevated blood alkaline phosphatase levels (p = .030).The presence of portal hypertension was associated with a higher mean fibrosis grade (1.6 vs. 0.8 in its absence; p = .026).In advanced SM, the presence of nodular regenerative hyperplasia (NRH) was associated with decreased overall survival (9.5 vs. 46.3months, p = .002).
Conclusions: MC infiltration induced polymorphic hepatic lesions and the degree of fibrosis is associated with portal hypertension.NRH identifies a poor prognosis subgroup of patients with advanced SM. Assessing liver histology can aid in SM prognostic evaluation.

K E Y W O R D S
advanced systemic mastocytosis, fibrosis, liver, mast cells, nodular regenerative hyperplasia, portal hypertension

Key points
Mastocytosis is a rare disease of an immune cell called a mast cell.Some forms of mastocytosis can affect the liver and may considerably reduce life expectancy.Our study investigates the characteristics of liver biopsies from patients with hepatic mastocytosis.We report hepatic features associated with ascites and one particular abnormality (nodular regenerative hyperplasia) reduces dramatically patients' life expectancy.Liver biopsy may be a useful tool in deciding the management of a subset of mastocytosis patients.
Given that (i) SM is a rare cause of liver dysfunction and (ii) specific histological characterization should help the pathologist to distinguish between SM (especially its aggressive forms) and other more frequent diagnoses of hepatomegaly and cirrhosis, we decided to characterize the histological features of the hepatic lesions associated with SM.We also studied the consequences of MC infiltration on clinical and laboratory variables and evaluated the histopathological features associated with the WHO classification, liver Cfindings, and outcomes.

| PATIENTS AND ME THODS
We used the database curated by the French nationwide network  For all LBs, we evaluated specific hepatic lesions: cirrhosis, fibrosis (according to the Metavir score), 13 portal or sinusoidal infiltration by lymphocytes or other cells, abnormal biliary ducts, hepatocyte necrosis, sinusoidal dilatation, cholestasis, or siderosis.The degree of MC infiltration was assessed with anti-c-kit or -tryptase antibodies and the number of MCs was counted on highpower field (HPF) (×40) for each LB.Overall survival (OS) analysis was considered from the date of liver biopsy to date of death or last visit.Statistical analyses were performed using the GraphPad Prism software (version 6.0; GraphPad Software, Inc, La Jolla, CA, USA).Groups were compared using Student's t test, a chi-squared test, or Fisher's exact test, as appropriate.Data were expressed as the mean, median [interquartile range], or median (range).OS probabilities were estimated using the Kaplan-Meier method and compared using the log-rank test.The threshold for statistical significance was set to p < .05.
In 5 of the 28 patients, the LB revealed a low degree of MC infiltration (1-5 MCs/HPF) (Table 2).The degree of MC infiltration was  the patients.When compared patients with vs. without C-findings, we observed a non-significant association between the presence of hepatic C-findings and the degree of mast cell infiltration (p = .08)(Table 4).The only marker significantly associated with the presence of hepatic C-findings was the severity of fibrosis (mean fibrosis stage: 1.6 vs. 0.8 in the absence of hepatic C-findings; p = .026).

| DISCUSS ION
Adv-SM is a rare cause of non-cirrhotic portal hypertension; hence, specific histological and immunohistochemical characterization is essential to distinguish between SM and other, more frequent etiologies of cirrhosis.This study is the first to have provided a detailed histological description of liver involvement in Adv-SM and to have assessed correlations between histological features on one hand and clinical and laboratory variables on the other hand.We observed correlations between CD30 expression by MCs, the blood ALP level, and the degree of MC infiltration.As blood ALP levels have already been linked to a poor outcome in SM, our observation suggests that the MC liver burden may be a prognostic marker. 14,15evious studies have investigated the features of LBs from patients with SM. [8][9][10][11][12] As we observed, most of these studies reported a high MC infiltrate in the liver in general and in the portal tract in particular.In contrast to the frequently observed fibrosis, cirrhosis was rare or absent.Our result confirmed the high prevalence of liver fibrosis in SM but also highlighted a correlation between the severity of fibrosis and the presence of hepatic C-finding.The presence of this correlation suggests that fibrosis is pathophysiologically involved in portal hypertension.
Finally, we found a significant decrease in the OS of Adv-SM patients with NRH.Interestingly, NRH have been previously reported in LB from patients with mastocytosis.Indeed, Mican et al  The management of advanced SM has been greatly improved with the approval of tyrosine kinase inhibitors (TKI) targeting the KIT D816V mutation (i.e.midostaurin and avapritinib).Phase II clinical trials highlighted that signs of liver dysfunction were responsive to treatment (including ascites and alkaline phosphatase levels). 16,17In addition, a recent study has shown that the severity of liver fibrosis may decrease with TKI. 18Interestingly, the authors analysed fibrosis in 2 patients using magnetic resonance elastography and repeated liver biopsies and the results appeared to be consistent between the two procedures.Magnetic resonance elastography may therefore be a tool for assessing liver stiffness and the degree of fibrosis in patients with advanced SM on therapy.
In conclusion, the hepatic lesions induced by MC infiltration are polymorphic and include abnormal biliary ducts, sinusoid dilatation, and fibrosis (the latter being associated with liver C-findings).The poor prognosis associated with NRH in advanced MS may argue for early intervention with dose-adapted kinase inhibitors and allogenous stem cell transplantation should be discussed if feasible in this context.
of mastocytosis reference centers (CEREMAST) to identify adult patients with a confirmed diagnosis of SM (according to the WHO 2016 classification) and an available liver biopsy.The database was registered with French National Data Protection Commission (Commission nationale de l'informatique et des libertés (Paris, France)); reference: 1445939, dated 29 October 2010).All identified patients had participated in the "Association for Research Initiatives on Mast Cells and Mastocytoses" (AFIRMM) study, which had been approved by the local investigational review board (Comité de Protection des Personnes Ile-de-France, Pitié-Salpétrière, Paris, France; reference: 93-00) in accordance with the Declaration of Helsinki.The CEREMAST centers were contacted and asked to provide the patients' clinical data and outcomes.In a detailed review, we checked for all possible confounding factors for abnormal hepatic features, including alcohol abuse, hepatitis B or C, chronic heart failure, autoimmune, and/or granulomatosis diseases with a hepatitis component, storage/metabolic diseases (such as hemochromatosis, Wilson disease, amyloidosis, glycogenosis, lysosomal diseases, and obesity/ metabolic syndrome), and hereditary or iatrogenic causes of liver disease.None of these conditions was considered to be a significant, a sole cause of liver function impairment.The diagnosis of portal hypertension was made by doppler ultrasonography.Overall, 28 patients were included in this study, and all the liver biopsies (LBs) were reviewed centrally by the same CEREMAST pathologist (DC, Figure 1).The liver biopsies were performed between December 1999 and March 2016.Confounding factors for abnormal liver characteristics were investigated and are described in the online repository.Each formalin-fixed biopsy was stained F I G U R E 1 Hepatic histological features of systemic mastocytosis.(A) Grade 2 fibrosis (formalin-fixed biopsy was stained with Red Sirius, ×100 magnification).(B) High mast cell infiltration in the liver (formalin-fixed biopsy was stained anti-CD117 antibody, ×100 magnification).(C) CD30-positive mast cells (formalin-fixed biopsy was stained anti-CD30 antibody, ×200 magnification).

F I G U R E 2
Overall survival according to the presence of nodular regenerative hyperplasia on LB in patients with Adv-SM.(A) Time from LB. Nodular regenerative hyperplasia was associated with decreased overall survival (9.5 vs. 46.3months, p = .002).(B) Time from diagnosis.Nodular regenerative hyperplasia was associated with decreased overall survival (16.7 vs. 49.92months, p = .035).OS probabilities were estimated using the Kaplan-Meier method and compared using the log-rank test.NRH: nodular regenerative hyperplasia.results suggest that SM should be assessed in patients with unexplained NRH, especially with signs of portal hypertension.
Clinical and laboratory data for the study cohort, overall, and as a function of the type of SM.

Table 3
Histopathological findings in liver biopsies from patients with SM.
Clinical, histologica,l and laboratory data as a function of hepatic MC infiltration.Overall survival according to hepatic histological features in advanced SM patients.OS analysis was considered from the date of liver biopsy to date of death or last visit.OS probabilities were compared using the log-rank test.
11have found NRH associated with fibrosis in 4/5 patients with mastocytosis TA B L E 3 Adv-SM; clinical studies, including allogenous haematopoietic stem cells transplantation, are critically needed to optimize the management of this specific population.Therefore, LB should be considered in Adv-SM patients with portal hypertension to assess the staging of liver involvement, including NRH presence.On the other hand, our TA B L E 4 Histological features, according to the presence or absence of hepatic C-findings.TA B L E 5Abbreviations: HPF, high-power field; MC, mast cell; ns, non-significant; OS, overall survival.