Nodular Regenerative Hyperplasia – an under-recognized vascular disorder of liver

Journal Pre-proof Nodular Regenerative Hyperplasia – an under-recognized vascular disorder of liver Neha Bakshi, Natasha Gulati, Archana Rastogi, Abhijit Chougule, Chhagan Bihari, Ankur Jindala

PII:

S0344-0338(19)32122-3

DOI:

https://doi.org/10.1016/j.prp.2020.152833

Reference:

PRP 152833

To appear in:

Pathology - Research and Practice

Received Date:

26 September 2019

Revised Date:

24 December 2019

Accepted Date:

18 January 2020

Please cite this article as: Bakshi N, Gulati N, Rastogi A, Chougule A, Bihari C, Jindala A, Nodular Regenerative Hyperplasia – an under-recognized vascular disorder of liver, Pathology - Research and Practice (2020), doi: https://doi.org/10.1016/j.prp.2020.152833

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TITLE PAGE

(A) Complete manuscript title: Nodular Regenerative Hyperplasia – an under-recognized vascular disorder of liver (B) Authors’ full names, highest academic degrees, and affiliations: Dr Neha Bakshia, MD DNB Senior resident (PDCC Fellow), Department of Pathology, a Institute of liver and biliary sciences, Vasant Kunj, New Delhi, India. [email protected]

2.

Dr Natasha Gulatia MD PDCC Hepatopathology, Department of Pathology, aInstitute of liver and biliary sciences, Vasant Kunj, New Delhi, India.

3.

Dr Archana Rastogia, MD DNB PDCC Professor, Department of Pathology, a Institute of liver and biliary sciences, Vasant Kunj, New Delhi, India. [email protected]

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Dr Chhagan Biharia, MD Associate Professor, Department of Pathology, a Institute of liver and biliary sciences, Vasant Kunj, New Delhi, India. [email protected] Dr Ankur Jindala, MD DM (Hepatology) Associate Professor, Department of Hepatology, a Institute of liver and biliary sciences, Vasant Kunj, New Delhi, India.

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Dr Abhijit Chougulea MD PDCC Hepatopathology, Department of Pathology, aInstitute of liver and biliary sciences, Vasant Kunj, New Delhi, India.

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(C) Name and address for correspondence Dr Archana Rastogi, MD DNB PDCC Additional Professor, Department of Pathology, Institute of liver and biliary sciences, Vasant Kunj, New Delhi, India. 1

[email protected]; Tel.: +91 11 46300000, Extn.: 6049

Abstract Introduction: Nodular regenerative hyperplasia (NRH) is a rare hepatic vascular disorder which is often associated with wide variety of systemic diseases. Intrahepatic microvascular injury and subsequent altered perfusion state leads to development of non-cirrhotic portal hypertension in

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many of these patients. Diagnosis of NRH often remains unsuspected clinically and liver biopsy is essential for the diagnosis and exclusion of fibrosis. We herein, present clinicopathological

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features of 22 NRH cases. In addition we assessed CK7 and CD34 expression in hepatocytes and sinusoidal endothelial cells respectively.

Results: Most of the cases were associated with systemic disorders, predominantly

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immunological, inflammatory and drug-related injuries. Signs and symptoms of portal hypertension were found in 86.4% patients. Majority of the patients showed a predominant mild

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cholestatic pattern of liver function tests. Nearly all the (21/22) cases showed CK7 positivity in centrizonal hepatocytes which ranged from <10% cells to diffuse perivenular positivity

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extending into the midzonal areas. CD34 positivity in sinusoidal endothelial cells was seen in all the cases, which was prominent in periportal areas in all cases; while perivenular (n=20) and midzonal (n=14) areas also showed CD34 positive sinusoidal endothelial cells.

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Conclusion: This study highlights the role of pathologist in the diagnosis of NRH and stresses upon the need for awareness of NRH as a cause of unexplained portal hypertension in patients with underlying systemic diseases. The altered perfusion state in NRH leads to phenotypic shift in centrizonal atrophic hepatocytes and sinusoidal endothelial cells (as depicted by IHC) which

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may be responsible for development of portal hypertension.

Key words: nodular regenerative hyperplasia; non-cirrhotic portal hypertension; histology; CK7; CD34.

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Introduction: Nodular regenerative hyperplasia (NRH) is a rare hepatic vascular disorder that usually occurs in association with various systemic diseases. NRH represents an adaptive hyperplastic reaction of hepatocytes to mechanical or functional abnormalities of hepatic microvasculature due to

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imbalances between arterial and portal venous blood flow [1-3] and is characterised by diffuse transformation of the hepatic parenchyma into small regenerative nodules in the absence of

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significant fibrosis. It is one of the important causes of non-cirrhotic portal hypertension and falls under the umbrella of idiopathic non-cirrhotic portal hypertension [4, 5]. The clinical

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presentation is variable from being asymptomatic to presentation with altered liver function tests or with the complications of portal hypertension such as bleeding oesophageal varices, splenomegaly and ascites. Histopathological examination plays a crucial role in providing

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definitive diagnosis and excluding cirrhosis. The currently used diagnostic criteria given by Wanless [1] are based on histopathological examination of autopsy liver specimens. But the

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subtle histopathological features of NRH are frequently overlooked or misinterpretated on liver biopsies. The awareness of NRH as one of the cause for non-cirrhotic portal hypertension is important and its early histological diagnosis can aid in timely patient management and

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prevention of the complications [5].

Normal undisturbed hepatocytes express cytokeratin (CK) 8 and CK18; while interlobular bile ducts additionally express CK7 and CK19. Aberrant expression of hepatocyte progenitor marker CK7 by periportal and midzonal hepatocytes is noted in chronic biliary tract diseases especially

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in later stages of primary biliary cirrhosis [6]. In periportal areas the CK7 positive hepatocytes are derived from the hepatic progenitor cells located in the canals of Herring, which have ability to differentiate into both cholangiocytes and hepatocytes. Recently a group of diseases associated with ‘centrizonal hepatocytic injury’ such as hepatic venous outflow obstruction, alcoholic and non-alcoholic steatohepatitis were shown to have aberrant CK7 expression in centrizonal hepatocytes [7-11]. Though NRH does not belong to this group of diseases, a similar CK7 expression pattern was observed in 4 NRH cases [10]. Normal endothelial cells of liver do not 3

express CD34; however under pathological perfusion states the sinusoidal endothelial cells can show CD34 expression [12]. As NRH is associated with altered portal venous perfusion, therefore the response of sinusoidal endothelial cells with respect to CD34 expression will be interesting to study. Herein, we describe the clinicopathological features of 22 histologically diagnosed NRH cases. We also studied the expression of CK7 and CD34 as markers of phenotypic shift in hepatocytes

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and sinusoidal endothelial cells respectively in NRH.

Methods & Materials:

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A total of 22 cases diagnosed as NRH between January 2011 and December 2016 were retrieved from the archives of the pathology department. Liver tissue was obtained by percutaneous needle

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biopsy (n=12, including one post-liver transplant allograft biopsy & one necropsy biopsy), transjugular approach (n=9) and right segment 6 hepatectomy specimen (n=1). Serial step

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sections stained with haematoxylin & eosin (H&E), Masson’s trichrome, reticulin silver and elastic van Gieson stain were examined. Clinical features, laboratory and imaging findings were

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retrieved from the patient files and hospital information system. None of these patients were diagnosed with or suspected to have hepatic vascular disorder prior to the index biopsy. All slides were reviewed and a classification system proposed by Wanless [1990] was applied as

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histological criteria for the diagnosis of NRH [1]. These included the presence of hepatocellular nodules less than 3 mm in diameter that were not surrounded by fibrosis (nodules graded 0-3 based on the extent of nodularity noted through all fields of the biopsy), and the presence of fibrous septa (graded 0-3) [1]. Biopsies that met the criteria of score 3 nodularity and score 0-1 fibrous septa were classified as NRH. Nodular transformation was recognized by regions of

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atrophy juxtaposed to normal or hyperplastic regions with a curved contour and no intervening fibrous septum. Microscopically, the nodules were composed of benign appearing hepatocytes often arranged in double-cell plates. The hepatocytes between nodules were atrophic with scanty cytoplasm. The other histological features assessed were: sinusoidal dilatation, perivenular and perisinusoidal fibrosis, portal vein dilatation, presence of atretic portal tracts and absence or sclerosis of portal vein radicals.

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Four micrometer sections were cut and immunohistochemistry was done using commercially available antibodies directed against: CK7 (OV-TL 12/30, Cell Marque, ready to use) and CD34 (QB End/10, Cell Marque, ready to use). The intensity of CK7 staining in hepatocytes and CD34 staining in sinusoidal endothelial cells was graded semi quantitatively as - 0: no staining; 1+: faint staining; 2+: moderate staining; 3+: strong staining. Also the number and location of hepatocytes and sinusoidal endothelial cells stained with CK7 and CD34 respectively were noted according to acinar zones. CK7 positive periportal hepatocytes were excluded while assessing

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the CK7 positivity.

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Results:

Of the total 10276 liver tissue specimens received during the study period, twenty-two cases

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(n=22; 0.21%) were diagnosed as NRH. Twelve of these patients were male and 10 were female, with a median age of 40 years (range: 16 years to 59 years). The main indications for liver

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biopsy were exclusion of chronic liver disease in view of clinical signs of portal hypertension, confirmation of clinical diagnosis and as part of work-up for liver function tests abnormalities. The commonest presenting symptom was abdominal pain (n=14; 63.6%); jaundice was seen in 3

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patients (13.6%). Signs and symptoms related to portal hypertension were seen in 19 (86.4%) patients (which included splenomegaly (n=16), hypersplenism (n=11), haematemesis due to

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bleeding esophageal varices (n=7) and ascites (n=2). Five patients had normal sized spleen while one patient had previously undergone splenectomy. Most of the patients with portal hypertension had grade I or II esophageal varices on upper gastrointestinal endoscopy. Associated systemic illness was recorded in 13 patients (n=13; 59%). These included present or past history or tuberculosis (n=5), diabetes mellitus (n=2), systemic lupus erythematosus (SLE;

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n=2), rheumatoid arthritis (n=1), congenital extrahepatic portocaval shunt (Abernethy type 1b; n=1) and schistosomiasis (n=1). Evidence of HCV-related and HBV-related chronic viral hepatitis was seen in 2 and one case respectively. History of drug intake was present in 12 patients [past or present history of antitubercular treatment (n=5), steroids (n=3), antidiabetics (n=2), complementary and alternative medicine (n=2), anticonvulsants (n=1), azathioprine (n=1) and immunosuppressants (tacrolimus, mycophenolate mofetil, prednisolone) for post-liver transplant (n=1)]. 5

Details of liver function test results in the study cohort are shown in table 1. Most patients showed a cholestatic pattern (mild to moderate elevation of alkaline phosphatase, gamma glutamyl transpeptidase and direct bilirubin levels); while some had mild transaminitis. Autoimmune markers were tested in 16 patients; antinuclear antibodies (ANA), perinuclear antineutrophil cytoplasmic antibodies (p-ANCA) and rheumatoid factor were detected in 4, 2 and 2 patients respectively. Serology for HIV I and II was negative in all 14 patients in whom it was tested; while serological markers for HBV & HCV were negative in all, except 3 patients in

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whom HCV genotype 3, HCV genotype 1b and HBV genotype B was confirmed by molecular analysis. Imaging studies (16- computed tomography (CT), 5-ultrasonography, 1- magnetic

hepatomegaly in 7 cases and splenomegaly in 16 cases.

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resonance imaging [MRI]) showed irregular lobulated outlines in most of the patients with mild

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Liver biopsy in all the cases showed vague parenchymal nodules composed of 2 cell thick cords of benign hepatocytes, with compressed curvilinear atrophic hepatocytes at the periphery of

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these nodules [Fig. 1A and 1B]. These findings were better highlighted on reticulin silver stain [Fig. 1C and 1D]. The atrophic hepatocytes were polygonal and considerably smaller compared

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with normal hepatocytes. Presence of histological features of the coexisting diseases such as compact epithelioid cell granulomas (in a case with sarcoidosis), schistosoma eggs with granulomatous reaction, portal inflammation (chronic viral hepatitis cases) were seen as

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expected. There was no evidence of cirrhosis. None of the patients with autoimmune markers positivity showed features of autoimmune liver diseases. We also assessed histopathological features of non-cirrhotic portal hypertension (NCPH) involving the portal and perivenular vasculature in our cases and noted variable presence of a spectrum of changes. These included sinusoidal dilatation (n=7), dilated portal vein radicals

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(n=13), portal vein remnants (n=6), compressed, atrophic or absent portal vein (n=11), and portal vein herniation into sinusoids (n=3). Centrilobular fibrosis was seen in one case and mild perisinusoidal fibrosis was noted in two cases. Such overlapping histological features indicate, NRH to be part of the spectrum of non-cirrhotic vascular disorders associated with portal hypertension.

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The clinicopathological profile and IHC findings are described in table 2. IHC for CK7 showed positivity in centrizonal hepatocytes (far away from the portal tracts) in 21 cases. Varying grades of staining intensity were noted and scored accordingly as 1+, 2+ and 3+ (n=4, 7 and 10 respectively). The number of CK7 positive hepatocytes ranged from <10% cells to diffuse perivenular positivity extending into the midzonal areas [Fig. 2A, B]. Most of the CK7 positive hepatocytes were considerably smaller than hepatocytes in the periportal areas and in centers of regenerating nodules suggesting them to be hepatocyte progenitor cells or intermediate

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phenotype hepatocytes. Periportal CD34 positive sinusoidal endothelial cells were seen in all cases with a variable

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staining intensity (1+, 2+ and 3+ in 1, 2 and 19 cases respectively). Perivenular (n=20) and midzonal (n=14) areas also showed CD34 positive endothelial cells with variable intensity (1+ to

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3+) (Table 2). The vascular histological features of non-cirrhotic portal hypertension such as portal vein dilatation, increased portal channels, herniation of portal vein into the lobules and

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sinusoidal dilatation were better appreciated by CD34 immunohistochemistry.

Discussion:

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NRH is a rare vascular disorder of liver, first described as “milliary hepatocellular adenomatosis” by Ranstrom in 1953 [13]. The term NRH was coined by Steiner in 1959 to distinguish this

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histopathologic entity from cirrhosis of liver [14]. Clinical diagnosis of this entity is challenging since the presentation is variable and the majority of patients present without symptoms or abnormalities in laboratory parameters. If symptoms are present, these are mainly due to portal hypertension, such as thrombocytopenia, oesophageal varices, splenomegaly and ascites [3, 5]. In our series 86.4% (n=19) patients presented with signs and symptoms of portal

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hypertension. Liver function tests are either normal or show mild derangements, which may be related to NRH or the underlying systemic disease in these patients. Many studies have reported a mild cholestatic pattern of liver biochemistry in NRH, which was also observed in our study [15, 16]. Imaging methods, such as MRI and CT can demonstrate nodularity and heterogeneous hepatic parenchyma suggestive of NRH, but non-invasive tests do not have adequate sensitivity or specificity and histological examination of liver tissue remains the cornerstone of diagnosis [2, 5]. However, NRH is characterized by subtle morphologic features and diagnosis may be easily missed even by experienced pathologists. Further, the morphological changes of NRH may not 7

be striking in needle core biopsies. A high index of suspicion, especially in patients of systemic diseases with portal hypertension in the absence of significant liver fibrosis is therefore vital for the accurate diagnosis of this entity. Though it is being increasingly reported recently, NRH remains a poorly understood entity, the current knowledge of which is limited to case reports, few small case series and literature reviews. Table no 3 depicts the few largest case series on NRH reported so far in literature. Our series has comparable number of cases. As discussed, the clinical presentation of NRH is variable, ranging from completely

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asymptomatic to non-specific symptoms related to underlying systemic diseases to presentation with features of portal hypertension. The incidence of portal hypertension has been variable in

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various studies, ranging from 4.7% in autopsy study by Wanless to around 50% in other studies [1, 3, 15]. In this study 86.4% (19/22) patients had evidence of portal hypertension, which is

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significantly more compared with other studies. This may be due to selection and referral bias, as only biopsy proven cases were studied in patients with clinically overt portal hypertension. Also NRH patients in current study probably represent the severe end of NRH spectrum, which might

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have been referred to our tertiary care specialist liver hospital. Several systemic disorders including diseases of autoimmune, inflammatory, neoplastic especially hematological

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malignancies, collagen vascular, metabolic, endocrine or idiopathic origin are associated with NRH [1-4, 15, 16]. The pathogenic role of the underlying systemic diseases may be related to factors such as hypercoagulability and thrombotic obliterative changes in small portal venules

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and cytotoxic cell and autoantibody mediated damage to sinusoids and endothelium [19]. Several drugs and medications were reported to have a more frequent association with NRH probably resulting from toxic endothelial damage in small venous channels and sinusoids. Present study also showed association of NRH with inflammatory, infective, autoimmune and rheumatologic

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conditions in the majority of patients. NRH is postulated to be a common cause of portal hypertension in patients with hematological disorders including myeloproliferative disorders and malignancies including Hodgkin’s disease, non-Hodgkin’s lymphoma chronic lymphocytic leukemia and multiple myeloma. Literature is scant on NRH in the setting of solid organ malignancies [20]. There are a few reported cases of NRH in patients with colorectal cancer in the setting of antineoplastic treatment, as well as a single case report of NRH associated with carcinoid tumor [21, 22]. Nzeako et al found that liver cell dysplasia occurred in a significantly greater proportion of those with NRH than those without; and 23 out of 342 Hepatocellular 8

carcinomas (HCC) arising in non-cirrhotic livers were associated with NRH [23]. They postulated that the association between HCC and NRH may be due to the development of HCC within the dysplastic foci that occur in livers with NRH or due to the converse possibility that NRH may also develop in a noncirrhotic liver with HCC, secondary to portal venous invasion with portal vein occlusion. The temporal relationship between HCC and NRH is probably determined in each case by the particular interaction of multiple pathogenetic factors. Turk et al reported a case of male breast carcinoma where portal vein occlusion by metastatic thrombosis

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led to NRH like picture on liver autopsy [24]. Recently, Dumas et al reported paraneoplastic NRH in association with Placental site trophoblastic tumor, the first report of its kind in literature [25]. In a recent report by Penrice et al, the development of primary liver malignancies (HCC

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and cholangiocarcinoma in 2 cases each) was identified in 4 of 183 cases of biopsy-proven NRH over a 15-year period [26]. None of the cases in our series had a present or past history of

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neoplasia.

NRH is considered as one of the morphologic manifestation of microvascular disorders of liver.

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Recent studies have also emphasized that NRH represents a pattern of injury in cases of noncirrhotic portal hypertension (NCPH), where normal blood flow through the liver is impeded in

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the absence of the architectural distortion and shunting that characterizes the cirrhosis [27, 28]. NCPH is a heterogeneous group of liver disorders of vascular origin, leading to portal hypertension with near normal HVPG. The diseases leading to NCPH are primarily vascular in

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nature and classified anatomically on the basis of site of resistance to blood flow, as prehepatic, hepatic, and post-hepatic – hepatic causes are further subdivided into pre-sinusoidal, sinusoidal and post-sinusoidal [29]. NRH comprises 27% of all cases of non-cirrhotic portal hypertension in Europe and about 14% in Japan [29]. Hemodynamically, portal hypertension in NRH is

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presinusoidal and characterized by an imbalance between arterial and portal venous blood flow [20]. Non-cirrhotic portal fibrosis (NCPF) is a clinical disorder of uncertain etiology characterized by features of portal hypertension in the absence of significant fibrosis, relatively preserved hepatic synthetic function, and patent portal and hepatic veins at radiological examination [29,30]. It is one of the commonest causes of portal hypertension in India and Japan; while prevalence in Western countries is low. The constellation of clinicopathological findings of this condition is known by different names in different regions which include NCPF in India, idiopathic portal hypertension (IPH) in Japan; while in western countries it is known by 9

various terms such as hepato-portal sclerosis (HPS), obliterative portal venopathy (OPV), nodular regenerative hyperplasia (NRH), and incomplete septal cirrhosis [29]. To avoid ambiguity, Schouten et al. proposed “idiopathic noncirrhotic portal hypertension (INCPH)” as uniform nomenclature for all these conditions, which was reiterated in Baveno VI consensus workshop which states that all these conditions are the same [31,32]. Histopathology of NCPF has a wide spectrum ranging from minor non-specific changes, obliterative sclerosis of portal vein branches, and portal fibrosis to NRH. Rastogi et al in their study evaluated in detail the

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spectrum of histopathological changes in 66 cases of NCPF and described the biopsy features and the approach to liver biopsies in these cases. They emphasized that due to relatively nonspecific and non-pathognomonic nature, a combination of different histological features in the

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absence of significant fibrosis and appropriate clinico-radiological background is needed for diagnosing NCPF [33]. Obliterative changes involving the small portal vein branches—the

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obliterative portal venopathy (OPV) is the predominant underlying histopathological lesion of all these conditions [33]. As emphasized by the recent study by Jharap et al [34], portal veins should

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be carefully examined in cases of NRH. In the present study, we examined all the biopsies for the portal and perivenular vasculature abnormalities. Portal vein obliteration, paraportal shunts,

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atretic portal tracts, dilated thin portal venules were seen in a significant number of cases, confirming the role of obliterative portal venopathy, as an underlying mechanism. These nodular regenerative nodules may be perpetuating the damage by further contributing to the presinusoidal

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resistance to hepatic blood flow.

Matsukuma et al. [35] studied CK7 expression in common non-neoplastic liver diseases such as chronic hepatitis (HBV and HCV related), non-alcoholic steatohepatitis, alcoholic liver disease, primary biliary cirrhosis, autoimmune hepatitis etc. In this study, CK7 expression in

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centrilobular hepatocytes was observed in around 50% cases with few cases showing positivity in rounded or cuboidal cells with scant cytoplasm morphologically resembling hepatic progenitor cells. Based on these findings, authors suggested centrilobular regions as another possible niche of hepatic progenitor cells. However, these findings need further corroboration by other studies using different hepatic progenitor cell markers. In this study, CK7 positivity was noted in perivenular atrophic hepatocytes in most of the cases. Whether this represents a protective metaplastic response of hepatocytes or migration of hepatic progenitor cells from their niche or activation of hepatic progenitor cells in the centrilobular niche is not known. A significant 10

association of CK7 positivity in centrilobular hepatocytes and cholestatic pattern of serum markers (elevated alkaline phosphatase, gamma glutamyl transpeptidase) was noted in various conditions in previous studies [7, 35]. This association favors CK7 positivity as a possible metaplastic response of centrizonal hepatocytes to cholestasis related damage. Also in the study by Matsukuma et al [35], 41.5% cases showed binucleated CK7 positive hepatocytes and these were associated with prominent thickening of hepatocytic plates, representing it to be active hepatocytic regeneration rather than regeneration through the activation of hepatic progenitor

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cells. Therefore in our opinion, CK7 positivity in atrophic perivenular hepatocytes of NRH might be an adaptive response to altered perfusion state rather than activation or migration of hepatic

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progenitor cells in these areas.

CD34 immunohistochemistry showed capillarization of periportal sinusoids in most of the cases

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along with capillarization in perivenular and midzonal location in some of the cases. This highlights phenotypic shift in the sinusoidal endothelial cells which are normally CD34 negative.

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CD34 is a transmembrane phosphoglycoprotein expressed on the luminal cell membrane of endothelial cells of small vessels and function as cell-cell adhesion molecule [12]. As mentioned

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above, imbalance between arterial and venous blood supply in NRH leads to hypertrophy of acinar hepatocytes with maintained or increased perfusion and atrophy of the peripheral hepatocytes corresponding to areas of hypoperfusion [28]. The expression of CD34 by the

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sinusoidal endothelial cells in NRH therefore might represent an attempt to stabilize and prevent the disruption of sinusoidal lining by altered perfusion states [12]. Whether the changes in local blood pressure or oxygenation status triggers this phenotypic shift needs further evaluation. Generally the sinusoidal capillarization precedes the fibrosis which subsequently leads to development of portal hypertension. But in most of our cases, there was no significant fibrosis,

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which might suggest the altered hemodynamics due to sinusoidal capillarization may be sufficient to produce portal hypertension. We have observed similar type of sinusoidal capillarization in small number of idiopathic non-cirrhotic portal fibrosis/hypertension cases (unpublished data). Therefore NRH truly belongs to morphological spectrum of conditions associated with clinically overt portal hypertension in the absence of significant parenchymal fibrosis.

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Conclusion: NRH is a rare vascular disease of liver that often remain unrecognized clinically. Liver biopsy is essential for the diagnosis and for excluding cirrhosis. NRH should be included in the differential diagnosis in patients with systemic inflammatory, toxic injury, immunological and infective diseases as a cause for portal hypertension. The altered perfusion state in NRH leads to phenotypic shift in centrizonal atrophic hepatocytes and sinusoidal endothelial cells which may

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be responsible for development of portal hypertension.

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(D) Sources of support that require acknowledgment

(E) Grant numbers and/or funding information None

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(F) Disclaimers, if any –

(H) Author contributors:

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None declared. (G) Word count: 2709 words

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None declared.

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Archana Rastogi designed the study and performed histopathological analysis, revision and improvement of the manuscript. Neha Bakshi, Abhijit Chougule and Natasha Gulati collected the clinical details from the hospital information system and assisted in the histopathology work and drafted the manuscript. Chhagan Bihari provided Pathology related inputs.

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Ankur Jindal provided Hepatology related inputs. Conflict of interest statement We certify that we have no conflict of interest to disclose.

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liver malignancies. Hepatology. 2019. doi: 10.1002/hep.30905. [Epub ahead of print]

Dis. 2015; 5: 123-6.

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27. Kleiner D. Noncirrhotic portal hypertension: pathology and nomenclature. Clin Liver

28. Lee H, Rehman AU, Fiel MI. Idiopathic noncirrhotic portal hypertension: an appraisal. J

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Pathol Transl Med. 2016; 50: 17-25.

29. Khanna R, Sarin SK. Non-cirrhotic portal hypertension—diagnosis and management. J Hepatol 2014;60:421–441.

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30. Goel A, Elias JE, Eapen CE, Ramakrishna B, Elias E. Idiopathic non-cirrhotic intrahepatic portal hypertension (NCIPH)-newer insights into pathogenesis and emerging newer treatment options. J Clin Exp Hepatol 2014; 4:247–56. 31. Schouten JN, Garcia-Pagan JC, Valla DC, Janssen HL. Idiopathic noncirrhotic portal hypertension. Hepatology 2011; 54: 1071–81.

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32. de Franchis R, Baveno VI. Faculty. Expanding consensus in portal hypertension: report of the Baveno VI consensus workshop: stratifying risk and individualizing care for portal hypertension.. J Hepatol 2015;63:743–752.

33. Chougule A, Rastogi A, Maiwall R, Bihari C, Sood V, Sarin SK. Spectrum of histopathological changes in patients with non-cirrhotic portal fibrosis. Hepatology International. https://doi.org/10.1007/s12072-018-9857-y(0123456789().,-volV)(

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34. Jharap B, van Asseldonk DP, de Boer NK, Bedossa P, Diebold J, Jonker AM, et al. Diagnosing Nodular Regenerative Hyperplasia of the Liver Is Thwarted by Low Interobserver Agreement. PLoS One. 2015; 10: e0120299. 35. Matsukuma S, Takeo H, Kono T, Nagata Y, Sato K. Aberrant cytokeratin 7 expression of

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centrilobular hepatocytes: a clinicopathological study. Histopathology. 2012; 61: 857-62.

Parameter (normal range)

Median

Range

Total bilirubin (0.3-1.2mg/dl)

0.7

0.6-19.4

AST (5-40 IU/L)

47

13-238

Jo

Table 1: Liver Function test values in the study population (n=22)

ALT (10-40 IU/L)

43.5

10-181

Alkaline phosphatase (32-92 IU/L)

167.5

23-550

Gamma glutamyl transpeptidase (7-64 IU/L)

46.5

12-194

Total proteins (6-8 g/dl)

7.3

3.9-8.8

Serum albumin (3.2-4.6 g/dl)

3.4

2-4.6

Prothrombin time (11-13 sec)

13.3

11.3-24.2

16

Hemoglobin (13-17 g/dl)

11.1

6.4-15.7

Total leukocyte count (4-11x109/L)

3.8

2.8-11.6

Platelet count (150-400x109/L)

120

17-391

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ALT- alanine aminotransferase, AST- aspartate aminotransferase,

Table 2: Clinicopathological and IHC findings in the study population (n=22) No. Age/

Clinical

Drug intake

CK7

of

diagnosis

(Past/present)

hepatocytes

Jo

sex

Type

33/F

PC

% positivity) Tuberculosis, ATT

PV (3+, 17%)

on ATT 2

55/M

PC

Location (intensity)

Location (intensity,

biopsy

1

positive CD34 positive SEC

-

PP (3+), few PV (3+) areas

PV (2+, 20%)

PP (3+), all PV (3+) areas

17

3

39/F

PC

-

Anti-

PV (3+, 15%)

PP (3+), 50% PV (3+),

convulsants 4

19/M

PC

few MZ (3+) areas

CAM

PV (2+, 12.5%)

PP (3+), few PV (1+) areas

6

25/M

31/M

TJLB

PM

Past

h/o ATT

PV (2+, 20%)

pulmonary

(3+), few MZ (3+)

Tuberculosis

areas

Abdominal

ATT

PV (3+, 60%)

tuberculosis

8

55/F

TJLB

32/M

PC

SLE

, few PV (3+) areas

Steroids

41/M

TJLB

HBV

PV (1+, 10%)

Tubercular

ATT

13

46/M

40/F

PC

TJLB

TJLB

Jo

14

54/F

PC

15

DM

on

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12

47/M

ATT

lP

ascites,

11

-p

PC

58/F

TJLB

Sarcoidosis

DM

PV (1+, 33%)

re

10

29/F

PV (2+, 50%)

Tuberculosis, ATT on ATT

9

PP (3+), few MZ (3+)

ro

7

PP (3+), >50% PV

of

5

Antidiabetics

Steroids

Negative

PP (3+), few MZ (3+) , few PV (3+) areas PP (3+), few PV (1+) areas PP (3+), few MZ (1+) & PV (1+) areas PP (3+), occasional MZ (1+) & PV (1+) areas

PV (3+, 40%)

PP (3+), few MZ (3+) & PV (3+) areas

PV (3+, 90%)

PP (3+), few MZ (3+) many PV (3+) areas

Antidiabetics

PV (1+, 10%)

PP (3+), few MZ (3+) & PV (3+) areas

HCV

PV (3+, 30%)

PP (3+) and PV (3+) areas

RA

Azathioprine

PV (3+, 30%)

PP (3+), few MZ (3+) & PV (3+) areas

16

59/M

PCx

-

Immunosuppresants

PV (3+, 40%)

PP (2+) and few PV (2+)

18

17

36/M

TJLB

HCV; CKD

PV (1+, 25%)

PP (3+), occasional

on MHD 18

42/M

TJLB

PV (2+) areas

-

PV (2+, 17%)

PP (3+), focal MZ (2+) & PV (2+) areas

19

55/F

TJLB

SLE

Steroids

PV (3+, 80%)

PP (3+), MZ (3+), few PV (3+) areas

20

48/F

PC

-

21

19/M

PC

Abernethy

PV

syndrome

MZ (3+, 50%)

Segmen

Schisto

PV (2+, <10%)

tectomy

somiasis

>90%), PP (3+), focal MZ

of

(3+,

PP (2+) areas

ro

16/F

PV (2+, 20%)

(2+)

Few PP (1+), MZ (1+) , minimal PV (1+)

-p

22

CAM

F- female, M- male, MZ- midzonal, PC- percutaneous liver biopsy, x – Post transplant, PM- post-

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disease, MHD-Maintenance hemodialysis.

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mortem, PP- periportal, PV- perivenular, TJLB- transjugular liver biopsy, CKD –Chronic kidney

Table 3: Details of previous reported large studies on NRH Study and year

Jo

S no 1 2

3 4

Colina et al (1989) [17] Wanless et (1990) [1]

No of Abnormal patients LFTs 24 87.5%

al 64 (of 2500 35.4% autopsies) (cholestatic)

Arvanitaki M et al 14 (2000) [4] Bissonnette et al 24 (2004) [18]

70% (cholestatic) 25% (cholestatic)

Portal Salient features hypertension 37.5% 2.6%

Proposed diagnostic criteria for NRH

Majority 100%

19

5

6

Vernier‐ Massouille 29 (+8) et al (2007) [19] 42 Morris et al (2010) [15]

70%

32%

76% (predominantly cholestatic)

31%

Only IBD patients treated with azathioprine

Figure legends:

of

Figure 1. A panel of microphotographs depicting: A, B: Nodules of hyperplastic hepatocytes (asterisk) surrounded by curvilinear cords of atrophic

ro

hepatocytes with compressed sinusoids (arrows) [A- HE, 10x; B-HE, 20x]

C, D: Reticulin stain highlighting hyperplastic (asterisk) and atrophic hepatocytes (arrows) [C-

Jo

ur na

lP

re

-p

10x; D-20x].

20

Figure 2. A panel of IHC microphotographs depicting: A: Focal CK7 positivity in few centrizonal hepatocytes [20x], B: Diffuse centrizonal hepatocytic CK7 positivity extending to midzonal areas [20x], C, D: CD34 positivity in periportal sinusoidal endothelial cells areas extending to midzonal areas [C-10x; D-20x]

Jo

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-p

ro

of

E, F: CD34 positivity in perivenular and midzonal areas [E-4x; F- 20x]

21