Histologic manifestations of ocrelizumab‐associated intestinal and hepatic injury in patients with multiple sclerosis

Ocrelizumab is a humanized anti‐CD20‐monoclonal antibody that has recently been approved for the treatment of certain types of multiple sclerosis. Isolated case reports of ocrelizumab‐associated colitis have been reported in the literature. We present a case series of ocrelizumab‐associated intestinal injury with a focus on histopathologic features and report a case of ocrelizumab‐associated hepatitis.

Aims: Ocrelizumab is a humanized anti-CD20monoclonal antibody that has recently been approved for the treatment of certain types of multiple sclerosis.Isolated case reports of ocrelizumab-associated colitis have been reported in the literature.We present a case series of ocrelizumab-associated intestinal injury with a focus on histopathologic features and report a case of ocrelizumab-associated hepatitis.Methods and results: A retrospective computerized search was conducted from 03/2017 to 08/2022, which identified six patients with suspected or clinically confirmed ocrelizumab-associated intestinal injury and one patient with hepatic injury.Pertinent clinical, endoscopic, and histopathologic findings were reviewed and recorded.Seven patients (six female, one male) were identified with ages ranging from 24 to 68 years.The presenting symptoms included diarrhoea (n = 5), abdominal pain (n = 3), hematochezia (n = 2), and vomiting (n = 1), nausea (n = 1) fever (n = 1), and weight loss (n = 1).Endoscopic findings ranged from normal (n = 1) to patchy colonic inflammation with or without ulceration (n = 4) and decreased mucosal vascular pattern in the rectum (n = 1).Crohn's disease was clinically suspected in two patients and ulcerative colitis in one patient.None of the patients had a prior confirmed diagnosis of inflammatory bowel disease.Histologic patterns of initial colonic injury included acute colitis/proctitis (n = 5), and chronic active colitis (n = 1).Follow-up ranged from 1 to 3 years and 10 months.All patients were alive at follow-up.Follow-up biopsies were available for four patients and findings included focal acute colitis (n = 1), apoptotic colopathy (n = 1) lymphocytic colitis (n = 1), and normal mucosa (n = 1).Four patients were treated with steroids and ocrelizumab was discontinued in three patients.Two patients were symptomatically managed with subsequent resolution of symptoms.The liver biopsy from the patient with a marked hepatic pattern of liver enzyme elevation showed an acute hepatitis pattern of injury with prominent centrilobular necrosis, which resolved upon discontinuation of the drug and treatment with steroids and azathioprine.Conclusions: The histologic manifestations of ocrelizumab-associated intestinal injury are variable and can mimic inflammatory bowel disease.Hepatic injury can rarely manifest as an acute hepatitis pattern of injury with necrosis.Identifying ocrelizumabassociated injury is paramount in determining management, which often includes discontinuation of ocrelizumab therapy, and/or administration of immunosuppressive therapy.

Introduction
Multiple sclerosis is a neurodegenerative and inflammatory disorder of the central nervous system affecting 2.8 million people worldwide. 1Relapsing-remitting multiple sclerosis (RRMS) is the most common phenotype, which, if untreated, over time can transform into a more progressive disease (secondary progressive multiple sclerosis).Primary progressive multiple sclerosis (PPMS), in which there is worsening of symptoms from the disease onset, is less common. 24][5][6] Ocrelizumab was approved for treatment in 2017 for RRMS and PPMS 7 and constitutes the first approved pharmacotherapy for PPMS. 3 It is available in liquid solution in single-use vials of 300 mg (30 mg/ml) under the brand name Ocrevus. 8he recommended dosage is 600 mg every 6 months, given intravenously as two 300-mg infusions (in 250 ml) 2 weeks apart for the first dose and single 600-mg infusions (500 ml) thereafter.In the US, the initial two infusions should each be given over ≥2.5 h.Subsequent doses can be given over ≥3.5 h. 4,5Unlike other anti-Cd20 therapies like rituximab, ocrelizumab is a humanized antibody conferring reduced immunogenicity.Studies show that circulating B cells and immunoglobulin levels can decrease postinfusion rapidly, and the effects can persist up to 6-18 months after the last dose. 9Although generally well-tolerated, side effects have been reported and include infusion reactions (most common), rash, diarrhoea, cough, infections like herpes simplex, urinary tract infections, pneumonia, and cellulitis.Rare serious side effects include adverse events seen with other anti-CD20 therapies like hepatitis B virus reactivation leading to hepatitis and liver failure, progressive multifocal leukoencephalopathy, focal segmental glomerulosclerosis, 10 psoriasiform dermatitis, 11 and refractory/severe colitis. 8,12Only isolated case reports of ocrelizumab-associated colitis and hepatitis have been reported in the literature.Herein, we report to our knowledge, the first case series of ocrelizumabinduced colitis and report a case of ocrelizumabassociated acute hepatitis.

Methods
The Institutional Review Board of the Ohio State University approved this study.A computerized search was performed to identify multiple sclerosis patients on ocrelizumab.The search was further refined to identify patients undergoing gastrointestinal tract and liver biopsies.Six patients with clinically confirmed cases of ocrelizumab-associated colitis and one patient with ocrelizumab-associated hepatitis were identified.Clinicopathologic data from the electronic medical record, including demographic data, presenting symptoms, colonoscopy and imaging findings, medication history, the timeline of ocrelizumab usage, treatment, and follow-up data were analysed.Glass slides and/or digitally scanned slides of 11 biopsies (10 colon and 1 liver) from the seven patients were reviewed to assess the pattern of injury.In colon biopsies, histopathologic features like cryptitis, crypt abscess, lamina propria inflammation, architectural changes, increased apoptosis, epithelial lymphocytosis, and ulceration were noted to identify the predominant pattern of injury.

C L I N I C A L F I N D I N G S
Six patients (five female, one male) with colitis and one female patient with hepatitis were identified.Ages ranged from 24 to 68 years.The presenting symptoms included diarrhoea (n = 5), abdominal pain (n = 3), hematochezia (n = 2), and vomiting (n = 1), nausea (n = 1) fever (n = 1), and weight loss (n = 1).The duration between the last dose of ocrelizumab and symptoms ranged from 14 days to 7 months.The number of ocrelizumab doses received prior to the development of symptoms ranged from one dose (n = 1), three doses (n = 1), four doses (n = 1), five doses (n = 1), and six doses (n = 2) (Table 1).
In the patient with hepatitis, the presenting symptoms were jaundice, dark urine, fatigue, shortness of breath, and nausea.The patient received one dose of ocrelizumab and developed symptoms 14 days after the infusion.Laboratory evaluation showed bimodal elevation of liver enzymes (aspartate aminotransferase peak 2081 U/l, alkaline phosphatase [ALP] peak 173 U/l) and increased total bilirubin (peak 12.7 mg/ dl).The autoimmune marker antinuclear antibody (ANA) was positive, with a titre of 1:80.Antismooth muscle actin antibodies (ASMA), and antimitochondrial antibodies (AMA) were negative.Serum quantitative immunoglobulins were in the normal range.Epstein-Barr virus (EBV) viral capsid antigen IgG antibody was found to be positive, but IgM was negative.Viral serologies for hepatitis A-E were negative.Serum quantitative viral loads for herpes simplex virus (HSV) I&II, cytomegalovirus (CMV), parvovirus B19, and enterovirus were negative.
Initial colonoscopy findings (Table 1) included unremarkable mucosa (n = 1), decreased vascularity in the rectum (n = 1), moderate inflammation and ulceration of the left colon (n = 1), patchy mild erythema in the rectum and rectosigmoid colon (n = 1), inflammation of the ileocecal valve (n = 1), and patchy mild to severe inflammation involving the entire colon and rectum (n = 1).
In the case of hepatitis, magnetic resonance imaging (MRI) of the abdomen showed a geographic area of hypoenhancement in the posterior right hepatic lobe with an associated likely unenhanced segment of the posterior branch of the right portal vein suggestive of segmental portal vein thrombosis.Hepatic venous pressure gradient measurements were found to be within normal limits (Table 1).

M I C R O S C O P I C F I N D I N G S
The most common pattern of colonic injury identified in our patients was acute colitis, identified in 7 of 10 biopsies.Other identified patterns of injury include chronic active colitis (n = 1) (Figure 1A,B), apoptotic colopathy (n = 1) (Figure 2B-D), and lymphocytic colitis (n = 1) (Figure 1C,D).Neutrophilic cryptitis/ crypt abscesses were found in all seven cases and ranged from minimal/focal (n = 3), mild (n = 3), and severe with surface erosions (n = 1) (Figure 3A,B).Immunostains for herpes simplex virus I&II (HSV), and adenovirus, and periodic acid Schiff (PAS) for fungus stains to rule out infection were done in two cases (one with mild colitis and one with mild to severe active colitis) were negative.Crypt architectural distortion and crypt atrophy were found in one case, and ulceration was found in one case.Increased lamina propria lymphoplasmacytic inflammation was seen in three cases and increased intraepithelial lymphocytosis was seen in one case and increased basal lymphoplasmacytic inflammation in one case (Table 2).Plasma cells were readily identified in all the patient's biopsies.Concurrent stomach and duodenal biopsies were performed in one patient (patient #5).These biopsies showed nonspecific mild chronic inactive gastritis and normal duodenal mucosa.
Transjugular biopsy from the patient with elevated liver enzymes showed acute hepatitis with lobular disarray, prominent centrilobular necrosis, and hepatocellular dropout (Figure 3C,D).There was diffuse lobular inflammatory infiltrate, which was mixed with predominantly lymphocytes, and occasional plasma cells, histiocytes, and eosinophils.There was mild macrovesicular steatosis (<10%).Trichrome and orcein stains did not reveal any significant fibrosis.Immunostains for CMV, adenovirus, HSV I&II, and Epstein-Barr encoding region (EBER) in situ hybridization were negative.

E X C L U S I O N O F O T H E R C A U S E S O F C O L I T I S
A thorough review of medication history showed that in 4/7 patients, there were no other medications that could be implicated in causing colitis/hepatitis.Patient# 2 was on fingolimod and natalizumab while receiving ocrelizumab infusions, but the lack of gastrointestinal and hepatic adverse effects from prior doses of fingolimod and natalizumab makes these causes less likely.Patient #4 was on long-term ibuprofen for 5 years, but the last dose was more than a year prior to the commencement of symptoms, making it less likely to be the cause of colitis.Patient #5 was found to be on metformin for the past 7 years.Although metformin has been associated with watery diarrhoea, the patient continued to have diarrhoea after discontinuation of metformin, making it unlikely to be the cause of diarrhoea. 13,14

R E A T M E N T A N D F O L L O W -U P D A T A
Among colitis patients, ocrelizumab was discontinued in three patients and four patients were treated with corticosteroids, leading to the resolution of symptoms.Two patients were symptomatically managed with subsequent resolution of symptoms.In the patient with hepatitis, resolution of symptoms was achieved by discontinuation of ocrelizumab and treatment with corticosteroids and azathioprine.
Follow-up data were available for all seven patients and ranged from 12 to 46 months (mean: 22.5 months).There was no recurrence of gastrointestinal or hepatic symptoms in these patients.Two Ocrelizumab-associated intestinal and hepatic injury 767 patients (#2 and #4) were symptomatically managed, with subsequent resolution of symptoms.Follow-up endoscopy with biopsies were available in 4/7 patients (patients #1, 3, 5, and 6).In patient #1, the follow-up endoscopy after stopping ocrelizumab and posttreatment with prednisone and mesalamine showed endoscopically normal colonic and rectal mucosa with random biopsies showing only mild acute colitis with rare foci of cryptitis.Patient #3 was found to have a complicated history.Two months after receiving her first dose of ocrelizumab, she developed abdominal pain and diarrhoea.Infectious workup revealed positive polymerase chain reaction (PCR) for Shigella, but the culture was negative.The patient was started on antibiotics, but the patient had persistent symptoms.Initial colon biopsies showed a mild acute cryptitis pattern of injury (Figure 2A).However, the patient received steroids to address her worsening arthralgias, which serendipitously improved her gastrointestinal symptoms.Medication-induced inflammatory bowel disorder-like illness secondary to ocrelizumab was considered at this point.However, given the symptomatic benefit of MS, ocrelizumab was not discontinued.On follow-up, the patient developed a perirectal abscess and fistula with multiple recurrences.A colonoscopy performed at this time showed patchy mucosal ulceration in the distal sigmoid colon and rectum, suspicious for Crohn's disease.The proximal and mid-sigmoid colon, transverse colon, right colon, and ileum were unremarkable.However, the biopsies from the rectum showed diffuse crypt atrophy, diffuse prominent crypt epithelial apoptosis, focal apoptotic microabscesses, and lamina propria oedema with a paucity of inflammatory cells, overall consistent with the apoptotic colopathy pattern of injury (Figure 2B-D).The immunohistochemical stain for CMV was negative.Biopsies from the terminal ileum, right colon, transverse colon, and left colon were normal.Given the persistent and worsening symptoms and development of perirectal fistula and abscess, ocrelizumab was discontinued.The presence of apoptotic colopathy on follow-up biopsy with a history of continued use of ocrelizumab and the absence of other chronic architectural changes seen with inflammatory bowel disease, ocrelizumab was favoured to be the cause of colonic injury.The patient was started on ustekinumab for gastrointestinal symptoms and fistula tract excision and subsequently received a loop-ileostomy with the resolution of perirectal tract/abscess.In patient #5, the presenting symptom was watery diarrhoea.The patient was on metformin for the past 7 years, so metformin-induced diarrhoea was Ocrelizumab-associated intestinal and hepatic injury 769 considered, but discontinuation of metformin failed to provide symptom resolution.Colon biopsies showed focal active cryptitis.The patient was treated with Imodium, which provided some relief, so ocrelizumab was not discontinued.Follow-up colon biopsy done 10 months later showed a lymphocytic colitis pattern of injury (Figure 1C,D) and was started on budesonide oral treatment.
In patient #6, ocrelizumab was discontinued after the initial presentation, and the patient was started on prednisolone followed by mesalamine and sulfasalazine, which led to the resolution of symptoms.At the 13-month follow-up, the colonoscopy showed normal colonic and ileal mucosa, which was confirmed on biopsies.

Discussion
Ocrelizumab (Ocrevus) is an intravenously administered IgG1 recombinant humanized monoclonal antibody that acts CD20-positive pre-B cells, mature B cells, and memory B cells and was approved for the treatment of RRMS and PPMS patients in 2017.Twelve cases of ocrelizumab-associated gastrointestinal tract injury and three cases of ocrelizumabassociated hepatitis have been reported in the literature.The clinical and histologic features of all reported cases of ocrelizumab-associated gastrointestinal tract injury [15][16][17][18][19][20][21][22][23][24][25] are summarized in Table 3.
Varied injury patterns that were reported in previous case reports were also observed in our case series.In our study, the predominant pattern of injury was acute colitis (4/6 cases).The injury was mild/focal in three cases.It is important to remember that multiple sclerosis patients are often on multiple immunosuppressive therapies, including corticosteroids, which might blunt some of the histopathological findings on biopsy, making the diagnosis even more challenging.The anti-CD20 immunosuppressive therapies also increase the risk of opportunistic infections producing a pattern of injury indistinguishable from druginduced colitis.So, from a clinical point of view, infections become a major differential diagnosis in these cases and should be excluded.Clinically, inflammatory bowel disease (IBD) was suspected in two patients on initial presentation and one on follow-up in our series.Only one patient's sigmoid and rectum biopsies at initial presentation showed features of chronic active colitis.Other patients showed features of severe active colitis and apoptotic colopathy.Ocrelizumab was discontinued in all three patients, in addition to immunosuppressive therapy.In one patient, while the clinical presentation and endoscopic findings indicated Crohn's disease, the histologic findings were of apoptotic colopathy.Apoptotic colopathy has been described in the context of graft-versus-host disease, drug-induced injury such as mycophenolate mofetil, checkpoint inhibitor therapy and others, infectious aetiologies such as CMV and adenovirus, and primary immune disorders. 26BD-like colitis with the usage of other closely related biological agents like rituximab has been the subject of few studies.8][29] It was found that patients developing IBDlike colitis received a higher number of doses than those who developed microscopic colitis, possibly indicating a dose-dependent propensity to develop more serious disease. 27Histopathologic findings in rituximab-associated colitis are variable and include apoptotic enteropathy with tubule dropout, loss of surface epithelium, marked depletion of mucin, and extensive apoptosis. 30In cases with an ulcerative colitis pattern, crypt distortion and crypt abscesses, 31 and crypt distortion with mixed inflammation in lamina propria 28 were identified.Crypt abscess with goblet cell depletion, active and chronic inflammation, 32 patchy active inflammation with ulceration, multiple small granulomas and multinucleated giant cells, 29 active ileitis with ulceration, focal active colitis with ulceration, and poorly formed granuloma 29 were noted in cases with Crohn's-type colitis.
The exact mechanism of anti-CD20-induced colitis is unknown.4][35] Studies have shown that anti-CD20 antibodies mainly target the circulating B-lymphocytes and a subset of CD20positive T cells, leading to near complete depletion of circulating CD20+ cells.Despite this effect, it is well demonstrated that gastrointestinal mucosal plasma  cells are persistent in patients on anti-CD20 antibody therapy.Plasma cells were also noted in all the mucosal biopsies in our patient population.The exact mechanism of persistent plasma cells in mucosa is unclear.][38] Only rare case reports of ocrelizumab-associated hepatitis have been reported.In Phase III clinical trials on ocrelizumab, serum liver enzyme elevations were noted in 1%-2% of the patients. 39,40Isolated cases of hepatitis B virus (HBV) reactivation have been reported in patients with resolved HBV infection (HBs antigen-negative/HBc antibody-positive). 41Therefore, screening for HBsAg and anti-HBc antibodies is required before starting ocrelizumab therapy. 8In patients with negative HBV serologies, monitoring of liver function tests is not required, but recommended before starting ocrelizumab. 6icolini et al. 42 reported a case of fulminant hepatitis owing to echovirus 25 in a 44-year female with RRMS who was receiving Ocrevus every 24 weeks for 4.5 years.Laboratory testing showed elevated serum AST and ALT without cholestasis.The patient eventually progressed to liver failure and received liver transplantation 11 days after admission.Plasma samples collected pretransplant detected enterovirus RNA that was subsequently identified as echovirus 25, which necessitated the permanent discontinuation of ocrelizumab treatment in this patient. 42ore recently, a bona fide fatal case of liver failure and invasive aspergillosis associated with ocrelizumab was reported by McEntire et al. 43 in a 21-year-old female with newly diagnosed RRMS.The patient developed jaundice and fatigue 2 weeks after receiving an ocrelizumab infusion.A liver biopsy showed Ocrelizumab-associated intestinal and hepatic injury 773 panlobular hepatitis with centrilobular necrosis.The patient was treated with N-acetylcysteine, lactulose, rifampin, and methylprednisolone, and eventually regained liver function within 2 weeks.Unfortunately, the patient developed opportunistic systemic aspergillosis 3 weeks later, leading to death. 43In our series, we report a second case of ocrelizumabassociated liver injury with histologic features of an acute hepatitis pattern of injury with prominent centrilobular necrosis, similar to the patient reported by McEntire et al.It was also interesting to note that both patients developed symptoms 2 weeks after the first ocrelizumab infusion.Our patient made a complete recovery with the discontinuation of ocrelizumab and immunosuppressive therapy.Currently, anti-CD20 drugs approved for patient use include rituximab, ocrelizumab, ofatumumab, obinutuzumab, ibritumomab, ublituximab, and tositumumab.As discussed, rituximab-associated colitis has been well established, and there are emerging reports of ocrelizumab-associated colitis; bona fide cases of colitis due to other anti-CD20 drugs have not been reported in the literature. 25Hepatitis and liver failure have been reported with rituximab and ocrelizumab, but liver injury associated with other anti-CD20 drugs is largely limited to reactivation of the hepatitis B virus 44,45 The exact pathophysiology of anti-CD20 colitis is largely unknown.][35] The study has a few limitations, including its retrospective nature.Furthermore, the data are limited to only a few cases.The presence of polypharmacy is quite common in patients with multiple sclerosis.Therefore, the possibility of another offending agent aggravating the effects of ocrelizumab-induced colitis/ hepatitis cannot be entirely ruled out.
In conclusion, the histologic manifestations of ocrelizumab-associated intestinal injury can be variable and even mimic IBD.Hepatic injury can rarely manifest as an acute hepatitis pattern of injury with necrosis.Identifying ocrelizumab-associated injury is of paramount importance in determining management, which often includes discontinuation of ocrelizumab therapy, and/or administration of immunosuppressive therapy.

Figure 1 .
Figure 1.(A,B) Patient #1 with chronic active colitis pattern of injury on initial biopsies.(C,D) Patient #5 with lymphocytic colitis on follow-up biopsy.

Figure 3 .
Figure 3. (A,B) Patient #6 with severe acute colitis with mucosal erosions on initial biopsies.(C,D) Patient #7 liver biopsy with acute hepatitis pattern of injury with prominent centrilobular necrosis.

Table 1 .
Clinical, endoscopic, and histopathologic findings on all patients in our series.

Table 2 .
Histopathologic findings of initial colon biopsies from patients with ocrelizumab-associated colitis.

Table 3 .
Summary of relevant clinical and histologic findings of previously reported ocrelizumab-associated gastrointestinal tract injury.