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Bone Marrow Transplantation in Congenital Erythropoietic Porphyria: Sustained Efficacy but Unexpected Liver Dysfunction
Bone marrow transplantation in CEP
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Bone marrow transplantation in congenital erythropoietic porphyria: Sustained efficacy but unexpected liver dysfunction Caroline Besnard , Caroline Schmitt , Louise Galmiche-Rolland , Dominique Debray , Monique Fabre , Thierry Molina , ´ Laurent Gouya , Cecile Ged , Martin Castelle , ´ edicte ´ Marina Cavazzana , Elisa Magrin , Ben Neven , ´ ´ Despina Moshous , Stephane Blanche , Marie-Louise Fremond PII: DOI: Reference:
S1083-8791(19)30867-5 https://doi.org/10.1016/j.bbmt.2019.12.005 YBBMT 55814
To appear in:
Biology of Blood and Marrow Transplantation
Received date: Accepted date:
30 October 2019 9 December 2019
Please cite this article as: Caroline Besnard , Caroline Schmitt , Louise Galmiche-Rolland , ´ Dominique Debray , Monique Fabre , Thierry Molina , Laurent Gouya , Cecile Ged , ´ edicte ´ Martin Castelle , Marina Cavazzana , Elisa Magrin , Ben Neven , Despina Moshous , ´ ´ Stephane Blanche , Marie-Louise Fremond , Bone marrow transplantation in congenital erythropoietic porphyria: Sustained efficacy but unexpected liver dysfunction, Biology of Blood and Marrow Transplantation (2019), doi: https://doi.org/10.1016/j.bbmt.2019.12.005
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Inc. on behalf of the American Society for Transplantation and Cellular Therapy
Highlights:
The very first still-living receivers of hematopoietic stem-cell transplantation (HSCT) for early and severe forms of congenital erythropoietic porphyria (CEP), 25 and 22 years ago, are healthy and asymptomatic, with sustained corrected UROS activity.
Intrinsic hepatic involvement of varying intensity is present at birth, with either a favorable or fatal outcome, despite HSCT engraftment.
Liver pathology revealed varying degrees of portal, centrilobular, and perisinusoidal fibrosis, clarification of hepatocytes, and cytosolic porphyrin deposits.
Due to the risks and difficulties of HSCT, the indication remains limited to the severe form of CEP.
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Bone marrow transplantation in congenital erythropoietic porphyria: sustained efficacy but unexpected liver dysfunction
Short title: Bone marrow transplantation in CEP Caroline Besnard1, Caroline Schmitt2, Louise Galmiche-Rolland3, Dominique Debray4, Monique Fabre3, Thierry Molina3, Laurent Gouya2, Cécile Ged5, Martin Castelle1, Marina Cavazzana6, Elisa Magrin6, Bénédicte Neven1, Despina Moshous1, Stéphane Blanche1, and Marie-Louise Frémond1*
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Pediatric Immuno-Hematology and Rheumatology unit, Necker Enfants Malades Hospital,
Assistance Publique-Hôpitaux de Paris, AP-HP, Paris, 2
French Center of Porphyrias, Louis Mourier Hospital, AP-HP, Colombes and Research
Center of Inflammation, UMR1149 INSERM, Université de Paris, Paris, 3
Pathology Department, Necker Enfants Malades Hospital, AP-HP, Paris,
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Paediatric Hepatology Unit, Necker Enfants Malades Hospital, AP-HP, Paris,
5
Biotherapy of Genetic Diseases, Inflammatory Disorders, and Cancers, U1035 INSERM,
Bordeaux University, Bordeaux 6
Biotherapy Unit, Necker Enfants Malades Hospital, AP-HP, Paris, France
*Present address: Laboratory of Neurogenetics and Neuroinflammation, Imagine Institute INSERM UMR1163, Paris, France
Corresponding author: Stéphane Blanche, Hôpital Necker, 149 rue de Sèvres, 75015 Paris, France [email protected]
Keywords: porphyria, long term, liver
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Abstract Congenital erythropoietic porphyria (CEP) is a rare disease characterized by erosive photosensitivity and chronic hemolysis due to a defect of the enzyme uroporphyrinogen-IIIsynthase (UROS). To date, hematopoietic stem-cell transplantation (HSCT) is the only curative therapy for the devastating early and severe form of the disease. We describe six CEP patients treated with HSCT – 3 of them twice after failure of a first graft - between 1994 and 2016 in our center, including two of the very first living patients treated more than 20 years ago. Four patients are doing well 6 to 25 years post HSCT, with near normal biochemical parameters of porphyrin metabolism without the cutaneous or hematological features of CEP. One patient died within the first year after HSCT from severe graft-versus host disease (GVHD). Finally, one child died of unexplained acute hepatic failure one year after HSCT, despite full donor chimerism. Retrospectively, it appears that all but one child had increased transaminase activity with onset from the early post-natal period, which was significantly more marked for the child who died of liver failure. In contrast, liver function values progressively normalized after engraftment for all other children. Liver pathology before HSCT for three patients revealed varying degrees of portal, centrilobular, and perisinusoidal fibrosis, clarification of hepatocytes, and cytosolic porphyrin deposits. Liver porphyrin content in biopsies was more than 60 times the normal values. Despite difficult engraftment, long-term efficacy of HSCT in CEP appears to be favorable and reinforces its benefits for the severe form of CEP. Hepatic involvement requires careful evaluation before and after HSCT and further investigation towards its pathophysiology and care.
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Introduction Congenital erythropoietic porphyria (CEP), formerly referred to as Günther’s disease, is a rare metabolic autosomal recessive inherited disorder of varying severity, but typically associated with early onset, devastating symptomatology, and poor prognosis1. CEP is due to the defective enzymatic activity of uroporphyrinogen III synthase (UROS), the 4th enzyme in the heme biosynthesis pathway. Approximately 50 different pathogenic mutations in UROS have been described thus far. They alter either the catalytic machinery, stability of the final conformation, or thermostability of the protein2. This defect results in the accumulation of the non-physiological and pathogenic porphyrin isomers uroporphyrinogen I and coproporphyrinogen I in the erythroid precursors of bone marrow, which are later released into the plasma and deposited in tissues (mainly skin, bones, and spleen). The accumulated porphyrins are then excreted in the urine (uroporphyrin I and coproporphyrin I) or feces (coproporphyrin I). They act as photoreactive intermediates, absorbing energy from long ultraviolet and visible light to release radicals or transfer electrons directly to targets, leading to a loss of integrity and function of the cell membrane, disruption of cell organelles, and cell death. Clinical presentations range from severe antenatal manifestations (hydrops fetalis, in utero death) to milder adult-onset phenotypes. In the early and severe infantile form, the accumulation of porphyrins in the skin combined with light exposure induces phototoxic lesions, such as vesicles, blisters, superinfections, and cartilage destruction, leading to epidermal atrophy and sclerodermoid changes with devastating mutilation. The anemia results from two mechanisms: i) an excess of porphyrins in the bone marrow precursor cells, which increases apoptosis, leading to ineffective erythropoiesis, and ii) accumulation of porphyrins in red blood cells, responsible for chronic intravascular hemolysis with variable bone-marrow compensation. Other CEP features include erythrodontia (i.e. reddish-brown coloration of the
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teeth in visible light) by deposits during dental development, scleritis associated with elevated porphyrin levels in teardrops, osteolysis, and osteoporosis. CEP diagnosis is based on the identification of high levels of urinary uroporphyrin I and coproporphyrin I, which are pathognomonic of CEP. Elevated levels of porphyrins in erythrocytes and plasma (uroporphyrin I and coproporphyrin I) or in feces (coproporphyrin I) are also observed. Assays demonstrating profoundly diminished UROS activity in erythrocytes or mutations in the UROS gene also confirm the diagnosis. Hematological complications and early onset of symptoms, before the age of five years, are the main predictive factors of a poor prognosis. Symptomatic treatment essentially consists of the exhaustive avoidance of visible light as the only way to avoid skin lesions. Repeated red blood cell transfusions and splenectomy can also be used to decrease endogenous heme synthesis and decrease the stimulation of erythropoiesis. Transfusion may also by beneficial through an exogenous transient supply of the normal enzyme. Hematopoietic stem-cell transplantation (HSCT) to replace the bone-marrow pool of defective enzymes is the only curative treatment to date . The proof of concept was established in 1991 by Kauffman et al., who succeeded in correcting the UROS activity in hematopoietic cells, but the patient died of infectious complications2. Five years later, we reported the first successful HSCT3 in a young girl suffering from a severe form of CEP. Since then, more than 20 other cases of HSCT for CEP have been reported4-17. Independently of the tolerance of the transplant procedure itself, HSCT for the treatment of congenital diseases requires a detailed description of the disease evolution, as some symptoms may not be improved, or only partially, by the supply of an enzyme via hematopoietic cells. In addition, certain symptoms, unknown during the natural course of the disease, can appear in the long term through the prolongation of the lifespan of transplanted patients. 5
Here, we present the outcome of all six patients who underwent HSCT for CEP in our center, including the two very first still-living patients transplanted in 1994 and 1997.
Methods Patients This retrospective and monocentric study included all children receiving HSCT to treat CEP between 1994 and 2016 in the Pediatric Immuno-Hematology and Rheumatology Unit of Necker-Enfants Malades Hospital (Paris, France). Clinical, biological, histological, and radiological data were collected until September 2018. Before transplantation, all parents gave informed consent for anonymous use of the clinical and biological data for research purposes. This consent was renewed when the patients became young adults, according to the French regulations on clinical research. Porphyria diagnosis Biochemical analysis Total urinary porphyrins were determined by spectrophotometry between 350 and 450 nm after acidification. The various porphyrins and their isomers were separated by reverse-phase high-performance liquid chromatography (HPLC) with fluorimetric detection. Total erythrocyte porphyrins were first extracted with ether/acetic acid and then extracted with 3N hydrochloric acid. The acid extract was compared with a protoporphyrin standard by scanning the fluorescence excitation signal between 380 and 440 nm (emission at 602 nm). Plasma porphyrins were measured, as already described, by dilution of the plasma in phosphate buffer18. The solution was compared with a uroporphyrin standard by scanning the fluorescence emission signal from 570 to 750 nm (excitation at 405 nm). For total liver porphyrins measurement, 50 mg tissue was carefully cut in 500 µl saline, then sonicated for three 12-s periods. Porphyrins were extracted from the suspension by ten-fold dilution with 6
1N perchloric acid:methanol (1:1 v/v). After mixing and centrifugation, the supernatant was isolated for quantification of porphyrins by spectrofluorometry, using a RF6000 Shimadzu spectrofluorometer fitted with a red sensitive photomultiplier (excitation at 410 nm, emission at 600 nm). The results are given in picomoles per gram of tissue. Uroporphyrinogen III synthase activity in erythrocytes was determined by an enzyme-coupled assay, as already described 19. Genetic analysis Signed consent was obtained for genetic diagnosis. Genetic diagnosis was performed by Sanger sequencing of the entire coding sequence of the UROS gene (NM_000375), including intron-exon boundaries and the erythroid specific promoter region. Compound heterozygosity was confirmed by the analysis of both parents. Histopathology Liver biopsies were fixed in 10% phosphate-buffered formalin, embedded in paraffin, and stained with hematoxylin-eosin-saffron (H.E.S), Masson’s trichrome, Picrosirius Red for collagen, Gordon-Sweet for reticulin, and PAS (Periodic-acid-Schiff) and Perls for porphyrin and iron quantification. Cytokeratin 7 (CK7) staining was also performed for the assessment of ductular reaction. Portal fibrosis was evaluated according to the METAVIR criteria 20 . The cellular distribution of iron deposits was assessed . The Rowe scoring system was used. Porphyrin deposits were assessed using HES and PAS. Polarized light was used to assess the characteristic Maltese cross shape of brilliant and birefringent crystalline pigment deposits. Ethics The study was approved by the ethics committee of Necker-Enfants Malades University Hospital.
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Results Initial clinical presentation Six children suffering from severe CEP were referred to our unit for HSCT between 1994 and 2014. There were four girls and two boys from unrelated families with no past history of porphyria (Table 1). Typical CEP skin lesions appeared at a median age of 2.1 [0-6.5] months, with active blisters and/or scars present for all patients at the time of HSCT. Hemolytic anemia and red urine occurred at a median age of 8 [0-13.5] and 1.6 [0-11.9] months, respectively. Hemolysis required one to two red-cell transfusions, except for one child who received nine red cell packs until the HSCT. Splenomegaly was either present at birth (5/6) or within the first two months (1/6). Before HSCT, a partial (n = 1) or total (n = 2) splenectomy was performed to relieve hemolysis but, above all, to prevent splenic sequestration of the graft. Porphyria diagnosis The diagnosis was confirmed at a median age of 7.1 [2.2-8] months, with very high levels of total urinary porphyrins (more than 100 times above normal) and characteristic high excretion of uroporphyrin I and coproporphyrin I. High amounts of porphyrins and characteristic profiles were also observed in feces, erythrocytes, and plasma. UROS activity was low in all five tested children (from 0.1 to 1.2, N > 6 U/mg Hb/h). All patients carried previously reported compound heterozygous (n = 5) or homozygous (n = 1) missense mutations in the UROS gene (Table 1). HSCT and related adverse events The median age at the first HSCT was 18.1 [9.5-22.4] months. The median time interval between diagnosis and first HSCT was 10.8 [4.6-14.4] months. All patients received a myeloablative conditioning regimen, including busulfan (empirical dose of 16 mg/kg orally for the
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two first patients, intravenous with drug monitoring thereafter, with a targeted area under the curve of 20,000 µM.min), 200 mg/kg cyclophosphamide (n = 4) or 160 mg/m² fludarabine (n = 2), and anti-thymocyte globulins (all but one). Four patients received a matched unrelated donor (MUD) graft, one, a graft from a matched sibling donor (MSD) who was a heterozygous carrier of CEP, and one, a haplo-identical graft from one parent (heterozygous carrier of CEP) after complete T-cell depletion by CD34 selection (methodology in practice at the time of this HSCT in 1997). GVHD prophylaxis was based on cyclosporine alone (n = 3), cyclosporine plus methotrexate (n = 1), or cyclosporine plus mycophenolate (n = 2). Three patients showed stable engraftment with full chimerism after the first transplantation. However, three patients required a second transplantation due to the absence of engraftment (n = 1) or early loss of chimerism (n = 2). In all three, a resurgence or the persistence of skin lesions was observed, together with worsening of the biochemical porphyria profile in blood and/or urine. Two children were re-transplanted with the same HLA-matched donor (MSD and MUD), but the alternate haplo-identical parent was selected for the third 32, 35, and 2 months after the first HSCT, respectively (Table 1). After the first or second transplantation, donor chimerism was evaluated by either i) the restoration of UROS activity, as indicated by decreased erythrocyte, plasma, or urine porphyrin levels, and/or ii) VNTR or X and Y chromosome FISH analysis in the blood for sex-mismatched HSCT, all confirming engraftment for all children. In addition to the usual neutropenia-related adverse events, one child presented with a posterior reversible encephalitis syndrome, controlled after cessation of cyclosporine and normalization of arterial blood pressure. Graft vs Host disease was observed in three children: skin and gut grade II for two children, leading to prolonged immunosuppressive treatment (until 16 months post HSCT) for one, and severe for a third child, with grade III/IV skin, liver (biopsy proven), gut, and eye involvement, requiring several lines of immunosuppressive 9
therapy, including high doses of methylprednisolone and anti-thymoglobulin. The child simultaneously developed severe thrombotic microangiopathy and died of multiorgan failure six months post HSCT, despite treatment with monoclonal anti-C5 antibody (eculizumab). No veno-occlusive disease (VOD) was observed. Of note, four of the six children received prophylactic defibrotide, because of their young age, the use of a full dose of busulfan for complete myeloablation, and preexisting liver dysfunction. Porphyrin metabolism and CEP symptomatology after HSCT Urinary porphyrin levels decreased rapidly after HSCT for all six patients, but never normalized (even for the oldest patient). Erythrocyte porphyrins were measured for five patients and showed normal or nearly normal levels. Evaluable UROS activities showed a strictly normal level for two patients and a nearly normal level for two others, as observed in heterozygous carriers of CEP, consistent with the fact that these two patients received a graft from a family member who was a heterozygous carrier. Enzyme activity remained stable 20 years after the HSCT for the two oldest patients (see below). Photosensitivity completely disappeared and no hemolysis was observed in any patient as soon as hematological recovery and donor chimerism were obtained. Liver biology and follow-up All but one child showed varying levels of elevated serum transaminase activities during the neonatal or early postnatal period, reaching up to more than ten times the upper limit of normal for one (Table 2). After HSCT, five children were evaluable, as one died at six months post HSCT from severe multipolar GVHD and severe thrombotic microangiopathy. Liver function values normalized progressively for four out of five children following HSCT. However, transaminase values did not normalize post-HSCT for one child, who developed unexplained acute liver failure (ALF) at five months after transplantation despite engraftment. 10
During this event, which is very uncommon in the course of HSCT, an extensive workup failed to detect any evidence of toxic or viral liver injury. There were no clinical signs of GVHD at this time. After spontaneous improvement (prothrombin times improving from 22% to 94% in one week), transaminases never normalized and ALF recurred seven months later, leading to death at one-year post-transplant. Liver histopathology and liver porphyrin levels Before HSCT Four liver biopsies were performed for three children. Liver biopsies were performed at the time of splenectomy for one and during the routine checkup before HSCT for the two most recent children. Portal fibrosis, confirmed by Masson trichome and Picrosirius Red stains, was present for all but one (patient 6), ranging from the F1 to F3 stage. In one case (patient 5), severe bridging fibrosis, stage F3 (porto-portal or portal-central linkage) was present but there was no obvious cirrhosis. Centrilobular fibrosis was a major feature present in all cases. In addition, sinusoidal fibrosis, of moderate to severe intensity, was always present and most often diffuse (3/4). Hepatocyte injury was constant and included clarification and ballooning degeneration of varying intensity and distribution, often in contact with portal and centrilobular fibrosis. The amount of steatosis ranged from 0 (patient 6) to 5%. Siderosis of Kupffer cells was always more marked than that of hepatocytes, varying between 0 and 3, whereas that in hepatocytes ranged between 0 and 1. Deposition of PAS-positive fibrillary clumps of dense material was mixed, more marked in hepatocytes than in Kupffer cells, detected in all biopsies, and detected in the cytoplasm of large bile ducts for one biopsy (patient 5) (Figure 1). The biliary ducts were normal in all but one case (patient 5), with a slight ductular reaction. Most portal tracts showed mild nonspecific lymphocytic inflammation. The portal veins and arteries were preserved in all cases. No bilirubinostasis was observed. 11
Porphyrin levels assessed in three of these biopsies showed extremely high values (26,490, 55,536, and 39,215 pmol/g tissue, normal < 400 pmol/g tissue). After HSCT A biopsy was performed for the child experiencing his first episode of ALF (patient 4): the histological picture excluded GVHD, veno-occlusive disease, and acute viral hepatitis (no necrosis, no apoptosis, and microvesicular steatosis not exceeding 5% in the centrilobular zone). Lobular fibrosis was graded 1 to 3. The major findings were portal fibrosis (F2) with mild lymphocytic infiltrate, severe disarray of lobular architecture with centrilobular fibrosis and perisinusoidal fibrosis (grade 3), moderate hepatocyte ballooning (30%), and numerous ground glass-like inclusions (20%), associated with mild porphyrin deposition in hepatocytes (Figure 2). Two biopsies were performed for the child with severe GVHD (patient 6). Histopathology confirmed the diagnosis (data not shown). Long-term outcome At last evaluation, all living patients had a normal physical examination, without hepatomegaly, splenomegaly, or photosensitivity. Blood counts and liver enzymes were in the normal range. More specifically, the two first still-living young women received transplants more than 20 years ago, were both healthy and asymptomatic, but definitive erythrodontia (including on permanent teeth which appeared after the HSCT) required esthetic care. Enzyme activity remained stable for the two oldest patients. No unexpected clinical or biochemical disorder was observed nor detected at the last check up, except for an asymptomatic restrictive syndrome, based on respiratory parameters, considered to be sequalae of double exposure to busulfan. The assessment of liver stiffness evaluated by transient elastography (FibroScan®) was normal (Table 3). Both lead well-balanced personal, social, and professional lives. Both were under hormonal substitution treatment for primary
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ovarian insufficiency attributed to the double conditioning regimen, but one gave birth to a healthy boy after a spontaneous pregnancy.
Discussion The benefit of bone marrow transplants for the severe form of CEP has been described in several case reports. We report here, for the first time, the long-term follow-up of two of the very first successfully treated patients more than 20 years post-transplant. The result for these two young women is remarkable, despite donor heterozygosity and reduced UROS activity; they are completely asymptomatic, and the follow-up showed no unexpected pathology related to late, undescribed expression of porphyria; these two women only suffer from the aesthetic disorder of their final teeth being colored reddish-brown, which requires regular dental care. Exposure to the sun, even without protection, does not cause any pathological photosensitivity and they do not have anemia. This finding reinforces the potential interest of bone marrow transplantation for this indication, even if lifelong efficacy is yet to be shown. Of note, urine porphyrin levels were still moderately elevated, despite full engraftment and normal levels of erythrocyte porphyrins. This remaining porphyrin excretion may be the consequence of porphyrin production in non-erythroid tissues, such as the liver. As the UROS gene defect is constitutively presented in all organs, the liver still produces and moderately accumulates uroporphyrin I and coproporphyrin I1. Aside from this reassuring long-term outcome, three points deserve special attention. The first is that half of the children had to be grafted twice to obtain hematopoietic chimerism and thus stable enzymatic correction. During the long study period, two elements could have been associated with some failures: T cell depletion methodology by CD34 selection used twenty years ago for the haploidentical HSCT, which today would be considered suboptimal, and
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oral busulfan without drug monitoring for two children. In addition, we did not consider the risk of trapping the stem cells in the splenomegaly for the first patient. Nevertheless, chronic hemolysis induces overactive marrow and myeloablative conditioning appears to be necessary to obtain optimal and durable enzyme correction. These observations can fuel recommendations, such as considering complete or partial splenectomy before HSCT in case of massive splenomegaly and proposing fully myeloablative conditioning similar to that proposed for other diseases associated with hyperactive erythropoiesis. Clearly, the recent in vivo or in vitro methodologies of selective T-cell depletion will improve the chance of success of haploidentical HSCT. The second important feature is the prevalence of liver involvement, observed here in all but one child prior to HSCT. In contrast to what is observed in the other form of erythropoietic porphyria, erythropoietic protoporphyria (EPP), liver dysfunction in CEP is uncommon. Liver enlargement and porphyrin-rich gallstones can be found in some patients. In a review of 29 cases of CEP, 11 had “abnormal liver function” values with onset from birth to the mid-30s, but this global figure also included hemolysis-related hyperbilirubinemiae21: although abnormally high liver transaminase activity was observed based on the presented median values, the incidence of specific liver dysfunction was not indicated. Severity ranged from spontaneous resolution to stability and cirrhosis in one case but no acute liver failure was described. A case of liver failure and hepatorenal syndrome was also described in a 35-year-old women22. The physiopathology of this liver dysfunction in CEP is yet to be elucidated. The pre-transplantation liver biopsies presented here show porphyrin deposition in hepatocytes and Kupffer cells, which could induce liver fibrosis via tissue damage from porphyrin-mediated oxidative stress. Liver function values gradually normalized following transplantation for four patients, while porphyrin levels in the erythrocytes and urine decreased. The two patients evaluated in the long term had normal liver enzymology and no signs suggestive of liver fibrosis on elastometry. However, the third
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unexpected event was the post-transplant occurrence of sudden fatal ALF in one child, despite full chimerism and decreased urine porphyrin levels. No cases of ALF have been reported in the post-transplant setting for CEP. This child had no clinical or biological signs of infection, toxic hepatitis, or other HSCT-related complications. However, a comparison of pretransplantation characteristics with those of the other children showed that the child presented the most severe liver dysfunction soon after birth and sustained transfusion-requiring hemolytic anemia until HSCT, suggesting a more severe disease. At diagnosis, this child had the most highly elevated levels of porphyrins in urine, plasma, and erythrocytes. No liver biopsy was available prior to HSCT for histological analysis and assessment of liver porphyrin content. Such fatal ALF, one year after the HSCT, could have been the consequence of massive accumulation of porphyrins insufficiently cleared by the enzyme originating from the engrafted cells, together with iron overload subsequent to his pretransplant sustained hemolysis. Macrophage and hepatocyte iron overload were noted on the post-HSCT biopsy. The joint action of iron and porphyrin overload may explain the development of intralobular fibrosis, especially in the centrilobular region, although urine porphyrin and serum ferritin levels were decreasing post-HSCT. Centrilobular fibrosis, such as perisinusoidal fibrosis, may be due to the effects of reactive oxygen species and oxidative stress23,24. An unidentified external triggering event that occurs in a chronically altered liver, such as drug-induced hepatitis, cannot be ruled out, as suggested by the presence of hepatocellular ballooning and numerous abnormal pseudo cytoplasmic ground-glass inclusions25. Finally, another genetic predisposition to liver damage could be hypothesized, as described in EPP, through the impairment of the expression of ATP-binding cassette transporter G226. In the future, massive accumulation of porphyrins in the liver could justify a preventive approach to protect the liver, as suggested, for example, with chloroquine for its anti-inflammatory action in porphyria cutanea tarda27 or, more recently, by an induced iron
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deficiency in CEP22. The overall morbidity and mortality of HSCT –even with potential suggested improvements make it necessary to restrict the indication of HSCT to the most severe form of the disease. Severity is sometimes obvious, such as in cases of early devastating skin photoreactivity in infancy. The evaluation of severity can also be based on the intensity of hemolysis, very high levels of urinary porphyrin, or the C73R mutation genotype. However, late onset worsening can also be observed in milder forms of the disease, with the exhaustion of erythropoiesis and secondary aplastic anemia. In addition to the severity of the disease, the place of HSCT will have to be evaluated in the light of donor characteristics (HLA identical sibling or unrelated haploidentical donor) and alternative treatment, such as induced iron deficiency or future gene therapy. In all cases, it will be based on a careful case-by-case evaluation and a very close collaboration between a porphyria specialist and the HSCT team. In conclusion, we strongly suggest that the initial workup before HSCT includes a liver biopsy to evaluate the level of liver damage and the level of porphyrin accumulation. Despite HSCT-related morbi-mortality, long-term data support HSCT as a life-changing treatment for patients presenting an early and severe form of CEP.
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Acknowledgments No funding support was provided for this study.
Authorship Contributions MLF, SB, and DB designed the study. CB collected the data and wrote the first version of the manuscript. MLF, SB, DM, BN, MC, and DD provided care to patients. LGR, MF, TM and DD analyzed the liver biopsies. CS and LG performed and analyzed the porphyrin assays and CG the genetic diagnosis. MC and EM (biotherapy) were in charge of the management and safety procedures of hematopoietic stem cells before infusion. All authors approved the final version of the manuscript.
Conflict of Interest Disclosures The authors have no conflict of interest to disclose.
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10. Dupuis-Girod S, Akkari V, Ged C et al. Successful match-unrelated donor bone marrow transplantation for congenital erythropoietic porphyria (Günther disease). European Journal of Pediatrics. 2005;164:104–7 11. Phillips JD, Steensma DP, Pulsipher MA, Spangrude GJ, Kushner JP. Congenital erythropoietic porphyria due to a mutation in GATA1: the first trans-acting mutation causative for a human porphyria. Blood. 2007;109:2618–21 12. Taibjee SM, Stevenson OE, Abdullah A et al. Allogeneic bone marrow transplantation in a 7-year-old girl with congenital erythropoietic porphyria: a treatment dilemma. British Journal of Dermatology. 2007;156:567–71 13. Faraci M, Morreale G, Boeri E et al. Unrelated HSCT in an adolescent affected by congenital erythropoietic porphyria. Pediatric Transplantation. 2008;12:117–20 14. Lebreuilly-Sohyer I, Morice A, Acher A et al. Congenital erythropoietic porphyria treated by haematopoietic stem cell allograft. Ann Dermatol Venereol. 2010;137:635–9 15. Singh S, Khanna N, Kumar L. Bone marrow transplantation improves symptoms of congenital erythropoietic porphyria even when done post puberty. Indian J Dermatol Venereol Leprol. 2012;78:108–11 16. Martinez Peinado C, Diaz de Heredia C, To-Figueras J et al. Successful Treatment of Congenital Erythropoietic Porphyria Using Matched Unrelated Hematopoietic Stem Cell Transplantation. Pediatric Dermatology. 2013;30:484–9 17. Karakurt N, Tavil B, Azik F, Tunc B, Karakas Z, Uckan-Cetinkaya D. Successful hematopoietic stem cell transplantation in a child with congenital erythropoietic porphyria due to a mutation in GATA-1. Pediatric Transplantation. 2015;19:803–5 18. Elder GH, Smith SG, Smyth SJ. Laboratory investigation of the porphyrias. Ann Clin Biochem. 1990;27:395-412
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19. Deybach JC, Grandchamp B, Grelier M et al. Prenatal exclusion of congenital erythropoietic porphyria (Günther's disease) in a fetus at risk. Hum Genet. 1980;53:21721. 20. The METAVIR cooperative group. Inter- and intra-observer variation in-the assessment of liver biopsy of chronic hepatitis C. Hepatology 1994; 1:15-20. 21. Katugampola RP, Anstey AV, Finlay AY et al. A management algorithm for congenital erythropoietic porphyria derived from a study of 29 cases: Management algorithm for congenital erythropoietic porphyria. British Journal of Dermatology. 2012;167:888–900 22. Egan DN, Yang Z, Phillips J, Abkowitz JL. Inducing iron deficiency improves erythropoiesis and photosensitivity in congenital erythropoietic porphyria. Blood. 2015;126:257-61 23. Boitnott J, Haas M, Anders RA et al. Glycogen pseudoground glass change in hepatocytes.Am J Surg Pathol. 2006;30:1085-90. Ground-glass, polyglucosan-like hepatocellular inclusions: A "new" diagnostic entity. 24. Lefkowitch JH, Lobritto SJ, Brown RS Jr et al. Ground-glass, polyglucosan-like hepatocellular inclusions: A "new" diagnostic entity. Gastroenterology. 2006;131:713-8 25. Yin C, Evason KJ, Asahina K, Stainier DY. Hepatic stellate cells in liver development, regeneration, and cancer. J Clin Invest. 2013;123:1902-10 26. Hagiwara S, Nishida N, Park AM, Sakurai T, Kawada A, Kudo M. Impaired expression of ATP-binding cassette transporter G2 and liver damage in erythropoietic protoporphyria. Hepatology. 2015;62:1638-9 27. González-Estrada A, Gomez-Morales LB, Gonzalez-Estrada A, García-Morillo JS. Sporadic porphyria cutanea tarda: treatment with chloroquine decreases hyperglycemia and reduces development of metabolic syndrome. Eur J Intern Med. 2014;25:e76-7
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Table 1. Patient characteristics
Patients Sex Age at diagnosis Photosensitivity Erythrodontia Red urine Splenomegaly Anemia Transfusions (n) Splenectomy Diagnosis Genotype
Erc-UROS activity
P1 F 7.6 months + + + + 0 +*
P2 F 8 months + + + + 0 -
P3 F 6.2 months + + + + 0 -
P4 M 5.6 months + + + + + 8 RBC & 2 platelets +
P5 F 2.2 months + + + + + 2 RBC & 10 platelets +
P6 M 8 months + + + + + 2 RBC -
[c.217T>C, p.(Cys73Arg)] [c.560A>C, p.(Gln187Pro)] 0.7
[c.217T>C, p.(Cys73Arg)] [c.217T>C, p.(Cys73Arg)] ND
[c.205G>A, p.(Ala69Thr)] [c.217T>C, p.(Cys73Arg)] 0.1
[c.10C>T, p.(Leu04Phe)] [c.673G>A, p.(Gly225Ser)] 0.9
[c.217T>C, p.(Cys73Arg)] [c.634T>C, p.(Ser212Pro)] 1.2
[c.205G>A, p.(Ala69Thr)] [c.244G>T, p.(Val82Phe)] 0.5
5 622
9 851
3 270
34 728
17 920
9 755
N>6U/mgHb/h)
Total U-porphyrins
N<30nmol/mmol creat)
Total Uroporphyrins
2 733
6 684
2 018
26 309
8 315
4 760
% Isomer I (N<30%)
ND
ND
ND
ND
97%
96%
Total Coproporphyrins
2 889
3 167
1 252
8 419
7 400
3 902
99% 66.9
96% 52.6
98% 17
ND 136
98% 39.2
98% 27.4
95
4 700
984
2007 13.9 months 20.1 months MUD
2009 4.6 months 10.1 months MUD
1st 2012 7.3 months 9.5 months MUD
2nd 2015 40.3 months 42.5 months MUD
10/10 IV Bu, Cy, ATG
10/10 IV Bu, Cy, ATG
14 106/kg
13.45 106/kg
10/10 IV Bu, Flu, ATG 10.66 106/kg
10/10 IV Bu, Flu, ATG 3.8 106/kg
CsA
CsA/MA
N<10 nmol/mmol creat)
N<20 nmol/mmol creat)
% Isomer I (N<30%) Erc-porphyrins
N<1.9µmol/LHb)
Plasma-porphyrins
ND
ND
667
N<20nmol/L)
HSCT Year Time Dg-HSCT Age Donor
1st 1994 13.7 months 21.6 months MSD
2nd 1995 21.7 months 29.6 months MSD
HLA compatibility Conditioning regimen
10/10 Oral Bu, Cy
10/10 Oral Bu, Cy
1st 1997 26.4 months 21.4 months Haplo T Ly depletion: In vitro CD34 selection 5/10 IV Bu, Cy
CD34+ cells
ND
ND
2.5 106/kg
2nd 1997 28.4 months 23.4 months Haplo T Ly depletion: In vitro CD34 selection 5/10 IV Bu, Cy, ATG 7 106/kg
Prophylaxis GVHD
CsA/MTX
CsA/MTX
CsA
CsA
CsA
CsA/MA
2014 8 months 16 months MUD
10/10 IV Bu, Flu, ATG 11.69 106/kg
CsA/MA
21
VOD
-
-
-
-
UA/Defibrotide
UA/Defibrotide
UA/Defibrotide
UA/Defibrotide
UA/Defibrotide
Abbreviations: ADV: adenovirus, ATG: anti-thymocyte globulin, Bu: busulfan, CsA: ciclosporin A, Cy: cyclophosphamide, Dg: diagnosis, F: female, Flu: fludarabine, GVHD: Graft versus Host disease, haplo: haplo-identical, HLA: Human Leukocyte Antigen, HSCT: hematopoietic stem cell transplantation, IV: intravenous, M: male, MA: mycophenolic acid, MSD: matched sibling donor, MUD: matched unrelated donor, MTX: methotrexate, n: number; ND: no data, RBC: red blood cells,, UA: ursodesoxycholic acid, VOD: vein occlusive disease. *splenectomy perfomed after the failure of the first graft.
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Table 2. Pre and post HSCT evolution of liver transaminases
Patients
P1
P2
P3
P4
P5
P6
Neonatal
+
NA
NA
+++
++
NA
Nl
Nl
+++
++
++
+
At HSCT
+
Nl
Nl
+
++
+
1 year post HSCT
Nl
Nl
Nl
Nl 24 Y post HSCT
Nl 22 Y post HSCT
Nl 3 Y post HSCT
Postnatal period until HSCT
Long term
Nl/++/+++* Death of acute liver failure at 1 year post HSCT _
++
Nl 3Y post HSCT
Non evaluable: Severe hepatic GVHD and TMA, Death at 6 Mo Post HSCT
_
HSCT: hematopoietic stem cell transplantation, NA: not available, GVHD: graft versus host disease, TMA: thrombotic micro angiopathy, transaminase values: Nl: normal value, + < 2x normal, ++ 2 to 10x normal, +++ > 10x normal, HSCT: hematopoietic stem cell transplantation, *fluctuating values.
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Table 3. Long-term outcome after HSCT
Follow-up after last HSCT Age at last visit Photosensitivity Scars Tooth color Red urine Anemia Thrombocytopenia Splenomegaly Urinary porphyrins (N<30nmol/mmol creat) Erythrocyte porphyrins (N<1.9µmol/LHb) Plasma porphyrins (N<20nmol/L) URO III synthase activity (N>6U/mgHb/h) Immunosuppressant treatment Professional life Height Weight Endocrine status
Heart ultrasonography Respiratory status Functional respiratory tests DXA femur Z-score DXA lumbar spine Z-score Abdominal ultrasonography Spleen Prothrombin time ASAT/ALAT Bilirubin Elastography (Fibroscan®, N<5kPa)
P16 24.3 years 26.1 years None None Reddish-brown None None None None 117
P221 20.9 years 22.4 years None None Reddish-brown None None None None 38
1.4 16 5* None Employed 169.8 cm 56 kg Ovarian insufficiency (one child natural fecundation) Normal Asymptomatic Normal -0.3 0.1 Normal Normal 98% 24/20 8 4.8 kPa
1.0 7 4.5* None Employed 160 cm 51 kg Ovarian insufficiency Normal Asymptomatic Restrictive syndrome -1.2 -1.5 Normal Normal 113% 26/30 10 4.8 kPa
Abbreviations: ALAT: alanine aminotransferase, ASAT: aspartate aminotransferase, DXA: dual X-ray absorptiometry, , HSCT: hematopoietic stem cell transplantation, N: normal values, nM: nmol/l, µM: µmol/l, *donor heterozygosity.
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Figure Legends Figure 1. Histopathology of liver biopsies before HSCT (A-H) A - Moderate bridging fibrosis, METAVIR score stage F2, with thin porto-portal linkage (Picrosirius Red stain). B - Bridging fibrosis, METAVIR score stage F3, with thick porto-portal and portal-central linkage, without cirrhosis, but note the variable thickness and orientation of the hepatocyte cords (Masson’s trichrome stain). C - Thickened centrilobular vein and sinusoidal fibrosis in zone 3 of the Rappaport acinus (Gordon-Sweet stain). D - Clarification and focal ballooning degeneration of hepatocytes, accompanied by vanishing or pyknosis of the nuclei (HES stain). E - Brown pigment of porphyrin in the cytoplasm of a large duct (HES stain). F - PAS-positive cytoplasmic fibrillary clumps of porphyrin in hepatocytes (Periodic-acid Schiff with diastase reaction). G – Diffuse brilliant and birefringent crystalline porphyrin deposits, showing the characteristic Maltese cross shape (polarized light microscopy). H - Mixed iron overload in hepatocytes and Kupffer cells. Intensity 2 (Perls stain).
Figure 2. Histopathology of liver biopsies after HSCT A - Severe disarray of lobular architecture with centrilobular fibrosis and perisinusoidal fibrosis, hepatocyte ballooning, and numerous ground glass-like inclusions in hepatocytes (Masson’s trichrome stain). B - Panlobular iron overload in hepatocytes and Kupffer cells (Perls stain).
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Figure 1
26
27
Figure 2
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Journal Pre-proof
Bone marrow transplantation in congenital erythropoietic porphyria: Sustained efficacy but unexpected liver dysfunction Caroline Besnard , Caroline Schmitt , Louise Galmiche-Rolland , Dominique Debray , Monique Fabre , Thierry Molina , ´ Laurent Gouya , Cecile Ged , Martin Castelle , ´ edicte ´ Marina Cavazzana , Elisa Magrin , Ben Neven , ´ ´ Despina Moshous , Stephane Blanche , Marie-Louise Fremond PII: DOI: Reference:
S1083-8791(19)30867-5 https://doi.org/10.1016/j.bbmt.2019.12.005 YBBMT 55814
To appear in:
Biology of Blood and Marrow Transplantation
Received date: Accepted date:
30 October 2019 9 December 2019
Please cite this article as: Caroline Besnard , Caroline Schmitt , Louise Galmiche-Rolland , ´ Dominique Debray , Monique Fabre , Thierry Molina , Laurent Gouya , Cecile Ged , ´ edicte ´ Martin Castelle , Marina Cavazzana , Elisa Magrin , Ben Neven , Despina Moshous , ´ ´ Stephane Blanche , Marie-Louise Fremond , Bone marrow transplantation in congenital erythropoietic porphyria: Sustained efficacy but unexpected liver dysfunction, Biology of Blood and Marrow Transplantation (2019), doi: https://doi.org/10.1016/j.bbmt.2019.12.005
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Inc. on behalf of the American Society for Transplantation and Cellular Therapy
Highlights:
The very first still-living receivers of hematopoietic stem-cell transplantation (HSCT) for early and severe forms of congenital erythropoietic porphyria (CEP), 25 and 22 years ago, are healthy and asymptomatic, with sustained corrected UROS activity.
Intrinsic hepatic involvement of varying intensity is present at birth, with either a favorable or fatal outcome, despite HSCT engraftment.
Liver pathology revealed varying degrees of portal, centrilobular, and perisinusoidal fibrosis, clarification of hepatocytes, and cytosolic porphyrin deposits.
Due to the risks and difficulties of HSCT, the indication remains limited to the severe form of CEP.
1
Bone marrow transplantation in congenital erythropoietic porphyria: sustained efficacy but unexpected liver dysfunction
Short title: Bone marrow transplantation in CEP Caroline Besnard1, Caroline Schmitt2, Louise Galmiche-Rolland3, Dominique Debray4, Monique Fabre3, Thierry Molina3, Laurent Gouya2, Cécile Ged5, Martin Castelle1, Marina Cavazzana6, Elisa Magrin6, Bénédicte Neven1, Despina Moshous1, Stéphane Blanche1, and Marie-Louise Frémond1*
1
Pediatric Immuno-Hematology and Rheumatology unit, Necker Enfants Malades Hospital,
Assistance Publique-Hôpitaux de Paris, AP-HP, Paris, 2
French Center of Porphyrias, Louis Mourier Hospital, AP-HP, Colombes and Research
Center of Inflammation, UMR1149 INSERM, Université de Paris, Paris, 3
Pathology Department, Necker Enfants Malades Hospital, AP-HP, Paris,
4
Paediatric Hepatology Unit, Necker Enfants Malades Hospital, AP-HP, Paris,
5
Biotherapy of Genetic Diseases, Inflammatory Disorders, and Cancers, U1035 INSERM,
Bordeaux University, Bordeaux 6
Biotherapy Unit, Necker Enfants Malades Hospital, AP-HP, Paris, France
*Present address: Laboratory of Neurogenetics and Neuroinflammation, Imagine Institute INSERM UMR1163, Paris, France
Corresponding author: Stéphane Blanche, Hôpital Necker, 149 rue de Sèvres, 75015 Paris, France [email protected]
Keywords: porphyria, long term, liver
2
Abstract Congenital erythropoietic porphyria (CEP) is a rare disease characterized by erosive photosensitivity and chronic hemolysis due to a defect of the enzyme uroporphyrinogen-IIIsynthase (UROS). To date, hematopoietic stem-cell transplantation (HSCT) is the only curative therapy for the devastating early and severe form of the disease. We describe six CEP patients treated with HSCT – 3 of them twice after failure of a first graft - between 1994 and 2016 in our center, including two of the very first living patients treated more than 20 years ago. Four patients are doing well 6 to 25 years post HSCT, with near normal biochemical parameters of porphyrin metabolism without the cutaneous or hematological features of CEP. One patient died within the first year after HSCT from severe graft-versus host disease (GVHD). Finally, one child died of unexplained acute hepatic failure one year after HSCT, despite full donor chimerism. Retrospectively, it appears that all but one child had increased transaminase activity with onset from the early post-natal period, which was significantly more marked for the child who died of liver failure. In contrast, liver function values progressively normalized after engraftment for all other children. Liver pathology before HSCT for three patients revealed varying degrees of portal, centrilobular, and perisinusoidal fibrosis, clarification of hepatocytes, and cytosolic porphyrin deposits. Liver porphyrin content in biopsies was more than 60 times the normal values. Despite difficult engraftment, long-term efficacy of HSCT in CEP appears to be favorable and reinforces its benefits for the severe form of CEP. Hepatic involvement requires careful evaluation before and after HSCT and further investigation towards its pathophysiology and care.
3
Introduction Congenital erythropoietic porphyria (CEP), formerly referred to as Günther’s disease, is a rare metabolic autosomal recessive inherited disorder of varying severity, but typically associated with early onset, devastating symptomatology, and poor prognosis1. CEP is due to the defective enzymatic activity of uroporphyrinogen III synthase (UROS), the 4th enzyme in the heme biosynthesis pathway. Approximately 50 different pathogenic mutations in UROS have been described thus far. They alter either the catalytic machinery, stability of the final conformation, or thermostability of the protein2. This defect results in the accumulation of the non-physiological and pathogenic porphyrin isomers uroporphyrinogen I and coproporphyrinogen I in the erythroid precursors of bone marrow, which are later released into the plasma and deposited in tissues (mainly skin, bones, and spleen). The accumulated porphyrins are then excreted in the urine (uroporphyrin I and coproporphyrin I) or feces (coproporphyrin I). They act as photoreactive intermediates, absorbing energy from long ultraviolet and visible light to release radicals or transfer electrons directly to targets, leading to a loss of integrity and function of the cell membrane, disruption of cell organelles, and cell death. Clinical presentations range from severe antenatal manifestations (hydrops fetalis, in utero death) to milder adult-onset phenotypes. In the early and severe infantile form, the accumulation of porphyrins in the skin combined with light exposure induces phototoxic lesions, such as vesicles, blisters, superinfections, and cartilage destruction, leading to epidermal atrophy and sclerodermoid changes with devastating mutilation. The anemia results from two mechanisms: i) an excess of porphyrins in the bone marrow precursor cells, which increases apoptosis, leading to ineffective erythropoiesis, and ii) accumulation of porphyrins in red blood cells, responsible for chronic intravascular hemolysis with variable bone-marrow compensation. Other CEP features include erythrodontia (i.e. reddish-brown coloration of the
4
teeth in visible light) by deposits during dental development, scleritis associated with elevated porphyrin levels in teardrops, osteolysis, and osteoporosis. CEP diagnosis is based on the identification of high levels of urinary uroporphyrin I and coproporphyrin I, which are pathognomonic of CEP. Elevated levels of porphyrins in erythrocytes and plasma (uroporphyrin I and coproporphyrin I) or in feces (coproporphyrin I) are also observed. Assays demonstrating profoundly diminished UROS activity in erythrocytes or mutations in the UROS gene also confirm the diagnosis. Hematological complications and early onset of symptoms, before the age of five years, are the main predictive factors of a poor prognosis. Symptomatic treatment essentially consists of the exhaustive avoidance of visible light as the only way to avoid skin lesions. Repeated red blood cell transfusions and splenectomy can also be used to decrease endogenous heme synthesis and decrease the stimulation of erythropoiesis. Transfusion may also by beneficial through an exogenous transient supply of the normal enzyme. Hematopoietic stem-cell transplantation (HSCT) to replace the bone-marrow pool of defective enzymes is the only curative treatment to date . The proof of concept was established in 1991 by Kauffman et al., who succeeded in correcting the UROS activity in hematopoietic cells, but the patient died of infectious complications2. Five years later, we reported the first successful HSCT3 in a young girl suffering from a severe form of CEP. Since then, more than 20 other cases of HSCT for CEP have been reported4-17. Independently of the tolerance of the transplant procedure itself, HSCT for the treatment of congenital diseases requires a detailed description of the disease evolution, as some symptoms may not be improved, or only partially, by the supply of an enzyme via hematopoietic cells. In addition, certain symptoms, unknown during the natural course of the disease, can appear in the long term through the prolongation of the lifespan of transplanted patients. 5
Here, we present the outcome of all six patients who underwent HSCT for CEP in our center, including the two very first still-living patients transplanted in 1994 and 1997.
Methods Patients This retrospective and monocentric study included all children receiving HSCT to treat CEP between 1994 and 2016 in the Pediatric Immuno-Hematology and Rheumatology Unit of Necker-Enfants Malades Hospital (Paris, France). Clinical, biological, histological, and radiological data were collected until September 2018. Before transplantation, all parents gave informed consent for anonymous use of the clinical and biological data for research purposes. This consent was renewed when the patients became young adults, according to the French regulations on clinical research. Porphyria diagnosis Biochemical analysis Total urinary porphyrins were determined by spectrophotometry between 350 and 450 nm after acidification. The various porphyrins and their isomers were separated by reverse-phase high-performance liquid chromatography (HPLC) with fluorimetric detection. Total erythrocyte porphyrins were first extracted with ether/acetic acid and then extracted with 3N hydrochloric acid. The acid extract was compared with a protoporphyrin standard by scanning the fluorescence excitation signal between 380 and 440 nm (emission at 602 nm). Plasma porphyrins were measured, as already described, by dilution of the plasma in phosphate buffer18. The solution was compared with a uroporphyrin standard by scanning the fluorescence emission signal from 570 to 750 nm (excitation at 405 nm). For total liver porphyrins measurement, 50 mg tissue was carefully cut in 500 µl saline, then sonicated for three 12-s periods. Porphyrins were extracted from the suspension by ten-fold dilution with 6
1N perchloric acid:methanol (1:1 v/v). After mixing and centrifugation, the supernatant was isolated for quantification of porphyrins by spectrofluorometry, using a RF6000 Shimadzu spectrofluorometer fitted with a red sensitive photomultiplier (excitation at 410 nm, emission at 600 nm). The results are given in picomoles per gram of tissue. Uroporphyrinogen III synthase activity in erythrocytes was determined by an enzyme-coupled assay, as already described 19. Genetic analysis Signed consent was obtained for genetic diagnosis. Genetic diagnosis was performed by Sanger sequencing of the entire coding sequence of the UROS gene (NM_000375), including intron-exon boundaries and the erythroid specific promoter region. Compound heterozygosity was confirmed by the analysis of both parents. Histopathology Liver biopsies were fixed in 10% phosphate-buffered formalin, embedded in paraffin, and stained with hematoxylin-eosin-saffron (H.E.S), Masson’s trichrome, Picrosirius Red for collagen, Gordon-Sweet for reticulin, and PAS (Periodic-acid-Schiff) and Perls for porphyrin and iron quantification. Cytokeratin 7 (CK7) staining was also performed for the assessment of ductular reaction. Portal fibrosis was evaluated according to the METAVIR criteria 20 . The cellular distribution of iron deposits was assessed . The Rowe scoring system was used. Porphyrin deposits were assessed using HES and PAS. Polarized light was used to assess the characteristic Maltese cross shape of brilliant and birefringent crystalline pigment deposits. Ethics The study was approved by the ethics committee of Necker-Enfants Malades University Hospital.
7
Results Initial clinical presentation Six children suffering from severe CEP were referred to our unit for HSCT between 1994 and 2014. There were four girls and two boys from unrelated families with no past history of porphyria (Table 1). Typical CEP skin lesions appeared at a median age of 2.1 [0-6.5] months, with active blisters and/or scars present for all patients at the time of HSCT. Hemolytic anemia and red urine occurred at a median age of 8 [0-13.5] and 1.6 [0-11.9] months, respectively. Hemolysis required one to two red-cell transfusions, except for one child who received nine red cell packs until the HSCT. Splenomegaly was either present at birth (5/6) or within the first two months (1/6). Before HSCT, a partial (n = 1) or total (n = 2) splenectomy was performed to relieve hemolysis but, above all, to prevent splenic sequestration of the graft. Porphyria diagnosis The diagnosis was confirmed at a median age of 7.1 [2.2-8] months, with very high levels of total urinary porphyrins (more than 100 times above normal) and characteristic high excretion of uroporphyrin I and coproporphyrin I. High amounts of porphyrins and characteristic profiles were also observed in feces, erythrocytes, and plasma. UROS activity was low in all five tested children (from 0.1 to 1.2, N > 6 U/mg Hb/h). All patients carried previously reported compound heterozygous (n = 5) or homozygous (n = 1) missense mutations in the UROS gene (Table 1). HSCT and related adverse events The median age at the first HSCT was 18.1 [9.5-22.4] months. The median time interval between diagnosis and first HSCT was 10.8 [4.6-14.4] months. All patients received a myeloablative conditioning regimen, including busulfan (empirical dose of 16 mg/kg orally for the
8
two first patients, intravenous with drug monitoring thereafter, with a targeted area under the curve of 20,000 µM.min), 200 mg/kg cyclophosphamide (n = 4) or 160 mg/m² fludarabine (n = 2), and anti-thymocyte globulins (all but one). Four patients received a matched unrelated donor (MUD) graft, one, a graft from a matched sibling donor (MSD) who was a heterozygous carrier of CEP, and one, a haplo-identical graft from one parent (heterozygous carrier of CEP) after complete T-cell depletion by CD34 selection (methodology in practice at the time of this HSCT in 1997). GVHD prophylaxis was based on cyclosporine alone (n = 3), cyclosporine plus methotrexate (n = 1), or cyclosporine plus mycophenolate (n = 2). Three patients showed stable engraftment with full chimerism after the first transplantation. However, three patients required a second transplantation due to the absence of engraftment (n = 1) or early loss of chimerism (n = 2). In all three, a resurgence or the persistence of skin lesions was observed, together with worsening of the biochemical porphyria profile in blood and/or urine. Two children were re-transplanted with the same HLA-matched donor (MSD and MUD), but the alternate haplo-identical parent was selected for the third 32, 35, and 2 months after the first HSCT, respectively (Table 1). After the first or second transplantation, donor chimerism was evaluated by either i) the restoration of UROS activity, as indicated by decreased erythrocyte, plasma, or urine porphyrin levels, and/or ii) VNTR or X and Y chromosome FISH analysis in the blood for sex-mismatched HSCT, all confirming engraftment for all children. In addition to the usual neutropenia-related adverse events, one child presented with a posterior reversible encephalitis syndrome, controlled after cessation of cyclosporine and normalization of arterial blood pressure. Graft vs Host disease was observed in three children: skin and gut grade II for two children, leading to prolonged immunosuppressive treatment (until 16 months post HSCT) for one, and severe for a third child, with grade III/IV skin, liver (biopsy proven), gut, and eye involvement, requiring several lines of immunosuppressive 9
therapy, including high doses of methylprednisolone and anti-thymoglobulin. The child simultaneously developed severe thrombotic microangiopathy and died of multiorgan failure six months post HSCT, despite treatment with monoclonal anti-C5 antibody (eculizumab). No veno-occlusive disease (VOD) was observed. Of note, four of the six children received prophylactic defibrotide, because of their young age, the use of a full dose of busulfan for complete myeloablation, and preexisting liver dysfunction. Porphyrin metabolism and CEP symptomatology after HSCT Urinary porphyrin levels decreased rapidly after HSCT for all six patients, but never normalized (even for the oldest patient). Erythrocyte porphyrins were measured for five patients and showed normal or nearly normal levels. Evaluable UROS activities showed a strictly normal level for two patients and a nearly normal level for two others, as observed in heterozygous carriers of CEP, consistent with the fact that these two patients received a graft from a family member who was a heterozygous carrier. Enzyme activity remained stable 20 years after the HSCT for the two oldest patients (see below). Photosensitivity completely disappeared and no hemolysis was observed in any patient as soon as hematological recovery and donor chimerism were obtained. Liver biology and follow-up All but one child showed varying levels of elevated serum transaminase activities during the neonatal or early postnatal period, reaching up to more than ten times the upper limit of normal for one (Table 2). After HSCT, five children were evaluable, as one died at six months post HSCT from severe multipolar GVHD and severe thrombotic microangiopathy. Liver function values normalized progressively for four out of five children following HSCT. However, transaminase values did not normalize post-HSCT for one child, who developed unexplained acute liver failure (ALF) at five months after transplantation despite engraftment. 10
During this event, which is very uncommon in the course of HSCT, an extensive workup failed to detect any evidence of toxic or viral liver injury. There were no clinical signs of GVHD at this time. After spontaneous improvement (prothrombin times improving from 22% to 94% in one week), transaminases never normalized and ALF recurred seven months later, leading to death at one-year post-transplant. Liver histopathology and liver porphyrin levels Before HSCT Four liver biopsies were performed for three children. Liver biopsies were performed at the time of splenectomy for one and during the routine checkup before HSCT for the two most recent children. Portal fibrosis, confirmed by Masson trichome and Picrosirius Red stains, was present for all but one (patient 6), ranging from the F1 to F3 stage. In one case (patient 5), severe bridging fibrosis, stage F3 (porto-portal or portal-central linkage) was present but there was no obvious cirrhosis. Centrilobular fibrosis was a major feature present in all cases. In addition, sinusoidal fibrosis, of moderate to severe intensity, was always present and most often diffuse (3/4). Hepatocyte injury was constant and included clarification and ballooning degeneration of varying intensity and distribution, often in contact with portal and centrilobular fibrosis. The amount of steatosis ranged from 0 (patient 6) to 5%. Siderosis of Kupffer cells was always more marked than that of hepatocytes, varying between 0 and 3, whereas that in hepatocytes ranged between 0 and 1. Deposition of PAS-positive fibrillary clumps of dense material was mixed, more marked in hepatocytes than in Kupffer cells, detected in all biopsies, and detected in the cytoplasm of large bile ducts for one biopsy (patient 5) (Figure 1). The biliary ducts were normal in all but one case (patient 5), with a slight ductular reaction. Most portal tracts showed mild nonspecific lymphocytic inflammation. The portal veins and arteries were preserved in all cases. No bilirubinostasis was observed. 11
Porphyrin levels assessed in three of these biopsies showed extremely high values (26,490, 55,536, and 39,215 pmol/g tissue, normal < 400 pmol/g tissue). After HSCT A biopsy was performed for the child experiencing his first episode of ALF (patient 4): the histological picture excluded GVHD, veno-occlusive disease, and acute viral hepatitis (no necrosis, no apoptosis, and microvesicular steatosis not exceeding 5% in the centrilobular zone). Lobular fibrosis was graded 1 to 3. The major findings were portal fibrosis (F2) with mild lymphocytic infiltrate, severe disarray of lobular architecture with centrilobular fibrosis and perisinusoidal fibrosis (grade 3), moderate hepatocyte ballooning (30%), and numerous ground glass-like inclusions (20%), associated with mild porphyrin deposition in hepatocytes (Figure 2). Two biopsies were performed for the child with severe GVHD (patient 6). Histopathology confirmed the diagnosis (data not shown). Long-term outcome At last evaluation, all living patients had a normal physical examination, without hepatomegaly, splenomegaly, or photosensitivity. Blood counts and liver enzymes were in the normal range. More specifically, the two first still-living young women received transplants more than 20 years ago, were both healthy and asymptomatic, but definitive erythrodontia (including on permanent teeth which appeared after the HSCT) required esthetic care. Enzyme activity remained stable for the two oldest patients. No unexpected clinical or biochemical disorder was observed nor detected at the last check up, except for an asymptomatic restrictive syndrome, based on respiratory parameters, considered to be sequalae of double exposure to busulfan. The assessment of liver stiffness evaluated by transient elastography (FibroScan®) was normal (Table 3). Both lead well-balanced personal, social, and professional lives. Both were under hormonal substitution treatment for primary
12
ovarian insufficiency attributed to the double conditioning regimen, but one gave birth to a healthy boy after a spontaneous pregnancy.
Discussion The benefit of bone marrow transplants for the severe form of CEP has been described in several case reports. We report here, for the first time, the long-term follow-up of two of the very first successfully treated patients more than 20 years post-transplant. The result for these two young women is remarkable, despite donor heterozygosity and reduced UROS activity; they are completely asymptomatic, and the follow-up showed no unexpected pathology related to late, undescribed expression of porphyria; these two women only suffer from the aesthetic disorder of their final teeth being colored reddish-brown, which requires regular dental care. Exposure to the sun, even without protection, does not cause any pathological photosensitivity and they do not have anemia. This finding reinforces the potential interest of bone marrow transplantation for this indication, even if lifelong efficacy is yet to be shown. Of note, urine porphyrin levels were still moderately elevated, despite full engraftment and normal levels of erythrocyte porphyrins. This remaining porphyrin excretion may be the consequence of porphyrin production in non-erythroid tissues, such as the liver. As the UROS gene defect is constitutively presented in all organs, the liver still produces and moderately accumulates uroporphyrin I and coproporphyrin I1. Aside from this reassuring long-term outcome, three points deserve special attention. The first is that half of the children had to be grafted twice to obtain hematopoietic chimerism and thus stable enzymatic correction. During the long study period, two elements could have been associated with some failures: T cell depletion methodology by CD34 selection used twenty years ago for the haploidentical HSCT, which today would be considered suboptimal, and
13
oral busulfan without drug monitoring for two children. In addition, we did not consider the risk of trapping the stem cells in the splenomegaly for the first patient. Nevertheless, chronic hemolysis induces overactive marrow and myeloablative conditioning appears to be necessary to obtain optimal and durable enzyme correction. These observations can fuel recommendations, such as considering complete or partial splenectomy before HSCT in case of massive splenomegaly and proposing fully myeloablative conditioning similar to that proposed for other diseases associated with hyperactive erythropoiesis. Clearly, the recent in vivo or in vitro methodologies of selective T-cell depletion will improve the chance of success of haploidentical HSCT. The second important feature is the prevalence of liver involvement, observed here in all but one child prior to HSCT. In contrast to what is observed in the other form of erythropoietic porphyria, erythropoietic protoporphyria (EPP), liver dysfunction in CEP is uncommon. Liver enlargement and porphyrin-rich gallstones can be found in some patients. In a review of 29 cases of CEP, 11 had “abnormal liver function” values with onset from birth to the mid-30s, but this global figure also included hemolysis-related hyperbilirubinemiae21: although abnormally high liver transaminase activity was observed based on the presented median values, the incidence of specific liver dysfunction was not indicated. Severity ranged from spontaneous resolution to stability and cirrhosis in one case but no acute liver failure was described. A case of liver failure and hepatorenal syndrome was also described in a 35-year-old women22. The physiopathology of this liver dysfunction in CEP is yet to be elucidated. The pre-transplantation liver biopsies presented here show porphyrin deposition in hepatocytes and Kupffer cells, which could induce liver fibrosis via tissue damage from porphyrin-mediated oxidative stress. Liver function values gradually normalized following transplantation for four patients, while porphyrin levels in the erythrocytes and urine decreased. The two patients evaluated in the long term had normal liver enzymology and no signs suggestive of liver fibrosis on elastometry. However, the third
14
unexpected event was the post-transplant occurrence of sudden fatal ALF in one child, despite full chimerism and decreased urine porphyrin levels. No cases of ALF have been reported in the post-transplant setting for CEP. This child had no clinical or biological signs of infection, toxic hepatitis, or other HSCT-related complications. However, a comparison of pretransplantation characteristics with those of the other children showed that the child presented the most severe liver dysfunction soon after birth and sustained transfusion-requiring hemolytic anemia until HSCT, suggesting a more severe disease. At diagnosis, this child had the most highly elevated levels of porphyrins in urine, plasma, and erythrocytes. No liver biopsy was available prior to HSCT for histological analysis and assessment of liver porphyrin content. Such fatal ALF, one year after the HSCT, could have been the consequence of massive accumulation of porphyrins insufficiently cleared by the enzyme originating from the engrafted cells, together with iron overload subsequent to his pretransplant sustained hemolysis. Macrophage and hepatocyte iron overload were noted on the post-HSCT biopsy. The joint action of iron and porphyrin overload may explain the development of intralobular fibrosis, especially in the centrilobular region, although urine porphyrin and serum ferritin levels were decreasing post-HSCT. Centrilobular fibrosis, such as perisinusoidal fibrosis, may be due to the effects of reactive oxygen species and oxidative stress23,24. An unidentified external triggering event that occurs in a chronically altered liver, such as drug-induced hepatitis, cannot be ruled out, as suggested by the presence of hepatocellular ballooning and numerous abnormal pseudo cytoplasmic ground-glass inclusions25. Finally, another genetic predisposition to liver damage could be hypothesized, as described in EPP, through the impairment of the expression of ATP-binding cassette transporter G226. In the future, massive accumulation of porphyrins in the liver could justify a preventive approach to protect the liver, as suggested, for example, with chloroquine for its anti-inflammatory action in porphyria cutanea tarda27 or, more recently, by an induced iron
15
deficiency in CEP22. The overall morbidity and mortality of HSCT –even with potential suggested improvements make it necessary to restrict the indication of HSCT to the most severe form of the disease. Severity is sometimes obvious, such as in cases of early devastating skin photoreactivity in infancy. The evaluation of severity can also be based on the intensity of hemolysis, very high levels of urinary porphyrin, or the C73R mutation genotype. However, late onset worsening can also be observed in milder forms of the disease, with the exhaustion of erythropoiesis and secondary aplastic anemia. In addition to the severity of the disease, the place of HSCT will have to be evaluated in the light of donor characteristics (HLA identical sibling or unrelated haploidentical donor) and alternative treatment, such as induced iron deficiency or future gene therapy. In all cases, it will be based on a careful case-by-case evaluation and a very close collaboration between a porphyria specialist and the HSCT team. In conclusion, we strongly suggest that the initial workup before HSCT includes a liver biopsy to evaluate the level of liver damage and the level of porphyrin accumulation. Despite HSCT-related morbi-mortality, long-term data support HSCT as a life-changing treatment for patients presenting an early and severe form of CEP.
16
Acknowledgments No funding support was provided for this study.
Authorship Contributions MLF, SB, and DB designed the study. CB collected the data and wrote the first version of the manuscript. MLF, SB, DM, BN, MC, and DD provided care to patients. LGR, MF, TM and DD analyzed the liver biopsies. CS and LG performed and analyzed the porphyrin assays and CG the genetic diagnosis. MC and EM (biotherapy) were in charge of the management and safety procedures of hematopoietic stem cells before infusion. All authors approved the final version of the manuscript.
Conflict of Interest Disclosures The authors have no conflict of interest to disclose.
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10. Dupuis-Girod S, Akkari V, Ged C et al. Successful match-unrelated donor bone marrow transplantation for congenital erythropoietic porphyria (Günther disease). European Journal of Pediatrics. 2005;164:104–7 11. Phillips JD, Steensma DP, Pulsipher MA, Spangrude GJ, Kushner JP. Congenital erythropoietic porphyria due to a mutation in GATA1: the first trans-acting mutation causative for a human porphyria. Blood. 2007;109:2618–21 12. Taibjee SM, Stevenson OE, Abdullah A et al. Allogeneic bone marrow transplantation in a 7-year-old girl with congenital erythropoietic porphyria: a treatment dilemma. British Journal of Dermatology. 2007;156:567–71 13. Faraci M, Morreale G, Boeri E et al. Unrelated HSCT in an adolescent affected by congenital erythropoietic porphyria. Pediatric Transplantation. 2008;12:117–20 14. Lebreuilly-Sohyer I, Morice A, Acher A et al. Congenital erythropoietic porphyria treated by haematopoietic stem cell allograft. Ann Dermatol Venereol. 2010;137:635–9 15. Singh S, Khanna N, Kumar L. Bone marrow transplantation improves symptoms of congenital erythropoietic porphyria even when done post puberty. Indian J Dermatol Venereol Leprol. 2012;78:108–11 16. Martinez Peinado C, Diaz de Heredia C, To-Figueras J et al. Successful Treatment of Congenital Erythropoietic Porphyria Using Matched Unrelated Hematopoietic Stem Cell Transplantation. Pediatric Dermatology. 2013;30:484–9 17. Karakurt N, Tavil B, Azik F, Tunc B, Karakas Z, Uckan-Cetinkaya D. Successful hematopoietic stem cell transplantation in a child with congenital erythropoietic porphyria due to a mutation in GATA-1. Pediatric Transplantation. 2015;19:803–5 18. Elder GH, Smith SG, Smyth SJ. Laboratory investigation of the porphyrias. Ann Clin Biochem. 1990;27:395-412
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19. Deybach JC, Grandchamp B, Grelier M et al. Prenatal exclusion of congenital erythropoietic porphyria (Günther's disease) in a fetus at risk. Hum Genet. 1980;53:21721. 20. The METAVIR cooperative group. Inter- and intra-observer variation in-the assessment of liver biopsy of chronic hepatitis C. Hepatology 1994; 1:15-20. 21. Katugampola RP, Anstey AV, Finlay AY et al. A management algorithm for congenital erythropoietic porphyria derived from a study of 29 cases: Management algorithm for congenital erythropoietic porphyria. British Journal of Dermatology. 2012;167:888–900 22. Egan DN, Yang Z, Phillips J, Abkowitz JL. Inducing iron deficiency improves erythropoiesis and photosensitivity in congenital erythropoietic porphyria. Blood. 2015;126:257-61 23. Boitnott J, Haas M, Anders RA et al. Glycogen pseudoground glass change in hepatocytes.Am J Surg Pathol. 2006;30:1085-90. Ground-glass, polyglucosan-like hepatocellular inclusions: A "new" diagnostic entity. 24. Lefkowitch JH, Lobritto SJ, Brown RS Jr et al. Ground-glass, polyglucosan-like hepatocellular inclusions: A "new" diagnostic entity. Gastroenterology. 2006;131:713-8 25. Yin C, Evason KJ, Asahina K, Stainier DY. Hepatic stellate cells in liver development, regeneration, and cancer. J Clin Invest. 2013;123:1902-10 26. Hagiwara S, Nishida N, Park AM, Sakurai T, Kawada A, Kudo M. Impaired expression of ATP-binding cassette transporter G2 and liver damage in erythropoietic protoporphyria. Hepatology. 2015;62:1638-9 27. González-Estrada A, Gomez-Morales LB, Gonzalez-Estrada A, García-Morillo JS. Sporadic porphyria cutanea tarda: treatment with chloroquine decreases hyperglycemia and reduces development of metabolic syndrome. Eur J Intern Med. 2014;25:e76-7
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Table 1. Patient characteristics
Patients Sex Age at diagnosis Photosensitivity Erythrodontia Red urine Splenomegaly Anemia Transfusions (n) Splenectomy Diagnosis Genotype
Erc-UROS activity
P1 F 7.6 months + + + + 0 +*
P2 F 8 months + + + + 0 -
P3 F 6.2 months + + + + 0 -
P4 M 5.6 months + + + + + 8 RBC & 2 platelets +
P5 F 2.2 months + + + + + 2 RBC & 10 platelets +
P6 M 8 months + + + + + 2 RBC -
[c.217T>C, p.(Cys73Arg)] [c.560A>C, p.(Gln187Pro)] 0.7
[c.217T>C, p.(Cys73Arg)] [c.217T>C, p.(Cys73Arg)] ND
[c.205G>A, p.(Ala69Thr)] [c.217T>C, p.(Cys73Arg)] 0.1
[c.10C>T, p.(Leu04Phe)] [c.673G>A, p.(Gly225Ser)] 0.9
[c.217T>C, p.(Cys73Arg)] [c.634T>C, p.(Ser212Pro)] 1.2
[c.205G>A, p.(Ala69Thr)] [c.244G>T, p.(Val82Phe)] 0.5
5 622
9 851
3 270
34 728
17 920
9 755
N>6U/mgHb/h)
Total U-porphyrins
N<30nmol/mmol creat)
Total Uroporphyrins
2 733
6 684
2 018
26 309
8 315
4 760
% Isomer I (N<30%)
ND
ND
ND
ND
97%
96%
Total Coproporphyrins
2 889
3 167
1 252
8 419
7 400
3 902
99% 66.9
96% 52.6
98% 17
ND 136
98% 39.2
98% 27.4
95
4 700
984
2007 13.9 months 20.1 months MUD
2009 4.6 months 10.1 months MUD
1st 2012 7.3 months 9.5 months MUD
2nd 2015 40.3 months 42.5 months MUD
10/10 IV Bu, Cy, ATG
10/10 IV Bu, Cy, ATG
14 106/kg
13.45 106/kg
10/10 IV Bu, Flu, ATG 10.66 106/kg
10/10 IV Bu, Flu, ATG 3.8 106/kg
CsA
CsA/MA
N<10 nmol/mmol creat)
N<20 nmol/mmol creat)
% Isomer I (N<30%) Erc-porphyrins
N<1.9µmol/LHb)
Plasma-porphyrins
ND
ND
667
N<20nmol/L)
HSCT Year Time Dg-HSCT Age Donor
1st 1994 13.7 months 21.6 months MSD
2nd 1995 21.7 months 29.6 months MSD
HLA compatibility Conditioning regimen
10/10 Oral Bu, Cy
10/10 Oral Bu, Cy
1st 1997 26.4 months 21.4 months Haplo T Ly depletion: In vitro CD34 selection 5/10 IV Bu, Cy
CD34+ cells
ND
ND
2.5 106/kg
2nd 1997 28.4 months 23.4 months Haplo T Ly depletion: In vitro CD34 selection 5/10 IV Bu, Cy, ATG 7 106/kg
Prophylaxis GVHD
CsA/MTX
CsA/MTX
CsA
CsA
CsA
CsA/MA
2014 8 months 16 months MUD
10/10 IV Bu, Flu, ATG 11.69 106/kg
CsA/MA
21
VOD
-
-
-
-
UA/Defibrotide
UA/Defibrotide
UA/Defibrotide
UA/Defibrotide
UA/Defibrotide
Abbreviations: ADV: adenovirus, ATG: anti-thymocyte globulin, Bu: busulfan, CsA: ciclosporin A, Cy: cyclophosphamide, Dg: diagnosis, F: female, Flu: fludarabine, GVHD: Graft versus Host disease, haplo: haplo-identical, HLA: Human Leukocyte Antigen, HSCT: hematopoietic stem cell transplantation, IV: intravenous, M: male, MA: mycophenolic acid, MSD: matched sibling donor, MUD: matched unrelated donor, MTX: methotrexate, n: number; ND: no data, RBC: red blood cells,, UA: ursodesoxycholic acid, VOD: vein occlusive disease. *splenectomy perfomed after the failure of the first graft.
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Table 2. Pre and post HSCT evolution of liver transaminases
Patients
P1
P2
P3
P4
P5
P6
Neonatal
+
NA
NA
+++
++
NA
Nl
Nl
+++
++
++
+
At HSCT
+
Nl
Nl
+
++
+
1 year post HSCT
Nl
Nl
Nl
Nl 24 Y post HSCT
Nl 22 Y post HSCT
Nl 3 Y post HSCT
Postnatal period until HSCT
Long term
Nl/++/+++* Death of acute liver failure at 1 year post HSCT _
++
Nl 3Y post HSCT
Non evaluable: Severe hepatic GVHD and TMA, Death at 6 Mo Post HSCT
_
HSCT: hematopoietic stem cell transplantation, NA: not available, GVHD: graft versus host disease, TMA: thrombotic micro angiopathy, transaminase values: Nl: normal value, + < 2x normal, ++ 2 to 10x normal, +++ > 10x normal, HSCT: hematopoietic stem cell transplantation, *fluctuating values.
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Table 3. Long-term outcome after HSCT
Follow-up after last HSCT Age at last visit Photosensitivity Scars Tooth color Red urine Anemia Thrombocytopenia Splenomegaly Urinary porphyrins (N<30nmol/mmol creat) Erythrocyte porphyrins (N<1.9µmol/LHb) Plasma porphyrins (N<20nmol/L) URO III synthase activity (N>6U/mgHb/h) Immunosuppressant treatment Professional life Height Weight Endocrine status
Heart ultrasonography Respiratory status Functional respiratory tests DXA femur Z-score DXA lumbar spine Z-score Abdominal ultrasonography Spleen Prothrombin time ASAT/ALAT Bilirubin Elastography (Fibroscan®, N<5kPa)
P16 24.3 years 26.1 years None None Reddish-brown None None None None 117
P221 20.9 years 22.4 years None None Reddish-brown None None None None 38
1.4 16 5* None Employed 169.8 cm 56 kg Ovarian insufficiency (one child natural fecundation) Normal Asymptomatic Normal -0.3 0.1 Normal Normal 98% 24/20 8 4.8 kPa
1.0 7 4.5* None Employed 160 cm 51 kg Ovarian insufficiency Normal Asymptomatic Restrictive syndrome -1.2 -1.5 Normal Normal 113% 26/30 10 4.8 kPa
Abbreviations: ALAT: alanine aminotransferase, ASAT: aspartate aminotransferase, DXA: dual X-ray absorptiometry, , HSCT: hematopoietic stem cell transplantation, N: normal values, nM: nmol/l, µM: µmol/l, *donor heterozygosity.
24
Figure Legends Figure 1. Histopathology of liver biopsies before HSCT (A-H) A - Moderate bridging fibrosis, METAVIR score stage F2, with thin porto-portal linkage (Picrosirius Red stain). B - Bridging fibrosis, METAVIR score stage F3, with thick porto-portal and portal-central linkage, without cirrhosis, but note the variable thickness and orientation of the hepatocyte cords (Masson’s trichrome stain). C - Thickened centrilobular vein and sinusoidal fibrosis in zone 3 of the Rappaport acinus (Gordon-Sweet stain). D - Clarification and focal ballooning degeneration of hepatocytes, accompanied by vanishing or pyknosis of the nuclei (HES stain). E - Brown pigment of porphyrin in the cytoplasm of a large duct (HES stain). F - PAS-positive cytoplasmic fibrillary clumps of porphyrin in hepatocytes (Periodic-acid Schiff with diastase reaction). G – Diffuse brilliant and birefringent crystalline porphyrin deposits, showing the characteristic Maltese cross shape (polarized light microscopy). H - Mixed iron overload in hepatocytes and Kupffer cells. Intensity 2 (Perls stain).
Figure 2. Histopathology of liver biopsies after HSCT A - Severe disarray of lobular architecture with centrilobular fibrosis and perisinusoidal fibrosis, hepatocyte ballooning, and numerous ground glass-like inclusions in hepatocytes (Masson’s trichrome stain). B - Panlobular iron overload in hepatocytes and Kupffer cells (Perls stain).
25
Figure 1
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27
Figure 2
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