—  SPECIALTY CONFERENCE  —

PEDIATRIC PATHOLOGY

Case 2 - X-linked Chronic Granulomatous Disease with Aspergillus fumigatus infection, associated to Infantile Myofibromatosis, familial type

Miguel Reyes-Múgica
Yale University School of Medicine
New Haven, Connecticut

Clinical summary
A 3 year-old African-American boy presented with limping due to a thigh mass of recent appearance. The lesion was initially painless and non-mobile, without erythema or swelling. There was no fever or other constitutional symptom. Four days before admission, a second tumor appeared on the left parietal region. The leg mass became erythematous and painful, and fever of 39.4°C ensued. The relevant past medical history includes three soft tissue nodules removed from the buttock on the third day of life, one of which is shown on shown on slides 6 and 7 below (the patient's father had an identical soft tissue lesion surgically removed as a newborn). In addition, there were frequent and recurrent infections including meningitis and pneumonias, and failure to thrive. At two years of age the patient developed an intra-abdominal lymphangioma which showed histological evidence of infection with multiple granulomata. At the time of admission the patient was at the 5th percentile for height and below the 3rd percentile for weight. A CT scan revealed a large soft tissue lesion in the scalp, eroding the parietal bone, and several small lesions involving the brain parenchyma. In addition, a large mass was found involving the posterior aspect of the right thorax. These findings were interpreted as suggestive of metastatic disease, possibly Ewing sarcoma or neuroblastoma. Pertinent lab results include normal levels of immunoglobulins and negative HIV testing. The leg and parietal lesions were surgically removed and drained. A representative sample from the leg is shown on slide 8 and 9 below.

Histologic findings
Slides 6 and 7 show a spindle cell proliferation with a storiform pattern, occasional mitoses, no pleomorphism and a slightly myxoid background. Identical findings were seen in the lesion excised from the father of the patient as a newborn. Both lesions exhibit a myofribroblastic phenotype, confirmed by immunohistochemistry; there are characteristic nodules of tumor cells pushing and protruding into the vascular lumen.

Slides 8 and 9 show acute and chronic inflammation with numerous granulomata. Foci of intense neutrophilic infiltration mixed with necrotic debris and fungal organisms consistent with Aspergillus are present. A few fungi are also seen within some granulomata. Microbiology cultures were positive for Aspergillus fumigatus.

There are two different and seemingly unrelated lesions in our patient, which have not been previously described occurring simultaneously. The first is a soft tissue tumor that features the distinctive appearance of infantile myofibromatosis (IM), which in this case has a familial presentation. Both, the father and son were "cured" (vide infra) from this condition with simple surgical excision of the tumors. The second set of lesions, with the characteristic granulomatous inflammation needs to be interpreted in the context of the clinical history. On that basis, the possibility of Chronic Granulomatous Disease (CGD) was suggested in the pathology report, and additional studies aimed towards securing the diagnosis were conducted.

Slides  (Click to enlarge)


Slide 6
Slide 7
Slide 8
Slide 9
Sections/images taken from the buttock on the third day of life, featuring a spindle cell proliferation with a vaguely myxoid background Sections/images taken from the leg at 3 years of age. There are multiple granulomata with associated acute inflammatory infiltrate. Special stain (GMS) shows fungal microorganisms consistent with Aspergillus in the center of a granuloma.

Ancillary tests
In order to assess the ability of the patient's neutrophils to produce a normal respiratory burst, a phorbol myristate acetate-stimulated nitroblue tetrazolium (NBT) test was conducted. In normal circumstances, when activated neutrophils are incubated with the yellow dye NBT, formazan is generated within the neutrophil cytoplasm producing a dark blue precipitate. Our patient's neutrophils failed to show blue pigment after the NBT test in comparison with normal controls; the tests were performed twice. Since interpretation of the NBT test is somewhat difficult and requires extensive experience, and because of the risk of false negative results, a second evaluation was conducted using the dye dihydrorhodamine (DHR) 123 to detect H202 generation during the oxidative burst. The results were consistent with those obtained from the NBT test. The patient's Stimulation Index (SI) was 1.04, compared with a SI of 76.6 in the control sample. Neutrophils from the patient's mother were also tested and showed two populations, one of which had a SI of 0.92, while the other displayed a SI of 20. These results are consistent with a carrier status for CGD. The DHR tests were kindly performed by Dr. Joe Rutledge, from the Children's Hospital & Regional Medical Center, in Seattle.

Flow cytometric analysis performed by Dr. Paul G. Heyworth at the Department of Molecular and Experimental Medicine of The Scripps Research Institute, revealed that the gp91phox subunit of the NADPH oxidase flavocytochrome ß558 (vide infra) appeared to be absent (or possibly present at very low levels) in our patient's neutrophil population. This result is indicative of X-linked CGD. p22phox - the smaller subunit of the flavocytochrome - was also abnormally low, and this secondary deficiency is common in X-linked CGD. When analyzed for gp91phox, the mother had two populations of cells, suggesting that she is a carrier of the X-linked variant of CGD and consistent with our NBT and DHR data. In both, our patient and his mother there were normal levels of p47phox, one of the cytosolic NADPH oxidase proteins. Furthermore, sequence analysis of the gp91phox gene, CYBB by Single Strand Conformation Polymorphism suggested that the patient's mutation involved exon 11, and it was identified as 1371G to A causing the change of Trp453 (TGG) to a STOP codon (TGA). This mutation would create a truncated (possibly unstable) mRNA transcript and a truncated, unstable, non-functional protein. According to Dr. Heyworth's lab database, this mutation has been previously identified in one patient out of 530 unrelated kindreds affected by X-linked CGD. The mother was heterozygous for the 1371G mutation, confirming her status as a carrier of the disease.

Diagnosis
X-linked Chronic Granulomatous Disease with Aspergillus fumigatus infection, associated to Infantile Myofibromatosis, familial type

Discussion
This patient presents an interesting diagnostic challenge due to the rare coincidence of two relatively uncommon problems. On one hand IM, apparently inherited from the father, manifested as the two congenital lesions for which the patient required neonatal surgery. IM is characterized by spindle cell proliferations that may occur as a solitary tumor or as groups of lesions involving soft tissues, bones or even viscera. It has been considered one of the most common soft tissue tumors of children. Familial presentation with a possible pattern of autosomal dominant inheritance has been recognized (1,2) but others have suggested an autosomal recessive inheritance.(3) Although localized lesions follow a benign course, recurrence occurs in bout 10% of cases ,(2) and it has been reported even 27 years after the initial presentation.(1) Multifocal, and specially visceral involvement portends an ominous prognosis.(1-3) The occurrence of these lesions in our patient set the stage for a more complicated clinical history.

The continuous development of repetitive infections ranging from pneumonia, to meningitis, to multisystemic aspergillosis, associated with failure to thrive and characteristic granulomatous lesions is the classic clinical presentation of CGD. With an estimated incidence between 1 in 200 000 in the US and 1 in 450 000 in Sweden,(4) CGD represents the archetype of inflammatory cellular phagocytic defects involving intracellular microbicidal mechanisms.(5) Described originally almost 50 years ago, research on this condition has contributed to our understanding of the physiology of leukocyte oxidant metabolism. The specific impairment lies on an enzymatic complex responsible for producing hydrogen peroxide: the NADPH oxidase. This system is responsible for the generation of superoxide, the precursor of several oxidant molecules necessary for adequate intracellular processing of phagocytized microbes. The enzymatic complex is most abundant in neutrophils, eosinophils and monocyte/macrophage cells, and it is composed of a membrane-bound flavocytochrome ß558 and four cytosolic proteins p47phox, p67phox, p40phox , and p21rac. Seventy percent of patients with CGD inherit the disorder on an X-linked pattern with a mutated gp91phox, the gene responsible for encoding the ßsubunit of the flavocytochrome. The second most common mutation involves p47phox, inherited in an autosomal recessive pattern.(4-7) Prenatal diagnosis may be established by analysis of neutrophils obtained from umbilical vein samples through fetoscopy; DNA from amniocytes and chorionic villous sampling may also be used.(4,6)

Patients with CGD are usually infected by catalase-positive micro-organisms, most frequently Aspergillus sp., S. aureus, Serratia sp., Nocardia sp. and B. cepacia.(5) Catalase degrades the small amounts of H2O2 produced by the infecting microbes, allowing the them to survive within the phagosome. Catalase-negative microorganisms apparently generate and maintain sufficient amounts of H2O2 from which other highly reactive compounds are produced within the phagocytic vacuole of the CGD neutrophils and macrophages, leading to effective microbial killing. Hence, these microorganisms are usually not seen in CGD patients.(4-7) Death is most commonly associated with Aspergillus infection.(8)

CGD patients may have muffled symptoms even when undergoing severe infections. Fever, leukocytosis and other indications of their septic state may be absent or subtle, which warrants a careful monitoring.(7) Additional complications of CGD patients are related to exuberant and persistent granulomatous inflammation, with extensive skin ulceration, systemic lupus erythematosus-like disease, pneumonitis, Crohn's-like disease, gastrointestinal and genitourinary tract obstruction.(5,6) Long-term antibiotic therapy, steroids, interferon-_, granulocyte transfusions, bone marrow transplantation and gene therapy, all have important roles in the adequate treatment of these patients.(5-7)

Survival of CGD patients has improved from only 21% of patients living beyond 5 years in 1967, to 92% reaching 8 years of age. In a report of patients born over a 32-year period, 50% survived through the third decade of live. The mean age of survivors in 1998 was 16 years. Difficulties with compliance of therapy in adolescents are thought to be responsible for the failure to improve the overall mortality rate.(4)

An extensive report on 368 patients included in the CGD registry has been recently published.(9)

References

  1. Jennings TA, Sabetta J, Duray PH, Enziger FM, Collins FS. Infantile myofibromatosis. Evidence for an autosomal-dominant disorder. Am J Surg Pathol 8:529-538,1984
  2. Bracko M, Cindro L, Golouh R. Familial occurrence of infantile myofibromatosis. Cancer 29:1294-1299,1992
  3. Narchi H. Four half-siblings with infantile myofibromatosis: a case for autosomal-recessive inheritance. Clin Genet 59:134-135,2001
  4. Goldblatt D, Thrasher AJ. Chronic granulomatous disease. Clin Exp Immunol 122:1-9,2000
  5. Segal BH, Holland SM. Primary phagocytic disorders of childhood. Pediatr Clin North Am. 47:1311-1338,2000
  6. Segal BH, Leto TL, Gallin JI, Malech HL, Holland SM. Genetic, biochemical and clinical features of chronic granulomatous disease. Medicine 79:170-200,2000
  7. Lekstrom-Himes JA, Gallin JI. Immunodeficiency diseases caused by defects in phagocytes. New Eng J Med 343:1703-1714,2000
  8. Liese J, Kloos S, Jendrossek V, Petropoulu T, Wintergerst U, Notheis G, Gahr M, Belohradsky BH. Long term follow-up and outcome of 39 patients with chronic granulomatous disease. J Pediatr 137:687-693,2000
  9. Winkelstein JA, Marino MC, Johnston RB Jr, Boyle J, Curnutte J, Gallin JI, Malech HL, Holland SM, Ochs H, Quie P, Buckley RH, Foster CB, Chanock SJ, Dickler H. Chronic granulomatous disease. Report on a national registry of 368 patients. Medicine 79:155-169, 2000