Occipital horn syndrome

* A 34-month-old black boy who had contracted acquired immunodeficiency syndrome from his mother presented with fever, vomiting, and cough. He was cachectic, hypertonic, and developmentally delayed. A brain computed tomography scan revealed masses in the left frontal horn, subependymal, and periventricular regions; secondary edema; and hydrocephalus. The differential diagnosis was cerebral lymphoma versus toxoplasmosis. The patient had disseminated Mycobacterium avium-intracellulare infection, lymphoid interstitial pneumonitis, as well as Pseudomonas and Klebsiella pneumonia. He died of respiratory insufficiency 53 days after admission. The autopsy confirmed a primary cerebral B-cell lymphoma, large cell type, which was positive for Epstein-Barr virus, latent phase, by in situ hybridization. Primary central nervous system lymphomas are rare in children, in contrast to adults. To our knowledge, only five well-documented cases of primary cerebral lymphomas in infants and children with acquired immunodeficiency syndrome have been reported previously. The current study shows that these childhood lymphomas are associated with and presumably caused by Epstein-Barr virus and thus have a pathogenesis similar to that of primary central nervous system lymphomas in adults. (Arch Pathol Lab Med. 1997;121:1287-1291)

Primary central nervous system lymphomas (PCNSL) are relatively common in adults affected with the acquired immunodeficiency syndrome (AIDS), with a reported incidence of 6% in a series of 153 adults in New York City, NY.1 They are rare in infants and children with AIDS, however. In the 12-year period from 1983 to 1995, a total of 98 autopsies were performed in infants and children with AIDS at the University of Miami/Jackson Memorial Medical Center, and we found only one patient with a PCNSL. The incidence of this malignant tumor in our population is therefore 1.02%. At the Pediatric Branch of the National Cancer Institute, more than 350 children infected with the human immunodeficiency virus (HIV) were evaluated, and only two had a malignant lymphoma of the brain, making the incidence for this large group even smaller (0.57%). The two cases are mentioned briefly in a comprehensive chapter about pediatric HIV infection,2 but they were not included in the Table because of lack of clinical and pathologic information. We searched the literature for reports of PCNSL in infants and children with HIV and AIDS, and we found only small series and isolated case reports (Table).

Herein to our knowledge we report the first case of Epstein-Barr virus (EBV-associated primary cerebral lymphoma, confirmed by autopsy in a child with AIDS in our institution, and we review the medical literature in relation to central nervous system (CNS) lymphoma in pediatric AIDS, compiling the reported cases in a single paper with emphasis on EBV and its association with the development of non-Hodgkin's lymphoma.

MATERIALS AND METHODS

The autopsy was performed according to the standard protocol for pediatric autopsies3 at Jackson Memorial Hospital. Systemic organs were fixed in 10% buffered formalin for 24 hours; the brain and spinal cord were fixed for 2 weeks. Representative sections of the organs and lesions of interest were sectioned after 24 hours of fixation, and hematoxylin-eosin-stained slides were prepared. Special stains were used on selected sections of tissues, including Wright-Giemsa and modified Steiner4 for bacteria, FiteFaraco for mycobacteria, and Gomori methenamine silver and periodic acid-Schiff for fungi. Immunoperoxidase techniques for the characterization of the lymphocytic infiltrate in the lungs and brain included T cells (CD45RO, UHCL1)5 and B cells (CD20, L26),6 both mouse monoclonal antibodies supplied in liquid form (Dako Corporation, Carpinteria, Calif). The sections were stained by the avidin-biotin method. The staining for T cells was done following enzymatic digestion.

Paraffin-embedded sections of lungs and brain were examined for EBV latent phase by in situ hybridization (Dako). This system uses fluorescein-labeled EBV oligonucleotide probes complimentary to two nuclear Epstein-Barr virus-encoded small RNAs. The slides with sections of the lung and brain were placed in an oven at 58degC for 30 minutes and were deparaffinized in two changes of xylene, 5 minutes each, and in two changes of 100% ethanol, 5 minutes each. They were rinsed in two changes of distilled water, 5 minutes each, immersed in warmed pepsin for 15 minutes (37oC), and kept at the same temperature. The slides were then washed in two changes of distilled water, 5 minutes each. Once the slides were removed from water, a drop of probe was placed on the section, and it was covered with a cover glass. The glass slides were placed on a flat heating block (90degC) for 5 minutes and then transferred to a humidity chamber for 60 minutes. The slides were removed from the humidity chamber and immersed in a tris-buffered saline (TBS) bath until the cover glass came off. They were immersed in warmed stringent wash solution for 30 minutes at 50degC and then washed in a TBS bath for 5 minutes. Detection reagent (alkaline phosphatase-conjugated rabbit F[ab'] antibody) was placed on every slide, completely covering the tissue, and the slides were then incubated for 20 minutes at room temperature. The slides were again immersed in a TBS bath for 5 minutes, and enough 5-bromo 4-chloro 3-indolyl phosphate/nitroblue tetrazolium substrate solution was placed on the slides to completely cover the tissue. The slides were then incubated for 20 minutes, placed in a water bath for 5 minutes, counterstained by immersion in nuclear fast red for 30 seconds, and rinsed in distilled water for 1 minute. The slides were dehydrated by immersing them in 95%-100% alcohol for 1 minute and in xylene for 1 minute and were then covered with a cover glass.

CLINICAL HISTORY

The deceased patient was a 34-month-old black boy with past medical history of maternally acquired HIV infection, thrombocytopenia, Mycobacterium avium-intracellulare infection, and Pneumocystis carinii pneumonia. Physical examination upon admission showed a small, cachectic, and hypertonic toddler with developmental delays. Neurological examination revealed generalized hypertonicity as well as poor head and trunk control. Lung auscultation disclosed bilateral end-expiratory wheezes and rales. A chest x-ray showed peribronchial thickening. The patient was admitted with a diagnosis of possible pneumonia and was treated with antibiotics and oxygen. Laboratory tests demonstrated leukopenia (3.4 x 10^sup 9^/L), thrombocytopenia (0.071 x 10^sup 12^/L), as well as decreased CD^sub 4^ count and CD^sub 4^/CD^sub 8^ (17 x 10^sup 6^/L and 0.7, respectively). Toxoplasma immunoglobulin M antibody was not detected. Numerous blood, urine, and stool cultures as well as gastric washings were negative for bacteria, mycobacteria, viruses, and fungi.

A computed tomography scan of the brain with and without contrast revealed a 4-cm mass in the left frontal cerebral hemisphere, involving the corpus callosum, periventricular white matter, septum, and basal ganglia, with secondary outlet hydrocephalus. The mass had a central hypodensity, surrounding edema, and areas of calcification in right frontal white matter and corona radiata. The differential diagnosis was between lymphoma and toxoplasmosis, but the former was favored. The boy adopted an opisthotonic posture occasionally, with spastic extremities when not posturing. He became unresponsive to pain. Ophthalmologic examination showed an afferent pupillary defect of the left eye, which was interpreted as secondary to old uveitis or brain lesion. A thallium spectrum study could not be done because of the child's clinical condition. He died 53 days after admission.

AUTOPSY FINDINGS

At autopsy, the patient was a cachectic black child, measuring 77.5 cm from crown to heel, and weighing 7.8 kg (normal for a 20-month-old boy). Internal examination showed a small thymus, generalized lymphadenopathy, as well as pleural, pericardial, and diaphragmatic adhesions. The lungs were heavier than expected and contained several well-circumscribed, round, soft, pale tan, necrotic areas ranging in size from 0.5 to 1.2 cm in diameter. On cut section, it was apparent that the above-mentioned areas were mainly peripherally distributed. The kidneys had few similar pale tan necrotic areas, from 0.2 to 0.5 cm in greatest dimension, preferentially located at the corticomedullary junction.

The weight of the brain was 900 g (normal is 1100 g). There was mild herniation of the left uncus. Coronal sections demonstrated a large, periventricular, partially necrotic mass replacing the left basal ganglia, anterior limb of the internal capsule, and left frontal white matter (Fig 1). The coronal measurements were 4 x 4 cm, and the anteroposterior length was 6 cm. Two other lesions, 0.5 and 1.0 cm, respectively, were in the right parietal centrum semiovale and right medial occipital subependymal white matter, forming well-circumscribed masses with necrosis and surrounded by a thin capsule. The ventricular system was compressed, and there was mild obstructive hydrocephalus of the occipital horns.

Microscopically, all examined lymph nodes were depleted of lymphocytes. In many, the parenchyma was replaced with numerous macrophages with crinkled, basophilic cytoplasm, which were loaded with acid-fast bacilli when stained with Fite-Faraco, as typically seen in Mycobacterium avium-intracellulare infections. The lungs showed abundant peribronchiolar lymphoid aggregates intermixed with areas of necrotizing bronchopneumonia; the lymphoid population was composed of large and small well-differentiated lymphocytes and plasma cells. In situ hybridization for EBV latent phase demonstrated focal positivity in lymphocytes. Immunoperoxidase for B-cell antigen stained the center of the lymphoid aggregates, while T-cell antigen was positive toward the periphery, as seen in germinal centers or reactive lesions. The lungs also disclosed areas of necrotizing vasculitis and bland tissue necrosis with a peculiar bluish hue, characteristic of Pseudomonas or Klebsiella bronchopneumonia.7 Brown-Brenn did not stain the organisms; however, Wright-Giemsa and modified Steiner stains confirmed the presence of rodshaped and filamentous organisms. The same organisms were found in the focally necrotic areas in the kidneys. Postmortem blood cultures grew Pseudomonas aeruginosa and Klebsiella pneumoniae.

The microscopic sections from the left cerebral mass demonstrated an infiltrating B-cell malignant lymphoma, largecell, immunoblastic type. The tumor cells had a tendency to infiltrate perivascular regions (Fig 2). At higher magnification, they were atypical medium and large lymphocytes exhibiting irregular chromatin pattern in the nucleus and increased nucleocytoplasmic ratio (Fig 3). There were also scattered well-differentiated lymphocytes. In situ hybridization for EBV latent phase was positive in tumor cells but was negative in well-differentiated lymphocytes (Fig 4). The two smaller necrotic masses in occipital lobes, one of which was calcified, depicted the same tumor cells. The brain also disclosed calcific vasculopathy in basal ganglia.

COMMENT

The relationship between immunosuppression and malignant lymphomas has been well documented for many years.8 Since early in the AIDS epidemic, an increase in the incidence of PCNSL was detected in the adult AIDS population,9 and this disease was included among the AIDS-indicator conditions by the Centers for Disease Control in 1985.10 For reasons not well understood, the inadence of CNS lymphomas in infants and children with AIDS is much lower. Several large series have reviewed the incidence of pediatric PCNSL.2,11,12 A report from the Centers for Disease Control up to December 31, 1995, stated that primary CNS lymphoma was found in only 27 of the 6948 total reported pediatric AIDS cases, resulting in an incidence of 0.4%. An extensive search of the medical literature revealed a paucity of articles discussing the subject in the pediatric population.

We have listed in the Table the reported pediatric AIDS cases with documented PCNSL. Several cases of pediatric AIDS were not listed in our Table because of the presence of lymphoma in other organs or the lack of exclusion of other organ involvement by autopsy.13-16 A total of five cases of primary brain lymphoma in infants and children with AIDS remained after an extensive search and exclusion of duplications (Table). The age of these patients ranges from 5 to 24 months, with an average age of 13.4 months. All acquired the HIV infection from their mothers.

The first pediatric HIV patient with a PCNSL was reported by Anderson et all7 in 1987. These authors presented a 20-month-old black boy with spasticity, seizures, and multiple enhancing lesions by computed tomography scan of the brain. The autopsy revealed multiple hemorrhagic soft areas in the brain, which were diagnosed microscopically as small, noncleaved, "Burkitt-like" lymphoma. No evidence of systemic involvement was found. Patient number 2 was described by Epstein and coworkers18 and later included in a chapter by DiCarlo and coworkers.ll The other two patients reported in the same article were excluded because no autopsy was performed to rule out the possibility of a primary systemic lymphoma with secondary CNS involvement. Although simultaneous coexistence of two lymphomas can occur, a different origin needs to be demonstrated by either in situ hybridization or polymerase chain reaction to prove they are two different primary tumors.19 This patient presented with the combination of progressive neurologic deficit secondary to multicentric masses in the brain and a B-cell lymphoma. Patient number 3 was extracted from a series of 26 infants and children with AIDS.14 Although these authors described a total of three children with CNS lymphomas, two had coexistent lymphoma elsewhere, and thus were excluded from our Table.

Patients 4 and 5 in the Table were 6 and 14 months at the time of death and had perinatally acquired HIV infection and cerebral non-Hodgkin's lymphoma at postmortem examination.20 The second case had been reported briefly by Cocchi and coworkers21 in a letter to the editor in 1988; however, the report by Del Mistro et al20 is more complete. The same two patients were also reported by Arico and collaborators12 from the Italian Multicenter Study on HIV infection in Children as part of a larger series dealing with malignancies in children with HIV infections. The two patients reported by DelMistro had Southern blot analysis for gene rearrangement, which was positive in one, but none of these children had in situ hybridization studies for latent EBV infection performed in tumor. Three of them had EBV antibodies detected in serum.

A high index of suspicion is required to diagnose a PCNSL while the patient is still alive, since the neurologic signs, available laboratory tests, and radiologic techniques may not be specific. These children may present with spasticity, seizures, developmental delays, and dementia, mimicking HIV-encephalopathy. Sometimes there are focal signs that could also be seen with cerebral toxoplasmosis. In adults, cerebral toxoplasmosis, which has similar clinical and radiologic presentations, is more common than PCNSL.20 However, since cerebral lymphoma is found more commonly than toxoplasmosis in children, a brain biopsy should be performed if a brain mass or enhancing lesion is found by computed tomography scan in a child with AIDS.

As has been reported previously, primary brain lymphomas tend to be multicentric.23 Our patient was no exception. This characteristic limits the therapeutic approach, since whole-brain irradiation in a child can produce long-term side effects, and surgical resection would be much more difficult. The other possibility would be chemotherapy alone, but the rate of success is lower.

Using an in vitro assay system to measure cellular responses to EBV-infected lymphocytes, it has been proven that patients with AIDS and AIDS-related disorders produce an abnormally low number of immunoglobulin-secreting cells, as compared with controls, and that T-cell immunity to EBV was profoundly disturbed.24

The staining pattern of the pulmonary lymphoid infiltrate is that of a reactive process, and we do not believe it to be a lymphoproliferative disease. The focal positivity for EBV as demonstrated by in situ hybridization is intriguing, and one could speculate whether the patient would have developed another EBV-induced lymphoma in the lung had he lived long enough.

The present case is the first to demonstrate the association between EBV and PCNSL by in situ hybridization in children. This virus has been closely associated with lymphomas in immunocompromised adult patients, including those with AIDS.25 In PCNSL of adult AIDS patients, the association between EBV and this neoplasm reaches 100% in several studies.25,26 According to McMahon and coworkers,25 there is a marked difference in the percent of EBV positivity by in situ hybridization between PCNSL in nonimmunocompromised patients (7%) versus patients with AIDS and systemic lymphomas (29%) versus individuals with AIDS and PCNSL (100%). The results obtained by Hamilton-Dutoit et al26 in another large study using paraffin-embedded material for in situ hybridization to detect latent EBV infection in tissues from AIDS patients with cerebral lymphomas, systemic Hodgkin's, and non-Hodgkin's lymphomas were very similar to results reported by the above-mentioned authors.

To study the relationship of EBV to different neoplasms, in situ hybridization offers several advantages over other molecular techniques. Formalin-fixed paraffin-embedded tissue can be used, thereby avoiding the use of frozen HIV-contaminated material. The brain can be cut in the conventional fashion after 2 weeks of fixation and carefully examined for multifocality, since some of the lesions are usually small. Also, archival material can be employed, making possible retrospective studies and use of stored controls. Another advantage is the combination of morphology and molecular analysis, which helps to dis

criminate between true positivity and background staining. For example, in our patient, the EBV positivity was confined mainly to tumor cells, sparing the surrounding non-neoplastic lymphocytes (Fig 4). Moreover, in a study comparing sensitivity and specificity between Southern blot analysis and in situ hybridization technique to detect EBV infection, the two methods were equally adequate, even with paraffin-embedded tissue.27

The outcome of PCNSL in children with AIDS is uniformly fatal, but with the advent of new antiviral therapy to decrease immunosuppression and to treat EBV infections, there are new possibilities for these patients in the not-too-distant future.

The authors thank Lilian Planas from the FISH Laboratory (Department of Pathology, University of Miami) for her technical support with the in situ hybridization for Epstein-Barr virus, as well as Mary Llosa and Blanca Cuenca from the Immunohistochemistry Laboratory (Department of Pathology, University of Miami) for performing the immunoperoxidase stains for B and lymphocytes.

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Accepted for publication June 11, 1997.

From the Department of Pathology, University of Miami (Fla), Jackson Memorial Medical Center.

Reprint requests to Department of Pathology (D-33), University of Miami School of Medicine, Jackson Memorial Hospital, 1611 NW 12 Ave, Miami, FL 33136 (Dr Rodriguez).

Copyright College of American Pathologists Dec 1997
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