1589
21, 1988, and her fever declined gradually. It disappeared completely on April 22,1988, when plasma retinol was 39-2 fig/dl. The patient reported that in previous years she had had long episodes of fever which had been diagnosed as being viral in origin, but it had always occurred concomitantly with retinol intake, usually prescribed for the treatment of respiratory tract infections. The fever has been attributed to retinol on the basis of (1) the temporal relation with retinol intake; (2) the exclusion of other possible causes of fever; and (3) the relation with high plasma
concentrations of retinol. Fever has been observed, but not commonly, in patients with hypervitaminosis A. It is usually part of a more complex clinical picture.1,2 In our patient it was practically the only symptom. Department of Internal Medicine, Hospital de Sant Joan de Déu, Martorell
Department of Clinical Pharmacology,
P. MUNTANER
Ciutat Sanitària Val d’Hebron,
C. RODIGUEZ
Barcelona, Spain
J. M. ARNAU
Severe hypervitaminosis A in siblings. Evidence of variable tolerance to retinol intake. J Pediatr 1987; 111: 507-12. 2. Helsing E. Vitamins. In: Dukes MNG, ed. Meyler’s side effects of drugs, 10th ed. Amsterdam: Elsevier, 1988: 799-811. 1.
Carpenter TO, Pettifor JM, Russell RM, et al.
mutation was further confirmed by dot-blot hybridisation analysis of genomic DNA from this cell culture with allele-specific oligonucleotide probes (figure, lane I). Positive hybridisation with only the mutant probe indicated that this patient was homozygous for the mutation. The same mutation, again in homozygous form, was also detected in cells from two other patients (figure, lanes II and III). In contrast, the mutation was not observed in 20 healthy caucasian and 6 healthy Japanese subjects). Since the three patients are unrelated, this observation suggested a high incidence of the Lys329-to-Glu329 mutation among caucasian patients with MCAD
deficiency. These data prompted us to screen more patients with MCAD deficiency for this mutation with DNA extracted from dried blood spots on Guthrie cards (data not shown). So far six additional patients with MCAD deficiency have been analysed and all have exhibited the same mutation in homozygous form, indicating the remarkably high frequency of this point mutation. Only a few laboratories can offer the confirmatory diagnosis of MCAD deficiency because of the difficulty of the methods.5,6 The dot-blot procedure described above, however, is easy to do in any molecular diagnostic laboratory and could be used to diagnose this disorder.
Tohoku University School of Medicine, 1-1 Seiryomachi, Sendai 980, Japan
YOICHI MATSUBARA KUNIAKI NARISAWA SHIGEAKI MIYABAYASHI KEIYA TADA
Department of Pediatrics. Children’s Hospital of Philadelphia, Philadelphia, USA
PAUL M. COATES
Departments of
Biochemical Genetics
and Paediatrics,
Molecular lesion in patients with mediumchain acyl-CoA dehydrogenase deficiency SIR,-Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is an autosomal recessive disorder that causes sudden infant death and Reye syndrome in children.1 The incidence is not certain, but may be as high as 1 in 10 000 births in caucasian subjects, and thus among the most common recessively inherited metabolic diseases.2 Strauss et aP have reported molecular defects that caused aberrant splicing and partial deletion of MCAD mRNA in a fibroblast culture from a patient with this disorder. However, their data from other patients with MCAD deficiency, as well as those reported by Ikeda et al,4 suggest that this type of mutation is probably not responsible for MCAD deficiency in most patients. We report here a new molecular lesion in the MCAD gene, which may be the most common cause of the disease. We first identified the mutation in fibroblast cultures from three caucasian patients with MCAD deficiency. These cultures were among those that Ikeda et al4 described as having immunoreactive MCAD protein of normal subunit size (MCAD activities ranging from 7 to 12% of the mean control value). The entire coding region of MCAD cDNA was synthesised from mRNA of one culture, amplified by the polymerase chain reaction (PCR), cloned, and sequenced. We identified an A to G nucleotide replacement which resulted in the substitution of lysine (codon AAA) by glutamic acid (codon GAA) at residue 329 of the enzyme. No other nucleotide replacement was seen in the sequenced clones. The presence of the
Detection of the point mutation by dot-blot analysis in PCRamplified DNA from three patients with MCAD deficiency.
(Normal probe. 5’-TGGCAATGAAAGTTGAA-3’; 5’ TTCAACTTCCATTGCCA-3’)
mutant
probe:
1. Editorial. Sudden infant death and inhented disorders of fat oxidation. Lancet 1986; ii: 1073-75. 2. Bennett MJ, Worthy E, Pollitt RJ. The incidence and presentation of dicarboxylic aciduria. J Inherited Metab Dis 1987; 10: 241-42. 3. Strauss AW, Duran M, Zhang Z, Alpers R, Kelly DP. Molecular analysis of medium chain acyl-CoA dehydrogenase deficiency. In: Tanaka K, Coates PM, eds. Fatty acid oxidation: clinical, biochemical, and molecular aspects. New York: Alan R. Liss, 1990: 609-23. 4. Ikeda Y, Hale DE, Keese SM, Coates PM, Tanaka K. Biosynthesis of variant medium chain acyl-CoA dehydrogenase m cultured fibroblasts from patients with medium chain acyl-CoA dehydrogenase deficiency. Pediatr Res 1986; 20: 843-47. 5. Roe CR, Coates PM. Acyl-CoA dehydrogenase deficiencies. In: Scriver CR, Beaudet AL, Sly WS, Valle D, eds. The metabolic basis of inherited disease, 6th ed. New York: McGraw-Hill, 1989: 889-914. 6. Rinaldo P, O’Shea JJ, Coates PM, Hale DE, Stanley CA, Tanaka K Medium-chain
acyl-CoA dehydrogenase deficiency diagnosis by stable-isotope dilution measurement of urinary n-hexanoylglycine and 3-phenylpropionylglycine. N Engl J Med 1988; 319: 1308-13.
Simplified detection of Nci mutation in Gaucher disease SIR,-In Gaucher disease, a sphingolipid storage disease characterised by accumulation of glucocerebroside, one mutation in the glucocerebrosidase gene has been observed in a heteroallelic or homoallelic state in most patients with the neuronopathic types III and III.",2 The substitution of a proline for a leucine creates a new site for the restriction enzyme Nezl (CCTGGCCCGG). The mutation occurs naturally in the glucocerebrosidase pseudogene. The Nezl site is exploited for diagnostic purposes by restriction enzyme digest of genomic DNA and Southern blot analysis.l Alternatively, specific amplification of the active gene and hybridisation with allele-specific oligonucleotides have been described.2,3 A large Swedish group of patients with type III Gaucher disease has been shown to carry the Nci mutation as the only sequence abnormality in the glucocerebrosidase gene.4 We observed that cleavage with Nczl might give false-negative results in heterozygous and homozygous individuals. A possible explanation is different methylation patterns in the recognition sequence. To overcome this the enzyme MspI was used which recognises the mutated but not the "wild type" sequence. Selective amplification of the active gene is followed by MspI cleavage and the fragments generated are then separated and detected on a 1-2% agarose gel (figure). The procedure is easy and quick and excludes the use of radioactivity.
1590
Three-generation family afflicted by Gaucher disease type III segregating for Nci mutation. Mutation traced back on both grandpaternal and grandmaternal side. 598 bp fragment specific for the active glucocerebrosidase gene was amplified under standard conditions using the primers 5’-TTGGGTGCGTAACTTTGTCGACAGT and 5’-TCACCATTGCCCTCACCGGTTTAGC. 20
was cleaved with the restriction enzyme Mspl and 1 2% agarose gel Two fragments were generated from normal allele, 580 and 18 bp Mutated allele generates fragments of 524, 56, and 18 bp.The 56and18 bpfragments have migrated through thegeL Lane 1 =control individual homozygous for the Nci mutation and affected by Gaucher disease Lane 2 and 10= norma controls. The sister (lane 6) of the affected family member is a carrier
separated
III
received five courses of epirubicin (100 mg per m2) every 3 weeks, but no further regression of tumour ensued. On Nov 16, 1989, we started a course of IL-2 (Laboratoires Roussel Uclaf, Romainville, France) by continuous infusion at a dose of 20 x 106 units/m2 daily for five consecutive days, the maximum tolerated dose as determined in a phase-I trial (unpublished). Eighteen days later a striking response was seen on chest radiography, confirmed by computed tomography on Dec 22. Investigation of peripheral lymphocytic subpopulations before and after IL-2 treatment showed an increase in T cells (mainly CD3 positive), mainly in natural killer type cells (NKH-1-CD56 positive), on day eight of IL-2 treatment. The patient received two additional courses of IL-2 and then had surgery for a residual mass which was mainly necrotic on
histopathology.
This encouraging result, of course, needs to be confirmed, but we can safely assume that invasive lymphoepithelial thymoma is another malignant tumour likely to respond to IL-2 given alone. We are investigating whether lymphocytic infiltration plays a part in IL-2 efficacy in such patients.
on a
Service of Medicine B, Institut Gustave-Roussy, 94805 Villejuif Cedex, France
1. 2
The test is being offered to relatives of patients in regions with a high estimated carrier frequency for the mutation (1in 20). No false-negatives have been detected so far. N. DAHL M. LAGERSTROM A. ERIKSON U. PETTERSSON
Department of Medical Genetics, Biomedical Centre, S-751 23 Uppsala, Sweden
Tsuji S, Choudary PV, Martin BM, et al. A mutation m the human glucocerebrosidase gene in neuronopathic Gaucher’s disease. N Engl JMed 1987; 316: 570-75. 2. Theophilus B, Latham T, Grabowsky GA, Smith FI. Gaucher disease: molecular heterogeneity and phenotype-genotype correlations. Am J Hum Genet 1989; 45: 1.
212-25. 3. Firon N, Eyal N, Kolodny EH, Horowitz M. Genotype assignment m Gaucher disease by selective amplification of the active glucocerebrosidase gene. Am JHum Genet 1990; 46: 527-32. 4. Dahl N, Lagerström M, Enkson A, Pettersson U. Gaucher disease type III (Norrbottnian type) is caused by a single mutation in exon 10 of the glucocerebrosidase gene. Am J Hum Genet (in press).
Effectiveness of interleukin-2 in invasive
lymphoepithelial thymoma SIR,-Human recombinant interleukin-2 (IL-2) given alone is effective in only a few solid tumours-namely, malignant melanoma and renal cell carcinoma.1 Since IL-2 is presumed to act by the in-vivo activation of cytotoxic lymphocytes, whether the presence of major lymphocytic infiltration within the tumour could be of interest in the selection of IL-2 sensitive malignancies is worthy of investigation. Thymomas are rare and usually confined to the mediastinum where they grow slowly. Invasive carcinomas of the thymus are a special subset with a poor prognosis, even when treated by surgical resection and radiotherapy. Promising results have been reported with regimens containing cisplatin and/or doxorubicin in patients with advanced disease or distant metastases/,4 but
most
tumours
have
primary
or
acquired
chemoresistance. We report the successful use of IL-2 in a patient with invasive thymoma. A 41-year-old man was referred to our institute in April, 1989, for treatment of a mediasdnal mass. Lymphoepithelial malignant thymoma was confirmed histologically.s The patient was negative for Epstein-Barr virus. Since the tumour had invaded the upper right ribs, pericardium, and diaphragmatic pleura, the patient was given a combination regimen containing cisplatin, vindesin, cyclophosphamide, and prednisone. No tumour response was observed after two cycles of this regimen. The patients then
3.
4. 5
PATRICE BERTHAUD THIERRY LE CHEVALIER THOMAS TURSZ
Rosenberg SA, Lotze MT, Yang JC,
et al. Experience with the use of high-dose interleukin-2 in the treatment of 652 cancer patients. Ann Surg 1989; 210: 474-84. Arriagada R, Bretel JJ, Caillaud JM, et al. Invasive carcinoma of the thymus: a multicentre retrospective review of 56 cases. Eur J Cancer Oncol 1984; 20: 69-74. Dy C, Calvo FA, Mindan JP, et al. Undifferentiated epithelial-rich invasive malignant thymoma: complete response to cisplatin, vinblastme and bleomycin therapy. J Clin Oncol 1988, 6: 536-42. Goldel N, Boning L, Fredrik A, et al. Chemotherapy of invasive thymoma a retrospective study of 22 cases. Cancer 1989, 63: 1493-500. Rosai R, Levine GD. Tumors of the thymus In Atlas of tumor pathology, fascicle 13. Washington, DC. Armed Forces Institute of Pathology, 1976: 34-37.
HIV-1 DNA proviral sequences in fresh urine pellets from HIV-1 seropositive persons SIR,-Proviral DNA sequences of the human immunodeficiency virus type-1 (HIV-1) have been detected by polymerase chain reaction (PCR) done on peripheral blood mononuclear cells (PBMC) from HIV-1 seropositive individuals, including symptom-free homosexual men, patients with AIDS-related complex (ARC), and those with the acquired immunodeficiency syndrome (AIDS).1,2 We have previously shown by ELISA and western blot the presence of specific IgG antibodies to HIV-1 in the urine of HIV-1 seropositive individuals.3 In this study, HIV-1 DNA proviral sequences were sought by use of PCR in centrifuged pellets of fresh urine specimens from 40 HIV-1 seropositive individuals and 6 HIV-1 seronegative homosexual men. Urine pellets from 24 healthy heterosexual men served as controls. Urine pellets containing mononuclear cells from 50 ml of fresh urine were obtained by centrifugation at 1200 g at 4°C and then washed thrice with phosphate-buffered saline to remove salts. Mononuclear cells were separated from blood by Ficoll-Hypaque gradient centrifugation. DNA was extracted from peripheral blood mononuclear cells (PBMC) and urine pellets as previously described.4 PCR was carried out with 1 f.lg of genomic DNA and 10 pmol of each of the primers (SK 38/39) in the buffer containing 10 mmol/1 "thus"HC1 (pH 8-3), 50 mmol/1 KCI,1 ’5mmol/1 MgCl,, 10 mg gelatin, 4 units of Taq polymerase, and 225 lunol/1 of each dNTP. Samples were cycled 30 times between 37°C and 94°C.’ Portions representing one-tenth of the amplified DNA product were denatured and neutralised with a filtration apparatus genescreen. After prehybridisation, the DNA was subjected to hybridisation with 32P end-labelled probe (SK-19) for HIV-1 gag region. The filters were washed and exposed to X-ray film (Kodak). Serum specimens from all the patient groups studied except the healthy controls were tested for p24 antigen and antibodies to HIV-1by ELISA (Abbott Laboratories, North Chicago, Illinois) (table). 31
All urine samples were within normal limits on routine urinalysis. out of 33 (94%) PBMC samples, 29 out of 40 (73%) urine pellets
21, 1988, and her fever declined gradually. It disappeared completely on April 22,1988, when plasma retinol was 39-2 fig/dl. The patient reported that in previous years she had had long episodes of fever which had been diagnosed as being viral in origin, but it had always occurred concomitantly with retinol intake, usually prescribed for the treatment of respiratory tract infections. The fever has been attributed to retinol on the basis of (1) the temporal relation with retinol intake; (2) the exclusion of other possible causes of fever; and (3) the relation with high plasma
concentrations of retinol. Fever has been observed, but not commonly, in patients with hypervitaminosis A. It is usually part of a more complex clinical picture.1,2 In our patient it was practically the only symptom. Department of Internal Medicine, Hospital de Sant Joan de Déu, Martorell
Department of Clinical Pharmacology,
P. MUNTANER
Ciutat Sanitària Val d’Hebron,
C. RODIGUEZ
Barcelona, Spain
J. M. ARNAU
Severe hypervitaminosis A in siblings. Evidence of variable tolerance to retinol intake. J Pediatr 1987; 111: 507-12. 2. Helsing E. Vitamins. In: Dukes MNG, ed. Meyler’s side effects of drugs, 10th ed. Amsterdam: Elsevier, 1988: 799-811. 1.
Carpenter TO, Pettifor JM, Russell RM, et al.
mutation was further confirmed by dot-blot hybridisation analysis of genomic DNA from this cell culture with allele-specific oligonucleotide probes (figure, lane I). Positive hybridisation with only the mutant probe indicated that this patient was homozygous for the mutation. The same mutation, again in homozygous form, was also detected in cells from two other patients (figure, lanes II and III). In contrast, the mutation was not observed in 20 healthy caucasian and 6 healthy Japanese subjects). Since the three patients are unrelated, this observation suggested a high incidence of the Lys329-to-Glu329 mutation among caucasian patients with MCAD
deficiency. These data prompted us to screen more patients with MCAD deficiency for this mutation with DNA extracted from dried blood spots on Guthrie cards (data not shown). So far six additional patients with MCAD deficiency have been analysed and all have exhibited the same mutation in homozygous form, indicating the remarkably high frequency of this point mutation. Only a few laboratories can offer the confirmatory diagnosis of MCAD deficiency because of the difficulty of the methods.5,6 The dot-blot procedure described above, however, is easy to do in any molecular diagnostic laboratory and could be used to diagnose this disorder.
Tohoku University School of Medicine, 1-1 Seiryomachi, Sendai 980, Japan
YOICHI MATSUBARA KUNIAKI NARISAWA SHIGEAKI MIYABAYASHI KEIYA TADA
Department of Pediatrics. Children’s Hospital of Philadelphia, Philadelphia, USA
PAUL M. COATES
Departments of
Biochemical Genetics
and Paediatrics,
Molecular lesion in patients with mediumchain acyl-CoA dehydrogenase deficiency SIR,-Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is an autosomal recessive disorder that causes sudden infant death and Reye syndrome in children.1 The incidence is not certain, but may be as high as 1 in 10 000 births in caucasian subjects, and thus among the most common recessively inherited metabolic diseases.2 Strauss et aP have reported molecular defects that caused aberrant splicing and partial deletion of MCAD mRNA in a fibroblast culture from a patient with this disorder. However, their data from other patients with MCAD deficiency, as well as those reported by Ikeda et al,4 suggest that this type of mutation is probably not responsible for MCAD deficiency in most patients. We report here a new molecular lesion in the MCAD gene, which may be the most common cause of the disease. We first identified the mutation in fibroblast cultures from three caucasian patients with MCAD deficiency. These cultures were among those that Ikeda et al4 described as having immunoreactive MCAD protein of normal subunit size (MCAD activities ranging from 7 to 12% of the mean control value). The entire coding region of MCAD cDNA was synthesised from mRNA of one culture, amplified by the polymerase chain reaction (PCR), cloned, and sequenced. We identified an A to G nucleotide replacement which resulted in the substitution of lysine (codon AAA) by glutamic acid (codon GAA) at residue 329 of the enzyme. No other nucleotide replacement was seen in the sequenced clones. The presence of the
Detection of the point mutation by dot-blot analysis in PCRamplified DNA from three patients with MCAD deficiency.
(Normal probe. 5’-TGGCAATGAAAGTTGAA-3’; 5’ TTCAACTTCCATTGCCA-3’)
mutant
probe:
1. Editorial. Sudden infant death and inhented disorders of fat oxidation. Lancet 1986; ii: 1073-75. 2. Bennett MJ, Worthy E, Pollitt RJ. The incidence and presentation of dicarboxylic aciduria. J Inherited Metab Dis 1987; 10: 241-42. 3. Strauss AW, Duran M, Zhang Z, Alpers R, Kelly DP. Molecular analysis of medium chain acyl-CoA dehydrogenase deficiency. In: Tanaka K, Coates PM, eds. Fatty acid oxidation: clinical, biochemical, and molecular aspects. New York: Alan R. Liss, 1990: 609-23. 4. Ikeda Y, Hale DE, Keese SM, Coates PM, Tanaka K. Biosynthesis of variant medium chain acyl-CoA dehydrogenase m cultured fibroblasts from patients with medium chain acyl-CoA dehydrogenase deficiency. Pediatr Res 1986; 20: 843-47. 5. Roe CR, Coates PM. Acyl-CoA dehydrogenase deficiencies. In: Scriver CR, Beaudet AL, Sly WS, Valle D, eds. The metabolic basis of inherited disease, 6th ed. New York: McGraw-Hill, 1989: 889-914. 6. Rinaldo P, O’Shea JJ, Coates PM, Hale DE, Stanley CA, Tanaka K Medium-chain
acyl-CoA dehydrogenase deficiency diagnosis by stable-isotope dilution measurement of urinary n-hexanoylglycine and 3-phenylpropionylglycine. N Engl J Med 1988; 319: 1308-13.
Simplified detection of Nci mutation in Gaucher disease SIR,-In Gaucher disease, a sphingolipid storage disease characterised by accumulation of glucocerebroside, one mutation in the glucocerebrosidase gene has been observed in a heteroallelic or homoallelic state in most patients with the neuronopathic types III and III.",2 The substitution of a proline for a leucine creates a new site for the restriction enzyme Nezl (CCTGGCCCGG). The mutation occurs naturally in the glucocerebrosidase pseudogene. The Nezl site is exploited for diagnostic purposes by restriction enzyme digest of genomic DNA and Southern blot analysis.l Alternatively, specific amplification of the active gene and hybridisation with allele-specific oligonucleotides have been described.2,3 A large Swedish group of patients with type III Gaucher disease has been shown to carry the Nci mutation as the only sequence abnormality in the glucocerebrosidase gene.4 We observed that cleavage with Nczl might give false-negative results in heterozygous and homozygous individuals. A possible explanation is different methylation patterns in the recognition sequence. To overcome this the enzyme MspI was used which recognises the mutated but not the "wild type" sequence. Selective amplification of the active gene is followed by MspI cleavage and the fragments generated are then separated and detected on a 1-2% agarose gel (figure). The procedure is easy and quick and excludes the use of radioactivity.
1590
Three-generation family afflicted by Gaucher disease type III segregating for Nci mutation. Mutation traced back on both grandpaternal and grandmaternal side. 598 bp fragment specific for the active glucocerebrosidase gene was amplified under standard conditions using the primers 5’-TTGGGTGCGTAACTTTGTCGACAGT and 5’-TCACCATTGCCCTCACCGGTTTAGC. 20
was cleaved with the restriction enzyme Mspl and 1 2% agarose gel Two fragments were generated from normal allele, 580 and 18 bp Mutated allele generates fragments of 524, 56, and 18 bp.The 56and18 bpfragments have migrated through thegeL Lane 1 =control individual homozygous for the Nci mutation and affected by Gaucher disease Lane 2 and 10= norma controls. The sister (lane 6) of the affected family member is a carrier
separated
III
received five courses of epirubicin (100 mg per m2) every 3 weeks, but no further regression of tumour ensued. On Nov 16, 1989, we started a course of IL-2 (Laboratoires Roussel Uclaf, Romainville, France) by continuous infusion at a dose of 20 x 106 units/m2 daily for five consecutive days, the maximum tolerated dose as determined in a phase-I trial (unpublished). Eighteen days later a striking response was seen on chest radiography, confirmed by computed tomography on Dec 22. Investigation of peripheral lymphocytic subpopulations before and after IL-2 treatment showed an increase in T cells (mainly CD3 positive), mainly in natural killer type cells (NKH-1-CD56 positive), on day eight of IL-2 treatment. The patient received two additional courses of IL-2 and then had surgery for a residual mass which was mainly necrotic on
histopathology.
This encouraging result, of course, needs to be confirmed, but we can safely assume that invasive lymphoepithelial thymoma is another malignant tumour likely to respond to IL-2 given alone. We are investigating whether lymphocytic infiltration plays a part in IL-2 efficacy in such patients.
on a
Service of Medicine B, Institut Gustave-Roussy, 94805 Villejuif Cedex, France
1. 2
The test is being offered to relatives of patients in regions with a high estimated carrier frequency for the mutation (1in 20). No false-negatives have been detected so far. N. DAHL M. LAGERSTROM A. ERIKSON U. PETTERSSON
Department of Medical Genetics, Biomedical Centre, S-751 23 Uppsala, Sweden
Tsuji S, Choudary PV, Martin BM, et al. A mutation m the human glucocerebrosidase gene in neuronopathic Gaucher’s disease. N Engl JMed 1987; 316: 570-75. 2. Theophilus B, Latham T, Grabowsky GA, Smith FI. Gaucher disease: molecular heterogeneity and phenotype-genotype correlations. Am J Hum Genet 1989; 45: 1.
212-25. 3. Firon N, Eyal N, Kolodny EH, Horowitz M. Genotype assignment m Gaucher disease by selective amplification of the active glucocerebrosidase gene. Am JHum Genet 1990; 46: 527-32. 4. Dahl N, Lagerström M, Enkson A, Pettersson U. Gaucher disease type III (Norrbottnian type) is caused by a single mutation in exon 10 of the glucocerebrosidase gene. Am J Hum Genet (in press).
Effectiveness of interleukin-2 in invasive
lymphoepithelial thymoma SIR,-Human recombinant interleukin-2 (IL-2) given alone is effective in only a few solid tumours-namely, malignant melanoma and renal cell carcinoma.1 Since IL-2 is presumed to act by the in-vivo activation of cytotoxic lymphocytes, whether the presence of major lymphocytic infiltration within the tumour could be of interest in the selection of IL-2 sensitive malignancies is worthy of investigation. Thymomas are rare and usually confined to the mediastinum where they grow slowly. Invasive carcinomas of the thymus are a special subset with a poor prognosis, even when treated by surgical resection and radiotherapy. Promising results have been reported with regimens containing cisplatin and/or doxorubicin in patients with advanced disease or distant metastases/,4 but
most
tumours
have
primary
or
acquired
chemoresistance. We report the successful use of IL-2 in a patient with invasive thymoma. A 41-year-old man was referred to our institute in April, 1989, for treatment of a mediasdnal mass. Lymphoepithelial malignant thymoma was confirmed histologically.s The patient was negative for Epstein-Barr virus. Since the tumour had invaded the upper right ribs, pericardium, and diaphragmatic pleura, the patient was given a combination regimen containing cisplatin, vindesin, cyclophosphamide, and prednisone. No tumour response was observed after two cycles of this regimen. The patients then
3.
4. 5
PATRICE BERTHAUD THIERRY LE CHEVALIER THOMAS TURSZ
Rosenberg SA, Lotze MT, Yang JC,
et al. Experience with the use of high-dose interleukin-2 in the treatment of 652 cancer patients. Ann Surg 1989; 210: 474-84. Arriagada R, Bretel JJ, Caillaud JM, et al. Invasive carcinoma of the thymus: a multicentre retrospective review of 56 cases. Eur J Cancer Oncol 1984; 20: 69-74. Dy C, Calvo FA, Mindan JP, et al. Undifferentiated epithelial-rich invasive malignant thymoma: complete response to cisplatin, vinblastme and bleomycin therapy. J Clin Oncol 1988, 6: 536-42. Goldel N, Boning L, Fredrik A, et al. Chemotherapy of invasive thymoma a retrospective study of 22 cases. Cancer 1989, 63: 1493-500. Rosai R, Levine GD. Tumors of the thymus In Atlas of tumor pathology, fascicle 13. Washington, DC. Armed Forces Institute of Pathology, 1976: 34-37.
HIV-1 DNA proviral sequences in fresh urine pellets from HIV-1 seropositive persons SIR,-Proviral DNA sequences of the human immunodeficiency virus type-1 (HIV-1) have been detected by polymerase chain reaction (PCR) done on peripheral blood mononuclear cells (PBMC) from HIV-1 seropositive individuals, including symptom-free homosexual men, patients with AIDS-related complex (ARC), and those with the acquired immunodeficiency syndrome (AIDS).1,2 We have previously shown by ELISA and western blot the presence of specific IgG antibodies to HIV-1 in the urine of HIV-1 seropositive individuals.3 In this study, HIV-1 DNA proviral sequences were sought by use of PCR in centrifuged pellets of fresh urine specimens from 40 HIV-1 seropositive individuals and 6 HIV-1 seronegative homosexual men. Urine pellets from 24 healthy heterosexual men served as controls. Urine pellets containing mononuclear cells from 50 ml of fresh urine were obtained by centrifugation at 1200 g at 4°C and then washed thrice with phosphate-buffered saline to remove salts. Mononuclear cells were separated from blood by Ficoll-Hypaque gradient centrifugation. DNA was extracted from peripheral blood mononuclear cells (PBMC) and urine pellets as previously described.4 PCR was carried out with 1 f.lg of genomic DNA and 10 pmol of each of the primers (SK 38/39) in the buffer containing 10 mmol/1 "thus"HC1 (pH 8-3), 50 mmol/1 KCI,1 ’5mmol/1 MgCl,, 10 mg gelatin, 4 units of Taq polymerase, and 225 lunol/1 of each dNTP. Samples were cycled 30 times between 37°C and 94°C.’ Portions representing one-tenth of the amplified DNA product were denatured and neutralised with a filtration apparatus genescreen. After prehybridisation, the DNA was subjected to hybridisation with 32P end-labelled probe (SK-19) for HIV-1 gag region. The filters were washed and exposed to X-ray film (Kodak). Serum specimens from all the patient groups studied except the healthy controls were tested for p24 antigen and antibodies to HIV-1by ELISA (Abbott Laboratories, North Chicago, Illinois) (table). 31
All urine samples were within normal limits on routine urinalysis. out of 33 (94%) PBMC samples, 29 out of 40 (73%) urine pellets
