- Email: [email protected]
Multiplex PCR analysis of RhD gene
devoid of anti-HCV is unknown. It is hoped that it will remain unknown because such products are not being distributed in the USA. *M W Yu, J S
Finlayson,
D L
Tankersley
Office of Blood Research and Review, Center for Biologics Evaluation and Research, Food and Drug Administration, Rockville, MD 20852, USA
Outbreak of hepatitis C associated with intravenous immunoglobulin administration-United States, October 1993-June 1994. MMWR
1
2
1994; 43: 505-09. Schiano TD, Bellary SV, Black M. Possible transmission of hepatitis C virus infection with intravenous immunoglobulin. Ann Intern Med 1995; 122: 802-03.
Clinical assessment of deep vein thrombosis SiR-In their report of the diagnosis of deep vein thrombosis (DVT) Wells and colleagues (May 27, p 1326) omit any discussion of doppler ultrasound. This is now widely used for the non-invasive diagnosis of DVT in both duplex and colour modes. Indeed in a meta-analysis of studies in symptom-free postoperative patients, essentially the same group acknowledged that the addition of doppler ultrasound offered theoretical advantages over the use of B-mode ultrasound alone.’ They showed that the use of duplex scanning increased the sensitivity of ultrasound from a mean of 61% to 79% in controlled bias studies, although the difference was not significant. Furthermore, although the results of colour doppler scanning were disappointing for proximal thromboses, Wells et al reviewed only three studies.’ Other workers have had very encouraging results with colour imaging.2,3 The exclusion of doppler imaging from the algorithm proposed by Wells and colleagues is of more than academic interest. An important suggestion from their report is that negative ultrasound scans should be repeated after a week, if there is a moderate pretest probability. Although this may be necessary if ultrasound is used to evaluate deep venous compressibility at only two points, as done by Wells, it is highly debatable if the combination of B-mode, duplex, and colour scanning has been used, especially when the entire deep venous system from the groin to at least the lower popliteal fossa has been satisfactorily imaged. We therefore feel the algorithm proposed by Wells and colleagues is of questionable clinical value in an institution where such fuller ultrasounds are done, especially in view of the cost implications. Unfortunately, the ultrasound technique used is mentioned only in the methods section; a casual reader concentrating on the abstract or algorithmic chart could be misled. Indeed, we have already had clinicians requesting serial doppler scans on the basis of this report without appreciating the difference between what are essentially two extremes of radiological practice-ie, a full study with colour doppler on the one hand and an inadequate examination by compression at only two sites on the other. *Martin
Blomley, David Cosgrove
Department of Diagnostic Radiology, Hammersmith Hospital and Royal Postgraduate Medical School, London W12 ONN, UK
1
2 3
Wells PS, Lensing AWA, Davidson BL, Prins MH, Hirsh J. Accuracy of ultrasound for the diagnosis of deep venous thrombosis in asymptomatic patients after orthopaedic surgery: a meta-analysis. Ann Intern Med 1995; 122: 47-53. Baxter GM, Duffy P, Partridge E. Colour flow imaging of calf vein thrombosis. Clin Radiol 1992; 46: 198-201. Bradley MJ, Spencer PA, Alexander L, Milner GR. Colour flow mapping in the diagnosis of the calf vein thrombosis. Clin Radiol 1993; 47: 399-402.
Multiplex
PCR
analysis of RhD gene
SIR—Determination of fetal rhesus D (RhD) type is of particular importance if there is substantial risk of disease from high levels of maternal anti-D and the partner is either unavailable or known to be heterozygous for the D gene. With the cloning of the RhD gene, it is now possible to use different sets of oligonucleotide primer pairs for PCR-based RhD typing.’,’ Bennett et al used primers A3 and A4 to amplify a region of DNA located in exon 10 of the RhD gene.’ Although A3 is common to the RhD and RhCE genes, A4 is specific for the RhD gene, and amplification yields a product of 186 basepairs (bp) from DNA samples obtained from RhD-positive subjects only. There is some doubt, however, about the reliability of results obtained with these primers since certain samples from RhD-negative donors, particularly r’r’ (Cde/Cde) subjects, have been found to contain fragments of the RhD gene including exon 10. In these donors a 186 bp PCR product could also be obtained.3 In addition, some RhD-positive donors have been found to contain rearranged genes and in some cases3 these rearrangements involve exon 10 giving negative results when Bennett and colleagues’ primers are used. Alternative primers (Rh 607 and Rh 768) have therefore been proposed by Simsek et al.2 These primers amplify a region of DNA between exons 4 and 5 of the RhD gene. The PCR product with these primers from RhD-positive samples is only 600 bp compared with a 1200 bp product obtained from RhDnegative samples. The disadvantage in using these primers is that discrepancies may occur, particularly since some RhD variants such as category VI have been shown to have rearrangements between exons 4 and 6.’ Therefore, it has been suggested that for safe and reliable RhD typing of the fetus at least two independent primer sets should be used.2
Figure: Amplification of donor control DNA and fetal DNA from amniocytes DNA samples (0-1 ug) were amplified in 25 pL reaction mixtures containing 2.51JL of 10 x Taq buffer, with 15 mmol/L MgCI2. 4 pL dNTP mixture (1-25 mmol/L), 1-5 uL each of primers Al and A2 (0-02 mg/mL), 3 uL each of pnmers A3, A4, Rh607, Rh768 (0-02 mg/mL),
and 0.16 uL of Taq polymerase (5 units/uL). Reactions were run under following conditions on a PTC-100 thermal cycler (M J Research Inc): 30 cycles of 92°C for 1 min, 49°C for 1 min, and 72°C for 1 min, followed by final extension step of 72°C for 9 mm. Lane l=posrtive control (RhD-posltive donor DNA) showing bands at 600 bp and 186 bp that correspond to amplification with Rh607 and Rh768 and A3 and A4 primers, respectively. The 136 bp band is product of amplification with pnmers Al and A2. These primers were used as internal control because they amplify region of DNA common to RhCE and RhD genes. Lane 2=negatlve control (RhD-negatlve donor DNA) showing control 136 bp band and no 186 bp or 600 bp bands. Lane 3=fetal DNA isolated from amniocytes. Lane 4=H20 control. Lane 5=DNA molecular weights control ladder. 1200 bp band should be present m all samples because It is product of amplification of DNA from RhCE gene with Rh607 and Rh768 primers.
However, since this band is not as clearly visible as 136 bp control band, we included Al and A2 primers in all our tests as internal control primers.
375
We have developed a multiplex PCR analysis, with the three sets of primers described by Bennett et al and Simsek et al which amplify DNA from different exons. The use of these three sets of primers, one of which amplifies DNA from exon 7 of the RhCE and RhD genes, provides an internal control and prevents discrepancies. With this approach we have RhD typed 22 amniotic fluid samples, including a fresh sample obtained at week 29 from an RhDnegative pregnant woman (figure). The subsequent serology the fetal blood samples from this patient’s fetus on confirmed these multiplex PCR results. We have also typed 20 DNA samples obtained from normal donors all of which gave results that were confirmed by serology. In addition, several rare Rh group samples have been typed with this method. These include, six Rvi r (CDvle/cde) and one R2VI r (cDvIE/cde) donor samples which were tested and gave positive results with the exon 10 primers but gave negative results with the exon 4-5 primers. These results may be due to a deletion or rearrangement between exons 4 and 6. Three r’r (Cde/cde) donor samples were also tested, two of which gave negative results with both sets of primers. The other, however, gave a positive result with the exon 10 primers, suggesting that in this case, a partial RhD gene may be present as previously shown with some r’r’ samples.5 Other samples tested, which gave negative results with both primer sets included one r"r" (cdE/cdE) and four r’r’ (Cde/Cde) samples. Positive results with both primer sets were also obtained from three Ro(cDe) and three Rour (cDue/cde) donor samples as expected.
*Jenny Pope, Cristina Navarrete, Ruth Warwick, Marcela Contreras *Department of Histocompatibility and Immunogenetics, North London Blood Transfusion Centre, Colindale Avenue, London NW9, UK 1 2 3
4 5
Bennett PR, Le Van Kim C, Colin Y, et al. Prenatal diagnosis of fetal RhD type by DNA amplification. N Engl J Med 1993; 329: 607-10. Simsek S, Bleeker PMM, von dem Borne AEG Kr. Prenatal determination of fetal RhD type. N Engl J Med 1994; 330: 795-96. Blunt T, Daniels G, Carritt B. Serotype switching in a partially detected RHD gene. Vox Sang 1994; 67: 397-401. Carritt B, Steers FJ, Avent ND. Prenatal determination of fetal RhD type. Lancet 1994; 344: 205-06. Mouro I, Le Van Kim C, Rouillac C, et al. Rearrangement of the blood group RhD gene associated with DV1 category phenotype. Blood 1994; 83: 1129-35.
Treatment of cutaneous T-cell retinoids and calcitriol
lymphoma by
SIR—Thomsen (June 17, p 1-583) reports no effect or even of disease during treatment of three patients with advanced cutaneous T-cell lymphoma by isotretinoin 40 mg daily and calcitriol 1 mg daily. Thomsen misinterpreted our report in two important points: one is the drugs used and the other is the type of cutaneous lymphoma treated. Acitretin, which we used, and isotretinoin, which Thomsen administered, are two retinoids that do not share a similar therapeutic profile; experts in the field of retinoids now carefully consider such potential differences within this class of drugs. The reasons for these differences are complex and include distinct nuclear receptor binding of the drug and its metabolites.’ Our patient, who responded to acitretin 10 mg per day and calcitriol 0-5 mg every other day,2 had pleomorphic small T-cell lymphoma, and she is still in complete remission 18 months after initiation of the combined treatment. The three patients reported by Thomsen had mycosis fungoides at a very advanced stage. The two diseases are not comparable, especially when evaluating
progression
376
differentiation
effect(s)
may
therapy with hormone-like compounds whose depend on phenotypic characteristics of the
lymphoma cells. In fact, we have treated seven additional patients with cutaneous T-cell lymphoma: six had mycosis fungoides and one pleomorphic small T-cell lymphoma. Of the six patients with mycosis fungoides there was no response in three, partial response in two, and progression of the disease in the only one with advanced disease. The patient with pleomorphic small T-cell lymphoma had a clear partial remission after two months of treatment, characterised by a decrease in the total number of skin lesions. We think that our observations warrant controlled studies that take into account the phenotype of the cutaneous lymphoma, its stage, and probably also the expression of nuclear receptors for retinoids and vitamin D in the malignant cells. *L E French, J-H Saurat Department of Dermatology, Geneva University Medical School, 1211 Geneva, Switzerland
1
2
Armstrong RB, Ashenfelter KO, Eckhoff C, Levin AA, Shapiro SS. General and reproductive toxicology of retinoids. In: Sporn MB, Roberts AB, Goodman DS, eds. The retinoids: biology, chemistry, and medicine, 2nd ed. New York: Raven Press, 1994: 545-72. French LE, Ramelet AA, Saurat J-H. Remission of cutaneous T-cell lymphoma with combined calcitriol and acitretin. Lancet 1994; 344: 686-87.
SIR—The report of remission of cutaneous T-cell lymphoma with combined calcitriol and acitretin provided an interesting new treatment modality in patients with lowgrade cutaneous T-cell lymphoma.’ However, Thomsen reports that three patients with mycosis fungoides treated with isotretinoin and calcitriol had a progression of their lesions. There are two differences between these controversial reports. First, the three patients reported by Thomsen had advanced-stage mycosis fungoides, whereas the initial patient had a cutaneous pleomorphic small T-cell lymphoma, presenting with multiple disseminated nodules and a non-epidermotropic granulomatous infiltrate. Second, Thomsen’s patients were treated with isotretinoin, whereas French and colleagues’ patient was given acitretin. We used the protocol initially reported, combining acitretin and calcitriol, in a patient with a cutaneous pleomorphic small T-cell lymphoma. A 70-year-old man had had a cutaneous pleomorphic small T-cell lymphoma for 3 years. He presented with multiple disseminated erythematous infiltrated nodules and tumours. Histology of a skin biopsy specimen showed a nonepidermotropic granulomatous infiltrate composed of small T-helper lymphocytes. PCR revealed a T-cell clone in the skin but not in the blood. There was no extracutaneous disease. Bone marrow biopsy was negative. Thoracoabdominal computed tomography scan was normal. The patient had been successively treated with local radiotherapy, cyclophosphamide, a-interferon, and miniCHOP and interferon. These treatments induced a partial or complete response but cutaneous lesions reappeared when the treatment was tapered. Combined systemic treatment with calcitriol (0-5 µg every other day) and acitretin (10 mg per day) was started. After 1 month, calcitriol was increased to 0-5 µg every day and acitretin to 20 mg per day. 2 months after beginning combined therapy, the patient presented with numerous cutaneous tumours, and with a rapidly
growing enlarged axillary lymph node, and treatment was withdrawn. Biopsy specimens of skin and lymph nodes showed a pleomorphic small T-cell lymphoma. In our patient, new cutaneous lesions and an extracutaneous lesion appeared and rapidly increased during treatment with retinoids and calcitriol. Therefore, this
Finlayson,
D L
Tankersley
Office of Blood Research and Review, Center for Biologics Evaluation and Research, Food and Drug Administration, Rockville, MD 20852, USA
Outbreak of hepatitis C associated with intravenous immunoglobulin administration-United States, October 1993-June 1994. MMWR
1
2
1994; 43: 505-09. Schiano TD, Bellary SV, Black M. Possible transmission of hepatitis C virus infection with intravenous immunoglobulin. Ann Intern Med 1995; 122: 802-03.
Clinical assessment of deep vein thrombosis SiR-In their report of the diagnosis of deep vein thrombosis (DVT) Wells and colleagues (May 27, p 1326) omit any discussion of doppler ultrasound. This is now widely used for the non-invasive diagnosis of DVT in both duplex and colour modes. Indeed in a meta-analysis of studies in symptom-free postoperative patients, essentially the same group acknowledged that the addition of doppler ultrasound offered theoretical advantages over the use of B-mode ultrasound alone.’ They showed that the use of duplex scanning increased the sensitivity of ultrasound from a mean of 61% to 79% in controlled bias studies, although the difference was not significant. Furthermore, although the results of colour doppler scanning were disappointing for proximal thromboses, Wells et al reviewed only three studies.’ Other workers have had very encouraging results with colour imaging.2,3 The exclusion of doppler imaging from the algorithm proposed by Wells and colleagues is of more than academic interest. An important suggestion from their report is that negative ultrasound scans should be repeated after a week, if there is a moderate pretest probability. Although this may be necessary if ultrasound is used to evaluate deep venous compressibility at only two points, as done by Wells, it is highly debatable if the combination of B-mode, duplex, and colour scanning has been used, especially when the entire deep venous system from the groin to at least the lower popliteal fossa has been satisfactorily imaged. We therefore feel the algorithm proposed by Wells and colleagues is of questionable clinical value in an institution where such fuller ultrasounds are done, especially in view of the cost implications. Unfortunately, the ultrasound technique used is mentioned only in the methods section; a casual reader concentrating on the abstract or algorithmic chart could be misled. Indeed, we have already had clinicians requesting serial doppler scans on the basis of this report without appreciating the difference between what are essentially two extremes of radiological practice-ie, a full study with colour doppler on the one hand and an inadequate examination by compression at only two sites on the other. *Martin
Blomley, David Cosgrove
Department of Diagnostic Radiology, Hammersmith Hospital and Royal Postgraduate Medical School, London W12 ONN, UK
1
2 3
Wells PS, Lensing AWA, Davidson BL, Prins MH, Hirsh J. Accuracy of ultrasound for the diagnosis of deep venous thrombosis in asymptomatic patients after orthopaedic surgery: a meta-analysis. Ann Intern Med 1995; 122: 47-53. Baxter GM, Duffy P, Partridge E. Colour flow imaging of calf vein thrombosis. Clin Radiol 1992; 46: 198-201. Bradley MJ, Spencer PA, Alexander L, Milner GR. Colour flow mapping in the diagnosis of the calf vein thrombosis. Clin Radiol 1993; 47: 399-402.
Multiplex
PCR
analysis of RhD gene
SIR—Determination of fetal rhesus D (RhD) type is of particular importance if there is substantial risk of disease from high levels of maternal anti-D and the partner is either unavailable or known to be heterozygous for the D gene. With the cloning of the RhD gene, it is now possible to use different sets of oligonucleotide primer pairs for PCR-based RhD typing.’,’ Bennett et al used primers A3 and A4 to amplify a region of DNA located in exon 10 of the RhD gene.’ Although A3 is common to the RhD and RhCE genes, A4 is specific for the RhD gene, and amplification yields a product of 186 basepairs (bp) from DNA samples obtained from RhD-positive subjects only. There is some doubt, however, about the reliability of results obtained with these primers since certain samples from RhD-negative donors, particularly r’r’ (Cde/Cde) subjects, have been found to contain fragments of the RhD gene including exon 10. In these donors a 186 bp PCR product could also be obtained.3 In addition, some RhD-positive donors have been found to contain rearranged genes and in some cases3 these rearrangements involve exon 10 giving negative results when Bennett and colleagues’ primers are used. Alternative primers (Rh 607 and Rh 768) have therefore been proposed by Simsek et al.2 These primers amplify a region of DNA between exons 4 and 5 of the RhD gene. The PCR product with these primers from RhD-positive samples is only 600 bp compared with a 1200 bp product obtained from RhDnegative samples. The disadvantage in using these primers is that discrepancies may occur, particularly since some RhD variants such as category VI have been shown to have rearrangements between exons 4 and 6.’ Therefore, it has been suggested that for safe and reliable RhD typing of the fetus at least two independent primer sets should be used.2
Figure: Amplification of donor control DNA and fetal DNA from amniocytes DNA samples (0-1 ug) were amplified in 25 pL reaction mixtures containing 2.51JL of 10 x Taq buffer, with 15 mmol/L MgCI2. 4 pL dNTP mixture (1-25 mmol/L), 1-5 uL each of primers Al and A2 (0-02 mg/mL), 3 uL each of pnmers A3, A4, Rh607, Rh768 (0-02 mg/mL),
and 0.16 uL of Taq polymerase (5 units/uL). Reactions were run under following conditions on a PTC-100 thermal cycler (M J Research Inc): 30 cycles of 92°C for 1 min, 49°C for 1 min, and 72°C for 1 min, followed by final extension step of 72°C for 9 mm. Lane l=posrtive control (RhD-posltive donor DNA) showing bands at 600 bp and 186 bp that correspond to amplification with Rh607 and Rh768 and A3 and A4 primers, respectively. The 136 bp band is product of amplification with pnmers Al and A2. These primers were used as internal control because they amplify region of DNA common to RhCE and RhD genes. Lane 2=negatlve control (RhD-negatlve donor DNA) showing control 136 bp band and no 186 bp or 600 bp bands. Lane 3=fetal DNA isolated from amniocytes. Lane 4=H20 control. Lane 5=DNA molecular weights control ladder. 1200 bp band should be present m all samples because It is product of amplification of DNA from RhCE gene with Rh607 and Rh768 primers.
However, since this band is not as clearly visible as 136 bp control band, we included Al and A2 primers in all our tests as internal control primers.
375
We have developed a multiplex PCR analysis, with the three sets of primers described by Bennett et al and Simsek et al which amplify DNA from different exons. The use of these three sets of primers, one of which amplifies DNA from exon 7 of the RhCE and RhD genes, provides an internal control and prevents discrepancies. With this approach we have RhD typed 22 amniotic fluid samples, including a fresh sample obtained at week 29 from an RhDnegative pregnant woman (figure). The subsequent serology the fetal blood samples from this patient’s fetus on confirmed these multiplex PCR results. We have also typed 20 DNA samples obtained from normal donors all of which gave results that were confirmed by serology. In addition, several rare Rh group samples have been typed with this method. These include, six Rvi r (CDvle/cde) and one R2VI r (cDvIE/cde) donor samples which were tested and gave positive results with the exon 10 primers but gave negative results with the exon 4-5 primers. These results may be due to a deletion or rearrangement between exons 4 and 6. Three r’r (Cde/cde) donor samples were also tested, two of which gave negative results with both sets of primers. The other, however, gave a positive result with the exon 10 primers, suggesting that in this case, a partial RhD gene may be present as previously shown with some r’r’ samples.5 Other samples tested, which gave negative results with both primer sets included one r"r" (cdE/cdE) and four r’r’ (Cde/Cde) samples. Positive results with both primer sets were also obtained from three Ro(cDe) and three Rour (cDue/cde) donor samples as expected.
*Jenny Pope, Cristina Navarrete, Ruth Warwick, Marcela Contreras *Department of Histocompatibility and Immunogenetics, North London Blood Transfusion Centre, Colindale Avenue, London NW9, UK 1 2 3
4 5
Bennett PR, Le Van Kim C, Colin Y, et al. Prenatal diagnosis of fetal RhD type by DNA amplification. N Engl J Med 1993; 329: 607-10. Simsek S, Bleeker PMM, von dem Borne AEG Kr. Prenatal determination of fetal RhD type. N Engl J Med 1994; 330: 795-96. Blunt T, Daniels G, Carritt B. Serotype switching in a partially detected RHD gene. Vox Sang 1994; 67: 397-401. Carritt B, Steers FJ, Avent ND. Prenatal determination of fetal RhD type. Lancet 1994; 344: 205-06. Mouro I, Le Van Kim C, Rouillac C, et al. Rearrangement of the blood group RhD gene associated with DV1 category phenotype. Blood 1994; 83: 1129-35.
Treatment of cutaneous T-cell retinoids and calcitriol
lymphoma by
SIR—Thomsen (June 17, p 1-583) reports no effect or even of disease during treatment of three patients with advanced cutaneous T-cell lymphoma by isotretinoin 40 mg daily and calcitriol 1 mg daily. Thomsen misinterpreted our report in two important points: one is the drugs used and the other is the type of cutaneous lymphoma treated. Acitretin, which we used, and isotretinoin, which Thomsen administered, are two retinoids that do not share a similar therapeutic profile; experts in the field of retinoids now carefully consider such potential differences within this class of drugs. The reasons for these differences are complex and include distinct nuclear receptor binding of the drug and its metabolites.’ Our patient, who responded to acitretin 10 mg per day and calcitriol 0-5 mg every other day,2 had pleomorphic small T-cell lymphoma, and she is still in complete remission 18 months after initiation of the combined treatment. The three patients reported by Thomsen had mycosis fungoides at a very advanced stage. The two diseases are not comparable, especially when evaluating
progression
376
differentiation
effect(s)
may
therapy with hormone-like compounds whose depend on phenotypic characteristics of the
lymphoma cells. In fact, we have treated seven additional patients with cutaneous T-cell lymphoma: six had mycosis fungoides and one pleomorphic small T-cell lymphoma. Of the six patients with mycosis fungoides there was no response in three, partial response in two, and progression of the disease in the only one with advanced disease. The patient with pleomorphic small T-cell lymphoma had a clear partial remission after two months of treatment, characterised by a decrease in the total number of skin lesions. We think that our observations warrant controlled studies that take into account the phenotype of the cutaneous lymphoma, its stage, and probably also the expression of nuclear receptors for retinoids and vitamin D in the malignant cells. *L E French, J-H Saurat Department of Dermatology, Geneva University Medical School, 1211 Geneva, Switzerland
1
2
Armstrong RB, Ashenfelter KO, Eckhoff C, Levin AA, Shapiro SS. General and reproductive toxicology of retinoids. In: Sporn MB, Roberts AB, Goodman DS, eds. The retinoids: biology, chemistry, and medicine, 2nd ed. New York: Raven Press, 1994: 545-72. French LE, Ramelet AA, Saurat J-H. Remission of cutaneous T-cell lymphoma with combined calcitriol and acitretin. Lancet 1994; 344: 686-87.
SIR—The report of remission of cutaneous T-cell lymphoma with combined calcitriol and acitretin provided an interesting new treatment modality in patients with lowgrade cutaneous T-cell lymphoma.’ However, Thomsen reports that three patients with mycosis fungoides treated with isotretinoin and calcitriol had a progression of their lesions. There are two differences between these controversial reports. First, the three patients reported by Thomsen had advanced-stage mycosis fungoides, whereas the initial patient had a cutaneous pleomorphic small T-cell lymphoma, presenting with multiple disseminated nodules and a non-epidermotropic granulomatous infiltrate. Second, Thomsen’s patients were treated with isotretinoin, whereas French and colleagues’ patient was given acitretin. We used the protocol initially reported, combining acitretin and calcitriol, in a patient with a cutaneous pleomorphic small T-cell lymphoma. A 70-year-old man had had a cutaneous pleomorphic small T-cell lymphoma for 3 years. He presented with multiple disseminated erythematous infiltrated nodules and tumours. Histology of a skin biopsy specimen showed a nonepidermotropic granulomatous infiltrate composed of small T-helper lymphocytes. PCR revealed a T-cell clone in the skin but not in the blood. There was no extracutaneous disease. Bone marrow biopsy was negative. Thoracoabdominal computed tomography scan was normal. The patient had been successively treated with local radiotherapy, cyclophosphamide, a-interferon, and miniCHOP and interferon. These treatments induced a partial or complete response but cutaneous lesions reappeared when the treatment was tapered. Combined systemic treatment with calcitriol (0-5 µg every other day) and acitretin (10 mg per day) was started. After 1 month, calcitriol was increased to 0-5 µg every day and acitretin to 20 mg per day. 2 months after beginning combined therapy, the patient presented with numerous cutaneous tumours, and with a rapidly
growing enlarged axillary lymph node, and treatment was withdrawn. Biopsy specimens of skin and lymph nodes showed a pleomorphic small T-cell lymphoma. In our patient, new cutaneous lesions and an extracutaneous lesion appeared and rapidly increased during treatment with retinoids and calcitriol. Therefore, this