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Hidden Chromosome Abnormalities in a Primary Central Nervous System Lymphoma Detected by Multicolor Spectral Karyotyping
Hidden Chromosome Abnormalities in a Primary Central Nervous System Lymphoma Detected by Multicolor Spectral Karyotyping H. Zattara-Cannoni, H. Dufour, H. Lepidi, C. Chatel, F. Grisoli, and A. M. Vagner-Capodano
ABSTRACT: Cytogenetic analysis provides important information for diagnosis and prognosis in some tumors. But karyotype analysis can be difficult in some cases, because metaphase chromosomes are contracted. New techniques, such as fluorescence in situ hybridization and, more recently, spectral karyotyping, or SKY, based on the hybridization of 24 fluorescently labeled chromosome painting probes, allow the detection and identification of complex chromosomal rearrangements. We report here a case of primary central nervous system lymphoma in which chromosomal rearrangements and marker chromosomes not identified by a routine cytogenetic technique were clarified by SKY. This shows the value of the SKY technique in the cytogenetic diagnosis of tumors. © Elsevier Science Inc., 1998 INTRODUCTION Karyotype analysis with conventional chromosome-banding techniques is often difficult in lymphomas. Poorly spread or contracted metaphase chromosomes obtained in highly rearranged karyotypes with numerous marker chromosomes are often difficult to interpret. The fluorescence in situ hybridization (FISH) technique may be used to visualize suspected aberrations, but it does not allow identification of marker chromosomes or complex abnormalities. Now, more information can be detected in complex karyotypes by the technique of multicolor spectral karyotyping (SKY), based on the hybridization of 24 fluorescently labeled chromosome painting probes. We report a case of primary central nervous system (PCNS) lymphoma with complex chromosomal rearrangements analyzed by SKY to complement conventional banding techniques. PCNS lymphomas are a rare entity accounting for 0.3– 1.5% of CNS tumors and 2% of malignant lymphomas [1]. Many cytogenetic analyses of lymphomas have been performed, but few have been reported for PCNS lymphomas [2–5]. In the cases reported, the karyotypes were complex. Analysis with the new technique SKY in these rare types
From the Cytogenetic Oncology Laboratory (H. Z.-C., C. C., A. M. V.-C.), the Department of Neurosurgery (H. D., F. G), and the Neuropathology Laboratory (H. L.), C. H. U. TIMONE, Marseille, France. Address reprint requests to: Dr. H. Zattara-Cannoni, Cytogenetic Oncology Laboratory, CHU TIMONE, 264, rue St-Pierre, 13385 Marseille Cedex 5, France. Received February 25, 1998; accepted May 6, 1998. Cancer Genet Cytogenet 107:98–101 (1998) Elsevier Science Inc., 1998 655 Avenue of the Americas, New York, NY 10010
of lymphomas seems important to allow one to identify the origin and the chromosomal mechanism of the chromosomal aberrations involved. CASE REPORT A 65-year-old woman developed a tumor in the temporal lobe. The histopathological diagnosis was a lymphoma with a B-cell IgM, high-grade malignancy in the Kiel classification [6]. The patient was given systematic chemotherapy (methotrexate-cytarabine-etoposide). Six months later, the tumor recurred in a new temporal localization. She died 1 month later. MATERIALS AND METHODS Histopathologic and Immunohistochemical Studies The tumor was paraffin embedded, and sections were stained by hematoxylineosin, Giemsa, and Gomori stains. A sample of fresh tissue was snap frozen and kept in liquid nitrogen at 2808C until immunophenotypic study. Immunophenotyping was performed on cryostat sections stained by the indirect immunoperoxidase method, with the use of monoclonal antibodies against B and T lymphocytes and antiimmunoglobulin heavy and light chains. Cytogenetic Studies Sterile tumor tissue biopsy of the first tumor was obtained from the operating room and immediately transported to the laboratory, where it was dissected. Cell suspension was seeded into 25-cm2 tissue culture flasks (Falcon) in Ham F10 medium, and antibodies were supplemented by 10%
0165-4608/98/$19.00 PII S0165-4608(98)00095-8
99
Chromosome Abnormalities Detected by SKY in Lymphoma fetal calf serum and incubated at 378C with 5% CO2 atmosphere for 7 days. Chromosome analysis was performed by routine cytogenetic techniques. The R-banding technique [7] was used for chromosomal identification. Spectral Karyotyping Slides for SKY were prepared by using chromosome preparations that we stored at 2208C. Seven metaphases were analyzed by SKY. For visualization by the SKY system, 24 chromosome-specific painting libraries are labeled in combinations of as many as four fluorochromes (SKY paint)
and hybridized simultaneously to metaphase chromosome preparations. The dyes that are used are SpectrumGreen, Cy3, Texas red, Cy5, and Cy55. Image acquisition was performed by using a SD200 spectratube (Applied Spectral Imaging Inc.) mounted on a Zeiss microscope (Axioplan 2) with a 75-W xenon excitation lamp. The filter used was a triple dichroic filter (SKY filter). A completely automatic classification of each pixel (point) along the chromosome was realized on the 24 chromosome color classes followed by a corresponding karyogram that resolves all markers chromosomes.
Figure 1 Karyotype (R-bands) of a cell from a lymphoma: 46,XX,add(1)(q34),add(6)(q44),del(7)(q22q36),del(9) (p13p24),212,212,214,1mar1,1mar2,1mar3.
100
H. Zattara-Cannoni et al.
RESULTS
DISCUSSION
Immunophenotypic Study Immunophenotypic characterization of the neoplastic cell population showed coexpression of pan B antigens (CD19, CD20, and CD22) and expression of monocytic surface immunoglobulins (k light chain).
We studied a case of PCNS lymphoma by routine karyotype and by the SKY technique. Chromosomes were identified as abnormal or missing by the standard technique, and SKY revealed the precise nature of the abnormalities, particularly those of chromosome 9, which were interpreted by cytogenetic analysis as a terminal deletion 9p13–pter, whereas SKY showed that part of chromosome 3 and part of chromosome 5 had substituted in the short arm of chromosome 9. Three marker chromosomes were identified as containing chromosome 12 material by SKY analysis. The two chromosomes 12 were missing on the standard karyotype. These observations showed the SKY technique to reveal hidden and unrecognized chromosomal aberrations. This information is important for recognition of chromosomes or part of chromosomes thus far unidentified in a specific pathology. The cytogenetic data in PCNS lymphomas are limited [2–5, 8, 9]. No specific abnormalities had ever been described in PCNS lymphomas type B. Structural abnormalities involving chromosome 14 at 14q11 or 14q32 were reported by some authors [2–4]. Trisomy 12 was reported in large B-cell lymphomas [10–12], and we observed it in a case of PCNS lymphoma type B [5]. In our present case, SKY showed that chromosome 12 was involved in several chromosome rearrangements. It is possible that a gene located on chromosome 12 might play a role in the pathogenesis of PCNS lymphomas B. These findings show that the SKY technique can detect specific chromosomal involvement in tumors that could not be detected by routine cytogenetic techniques. We already know that FISH enabled one to discover a structural abnormality t(12;21) unrecognized by standard cytogenetic techniques in childhood B-cell acute lymphocytic leukemia [13,14]. Veldman
Cytogenetic Study A total of 16 metaphases were analyzed by conventional banding techniques. Several chromosomal aberrations were detected by means of R-banding and the karyotype was interpreted as: 46,XX,add(1)(q44),add(6)(p25),del(7) (q22q36),del(9)(p13p24),212,212,214,1mar1,1mar2, 1mar3 (Fig. 1). The metaphases were rekaryotyped on the basis of the spectral classification (Fig. 2) revealed by SKY analysis: • Additional material on chromosome 1 is duplication of part of chromosome 1. • Additional material on chromosome 6 came from chromosome 12. • Deletion of chromosome 7 is visualized. • Abnormalities of chromosome 9p are identified as a loss of 9p, which is substituted with chromosomes 3 and 5 material. • Additional material on chromosome 17 came from chromosome 15. This abnormality was not seen in the standard karyotype. • The first marker (mar1) is identified as containing chromosomes 5 and 14 material. • The second marker (mar2) is identified as containing chromosomes 5 and 12 material. • The third marker (mar3) is revealed as a part of a chromosome 12.
Figure 2 Spectral karyotyping. After spectral based classification, chromosomes were assigned a pseudocolor according to the measured spectrum. Aberrant chromosomes with pseudocolor are highlighted by arrows.
101
Chromosome Abnormalities Detected by SKY in Lymphoma et al. [15] showed the use of SKY in the clarification of complex rearrangements in hematological malignancies. Our report showed the capability of the SKY technique in the identification of marker chromosomes and the delineation of complex chromosome aberrations in tumors. Only few specific chromosomal abnormalities have been described in solid tumors. The SKY technique will certainly permit one to detect specific chromosomal abnormalities in some tumors. It seems to us that the cytogenetic diagnosis of tumors requires a necessary combination of the conventional banding technique and the SKY technique.
6.
7.
8.
9.
We are grateful to Applied Spectral Imaging Inc for image acquisition. We thank Dr. Marina Lafage for her collaboration. This work was supported by Ligue Nationale contre le cancer du Var.
10.
REFERENCES
11.
1. Xerri L, Gambarelli D, Horschowski N, Andrac L, Hassoun J (1990): What’s new in primary central nervous system lymphoma? Pathol Res Pract 186:809–816. 2. Yamada K, Yoshioka M, Dami H (1977): A 14q1 marker and a late replicating chromosome #22 in a brain tumor: brief communication. J Natl Cancer Inst 59:1193–1195. 3. Poissonnier M, Casassus P, Noel L (1989): Primary cerebromeningeal lymphoma with granulated CSF blast cells showing the (8;14) chromosome translocation. Nouv Rev Fr Hematol 13:272. 4. Itoyama T, Sadamori N, Tsutsumi K, Tokunaga Y, Soda H, Tomonaga H, Yamamori S, Masuda Y, Oshima K, Kikuchi M (1994): Primary central nervous system lymphomas: immunophenotypic, virologic, and cytogenetic findings of three patients without immune defects. Cancer 73:455–463. 5. Zattara-Cannoni H, Horschowski N, Figarella-Branger D, Dufour H, Grisoli F, Vagner-Capodano AM (1996): Unusual
12.
13.
14.
15.
chromosome abnormalities in primary central nervous system lymphoma. Leuk Lymphoma 21:515–517. Stansfeld AG, Diebold J, Kapanci Y, Kelenyi G, Lennert K, Mioduszewska O, Noel H, Rilke F, Sundstrom C, Van Unnik J, Wright D (1988): Updated Kiel classification for lymphomas. Lancet 1:292–293. Dutrillaux B, Lejeune J (1971): Sur une nouvelle technique d’analyse du caryotype humain [A new technic of human karyotype analysis]. Cr Acad Sci (Paris) 272:2638–2640. Miyoshi I, Kubonischi I, Yoshinoto S, Hikita T, Dabasaki H, Tanaka T (1982): Characteristics of a brain lymphoma cell line derived from primary intracranial lymphoma. Cancer 49:456–459. Van Diemen-Steenvoorde R, Donckerwolcker AMG, Kluin PM, Kapsenberg JG, Lepoutre JMM, Fleer A (1986): Epstein-Barr virus related central nervous system lymphoma in a child after renal transplantation. Int J Pediatr Nephrol 7:55–58. Konduru PRK, Filippa DA, Richarson ME, Jhanwar SC, Chaganti SR, Koziner B (1987): Cytogenetic and histologic correlations in malignant lymphoma. Blood 69:97–102. Cabanillas F, Pathak S, Trujillo J, Manning J, Katz R, McLaughlin P (1988): Frequent nonrandom chromosome abnormalities in 27 patients with untreated large cell lymphoma and immunoblastic lymphoma. Cancer Res 48:5557–5564. Offit K, Jhanwar SC, Ladanyi M, Fillippa DA, Chaganti RSK (1991): Cytogenetic analysis of 434 consecutively ascertained specimens of non-Hodgkin’s lymphoma: correlations between recurrent aberrations, histology, and exposure to cytotoxic treatment. Genes Chromosom Cancer 3:189–201. Golub TR, Barker GF, Bohlander SK (1995): Fusion of the TEL gene on 12p13 to the AML1 gene on 21q22 in acute lymphoblastic leukemia. Proc Natl Acad Sci USA 92:4917–4921. Romana SP, Mauchauffe M, Le Coniat M (1995): The t(12;21) of acute lymphoblastic leukemia results in a TEL–AMLI gene fusion. Blood 85:3662–3670. Veldman T, Vignon C, Schrock E, Rowley J, Ried T (1997): Hidden chromosome abnormalities in hematological malignancies detected by multicolour spectral karyotyping. Nat Genet 15:406–410.
ABSTRACT: Cytogenetic analysis provides important information for diagnosis and prognosis in some tumors. But karyotype analysis can be difficult in some cases, because metaphase chromosomes are contracted. New techniques, such as fluorescence in situ hybridization and, more recently, spectral karyotyping, or SKY, based on the hybridization of 24 fluorescently labeled chromosome painting probes, allow the detection and identification of complex chromosomal rearrangements. We report here a case of primary central nervous system lymphoma in which chromosomal rearrangements and marker chromosomes not identified by a routine cytogenetic technique were clarified by SKY. This shows the value of the SKY technique in the cytogenetic diagnosis of tumors. © Elsevier Science Inc., 1998 INTRODUCTION Karyotype analysis with conventional chromosome-banding techniques is often difficult in lymphomas. Poorly spread or contracted metaphase chromosomes obtained in highly rearranged karyotypes with numerous marker chromosomes are often difficult to interpret. The fluorescence in situ hybridization (FISH) technique may be used to visualize suspected aberrations, but it does not allow identification of marker chromosomes or complex abnormalities. Now, more information can be detected in complex karyotypes by the technique of multicolor spectral karyotyping (SKY), based on the hybridization of 24 fluorescently labeled chromosome painting probes. We report a case of primary central nervous system (PCNS) lymphoma with complex chromosomal rearrangements analyzed by SKY to complement conventional banding techniques. PCNS lymphomas are a rare entity accounting for 0.3– 1.5% of CNS tumors and 2% of malignant lymphomas [1]. Many cytogenetic analyses of lymphomas have been performed, but few have been reported for PCNS lymphomas [2–5]. In the cases reported, the karyotypes were complex. Analysis with the new technique SKY in these rare types
From the Cytogenetic Oncology Laboratory (H. Z.-C., C. C., A. M. V.-C.), the Department of Neurosurgery (H. D., F. G), and the Neuropathology Laboratory (H. L.), C. H. U. TIMONE, Marseille, France. Address reprint requests to: Dr. H. Zattara-Cannoni, Cytogenetic Oncology Laboratory, CHU TIMONE, 264, rue St-Pierre, 13385 Marseille Cedex 5, France. Received February 25, 1998; accepted May 6, 1998. Cancer Genet Cytogenet 107:98–101 (1998) Elsevier Science Inc., 1998 655 Avenue of the Americas, New York, NY 10010
of lymphomas seems important to allow one to identify the origin and the chromosomal mechanism of the chromosomal aberrations involved. CASE REPORT A 65-year-old woman developed a tumor in the temporal lobe. The histopathological diagnosis was a lymphoma with a B-cell IgM, high-grade malignancy in the Kiel classification [6]. The patient was given systematic chemotherapy (methotrexate-cytarabine-etoposide). Six months later, the tumor recurred in a new temporal localization. She died 1 month later. MATERIALS AND METHODS Histopathologic and Immunohistochemical Studies The tumor was paraffin embedded, and sections were stained by hematoxylineosin, Giemsa, and Gomori stains. A sample of fresh tissue was snap frozen and kept in liquid nitrogen at 2808C until immunophenotypic study. Immunophenotyping was performed on cryostat sections stained by the indirect immunoperoxidase method, with the use of monoclonal antibodies against B and T lymphocytes and antiimmunoglobulin heavy and light chains. Cytogenetic Studies Sterile tumor tissue biopsy of the first tumor was obtained from the operating room and immediately transported to the laboratory, where it was dissected. Cell suspension was seeded into 25-cm2 tissue culture flasks (Falcon) in Ham F10 medium, and antibodies were supplemented by 10%
0165-4608/98/$19.00 PII S0165-4608(98)00095-8
99
Chromosome Abnormalities Detected by SKY in Lymphoma fetal calf serum and incubated at 378C with 5% CO2 atmosphere for 7 days. Chromosome analysis was performed by routine cytogenetic techniques. The R-banding technique [7] was used for chromosomal identification. Spectral Karyotyping Slides for SKY were prepared by using chromosome preparations that we stored at 2208C. Seven metaphases were analyzed by SKY. For visualization by the SKY system, 24 chromosome-specific painting libraries are labeled in combinations of as many as four fluorochromes (SKY paint)
and hybridized simultaneously to metaphase chromosome preparations. The dyes that are used are SpectrumGreen, Cy3, Texas red, Cy5, and Cy55. Image acquisition was performed by using a SD200 spectratube (Applied Spectral Imaging Inc.) mounted on a Zeiss microscope (Axioplan 2) with a 75-W xenon excitation lamp. The filter used was a triple dichroic filter (SKY filter). A completely automatic classification of each pixel (point) along the chromosome was realized on the 24 chromosome color classes followed by a corresponding karyogram that resolves all markers chromosomes.
Figure 1 Karyotype (R-bands) of a cell from a lymphoma: 46,XX,add(1)(q34),add(6)(q44),del(7)(q22q36),del(9) (p13p24),212,212,214,1mar1,1mar2,1mar3.
100
H. Zattara-Cannoni et al.
RESULTS
DISCUSSION
Immunophenotypic Study Immunophenotypic characterization of the neoplastic cell population showed coexpression of pan B antigens (CD19, CD20, and CD22) and expression of monocytic surface immunoglobulins (k light chain).
We studied a case of PCNS lymphoma by routine karyotype and by the SKY technique. Chromosomes were identified as abnormal or missing by the standard technique, and SKY revealed the precise nature of the abnormalities, particularly those of chromosome 9, which were interpreted by cytogenetic analysis as a terminal deletion 9p13–pter, whereas SKY showed that part of chromosome 3 and part of chromosome 5 had substituted in the short arm of chromosome 9. Three marker chromosomes were identified as containing chromosome 12 material by SKY analysis. The two chromosomes 12 were missing on the standard karyotype. These observations showed the SKY technique to reveal hidden and unrecognized chromosomal aberrations. This information is important for recognition of chromosomes or part of chromosomes thus far unidentified in a specific pathology. The cytogenetic data in PCNS lymphomas are limited [2–5, 8, 9]. No specific abnormalities had ever been described in PCNS lymphomas type B. Structural abnormalities involving chromosome 14 at 14q11 or 14q32 were reported by some authors [2–4]. Trisomy 12 was reported in large B-cell lymphomas [10–12], and we observed it in a case of PCNS lymphoma type B [5]. In our present case, SKY showed that chromosome 12 was involved in several chromosome rearrangements. It is possible that a gene located on chromosome 12 might play a role in the pathogenesis of PCNS lymphomas B. These findings show that the SKY technique can detect specific chromosomal involvement in tumors that could not be detected by routine cytogenetic techniques. We already know that FISH enabled one to discover a structural abnormality t(12;21) unrecognized by standard cytogenetic techniques in childhood B-cell acute lymphocytic leukemia [13,14]. Veldman
Cytogenetic Study A total of 16 metaphases were analyzed by conventional banding techniques. Several chromosomal aberrations were detected by means of R-banding and the karyotype was interpreted as: 46,XX,add(1)(q44),add(6)(p25),del(7) (q22q36),del(9)(p13p24),212,212,214,1mar1,1mar2, 1mar3 (Fig. 1). The metaphases were rekaryotyped on the basis of the spectral classification (Fig. 2) revealed by SKY analysis: • Additional material on chromosome 1 is duplication of part of chromosome 1. • Additional material on chromosome 6 came from chromosome 12. • Deletion of chromosome 7 is visualized. • Abnormalities of chromosome 9p are identified as a loss of 9p, which is substituted with chromosomes 3 and 5 material. • Additional material on chromosome 17 came from chromosome 15. This abnormality was not seen in the standard karyotype. • The first marker (mar1) is identified as containing chromosomes 5 and 14 material. • The second marker (mar2) is identified as containing chromosomes 5 and 12 material. • The third marker (mar3) is revealed as a part of a chromosome 12.
Figure 2 Spectral karyotyping. After spectral based classification, chromosomes were assigned a pseudocolor according to the measured spectrum. Aberrant chromosomes with pseudocolor are highlighted by arrows.
101
Chromosome Abnormalities Detected by SKY in Lymphoma et al. [15] showed the use of SKY in the clarification of complex rearrangements in hematological malignancies. Our report showed the capability of the SKY technique in the identification of marker chromosomes and the delineation of complex chromosome aberrations in tumors. Only few specific chromosomal abnormalities have been described in solid tumors. The SKY technique will certainly permit one to detect specific chromosomal abnormalities in some tumors. It seems to us that the cytogenetic diagnosis of tumors requires a necessary combination of the conventional banding technique and the SKY technique.
6.
7.
8.
9.
We are grateful to Applied Spectral Imaging Inc for image acquisition. We thank Dr. Marina Lafage for her collaboration. This work was supported by Ligue Nationale contre le cancer du Var.
10.
REFERENCES
11.
1. Xerri L, Gambarelli D, Horschowski N, Andrac L, Hassoun J (1990): What’s new in primary central nervous system lymphoma? Pathol Res Pract 186:809–816. 2. Yamada K, Yoshioka M, Dami H (1977): A 14q1 marker and a late replicating chromosome #22 in a brain tumor: brief communication. J Natl Cancer Inst 59:1193–1195. 3. Poissonnier M, Casassus P, Noel L (1989): Primary cerebromeningeal lymphoma with granulated CSF blast cells showing the (8;14) chromosome translocation. Nouv Rev Fr Hematol 13:272. 4. Itoyama T, Sadamori N, Tsutsumi K, Tokunaga Y, Soda H, Tomonaga H, Yamamori S, Masuda Y, Oshima K, Kikuchi M (1994): Primary central nervous system lymphomas: immunophenotypic, virologic, and cytogenetic findings of three patients without immune defects. Cancer 73:455–463. 5. Zattara-Cannoni H, Horschowski N, Figarella-Branger D, Dufour H, Grisoli F, Vagner-Capodano AM (1996): Unusual
12.
13.
14.
15.
chromosome abnormalities in primary central nervous system lymphoma. Leuk Lymphoma 21:515–517. Stansfeld AG, Diebold J, Kapanci Y, Kelenyi G, Lennert K, Mioduszewska O, Noel H, Rilke F, Sundstrom C, Van Unnik J, Wright D (1988): Updated Kiel classification for lymphomas. Lancet 1:292–293. Dutrillaux B, Lejeune J (1971): Sur une nouvelle technique d’analyse du caryotype humain [A new technic of human karyotype analysis]. Cr Acad Sci (Paris) 272:2638–2640. Miyoshi I, Kubonischi I, Yoshinoto S, Hikita T, Dabasaki H, Tanaka T (1982): Characteristics of a brain lymphoma cell line derived from primary intracranial lymphoma. Cancer 49:456–459. Van Diemen-Steenvoorde R, Donckerwolcker AMG, Kluin PM, Kapsenberg JG, Lepoutre JMM, Fleer A (1986): Epstein-Barr virus related central nervous system lymphoma in a child after renal transplantation. Int J Pediatr Nephrol 7:55–58. Konduru PRK, Filippa DA, Richarson ME, Jhanwar SC, Chaganti SR, Koziner B (1987): Cytogenetic and histologic correlations in malignant lymphoma. Blood 69:97–102. Cabanillas F, Pathak S, Trujillo J, Manning J, Katz R, McLaughlin P (1988): Frequent nonrandom chromosome abnormalities in 27 patients with untreated large cell lymphoma and immunoblastic lymphoma. Cancer Res 48:5557–5564. Offit K, Jhanwar SC, Ladanyi M, Fillippa DA, Chaganti RSK (1991): Cytogenetic analysis of 434 consecutively ascertained specimens of non-Hodgkin’s lymphoma: correlations between recurrent aberrations, histology, and exposure to cytotoxic treatment. Genes Chromosom Cancer 3:189–201. Golub TR, Barker GF, Bohlander SK (1995): Fusion of the TEL gene on 12p13 to the AML1 gene on 21q22 in acute lymphoblastic leukemia. Proc Natl Acad Sci USA 92:4917–4921. Romana SP, Mauchauffe M, Le Coniat M (1995): The t(12;21) of acute lymphoblastic leukemia results in a TEL–AMLI gene fusion. Blood 85:3662–3670. Veldman T, Vignon C, Schrock E, Rowley J, Ried T (1997): Hidden chromosome abnormalities in hematological malignancies detected by multicolour spectral karyotyping. Nat Genet 15:406–410.