Clinicopathological Study of Ki-1-Positive Lymphomas

Path. Res. Pract. 185,461-467 (1989)

Clinicopathological Study of Ki-l-Positive Lymphomas K. Tashiro, M. Kikuchi, M. Takeshita, 1. Yoshida and K. Ohshima First Department of Pathology, School of Medicine, Fukuoka University, Fukuoka, Japan

SUMMARY We examined an antibody against Ki-1 antigen in 161 cases ofmalignant lymphoma, four of histiocytic sarcoma, and six of nonspecific lymphadenitis, using monoclonal antibody Ki-1, which is known to react selectively with activated lymphocytes, Reed-Sternberg cells, and Hodgkin's cells. Among them, 12 cases of malignant lymphoma demonstrated a diffuse positive cell membrane and/or cytoplasmic reaction of tumor cells and were categorized as Ki-1-positive lymphoma. Nine of these cases exhibited large cells with indented nuclei, distinct nucleoli, and abundant basophilic or amphophilic cytoplasm. Of the remaining three cases, two were of medium-sized and one of small-cell type. Immunologically, the 12 cases of malignant lymphoma demonstrated T-helper/inducer phenotype in six cases, B-cell in two case, and non- T, non-B in four cases. Tac and HLADR were positive in 9/12 and 4/5, respectively, and markers for histiocytes (lysozyme, a-1 anti-chymotrypsin, and OK-M1) were usually negative. Clinically, T-cell Ki-1-positive lymphoma was most likely to occur in the elderly, at extranodal sites, and had a rather poor prognosis (mean survival 35.5 months) as compared with B-cell and non-T, non-B lymphoma (7-52 months survival).

Introduction Since the establishment of monoclonal antibody Ki-l against the Hodgkin's cell line L 428 11 , several cases of non-Hodgkin's lymphoma with diffuse proliferation of Ki-l-positive cells have been found and reported as Ki-l lymphomas?' 8, 12. However, precise clinical characterization of this lymphoma was lacking8 • The purpose of our study was to clarify the clinicopathological characteristics of Ki-l-positive lymphoma as compared with other lymphomas and reactive lesions of the lymph nodes. Material and Methods The patients examined for Ki-l antigen were selected from cases of malignant lymphoma and allied disorders who presented at the Department of Pathology, Fukuoka University School of Medicine, from 1982 to 1986. They comprised 161 cases of malignant lymphoma (T-cell 103, B-cell 45, non-T, non-B 6, © 1989 by Gustav Fischer Verlag, Stuttgart

Hodgkin's disease 7), and four cases of histiocytic sarcoma. Six cases of nonspecific lymphadenitis served as a control group. Clinical information, including age, sex, presenting signs and symptoms, laboratory data, treatment, and follow-up data, was obtained from questionnaires which were sent to clinicians. Tissues obtained at biopsy of lymph nodes, skin, etc. were usually divided into three portions for light microscopy, immunohistochemistry, and electron microscopy. Paraffin sections for light microscopy were fixed in B-5 solution (10% neutral formalin with 6% w/v mercuric chloride) for routine staining with H & E, Giemsa, PAS, and silver impregnation. These sections were additionally stained for intracytoplasmic immunoglobulins, lysozyme, a-I anti-chymotrypsin, epithelial membrane antigen (EMA), common leukocyte antigen (CLA), and nonspecific esterase. Unfixed tissues for immunohistochemical study were embedded in OCT compound and kept in liquid nitrogen until examination. Frozen sections (5 !tm thick) were examined with anti-human monoclonal antibody against Ki-l antigen (Source DAKO Japan; Dilution X 10; Detection of Reed-Sternberg cells). In additon, lymphocyte activation antigens (HLADR, Tac 14 ), T-cell antigens (CD 2, CD 4, CD 8), B-cell 0344-0~38/89/0185-0461$3.5010

• 462 . K. Tashiro, M. Kikuchi, M. Takeshita, T. Yoshida and K. Ohshima antigens (B-1, Leu-14), macrophage antigen (OK-M1), and surface immunoglobulin (x, A, M, G, A, 0, E) were investigated using indirect three-step immunoperoxidase methods. For electron microscopy, the specimens were fixed in 2% glutaraldehyde and postfixed in 1% OS04, routinely processed, and examined with a.JEM 100 CX electron microscope. Gene rearrangement studies were kindly provided by Dr RyuZQ Ueda (Laboratory of Chemotherapy, Research Institute, Aichi Cancer Center, Nagoya, Japan) in three cases.

Results Clinical and Laboratory Findings

Twelve cases of malignant lymphoma were identified as Ki-1-positive lymphoma on the basis of a strong, diffuse positive reaction in the cell membrane and/or cytoplasm. The main clinical and laboratory findings are summarized in Table 1. Eight patients were male and four were female. Ages ranged from 13 to 77 years, with a mean age of 51.3. Nine patients had lymphadenopathy, three of these representing systemic enlargement. None of the patients had hepatosplenomegaly at the time of presentation. In cases of T-cell lymphoma, subcutaneous nodules were found in four and cutaneous nodules in one. Tumor formation was found in two cases of B-cell phenotype, one at the palpebral conjunctiva and another at the tonsil. The bone marrow was involved in two cases of T-cell lymphoma. Atypical cells in the p~ripheral blood were found in one

case each of T- and non-T, non-B. In 10 cases examined, serum antibodies against adult T-cell leukemia virus (ATLV) were seen in three cases of T- and two of non-T, non-B. An increased LDH level was found in nine of 12. In 11 patients, chemotherapy or combined therapy (radiation + chemotherapy) was performed; one patient of Bcell type with a conjunctival mass underwent only excision. Of the cases with the T-cell phenotype, four of six patients died between 3 and 69 months after presentation, with a mean survival of 35.5 months. Five of six patients with B-cell and non-T, non-B phenotypes are stilI alive, without recurrence. Histology

Histologically, nine of the cases with Ki-1-positive lymphoma were large-cell lymphomas, composed of large neoplastic cells with round or indented nuclei, distinct nucleoli, and abundant basophilic or amphophilic cytoplasm (Figs. la, 2a). Eight of these cases showed diffuse proliferation of the tumor cells; in one (Case 5), however, large neoplastic cells infiltrated the paracortical region and subcapsular sinus, giving the appearance of metastatic tumor or malignant histiocytosis (Fig. 1b). Partly follicular growth patterns of tumor cells were seen in one instance (Case 12). Two cases (Cases 7,8) exhibited diffuse, medium-sized cells (Fig. 3a) with round or oval nuclei. The conjunctival tumor (Case 11) showed diffuse proliferation of small neoplastic cells with irregular nuclei, indistinct

Table 1. Clinical and Laboratory Findings of Ki-1-Positive Lymphomas (12 Cases) Phenotype

No.

13

M

2

24

F

3

24

F

4

76

F

5

72

M

6

77

F

7

56

M

8

53

M

9

76

M

10

66

M

11

35

M

12

43

M

Non-T, Non-B

T

B

t

Age Sex (Y.O.)

Initial Diagnosis

Lymphadenopathy

ML, diffuse, , Systemic large ML, diffuse, Local (1t. axilla) large ML, diffuse, Local large (rt. neck) ML, diffuse, Local (rt. neck) large ML, diffuse, large ML, diffuse, large ML, diffuse, medium ML, diffuse, medium ML, diffuse, large ML, diffuse, large ML, diffuse, small ML, diffuse, large

= Dead, ML = Malignant lymphoma.

Hepato- Tumor splenomegaly

Leukemic B. M. Changes Involv.

+

ATLAAb.

+

+

Subcut.

+

Subcut.

Local (1t. axilla) Local (rt. neck)

Local (1t. neck)

7

+

26 28

+

Subcut. Bone Subcut.

Survival (Mos)

+

Mediast.

Systemic Systemic

High LDH

+

+

+ +

2

+

3t

+

63t

+

+

+

+

69t

Skin

21

.Conj. palp. Tonsil

55

+

43

Ki-l-Positive Lymphomas· 463

Fig. la. Diffuse ptoliferation of large neoplastic cells with round or indented nuclei, distinct nucleoli, and abundant cytoplasm (Case 4). H & E stain, x 480. - Fig. 1b. The neoplastic cells were infiltrating to the marginal sinus. H & E stain, x 240. Fig. 1e. Ki-l positivity. Frozentissue, x 480. - Fig. 2a. Diffuse proliferation of large atypical lymphoid cells with round or indented nuclei, one or more prominent nucleoli, and fine chromatin (Case 5). H & E stain, X 480. - Fig. 2b. Ki-l positivity. Frozen tissue, X 480. - Fig. 2e. CD-4 positive cells. Frozen tissue, x 480.

464 . K. Tashiro, M. Kikuchi, M. Takeshita, T. Yoshida and K. Ohshima

6a Fig. 3 a. Medium-sized atypical lymphoid cells are diffusely present (<::ase 7). H & E stain, X 480. - Fig. 3 b. Ki-1 positivity. Frozen tissue, X 480. - Fig. 3c. CD 2 (pan-T cells)-positive cells. Frozen tissue, X 480. - Fig.4a. Conjunctival mass showing diffuse proliferation of small atypical lymphoid cells with irregular nuclei and occasional plasma cell differentiation (Case 11). H & E stain, X 480. - Fig. 4b. Ki-1 -positivity. Frozen tissue, X 480. - Fig. 4c. Leu-14 (pan-B cells) positive cells. Frozen tissue, x 480. Fig. 5. The neoplastic cells usually have scanty intracytoplasmic organelles (Case 9). Electron microscopy, X 1820. - Fig. 6a. A few Ki-1-positive cells are seen in pleomorphic T-cell lymphoma. Frozen tissue, X 480. - Fig. 6b. A few Ki-1 positive cells in Hodgkin's disease, mixed cellularity. Frozen tissue, X 480.

Ki-1-Positive Lymphomas' 465 Table 2. Immunohistological Study of Ki-1-Positive Lymphomas (12 Cases) Pheno- Case Age Sex Biopsy Ki-l HLADR CD-4 -8 type (Y.O.) Site Non-T, 1 Non-B 2 3 4

i3 24 24 76

M F

F F

LN LN LN LN

T

5 6 7 8 9 10

72 77 56 53 76 66

M F M M M M

LN Subcut. Subcut. Subcut. Subcut. Skin

11 12

35 43

M M

B

CD-lB-l

S-Ig

M-l CD-25 Lyso- a-I Clg a-NBE GR zyme ACT (NaF) Tcr~

+ + + +

+ + + + + + Subcut. + LN +

+ + + + + + +

+ + + + + +

+ + + + + +

+

+

Immunohistochemistry

The immunohistological findings of the Ki-l-positive lymphomas are summarized in Table 2. They demonstrated a diffuse and strong positive cell membrane andlor cytoplasmic reaction for Ki-l antigen in almost all tumor cells (Figs. 1c, 2 b, 3 b, 4 b). Six cases exhibited positive reactions for CD 2 (Fig.3c) and CD-4 (Fig.2c). B-cell markers were noted in two cases, but in four cases no positive markers for either T- or B-cells were detected. Both CD 25 (antibody against a receptor for interleukin II) and HLADR were positive in 9/12 and 4/5, respectively. Markers for histiocytes were usually negative (no positive reaction for lysozyme and a-I anti-chymotrypsin, and only one case showed positive reaction against OK-Ml in the 12 cases examined). Surface or intracytoplasmic immunoglobulins were detected in two cases (Cases 11, 12). In five

G

R

G

G

EMACLA

+ + + +

+(Inh) +

+

a-I ACT = a-I anti-chymottypsin, a-NBE = a-naphthol butyrate esterase, Inh = Inhibited by NaF, GR G = Germline, EMA = Epithelial membrane antigen, CLA = Common leukocyte antigen.

nucleoli, stippled chromatin, and occasional plasma cell differentiation (Fig. 4a). Cells with giant nuclei were present in five cases (Cases 3, 4, 5, 9, and 10). Only in one (Case 5) massive necrosis was seen.

+(Inh) R +(Inh) +(Inh) R

+ + + + +

+ +

JH

+ +

+(Inh)

= Gene rearrangement, G = Rearranged,

cases, tumor cells showed diffuse staining for a-naphthol butyrate esterase. In 11 cases examined, epithelial membrane antigen (EMA) was negative, but six cases showed positive reactions against common leukocyte antigen (CLA). Electron Microscopy

We examined two cases of Ki-l positive lymphoma. Cytoplasmic organelles tended to cluster near the nuclear concavity and to be sparse at the cell periphery in one (Case 2). Many neoplastic cells usually had scanty intracytoplasmic organelles in another (Case 9) (Fig. 5). Gene Rearrangement

As shown in Table 2, three cases were examined for rearrangement of the ~ chain of T-cell antigen receptor (TcR) gene 4 and immunoglobulin gene of JH. Rearrangement of the TcR ~ gene was seen in all three cases, but no rearrangement of Ig gene was detected.

Table 3. Ki-1 Antigen in Other Non-Hodgkin's Lymphomas (142 cases) Non-Hodgkin's Lymphoma Follicular med. mix. Diffuse small med. large pleo. lbl. Burkitt with AILDfeature

No.of Cases

(-)

T (97) (+-) (++)

(--)

Ki-1 reactivity B (43) (+-) (++)

Non-T, Non-B (2) (--) (+-) (++)

4 1

0 0

0 0

0 0

4 1

0 0

0 0

0 0

0 0

0 0

7 57 45 14 5 2 7

6 34 15 10 4 0 0

0

0 5 1 2 0 0 4

1 10 24 0 1 2 0

0 0 0 0 0 0 0

0 0 0 0 0 0 0

0 0 2 0 0 0 0

0 0 0 0 0 0 0

0 0 0 0 0 0 0

8

3 2 0 0 3

466 . K.Tashiro, M. Kikuchi, M. Takeshita, T. Yoshida and K. Ohshima

Ki-l Antigen in Other Lymphomas and Lymphadenopathies Ki-l antigen was found in other cases of lymphomas, too, with various populations of tumor cells (Fig. 6a). No positive cells were opserved in 43 cases ofB-celllymphoma and two cases of non-T, non-B. The results are shown in Table 3. As demonstrated in Table 4, the same Ki-l reactivity was present in Reed-Sternberg and/or Hodgkin's like cells in seven cases of Hodgkin's disease (Fig. 6b). No positive reactions were seen for Ki-l antigen in all four cases of histiocytic sarcoma. In cases of nonspecific lymphadenitis, a few Ki-l positive large cells were usually seen in the perifollicular area. The pattern and frequency of reaction against Ki-l antigen were nearly the same in both ATLA-positive and -negative groups in malignant lymphoma. Discussion Ki-1 antigen was initially considered as the specific marker for Hodgkin's disease by Stein et al. l2 , but strong and diffuse expression of the antigen in a specific type of non-Hodgkin's lymphoma has recently been reported by Stein et al. 13 . This lymphoma is characterized by sheets of large anaplastic (bizarre), sometimes multinucleated cells, which often resemble Reed-Sternberg cells and tend to infiltrate sinuses and paracortical regions of lymph nodes. These authors classified this lymphoma as large anaplastic type or Ki-l lymphoma. The cases also possessed HLA class II antigens and, in most instances, surface expression of the IL-2 receptor. No cases expressed macrophageassociated antigen, lysozyme, or cytokeratin, but some were positive for intracytoplasmic a-I anti-trypsin. These observations, in association with the presence of HLA class II and a variable or limited expression of cell lineage antigen, suggested that many of these cases would previously have been considered as being of histiocytic origin and, indeed, this diagnosis was made on the original biopsy material of many of these cases. Stein et al. 13 also described that 26 of 45 of these lymphomas possessed the T-cell phenotype and occurred primary in adults, with only rare cases in children. The B-cell phenotype was found in seven, and the remaining patients were of mixed phenotype or displayed no markers. Kadin et al. 8 described six patients with childhood Ki-l lymphoma who presented with skin and peripheral lymphadenopathy and sinus infiltration of lymph nodes. T-cell lineage was found in three of these, with surface phenotype or rearrangement of T-cell receptors. All expressed HLADR and CD 25. In the present series, we selected the Ki-l-positive lymphoma on the basis of strong and diffuse reaction of Ki-l antigen in almost all tumor cells, and this was found only in malignant lymphoma. No Ki-l positive cases were present in histiocytic sarcoma and reactive lymphadenopathy (Table 4). Nine of 12 cases were largecell type, with large round or indented vesicular nuclei, prominent nucleoli, and abundant basophilic or amphophilic cytoplasm, presenting diffuse proliferation in the lymph nodes (Figs. 1a, 2a) except for one' with paracortical

Table 4. Ki-1 Antigen in Hodgkin's Disease, Histiocytic Sarcoma, and Lymphadenopathies No. of cases Ki-1 positive lymphoma

12

(-)

Ki-1 Reactivity (+) (++) (+++)

0

0

0

12 0

Hodgkin's disease

7

0

7'"

0

Histiocytic sarcoma

4

4

0

0

0

Nonspecific lymphadenitis

6

0

5

1

0

* = Reed-Sternberg and Hodgkin's-like cells, (+) = A few (less than 5% of the cells), (+ +) = Scattered (10% of the cells), (+ + +) = Diffuse. and intrasinusoidal involvement (Fig. 1b). The histology was consistent with that of large anaplastic lymphoma or Ki-l lymphoma. In addition, three cases of malignant lymphoma also displayed a diffuse and strong reaction with Ki-l antigen, two of medium-sized type and one of small-cell type. The tumor cells were of T-cell lineage in eight cases, which was proved by surface phenotype in six and rearrangement ofT-cell receptors in two. B-celilineage was seen in two cases, and the other two had no lymphocyte markers (Table 2). The results were similar to those of Stein et al. 13. In the T-cell group, five of eight had cutaneous and/or subcutaneous masses. Such extranodal involvement was reported in three patients by Stein et al. 13 and in five patients by Kadin et al. 8 . Both cases of B-cell type occurred in extranodal sites (palpebral conjunctiva and tonsil). In our series, the patients of the T-cell group were usually over 50 years old, with a male predominance, but the cases reported by Kadin et al. 8 were children. Both B-cell type and cases with no lymphoid markers occurred in younger age groups than T-cell type, but no children were included. Among 12 patients, five died after a short clinical course (less than 15 months). The remainder survived much longer (over 60 months). All belonged to the T-cell group. All patients with other phenotypes were alive after 2 to 55 months, without recurrence. These results indicate a rather better prognosis for Ki-l-positive lymphoma than for malignant lymphoma, especially Band non-T, non-B-cell groups. The cases reported previously were histologically of the large-cell type l , but we found two cases of medium-sized cell type with T-cell markers and one case of small-cell type with B-cell markers, presenting the same strong and diffuse staining patterns in the tumor cells. A few cases of T cell lymphoma with medium-sized nuclei (Stein et al. l l ) as these cases were reported previously, but no B cell lymphoma of small cell type were found in the literature. The case we found originated from conjunctiva palpebrae and had an excellent clinical course. It is suggested that the K-l antigen was expressed in a few peculiar types of slow growing extranodal lymphoma. CLA was positive in six cases. HLADR and CD 25, markers for activated lymphocytes, were

Ki-l-Positive Lymphomas· 467

positive in 9/12 and 4/5, respectively. The results were similar to those of Stein et al. 13 , Kadin et al. 8, and Delsol et al}, but markers for histiocytes such as lysozyme, a-I anti-chymotrypsin, OK-Ml (Mendelsohn et al. IO ) were negative (Table 2). Delsol et al. 3 reported a frequent positive reaction for EMA in large anaplastic celllymphorna, but in the present series no EMA-positive cases were seen (Table 2). This discrepancy might be due to a difference in the tumor population based on race or other hereditary factors. Few electron microscopic studies of Ki-l lymphoma have been performed5 ,9. Fujimoto et aLS described remarkable invaginations of the cell membrane similar to those seen in interdigitating reticulum cells, but in our series the two cases of large-cell type examined exhibited no such invaginations. One case had the T-cell phenotype and another showed rearrangement of T-cell receptors. These findings indicate the existence of a heterogenous cell lineage in Ki-l-positive lymphoma. Ki-l antigen was also expressed in other cases of lymphoma and reactive lymphadenopathy, with varying proportions of less than 10% of tumor cells 2,6,13. Our series was positive for Ki-l antigen in 28 among 97 cases of T-cell lymphoma (Fig. 6a) and in all cases of nonspecific lymphadenitis, but no positive reaction was seen in 43 cases of B-cell lymphoma and four cases of histiocytic sarcoma (Tables 3,4). Histologically, there were no positive cases in lymphobl'l!'tic and follicular types. These results suggest that the antigen cannot usually be expressed in slow-growing lymphomas of follicular center cells and in precursors of T- and B-cells.

Acknowledgements The data on gene rearrangement were a generous gift from Dr Ryuzo Ueda (Laboratory of Chemotherapy, Research Institute, Aichi Cancer Center, Nagoya, Japan). We wish to thank Ms Midori Sugihara, Mr Katsumi Kobata, Mr Masami Nambu, Ms Yumiko Hirose, Ms Tomoko Ichihara, and Mr Toshihiko Nishimura for technical assistance, and Ms Kaoruko Kawasaki for typing the manuscript.

References 1 Agnarsson BA, Kadin ME (1988) Ki-l positive large cell lymphoma. A morphologic and immunologic study of 19 cases. Am] Surg Pathol12: 264-274

2 Andreesen R, Osterholz ], L6hr GW, Bross K] (1984) A Hodgkin cell-specific antigen is expressed on a subset of auto- and allo-activated T (helper) lymphoblasts. Blood 63: 1299-1302 3 Delsol G, Al Saati T, Garter KC, Gerdes ], Schwarting R, Caveriviere P, Rigal-Huguet F, Robert A, Stein H, Mason DY (1988) Coexpression of epithelial membrane antigen (EMA), Ki-l and interleukin-2 receptor by anaplastic large cell lymphomas. Diagnostic value in so-called malignant histiocytosis. Am] Pathol 130: 59-70 4 Duby AD, Klein KA, Murre C, Seidman]G (1985) A novel mechanism of somatic rearrangement predicted by a human T-cell receptor ~-chain complementary DNA. Science 228: 1204 5 Fujimoto ], Hata ], Ishii E, Kiyokawa N, Tanaka S, Morikawa Y, Shimizu K, Hajikano H (1988) Ki-llymphomas in children: immunohistochemical analysis and significance of epithelial membrane antigen (EMA) as a new marker. Virchows Arch (A) 412: 307-314 6 Gerdes], Schwarting R, Stein H (1986) High proliferative activity of Reed Sternberg associated antigen Ki-l positive cells in normal lymphoid tissue. ] Clin Pathol 39: 993-997 7 Jones DB, Geredes ], Stein H, Wright DH (1986) An investigation of Ki-l positive large cell lymphomas with antibodies reactive with tissue macrophages. Hematol Oncol 4: 315-322 8 Kadin ME, Sako D, Berliner N, Flanklin W, Woda B, Borowitz M, Ireland K, Schweid A, Herzog P, Lange B, Dorfman R (1986) Childhood Ki-llymphoma presenting with skin lesions and peripheral lymphadenopathy. Blood 68: 1042-1049 9 LeTourneau AL, Audouin], Diebold] (1988) Ultrastructural study of 4 cases of Ki-l positive large anaplastic cell malignant lymphoma. Virch Arch A Pathol Anat 413: 215-222 10 Mendelsohn G, Eggleston ]C, Mann RB (1980) Relationship of lysozyme (muramidase) to histiocytic differentiation in malignant histiocytosis. An immunohistochemical study. Cancer 45:273-279 11 Schwab U, Stein H, Gerdes], Lemke H, Kirchner H, Schaadt M, Diehl V (1982) Production of monoclonal antibody specific for Hodgkin's disease and a subset of normal lymphoid cells. Nature 299: 65 12 Stein H, Gerdes], Schwab U, Lemke H, Mason DY, Ziegler A, Schienle W, Diehl V (1982) Identification of Hodgkin and Sternberg-Reed cells as a unique cell type derived from a newly-detected small cell population. Int] Cancer 30: 445 13 Stein H, Mason DY, Gerdes], O'Connor N, Wainscoat], Pallesen G, Gartler K, Falini B, Belsol G, Lemke H, Schwarting R, Lennert K (1985) The expression of the Hodgkin's disease associated antigen Ki-l in reactive and neoplastic tissue. Evidence. that Reed-Sternberg cells and histiocytic malignancies are derived from activated lymphoid cells. Blood 66: 848-858 14 Uchiyama T, Broder S, Waldmann TA (1981) A monoclonal antibody (anti-Tac) reactive with activated and functionally mature human T cells. I. Production of anti-Tac monoclonal antibody and distribution of Tac( +) cells. ] Immunol 126: 1393-1397

Received March 16, 1989 . Accepted in revised form July 9, 1989

Key Words: Malignant lymphoma - Ki-l (CD 30) - Lymphoma types - Histiocytic sarcoma

Prof. Masahiro Kikuchi, Nanakuma 7-45-1, ]onari-ku Fukuoka 814-01, Japan