Frequent microsatellite instability in non-Hodgkin lymphomas irresponsive to chemotherapy

Available online at www.sciencedirect.com

Leukemia Research 32 (2008) 1183–1195

Frequent microsatellite instability in non-Hodgkin lymphomas irresponsive to chemotherapy Kaname Miyashita a,b,c , Kei Fujii d , Yu Yamada b,e , Hiroyoshi Hattori f , Kenichi Taguchi a , Takeharu Yamanaka a , Mitsuaki A. Yoshida g , Jun Okamura a , Shinya Oda a,∗ , Koichiro Muta c , Hajime Nawata c , Ryoichi Takayanagi c , Naokuni Uike b a

c

Institute for Clinical Research, National Kyushu Cancer Center, 3-1-1 Notame, Fukuoka 811-1395, Japan b Department of Hematology, National Kyushu Cancer Center, Fukuoka 811-1395, Japan Department of Medicine and Bioregulatory Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan d Department of Anatomic Pathology, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan e Department of Psychosomatic Medicine, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan f Department of Pediatrics, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan g Biological Dose Section, Department of Dose Assessment, Research Center for Radiation Emergency Medicine, National Institute of Radiological Sciences, Chiba 263-8555, Japan Received 28 July 2007; received in revised form 13 November 2007; accepted 14 November 2007 Available online 4 January 2008

Abstract Microsatellite instability (MSI) in haematopoietic malignancies has been controversial. Particularly in non-Hodgkin lymphoma, the data published to date lack unity. Using a unique fluorescent technique, we found MSI in eight (14%) tumours in a panel of 59 carefully selected non-Hodgkin lymphoma patients. Our fluorescent technique also reveals two qualitatively distinct modes of MSI, i.e. Type A and Type B. Based on our previous studies using DNA mismatch repair (MMR) gene-knock out animals, we have concluded that Type A MSI is a direct consequence of defective MMR. MSI observed in non-Hodgkin lymphomas was uniformly Type A, which implies that MMR deficiency occurs in this malignancy. Intriguingly, in non-Hodgkin lymphoma patients treated by CHOP/VEPA-based therapies, response to chemotherapy was significantly worse in those with microsatellite-unstable tumours (p = 0.027). As a consequence, the patient outcomes at 1 year after treatment were significantly less favourable in this population (p = 0.046), although the survival difference was not statistically confirmed in a longer term. These findings suggest that in some non-Hodgkin lymphomas MMR deficiency may lead to drug resistance in tumour cells and, consequently, to poor patient outcomes. In non-Hodgkin lymphoma, MSI may be a potential biomarker that predicts the tumour response against chemotherapy. © 2007 Elsevier Ltd. All rights reserved. Keywords: Microsatellite instability; Mismatch repair; Non-Hodgkin lymphoma; Genetic instability; Chemotherapy

1. Introduction Microsatellites are repetitive DNA sequences comprised of short reiterative motifs. Numerous microsatellites have been mapped throughout the eukaryotic genome. Microsatellite lengths are highly polymorphic in human populations. Although this polymorphism may derive from the instability ∗

Corresponding author. Tel.: +81 92 541 3231; fax: +81 92 542 8534. E-mail address: [email protected] (S. Oda).

0145-2126/$ – see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.leukres.2007.11.024

of these sequences over generations, microsatellites appear stable during a relatively short time such as the life span of the individual. The phenomenon of unstable microsatellites at the somatic level, i.e. microsatellite instability (MSI), was initially reported in human colorectal cancer [1,2], and, subsequently, in the familial cancer-prone syndrome, hereditary non-polyposis colorectal cancer (HNPCC) [3,4]. In 1993, mutations in one of the genes functioning in DNA mismatch repair (MMR) were found in HNPCC individuals [5,6]. In repetitive sequences such as microsatellites,

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K. Miyashita et al. / Leukemia Research 32 (2008) 1183–1195

Table 1 Clinicopathological characteristics, microsatellite instability and the response to chemotherapy in 59 non-Hodgkin lymphoma patients Case

Subtype

Stage

Initial treatment

Response

D2S123

D5S107

D10S197

D11S904

D13S175

ML04 ML56 ML06 ML19 ML22 ML55 ML20 ML67 ML01 ML02 ML05 ML07 ML10 ML13 ML15 ML16 ML17 ML24 ML28 ML31 ML33 ML34 ML36 ML37 ML38 ML45 ML47 ML53 ML54 ML58 ML59 ML60 ML62 ML46 ML03 ML08 ML09 ML14 ML25 ML29 ML30 ML39 ML48 ML52 ML57 ML27 ML51 ML63 ML64 ML50 ML42 ML49 ML44 ML40 ML18 ML21 ML26 ML61 ML41

AILT AILT ALCL ALCL ALCL ALCL BL BL DLBCL DLBCL DLBCL DLBCL DLBCL DLBCL DLBCL DLBCL DLBCL DLBCL DLBCL DLBCL DLBCL DLBCL DLBCL DLBCL DLBCL DLBCL DLBCL DLBCL DLBCL DLBCL DLBCL DLBCL DLBCL FL (Grade 1) FL (Grade 2) FL (Grade 2) FL (Grade 2) FL (Grade 2) FL (Grade 2) FL (Grade 2) FL (Grade 2) FL (Grade 2) FL (Grade 2) FL (Grade 2) FL (Grade 2) FL (Grade 3a) FL (Grade 3a) FL (Grade 3a) FL (Grade 3a) FL (Grade 3b) LBL LBL LDL low grade B MALT MALT NKTCL NKTCL PTL unspec

III III IV III IIIS II II IV IV II IV IV IV IV III IV II IE II I II IV III III IV IV III II III III IE III II IV IV IV III III III IV I III IV III IV IV III IV IV IV II IV IV II IE IIE IIE IIE III

VEPA CHOP VEPA CHOP CHOP CHOP B-ALL protocol B-ALL protocol VENP + RT RT mLSG9#1 LSG9 + RT VEPA VEPA + RT VEPA + RT VEPA VEPA + RT VEPA + RT mEPOCH + RT CHOP + RT CHOP + RT CHOP CHOP CHOP CHOP CHOP CHOP CHOP CHOP CHOP CHOP CHOP CHOP CHOP LSG4 mLSG9#2 + RT mLSG9#1 CHOP mEPOCH CHOP CHOP + RT CHOP CHOP CHOP CHOP + R CHOP CHOP + R CHOP + R CHOP + R CHOP CHOP CHOP Fludarabine CHOP + RT VEPA + RT Operation VEPA DeVIC + RT CHOP

PD CR PD CR CR PD CR CR PD CR CR CR PD PD CR PR CR CR CR CR PR PD CR CR CR PD CR CR CR PD CRu CR CR SD CR CR CR CR CR CR CRu CR CR CR PR PD CR PR CR PD PD PD PR CR CR CR PD PD CR

– – – – – – MSI – – – – – MSI L – – – – – – – – – – L – – – – – – L L – – – MSI – – – – – – – – – – – – – – – L – – – – – L

– – – – – – – – – MSI – – – – – – – – – – – – – – L – – – – – – – – – – – – – – – – – – – – – – L – – – – – – – – – – L

– – – – – MSI – – – – – – – – – – – – – L – – – – L – – – – – – – MSI – – – – – – – – – – – – – – – – – – MSI – – – – – – –

– – – – – – – – – – L – – – L – – L – – – – – – – – – L – – – – L L – – L – – – – – – – – – – – – – – L – – – – – – –

– – – – – MSI – – – – – – – L – – – – – – – – – – – – – – – – L – – – – – – – – – – – – – – – – – – – – – – – – – MSI – –

All the disease subtypes according to the Working Formulation [66] or REAL [67] Classification were translated to fit the WHO Classification. AILT: angioimmunoblastic T-cell lymphoma; ALCL: anaplastic large cell lymphoma; BL: Burkitt lymphoma; DLBCL: diffuse large B-cell lymphoma; FL: follicular lymphoma; LBL: lymphoblastic lymphoma; LDL: lymphoplasmacytic lymphoma; low grade B: low grade B-cell lymphoma unclassifiable; MALT: extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue; NKTCL: extranodal NK/T-cell lymphoma, nasal type; PTL unspec: peripheral T-cell lymphoma, unspecified; CHOP or VEPA: cyclophosphamide, doxorubicin, vincristine and prednisolone; VENP: cyclophosphamide, procarbazine, vincristine and prednisolone; mEPOCH: etoposide, vincristine, carboplatin, doxorubicin and prednisolone; DeVIC: etoposide, ifosfamide, carboplatin and dexamethasone; B-ALL protocol: cyclophosphamide, doxorubicin, vincristine, methotrexate, etoposide, prednisolone and dexamethasone; LSG4: VEPAB (cyclophosphamide, doxorubicin, vincristine, prednisolone and bleomycin) + M-FEPA (methotrexate, vindesine, cyclophosphamide, doxorubicin and prednisolone) + VEPP-B (vincristine, etoposide, procarbazine, prednisolone and bleomycin); LSG9: VEPA-B + M-FEPA + FEPP-AB (vindesine, etoposide, procarbazine, prednisolone, doxorubicin and bleomycin); mLSG9#1: VEPA + FEPP (vindesine, etoposide, procarbazine and prednisolone); mLSG9#2: VEPA + FEPP-AB; R: rituximab; RT: radiotherapy; CR: complete response; CRu: complete response unconfirmed; PR: partial response; SD: stable disease; PD: progressive disease; MSI: microsatellite instability; L: loss of heterozygosity (LOH); L : microsatellite change ambiguous for LOH or MSI; –: no change.

K. Miyashita et al. / Leukemia Research 32 (2008) 1183–1195

DNA polymerases are particularly prone to slippage and, consequently, misalignments are formed between the template and nascent strands. MMR counteracts such strand misalignments that occur during DNA replication. If uncorrected, these errors are fixed after a next round of replication as addition or deletion of one or more repeat units. Thus, MSI, in which tumour cells accumulate this type of microsatellite length alterations, is considered to reflect cellular MMR deficiency. As the MSI+ phenotype is frequently associated with various human malignancies, analyses of microsatellite instability have been prevalent in the field of oncology. However, the reported frequency for MSI+ tumours is not uniform in each malignancy. Also in non-Hodgkin lymphoma, the reported frequencies differ widely in the literature. Although a number of studies have been done to address this subject, the MSI frequency varies from 0% to 100% (see Section 4). This diversity in the data may partly derive from the heterogeneity in the disease subtypes or ethnical and/or geographical differences in the patient populations examined. However, we believe that, in addition, methodological problems left in assay techniques may also account for some of the variability. Although analysis of MSI is now commonplace, a precise designation of MSI+ is in fact difficult. Changes in microsatellite lengths are sometimes as minimal as a single repeat unit. In addition, cells carrying microsatellite alterations are not always major in a given sample. In an assay technique using the conventional sequencing gel electrophoresis and autoradiography, it appears difficult to resolve polymerase chain reaction (PCR) products of microsatellite sequences, precisely and quantitatively. PCR itself is also not free from variability. The most widely used thermostable DNA polymerase, Taq, has a terminal deoxynucleotidyl transferase (TDT) activity, which adds one additional nucleotide to PCR products. TDT activity of Taq polymerase is variably expressed, depending on the conditions used, and, consequently, increases the complexity of PCR products. In the conventional microsatellite analysis, the frequency for minor microsatellite changes, such as alterations of limited numbers of repeat units, may have been underestimated. We have applied our fluorescent technique [7] to determine the frequency for MSI in non-Hodgkin lymphomas. In this technique, several devices have been made to improve the detection characteristics, the migration accuracy of electrophoresis and the reproducibility of PCR. Furthermore, we focused our attention on the relationship between the MSI+ phenotype and the response to chemotherapy, since it is now widely accepted that MMR deficiency confers resistance to antineoplastic agents on tumour cells [8,9]. In colorectal cancer, MSI, reflecting cellular defects in MMR, is now suggested to be an important biomarker that predicts the tumour response against chemotherapy [10]. Here, we report an MSI frequency in non-Hodgkin lymphomas determined using our fluorescent technique. Our study also revealed an intriguing relationship between the MSI+ phenotype and the response to chemotherapy in lymphoma patients.

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2. Materials and methods 2.1. Patients and tissue specimens Tumour specimens were collected from 59 non-Hodgkin lymphoma patients who underwent treatment in the Department of Hematology, National Kyushu Cancer Center from 1987 to 2003. The patient characteristics are summarised in Table 1. The male and female patients were 31 and 28, respectively. The age at diagnosis ranged from 22 to 86 years old, with a mean of 59 years old. All the patients were not associated with any forms of immunodeficiency. The histological subtype of each tumour was determined according to the World Health Organization (WHO) Classification [11]. Forty-five patients received CHOP or VEPA-based initial chemotherapy [12,13]. In elderly patients or those with an impaired cardiac function, pirarubicin was administered instead of doxorubicin [14]. Most of the patients at stage I or II received several courses of chemotherapy followed by radiotherapy. Some responders at stage III or IV received radiotherapy after chemotherapy. Some follicular lymphoma patients who responded to initial CHOP-therapy were treated with a combination of CHOP and rituximab. The response to initial treatments was judged according to the International Workshop criteria [15]. Corresponding normal tissues as a control for MSI analysis were collected from either the bone marrow or the peripheral blood that did not contain tumour cells more than 10% of the total mono-nuclear cell number. Written informed consent for studies using the tissues was obtained from each patient. Ethical approval was obtained from the IRB of National Kyushu Cancer Center. 2.2. DNA extraction Tumour specimens were lysed in digestion buffer (10 mM Tris–Cl pH 8.0, 0.1 M EDTA pH 8.0, 0.5% SDS, 20 ␮g/ml pancreatic RNase). After treatment with proteinase K and extraction with phenol, DNA was precipitated with ethanol, then dissolved in 1X TE (10 mM Tris–Cl pH 7.5, 1 mM EDTA). The concentration of DNA was determined by OD260 using a spectrophotometer. The quality of DNA was checked by agarose gel electrophoresis. 2.3. Microsatellite instability Microsatellite analysis using fluorescence-labelled primers and an automated DNA sequencer has been described in detail [7]. Briefly, five human dinucleotide microsatellites, D2S123, D5S107, D10S197, D11S904 and D13S175, were amplified by polymerase chain reaction (PCR). Forward primers were labelled with the fluorescent compound, 6-FAM (6-carboxyfluorescein) or HEX (6carboxy-2 ,4 ,7 ,4,7-hexachloro-fluorescein). TaKaRa Taq (TaKaRa Co. Ltd., Tokyo, Japan) was used as a thermostable polymerase. To compare the electrophoretic profiles between two samples, 6-FAM-labelled products and HEX-labelled

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Fig. 1. Microsatellite instability observed in non-Hodgkin lymphomas. The amount of each DNA fragment is quantitatively detected and its size is standardised with an accuracy of one base pair, using co-electrophoresed size markers. Representative results for MSI are shown: red lines, tumour; green lines, normal control. All the microsatellite changes observed in non-Hodgkin lymphoma were Type A; (A) ML20; (B) ML02; (C) ML10; (D) ML49. Patient codes in the parenthesis correspond to those used in Table 1.

products were mixed and co-electrophoresed in the ABI310 sequencer (Applied Biosystems, Foster City, CA, USA). The data were processed using the GeneScan software (Applied Biosystems). 2.4. hMSH2 and hMLH1 sequencing All the exons and exon-intron junctions of hMSH2 and hMLH1 were amplified by PCR using Taq polymerase with 3 exonuclease activity, TaKaRa Ex Taq (TaKaRa Co. Ltd.). The sequencing strategy used is the same as the one reported by Kolodner et al. [16,17], except that the sequence complementary for M13 universal primer was deleted from each of the primer sequences, and that one-step PCR was employed. PCR products were used as a template for cycle sequencing reactions using BigDye Terminator Cycle Sequencing Kit (Applied Biosystems). Mutations found in one PCR product were verified by reverse sequencing and finally confirmed in two independently amplified PCR products.

Cruz Biotechnology Inc., Santa Cruz, CA, USA), anti-PCNA; PC10 (DakoCytomation California Inc., Carpinteria, CA, USA), non-specific IgG; X931 and X936 (DakoCytomation California Inc.). 2.6. Statistical analysis To examine the correlation between the clinicopathological variables of the patients and the MSI status, Fisher’s exact probability test was used. Correlations between the MSI status and the patient response to chemotherapy were also examined using Fisher’s exact probability test. The cumulative survival curves were calculated using the Kaplan–Meier method and compared using the logrank (Mantel–Cox) method.

2.5. hMSH2 and hMLH1 immunohistochemistry Tissue specimens were fixed in buffered 10% formaldehyde and embedded in paraffin. Prior to the assay, the specimens were sectioned at 4 ␮m and deparaffinised using xylene. Immunohistochemistry was performed using the HISTOFINE SAB-PO(M)/(R) Kit (NICHIREI CORPORATION, Tokyo, Japan). Three independent antibodies against MSH2 and MLH1 were used to confirm the results. Expression of PCNA was also used as a positive control for nuclear staining. The antibodies used were as follows: anti-MSH2; Ab-1, Ab-2 (Oncogene Research Products, Cambridge, MA, USA) and MSH-2 (BD Biosciences Pharmingen, Hamburg, Germany), anti-MLH1; Ab-1 (Oncogene Research Products), MLH-1 (BD Biosciences Pharmingen) and C-20 (Santa

Fig. 2. hMSH2 and hMLH1 sequence alterations in non-Hodgkin lymphomas exhibiting MSI. Sequences for all the exons including exon-intron boundaries of hMSH2 and hMLH1 were determined using an automated sequencer. The sequence alterations found at (A) codon 390 of hMSH2 (patient ML49) and (B) codon 219 of hMLH1 (patient ML55) are shown.

K. Miyashita et al. / Leukemia Research 32 (2008) 1183–1195

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Table 2 Mismatch repair gene alterations found in the non-Hodgkin lymphomas exhibiting MSI Case

ML02 ML09 ML10 ML20 ML26 ML49 ML55 ML62

hMSH2

hMLH1

Sequence

Nuclear expression

Sequence

Nuclear expression

– – – – – L390F (CTT to TTT) – L390F (CTT to TTT)

P P P P P P P P

– – – – – – I219V (ATC to GTC) –

P P P P P P P P

MSI: microsatellite instability; –: no alterations in sequence; P: positive nuclear staining in immunohistochemistry.

3. Results 3.1. Detection of microsatellite instability in non-Hodgkin lymphomas using High-Resolution Fluorescent Microsatellite Analysis (HRFMA) We have established a sensitive fluorescent technique for microsatellite analysis, High-Resolution Fluorescent

Microsatellite Analysis (HRFMA) [7]. Application of this technique to non-Hodgkin lymphomas revealed that microsatellite instability (MSI) does occur in this malignancy. In our panel of 59 non-Hodgkin lymphomas, eight tumours (8/59, 14%) exhibited significant length changes in the dinucleotide microsatellite sequences. Examples are shown in Fig. 1. These alterations were noted mainly in one locus among the five microsatellites examined. Established

Fig. 3. hMSH2 and hMLH1 immunohistochemistry in MSI+ lymphomas. Expressions of hMSH2 and hMLH1 in lymphoma cells were examined by immunohistochemistry using three independent antibodies. Representative results obtained using anti-hMSH2 (Oncogene Research Products, Ab-1), anti-hMLH1 (Oncogene Research Products, Ab-1) and non-specific IgG are shown (patient ML10 and ML62). No abnormal expression of these proteins was observed in the MSI+ lymphomas.

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guidelines for classification of MSI utilise the frequency of changes in a set of microsatellite markers, i.e. MSI-H and -L [18]. According to this classification, MSI-L predominated in this panel. Among the MSI+ lymphomas, microsatellite changes at more than one locus, i.e. MSIH, were observed in only one tumour (1/59, 2%), while seven were categorised as MSI-L (7/59, 12%) (Table 1). In other human malignancies, typically in colorectal cancer, use of HRFMA reveals two qualitatively distinct modes of dinucleotide microsatellite changes, i.e. Type A and Type B [19–21]. In Type A instability, length changes are relatively small and affect ≤6 base pairs, whereas in Type B more dramatic changes involving ≥8 base pairs are observed. From this point of view, MSI observed in non-Hodgkin lymphomas was uniformly Type A (Fig. 1). Throughout these analyses using this technique, results were highly reproducible in several independent experiments. Neither additional peaks nor changes in the ratio between peaks were noted. Microsatellite instability (MSI) is generally regarded as reflecting a cellular deficiency in DNA mismatch repair (MMR). We further investigated the relationship between MSI and MMR gene inactivation in our panel of non-Hodgkin lymphomas. The two major MMR genes, hMSH2 and hMLH1, of eight MSI+ tumours were sequenced. Sequence alterations causing amino acid substitutions were identified in three of the eight lymphomas (Fig. 2 and Table 2). They all were single-base substitutions, including one acknowledged as a polymorphism, I219V, in hMLH1 [22]. We next tested the possibility of epigenetic silencing of these genes using immunohistochemistry, since hMLH1 gene silencing often occurs by altered methylation in the promoter sequences [23–25]. The eight MSI+ lymphomas exhibited neither a loss of nuclear expression nor an abnormal subcellular distribution of hMSH2 and hMLH1 (Fig. 3 and Table 2).

Table 3 Relationship between MSI and the response to CHOP or VEPA-based initial therapies CR/PR

SD/PD

Subtotal

MSI+ MSI−

1 30

4 10

5 40

Subtotal

31

14

45

p = 0.027. MSI: microsatellite instability; CR: complete response and complete response unconfirmed; PR: partial response; SD: stable disease; PD: progressive disease.

cal variables, i.e. age, gender, clinical stage, International Prognostic Index (IPI) and histological subtype. However, regarding the clinical stage, we noted that MSI+ tumours tended to be more frequent in patients at an early stage (p = 0.051). More significantly, in lymphoma patients initially treated by CHOP/VEPA-based therapies, the response to chemotherapy was significantly poor in those with MSI+ tumours (p = 0.027, Tables 1 and 3). In patients with microsatellite-stable tumours, 75% (30/40) were judged to be CR/PR, whereas only one (20%, 1/5) responded to the treatment in MSI+ lymphoma patients (Table 3). Limited response to chemotherapy in patients with MSI+ tumours suggests a poor patient outcome in this group. Cumulative overall survival for patients with MSI+/− lymphomas was determined, using the Kaplan–Meier method (Fig. 4). As expected, the patient survival at 1 year after treatment was significantly different between these two populations (logrank test, p = 0.046) (Fig. 4). However, the difference at 4 years after treatment was not statistically significant. These findings suggest a possibility that MMR defects in tumour cells, manifested as MSI, influence the tumour sensitivity against antineoplastic agents and, consequently, lead to poor clinical outcomes in non-Hodgkin lymphoma patients.

3.2. Poor response to chemotherapy in non-Hodgkin lymphoma patients with MSI+ tumours It is now widely accepted that DNA mismatch repair (MMR) activity modulates the cellular sensitivity against various anticancer drugs, including alkylating agents [8], fluorouracil (5-FU) [26–28], cis-platinum (CDDP) [29–31], camptothecin [32–34] and etoposide [30,34,35]. In colorectal cancer, MSI, reflecting a cellular MMR deficiency, is now suggested to be an important biomarker to predict the tumour response against chemotherapy [10]. Since antineoplastic agents play a major role in the treatment of lymphoma patients, we next examined the relationship between the MSI status in tumours and their response to chemotherapy. In the panel of non-Hodgkin lymphoma patients treated by CHOP/VEPA-based initial therapies, including those plus irradiation or rituximab, there was no significant difference between MSI+ tumours and those with stable microsatellites in commonly used clinicopathologi-

Fig. 4. Overall survival of patients with MSI+ non-Hodgkin lymphomas. In patients with MSI+ lymphomas, the 1-year overall survival was significantly worse than in those without MSI (probability of survival: MSI+ ; 0.400, MSI− ; 0.771, p = 0.046). However, this survival difference was not statistically confirmed at 4 years after the initial CHOP or VEPA-based therapies (probability of survival: MSI+ ; 0.400, MSI− ; 0.536, p = 0.187).

Table 4 Frequencies of MSI in non-Hodgkin lymphoma: a survey of the literature Author

Year

Subtype

No. of patients

Marker No.

Bedi et al.

1995

NHL NHL

Robledo et al.

1995

NHL

Peng et al. Randerson et al.

1996 1996

MALT FL

Gartenhaus et al.

1996

Volpe et al.

1996

(HIV−) (HIV+)

%H

%L

Positive marker

Ref. No.

0 67

0 50

0 17

d

10

d, tr

E, R

10

0

10

d

40 40

5 12

d d, te, p

F F

83 18

53 5

30 13

d d, te, p

[69] [42]

CLL Richter

27 2

10

m, d, tr, te

R

11 50

7 0

4 50

m, d, tr, te

[70]

CLL Richter

18 1

9

d, tr, te

R

0 100

0 100

0 0

tr, te

[71]

1996

NHL

(PTLD)

1997

NHL NHL BL FLa FLb

(HIV−) (HIV+) (cell line)

7

6

d

R

29

29

0

29 17 12 8 3

5

d, tr, te

R

14 6 8 25 0

0 6 0 0 0

14 0 8 25 0

Larson et al.

1997

NKTCL NKTCL

(IC) (ID)

3 3

6

d

R

0 33

0 33

[68] hMSH2 0% (exon 13)

[39]

d

[72]

d, tr, te

[40]

0 0

d

[73]

De Vita et al.

1997

NHL

(HCV+)

8

15

d, tr, te

R

25

0

25

d

[74]

Chong et al.

1997

MALT DLBCL

(gastric) (gastric)

6 14

13

m, d

R

67 100

0 7

67 93

m, d

[75]

(gastric)

Xu et al.

1998

MALT

33

9

d, te

R

21

0

21

d, te

[43]

Sol Mateo et al.

1998

SMZL MALT

20 22

7

te

R

10 32

0 5

10 27

te

[76]

Indraccolo et al.

1999

NHL

21

10

d, tr, te

R

19

0

19

d

Hodges et al. Takakuwa et al.

1999 1999

ALCL NKTCL

(nasal)

5 5

7 15

d m, d

R R

0 20

0 0

0 20

– d

[77] [78]

Hoeve et al.

1999

MALT DLBCL

(gastric) (gastric)

23 3

17

m, d, tec

F, R

13 0

0 0

13 0

d, te

[79]

Furlan et al. Herranz et al. Starostik et al.

1999 1999 2000

MALT NHL DLBCL

(gastric) (gastric)

14 30 31

10 6 73

m, dc m, d m, d, tr, te, p

S S F

29 17 87

0 7 0

29 10 87

dc m, d U

[80] [81] [48]

Starostik et al.

2000

MALT DLBCL

(gastric) (gastric)

25 31

29 118

d, tr, te m, d, tr, te, p

F

12 90

0 0

12 90

U

Takakuwa et al.

2000

NHL

(thyroid)

19

16

Scarisbrick et al.

2000

MF SS CTCL

44 6 4

8

[41]

hMSH2 0% (exon 5 &13) hMLH1 0% (exon 9 &16)

hMSH2 0% hMLH1 0% hMSH2 0% hMLH1 0%

K. Miyashita et al. / Leukemia Research 32 (2008) 1183–1195

Larson et al.

10

%MSI

MMR gene alterations in MSI+ tumours Mutation IHC

R

Gamberi et al.

8

Variety

MSI

d

(gastric)

6 6

Methods for detection

[49]

F

58

5

53

d, p

[82]

R

27 17 0

7 17 0

20 0 0

d

[83]

1189

m, d, p d

1190

Table 4 (Continued ) Author

Year

Subtype

No. of patients

2000

FLb

Sanz-Vaque et al.

2001

MCL CLL Richter

Skacel et al.

2002

MALT DLBCL

Teruya-Feldstein et al. Scarisbrick et al.

2002 2003

NHLd MF

Hiyama et al.

2003

MALT DLBCL

Fulop et al.

2003

CLLa Richter

Baumgartner et al. Jordanova et al.

2003 2003

ETL DLBCL

(testis & CNS)

Duval et al.

2004

NHL NHL

(IC) (ID)

Rubben et al.

2004

CTCL

Niv et al.

2004

MALT

(gastric) (gastric)

(gastric) (gastric)

(gastric)

Methods for detection

No.

Variety

8

8

d, tr, te

R

29 24 4

10

d, p

10 5

5

6 51

MSI %MSI

MMR gene alterations in MSI+ tumours

Ref. No.

%H

%L

Positive marker

Mutation

63

13

50

d, tr, te

hMSH2 0% (exon 13) hMLH1 0% (exon 9, 11, 14, 15 &16)

F

0 13 0

0 0 0

0 13 0

d

[84]

d

F

40 0

20 0

20 0

d

[85]

5 9

m, d m, dc

R R

0 28

0 8

0 20

– m, dc

24 20

5

m, d, te

R

25 10

0 5

25 5

d, te

10 9

8

d, tr, te

R

30 77

0 44

30 33

d, tr, te

26 15

47 8

d, tr, te, p m, dc

F F

69 33

0 0

69 33

U m, dc

364 239

2

m

F

0 5

0 5

NE NE

m

10

6

d

F

100

NE

NE

d

13

5

m, m + tr, d

F

77

54

23

m + tr, d

hMLH1 0% (exon 2, 9, 15 &16)

IHC [44]

hMSH2 0% hMLH1 45%

[86] [52]

[87] hMSH2 0% (exon 13) hMLH1 0% (exon 9, 11, 14, 15 &16)

[88]

hMSH2 0% hMLH1 0%

[89] [50] [45]

hMSH2 0% hMLH1 0%

[51] [90]

IC: immunocompetent; ID: immunodeficient; CNS: central nerve system; m: mononucleotide; d: dinucleotide; tr: trinucleotide; te: tetranucleotide; p: pentanucleotide; E: ethidium bromide staining; F: fluorescent system; R: radiolabelling; S: silver staining; %MSI: % of microsatellite instability; %H: % of MSI-H; %L: % of MSI-L; U: unknown; NE: not evaluable; IHC: immunohistochemistry. a Cases without histological transformation at progression. b Cases with histological transformation at progression. c Including NCI working reference panel. d Cases from 21 hereditary non-polyposis colorectal cancer (HNPCC) kindreds.

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Nagy et al.

Marker

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4. Discussion The fluorescent technique used herein enabled us the unequivocal designation of the MSI phenomenon in a panel of 59 non-Hodgkin lymphoma patients, and has revealed the existence of MSI+ tumours in this malignancy. In our panel, eight MSI+ tumours were found (8/59, 14%). All the observed changes were Type A and noted mainly at one locus of the examined microsatellites, which implies that MSI-L predominates in non-Hodgkin lymphoma. In this panel, the MSI+ phenotype is associated neither with deleterious mutations in hMSH2 and hMLH1, nor with an epigenetic silencing of hMLH1 gene expression. Instead, some missense sequence alterations with an unknown pathogenetic significance were found. Intriguingly, the MSI+ phenotype correlates with a poor response to chemotherapy in these lymphoma patients (p = 0.027). Consequently, the clinical outcomes at 1 year after treatment were significantly less favourable in MSI+ lymphoma patients than in those with microsatellite-stable tumours (p = 0.046), although the survival difference at 4 years after treatment was not statistically confirmed. In the literature, the data reported to date concerning MSI in non-Hodgkin lymphoma are indeed diverse (Table 4). This may be partly due to the heterogeneity in the patient panels used that differ in the disease subtype. In addition to this, the variety in methods used to detect MSI, including microsatellite markers and detection techniques, may also contribute to this diversity in the data. We have established a new fluorescent technique to detect microsatellite changes sensitively and quantitatively [7], and we have approached MSI in various human malignancies, using this technique [20,21,36–38]. In the present study, we applied this technique to non-Hodgkin lymphoma, to determine the frequency of MSI in this malignancy. In our panel, patients were carefully selected so that the panel may not be biased regarding the disease subtype and reflect the actual frequencies of each subtype. In addition, no patients associated with immunodeficiency was included in this panel. As a result, we detected MSI in eight tumours (8/59, 14%) and this frequency appears to be highly consistent with those thus far reported using similar patient panels [39–41]. One important finding of our study is that MSI-L predominated in our panel of non-Hodgkin lymphomas. This conclusion is also supported by an inspection of published data (Table 4). Throughout the intensive analyses using the fluorescent technique, we found that there are at least two qualitatively different modes of dinucleotide microsatellite changes in human malignancies. We designate them as Type A and Type B [19–21]. In Type A instability, length changes are limited to within 6 base pairs, whereas Type B alterations involve ≥8 base pairs. From our previous studies, it has become evident that Type A MSI tends to occur in a limited number of microsatellite markers examined and, consequently, correspond to MSI-L, whereas Type B changes are always noted in a majority of the markers and are connected to MSI-H [20]. Also in this study, all the observed microsatellite changes were uniformly Type

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A. Indeed, an examination of published data reveals that in some reports Type A MSI was observed [39,40,42–44]. Therefore, we conclude that MSI-L/Type A instability predominates in non-Hodgkin lymphomas, in particular ones occurred in immunocompetent patients, since Duval et al. have reported the MSI-H phenotype in immunodeficiencyassociated lymphomas [45]. Another important implication of our results is that some non-Hodgkin lymphomas may harbour a defect in DNA mismatch repair (MMR), since the observed microsatellite changes were Type A. Based on the analyses using human and mouse cell lines with a known defect in MMR genes and tumours that occurred in MMR gene-knock out animals, we have concluded that Type A MSI is a direct consequence of defective MMR [21,36]. Uniform Type A microsatellite changes in nonHodgkin lymphomas strongly suggest that MMR deficiency occurs in this malignancy. In general, MSI is connected to a MMR defective phenotype. However, deleterious mutations, i.e. nonsense or frameshift mutations, are rare in sporadic MSI+ tumours and, instead, missense mutations with an unknown significance are found, while the former mutations are relatively frequent in MSI+ tumours with a genetic background such as ones occurring in hereditary non-polyposis colorectal cancer (HNPCC). Our present study is the first that sequenced full lengths of the two major MMR genes, hMSH2 and hMLH1, in MSI+ non-Hodgkin lymphomas, and the results of sequencing confirmed a similar tendency of mutation. Genomic rearrangements such as a large deletion are recently regarded as a possible cause for defective MMR. However, using the multiple ligation-dependent probe amplification (MLPA) method, these events were reported to be relatively less frequent even in HNPCC [46,47]. An LOH event in the D2S123 marker was indeed found in one MSI+ tumour of our panel (data not shown). However, this tumour (ML62) harboured a missense mutation in hMSH2 (Table 2). Immunohistochemistry also revealed no negative staining that suggests structural abnormalities in MMR gene products or an epigenetic silencing of the hMLH1 gene. Some groups similarly reported no case of possible hMLH1 gene silencing in non-Hodgkin lymphomas [48–51], whereas Scarisbrick et al. reported a relatively high incidence of loss of hMLH1 expression [52]. Existence of MMR defective lymphomas may also provide insight into the mechanism of genetic instability in lymphomagenesis. Genetic instability in tumours has been regarded as deriving from several pathways, (a) chromosomal instability (CIN) with a globalised numerical and structural abnormalities in chromosomes, (b) disease-specific chromosomal rearrangement (DSCR), a limited form of CIN to yield a disease-specific fusion gene or an abnormal chromosomal context mainly by chromosomal translocation and (c) ‘microsatellite mutator phenotype (MMP) [53,54]’ characterised by MSI, particularly MSI-H/Type B instability. From our previous observations, it is suggested that tumours exhibiting Type A MSI may arise via the CIN pathway, since mutations in the tumour suppressor genes such as p53 are

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frequently associated with CIN, and Type A strongly correlated with p53 mutation [21]. This conclusion may also be supported by several recent reports that suggest a connection between MSI-L and p53 mutation [55–57]. Our findings in the present study suggest that some of nonHodgkin lymphomas, i.e. MSI+ lymphomas, may arise via the CIN pathway as do colorectal carcinomas. Indeed, karyotype analyses revealed that they harbour various numerical and structural abnormalities in the chromosomes (data not shown). Furthermore, we sequenced the p53 gene in the eight MSI+ lymphomas, to test this hypothesis. Mutation in an acknowledged hot spot, R273H, was indeed found in one (data not shown). More importantly, MSI+ tumours include follicular or Burkitt lymphomas characterised by DSCR such as t(14;18) or t(8;14) (Table 1), which implies that in non-Hodgkin lymphoma the CIN and DSCR pathways may overlap. In colorectal cancer, it was initially proposed that the CIN and MMP pathways are mutually exclusive [58,59]. However, several recent reports suggest that there might be an oversimplification in this distinction [21,60–63]. Similarly in non-Hodgkin lymphoma, DSCR has been regarded as a major aetiology in some disease subtypes. Non-Hodgkin lymphoma may in fact arise via several complicated pathways that mutually overlap. One important clinical implication of our results is that MMR deficiency influences tumour sensitivity against antineoplastic agents and, consequently, leads to poor clinical outcomes in non-Hodgkin lymphoma patients. In 1993, Karran and colleagues [8] first demonstrated that cells defective in MMR are highly resistant to mono-functional alkylating agents such as N-methyl-N -nitro-N-nitrosoguanidine (MNNG), methyl methanesulfonate (MMS) or N-methyl-Nnitrosourea (MNU). In cells treated with alkylating agents, base mismatches are formed in DNA replication, because alkylated bases pair incorrectly, and MMR reacts with these mismatches. In case that base alkylation is frequent on the genome, reiterative and futile repair processes are continued, and, as a consequence, cell death is induced. Therefore, MMR activity is regarded as one of the most important determinants of cellular sensitivity against alkylating agents. To date, it has been reported that MMR deficiency also leads to an acquired resistance to various other antineoplastic agents including fluorouracil (5-FU) [26–28], cis-platinum (CDDP) [29–31], camptothecin [32–34] and etoposide [30,34,35], although molecular mechanisms for such resistance remain unclear. In colorectal cancer, it has been accepted that patients with MSI+ tumours generally exhibit favourable clinical outcomes [2,64]. However, this is not consistent with the cellular resistance conferred by defective MMR. Ribic et al. has recently shown that in patients with microsallite-stable, i.e. MMRproficient, tumours the survival was improved by adjuvant chemotherapy, whereas the survival of patients with MSI+ tumours did not differ between the populations with/without chemotherapy [10]. In non-Hodgkin lymphoma, Scarisbrick et al. reported that the survival in patients with MSI+ tumours was significantly worse [52]. They connected the

less favourable patient outcomes to a relatively higher age and more frequent advanced disease in the population. In the present study, we have shown that the clinical outcomes were significantly less favourable in MSI+ lymphoma patients than those with microsatellite-stable tumours, although the survival difference was confirmed only at 1 year after treatment. This may, however, appear rather rational, considering that in this study the microsatellites were analysed in tumour cells obtained at the time of diagnosis, i.e. right before the initial treatments, and that long-term patient survivals obviously depend on various conditions including sensitivities to subsequent treatment. We therefore propose that, in addition to the clinicopathological backgrounds, sensitivity to chemotherapy may contribute to the survival difference between patients with MSI+ lymphomas and those with microsatellite-stable tumours. From this point of view, our present report is the first that has discussed the connection between cellular resistance derived from MMR deficiency and the clinical outcomes in non-Hodgkin lymphoma patients. CHOP/VEPA-based therapies are comprised of three antineoplastic agents, i.e. cyclophosphamide (CPA), doxorubicin (DXR) and vincristine. Interstrand cross-linking formed by CPA or intercalation of DXR to DNA may cause double strand break (DSB) on the genomic DNA in cells treated with these agents. MMR counteracts erroneous DNA recombination induced as a process of DSB repair [65]. In MMR defective tumour cells, efficiency in recombinational DSB repair may be abnormally elevated and this may lead to a survival advantage. Precise assessment of the MSI status in non-Hodgkin lymphomas will provide important information to rationalise the treatment choice, and, consequently, improve the survival of the patient populations. Such efforts may finally realise algorithmic approaches for the management of lymphoma patients, in which more individualised treatment will thus be possible.

Acknowledgements We are most grateful to P. Karran and M. Sekiguchi for their helpful advice. The expert assistance in DNA extraction, DNA sequencing and immunohistochemistry by Y. Ogata, M. Hanaki, S. Kato and Y. Kubota is also gratefully acknowledged. We also thank K. Nishiyama and H. Yamamoto for their histopathological expertise. This study was supported by a Grant-in-aid for Cancer Research from the Ministry of Health, Labour and Welfare and grants from the Ministry of Education, Science, Sports and Culture of Japan. K.M. was supported by a grant from the Foundation for Promotion of Cancer Research (Japan) for the 3rd Term Comprehensive 10-Year-Strategy for Cancer Control. Contributions. Kaname Miyashita performed all the microsatellite/gene analyses and prepared the manuscript. Kei Fujii performed the immunohistochemical analyses. Yu Yamada commented on the microsatellite/gene analyses. Hiroyoshi Hattori commented on the microsatellite/gene

K. Miyashita et al. / Leukemia Research 32 (2008) 1183–1195

analyses. Kenichi Taguchi directed the immunohistochemical analyses. Takeharu Yamanaka commented on the statistical analyses. Mitsuaki A. Yoshida commented on the cytogenetic aspects of the study. Jun Okamura supported and commented on the study. Shinya Oda designed and organised the study. Koichiro Muta commented on the study. Hajime Nawata commented on the study. Ryoichi Takayanagi commented on the study. Naokuni Uike proposed the study and provided the materials. References [1] Ionov Y, Peinado MA, Malkhosyan S, Shibata D, Perucho M. Ubiquitous somatic mutations in simple repeated sequences reveal a new mechanism for colonic carcinogenesis. Nature 1993;363:558–61. [2] Thibodeau SN, Bren G, Schaid D. Microsatellite instability in cancer of the proximal colon. Science 1993;260:816–9. [3] Aaltonen LA, Peltomaki P, Leach FS, Sistonen P, Pylkkanen L, Mecklin JP, et al. Clues to the pathogenesis of familial colorectal cancer. Science 1993;260:812–6. [4] Peltomaki P, Lothe RA, Aaltonen LA, Pylkkanen L, Nystrom-Lahti M, Seruca R, et al. Microsatellite instability is associated with tumors that characterize the hereditary non-polyposis colorectal carcinoma syndrome. Cancer Res 1993;53:5853–5. [5] Fishel R, Lescoe MK, Rao MR, Copeland NG, Jenkins NA, Garber J, et al. The human mutator gene homolog MSH2 and its association with hereditary nonpolyposis colon cancer. Cell 1993;75:1027–38. [6] Leach FS, Nicolaides NC, Papadopoulos N, Liu B, Jen J, Parsons R, et al. Mutations of a mutS homolog in hereditary nonpolyposis colorectal cancer. Cell 1993;75:1215–25. [7] Oda S, Oki E, Maehara Y, Sugimachi K. Precise assessment of microsatellite instability using high resolution fluorescent microsatellite analysis. Nucl Acids Res 1997;25:3415–20. [8] Branch P, Aquilina G, Bignami M, Karran P. Defective mismatch binding and a mutator phenotype in cells tolerant to DNA damage. Nature 1993;362:652–4. [9] Swann PF, Waters TR, Moulton DC, Xu YZ, Zheng Q, Edwards M, et al. Role of postreplicative DNA mismatch repair in the cytotoxic action of thioguanine. Science 1996;273:1109–11. [10] Ribic CM, Sargent DJ, Moore MJ, Thibodeau SN, French AJ, Goldberg RM, et al. Tumor microsatellite-instability status as a predictor of benefit from fluorouracil-based adjuvant chemotherapy for colon cancer. N Engl J Med 2003;349:247–57. [11] World Health Organization Classification of Tumours. In: Jaffe ES, Harris NL, Stein H, Vardiman JW, editors. Pathology and genetics of tumours of haematopoietic and lymphoid tissues. Lyon: IARC Press; 2001. [12] Fisher RI, Gaynor ER, Dahlberg S, Oken MM, Grogan TM, Mize EM, et al. Comparison of a standard regimen (CHOP) with three intensive chemotherapy regimens for advanced non-Hodgkin’s lymphoma. N Engl J Med 1993;328:1002–6. [13] Shimoyama M, Ota K, Kikuchi M, Yunoki K, Konda S, Takatsuki K, et al. Chemotherapeutic results and prognostic factors of patients with advanced non-Hodgkin’s lymphoma treated with VEPA or VEPA-M. J Clin Oncol 1988;6:128–41. [14] Miller AA, Schmidt CG. Clinical pharmacology and toxicity of 4 -Otetrahydropyranyladriamycin. Cancer Res 1987;47:1461–5. [15] Cheson BD, Horning SJ, Coiffier B, Shipp MA, Fisher RI, Connors JM, et al. NCI Sponsored International Working Group. Report of an international workshop to standardize response criteria for non-Hodgkin’s lymphomas. J Clin Oncol 1999;17:1244–53. [16] Kolodner RD, Hall NR, Lipford J, Kane MF, Rao MR, Morrison P, et al. Structure of the human MSH2 locus and analysis of two Muir-Torre kindreds for msh2 mutations. Genomics 1994;24:516–26.

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