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Minimal residual disease in canine lymphoma: An objective marker to assess tumour cell burden in remission
Accepted Manuscript Title: Minimal residual disease in canine lymphoma: An objective marker to assess tumour cell burden in remission Author: Masahiko Sato, Jumpei Yamazaki, Yuko Goto-Koshino, Asuka Setoguchi, Masashi Takahashi, Kenji Baba, Yasuhito Fujino, Koichi Ohno, Hajime Tsujimoto PII: DOI: Reference:
S1090-0233(16)30064-8 http://dx.doi.org/doi: 10.1016/j.tvjl.2016.05.012 YTVJL 4821
To appear in:
The Veterinary Journal
Accepted date:
24-5-2016
Please cite this article as: Masahiko Sato, Jumpei Yamazaki, Yuko Goto-Koshino, Asuka Setoguchi, Masashi Takahashi, Kenji Baba, Yasuhito Fujino, Koichi Ohno, Hajime Tsujimoto, Minimal residual disease in canine lymphoma: An objective marker to assess tumour cell burden in remission, The Veterinary Journal (2016), http://dx.doi.org/doi: 10.1016/j.tvjl.2016.05.012. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Review Article
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Minimal residual disease in canine lymphoma: An objective marker to assess tumour
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cell burden in remission
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Masahiko Sato a, Jumpei Yamazaki b, Yuko Goto-Koshino c, Asuka Setoguchi d, Masashi
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Takahashi c, Kenji Baba e, Yasuhito Fujino f, Koichi Ohno f, Hajime Tsujimoto c,f,*
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a
Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences,
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Colorado State University, Fort Collins, CO 80523, USA
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b
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University, Sapporo, Hokkaido 060-0818, Japan
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c
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University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan
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d
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Kagoshima University, Kagoshima, Kagoshima 890-0065, Japan
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e
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University, Yamaguchi, Yamaguchi 753-8515, Japan
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f
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Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan
Laboratory of Molecular Medicine, Graduate School of Veterinary Medicine, Hokkaido Veterinary Medical Centre, Graduate School of Agricultural and Life Sciences, The Laboratory of Small Animal Internal Medicine, Joint Faculty of Veterinary Medicine, Department of Veterinary Medicine, Joint Faculty of Veterinary Medicine, Yamaguchi Department of Veterinary Internal Medicine, Graduate School of Agricultural and Life
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* Corresponding author: Tel.: +81 3 58415402. E-mail address: [email protected] (H. Tsujimoto). Highlights
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Minimal residual disease (MRD) can be measured in the peripheral blood of dogs with lymphoma (lymphosarcoma).
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The efficacy of chemotherapy in canine lymphoma can be objectively compared by detection of MRD.
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The level of MRD is predictive of relapse and prognosis of canine lymphoma.
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MRD monitoring by real-time quantitative PCR will be of value in evaluating novel
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therapies for canine lymphoma. Abstract
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Lymphoma is the most common haematopoietic malignancy in dogs. Since a high
34
proportion of dogs with lymphoma achieve remission soon after initiation of chemotherapy,
35
an objective marker assessing treatment efficacy is required. Following clinical remission, the
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residual population of tumour cells can be referred to as the minimal residual disease (MRD).
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MRD traditionally has been detected by cytology and flow cytometry; however, if the burden
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of malignant cells is low, these methods might not be sufficiently sensitive to detect MRD. As
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an extension of the development of PCR for antigen receptor gene rearrangements (PARR) in
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dogs, there has been recent progress in the application of real-time quantitative PCR
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(RT-qPCR) to canine lymphoma. With the RT-qPCR system, a very high sensitivity (1 cell per
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10,000 cells) has been achieved by preparing allele-specific oligonucleotide primers and
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probes designed from neoplastic clones of each dog. A series of MRD diagnostics studies
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employing the RT-qPCR system have revealed its usefulness as a prognostic indicator, an
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objective marker of treatment efficacy and a predictor of relapse for dogs with lymphoma
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receiving chemotherapy. Introduction of the MRD monitoring system will provide an
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innovative scientific tool in the development of superior treatments and monitoring strategies
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for canine lymphoma.
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Keywords: Canine; Lymphoma; Chemotherapy; Minimal residual disease; Real-time
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quantitative PCR
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Introduction Lymphoma (lymphosarcoma), the most common haematopoietic malignancy in
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dogs, is characterised by the clonal proliferation of lymphoid cells. In most cases, such as in
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high grade multicentric B cell lymphoma, the disease is initially responsive to chemotherapy.
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However, high rates of relapse and mortality are evident in nearly all cases as a result of
58
disease progression.
59 60
Cyclophosphamide, doxorubicin, vincristine and prednisolone (CHOP)-based
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protocols were introduced in the treatment of canine lymphoma (Greenlee et al., 1990; Keller
62
et al., 1993) as a superior alternative to COP-based protocols (Cotter et al., 1983). The
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combination of CHOP-based protocols with L-asparaginase as adjunct therapy is referred to
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as L-CHOP. Overall response (OR) rate, progression-free survival (PFS) and overall survival
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(OS) in dogs with lymphoma treated with L-CHOP-based protocols (Greenlee et al., 1990;
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Keller et al., 1993; Vail et al., 1996; Myers et al., 1997; Zemann et al., 1998; Khanna et al.,
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1998) were 69-87%, 7.3-12.8 months and 10.1-17.0 months, respectively. In these protocols,
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antineoplastic agents were continuously administered for up to 2-3 years; however,
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subsequent studies have indicated that similar treatment outcomes were achievable with
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maintenance-free L-CHOP-based protocols in up to 25 weeks (Garrett et al., 2002;
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MacDonald et al., 2005; Simon et al., 2006; Hosoya et al., 2007; Burton et al., 2012; Curran
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and Thamm, 2015). The influence of L-asparaginase on the efficacy of the CHOP/COP
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protocols was investigated in dogs with multicentric high grade lymphoma in two
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independent studies, in which outcomes following use of a multi-agent chemotherapy
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protocol, with or without the addition of L-asparaginase (depending on commercial
76
availability), were compared (Jeffreys et al., 2005; MacDonald et al., 2005). Both of these
77
studies suggested that addition of L-asparaginase to CHOP/COP protocols may not improve
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treatment outcomes, suggesting that L-asparaginase could be reserved for rescue protocols.
79 80
Recent studies on the clinical application of chemoimmunotherapy (i.e.
81
chemotherapy combined with an autologous tumour cell vaccine) revealed clinical benefits
82
without incidents of increased toxicity in dogs with B cell lymphomas (Aresu et al., 2014;
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Marconato et al., 2014). Most of the dogs enrolled in these studies had multicentric high
84
grade B cell lymphomas. However, many studies have reported poorer prognoses for high
85
grade T cell lymphomas, indicating the need for alternative protocols. For T cell lymphomas,
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the use of alkylating agent-based protocols, such as L-asparaginase combined with
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mechlorethamine, oncovin, procarbazine and prednisone (L-asparaginase-MOPP) was
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superior to a classic CHOP-based protocol (Brodsky et al., 2009). However, another study
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reported that the use of a generally applied CHOP-based protocol to dogs with T cell
90
lymphoma produced results comparable to those for B cell lymphomas (Rebhun et al., 2011).
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L-CHOP can induce complete remission (CR) rapidly in a large proportion of dogs
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with lymphoma; the median time from the first day of L-CHOP to CR was 11 days (Simon et
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al., 2006). After achieving CR, chemotherapy is usually continued for a certain period, e.g. up
95
to 25 weeks in UW-25 protocol (Garrett et al., 2002). However, most dogs experience relapse
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after termination of, or during, the protocol. This is indicative of a small, residual, population
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of malignant tumour cells that persist within the body, referred to as the minimal residual
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disease (MRD), which are implicated as the source of tumour relapses. In general, following
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initiation of chemotherapy, the amount of detectable malignant cells within the body
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decreases to subthreshold detection limits of clinical diagnostic techniques (clinical
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remission) (Fig. 1). Detection of MRD is facilitated by a sensitive, molecular biological
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technique that may be subject to, in some cases, further subthreshold decreases to levels
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below that of its detection limit (molecular remission) following successful remission
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induction therapy or consolidation therapy. Consequently, the residual malignant cells
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proliferate after a certain period, resulting in molecular relapse leading to clinical relapse (Fig.
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1).
107 108
In human haematological malignancies, the quantity of MRD after treatment can be
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indicative of treatment outcome. High MRD after chemotherapy indicates a poor prognosis
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for follicular lymphoma (Rambaldi et al., 2002), mantle cell lymphoma (Pott et al., 2006),
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adult acute lymphoblastic leukaemia (Bruggemann et al., 2006), and acute promyelocytic
112
leukaemia (Miller et al., 1993). Several reports have documented the usefulness of MRD in
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the molecular assessment of treatment efficacy for diffuse large B cell lymphoma (DLBCL)
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in human beings (Mitterbauer et al., 2001; Uchiyama et al., 2003; Yashima et al., 2003).
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DLBCL is the most common histological subtype of canine lymphoma (Valli et al., 2011) and
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comparative studies between human and canine populations would be possible for this type of
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lymphoma. MRD diagnostics is of high clinical relevance to human patients affected with B
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cell non-Hodgkin lymphomas. This may serve as a surrogate parameter in the evaluation of
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treatment effectiveness and long term prognosis (Ladetto et al., 2013). Subsequently, MRD
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diagnostics performed by real-time quantitative PCR (RT-qPCR) is now the gold-standard
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and currently the most sensitive and broadly applied method for the staging, classification and
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treatment response assessment of human non-Hodgkin lymphoma (Pott et al., 2013; Rosolen
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et al., 2015; Sandlund et al., 2015).
124 125
The assessment of MRD in human patients with lymphoid malignancies has shown
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that the prognoses of patients were strongly influenced by the MRD quantities following
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chemotherapy (or other therapeutic modalities), but were unrelated to the tumour cell burden
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prior to treatment (Mitterbauer et al., 2001; Rambaldi et al., 2002; Pott et al., 2006; Ladetto et
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al., 2013). Although available data for canine lymphomas are limited, there are several studies
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comparing the prognostic values of tumour cell burden amounts before and after
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chemotherapy. Results of the qualitative status (positive or negative) in the PCR for antigen
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receptor gene rearrangements (PARR) before chemotherapy were not related to the prognosis
133
of dogs with lymphoma (Lana et al., 2006). Similarly, a correlative, prognostic significance
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for tumour cell burden prior to the initiation of RT-qPCR-assessed chemotherapy for the
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immunoglobulin heavy chain (IgH) gene was lacking in dogs with multicentric high grade B
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cell lymphoma (Sato et al., 2013). However, MRD quantities as assessed by RT-qPCR at
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weeks 11 and 25 of a standard CHOP protocol (UW-25) (Garrett et al., 2002) were correlated
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with prognosis in dogs with lymphoma (Yamazaki et al., 2010; Sato et al., 2013). On the basis
139
of these observations, MRD quantity after treatment for a certain period (e.g. 11 weeks or
140
more with the UW-25 protocol) could be used as an objective prognostic marker in canine
141
lymphoma.
142 143
Molecular biological detection of tumour cells in lymph nodes and the peripheral
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blood of dogs with lymphoid neoplasia has been reported using PARR, resulting in the
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assessment of clonality and T/B cell lineage of the lymphoid cells (Burnett et al., 2003; Keller
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et al., 2004; Calzolari et al., 2006; Tamura et al., 2006; Valli et al., 2006; Lana et al., 2006;
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Yagihara et al., 2007). PARR has also been used for the detection of MRD in dogs with
148
lymphoma that achieved remission after chemotherapy (Thilakaratne et al., 2010; Manachai et
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al., 2014). Aresu et al. (2014) reported that the combination of PARR and flow cytometry for
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large CD21+ cells was more sensitive than either technique alone in predicting outcomes of
151
dogs with DLBCL after chemotherapy. All of these studies employed the conventional PCR
152
method using universal primers to detect for the rearrangements of antigen receptor genes.
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The sensitivity for detecting neoplastic lymphoid cells was shown to be ~1 per 100 (Burnett et
154
al., 2003). Therefore, it is conceivable that tumour cells with clonal rearrangement can
155
become undetectable during remission.
156 157
In a study by Yamazaki et al. (2008), MRD in the peripheral blood was successfully
158
detected and quantified by RT-qPCR using allele-specific primers and probes in most canine
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lymphoma patients in CR after chemotherapy. Subsequent studies using RT-qPCR procedures
160
enabled the dynamic exploration of tumour cell burden in dogs with lymphoma before, during
161
and after chemotherapy, as well as prior to and during episodes of relapse (Yamazaki et al.,
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2010; Sato et al., 2011a, b, 2013).
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Quantitative assessment of minimal residual disease in canine lymphoma
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A quantitative detection system for MRD in canine lymphoma was developed by
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our group using RT-qPCR (Yamazaki et al., 2008). UL-1, a canine T cell lymphoma-derived
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cell line, was used to examine the specificity and sensitivity of the assay for detecting MRD.
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Allele-specific oligonucleotide primers and probes were designed based on the rearranged T
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cell receptor γ chain (TCRγ) gene sequence of UL-1 cells in conjunction with its downstream
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sequence obtained from the dog genome database. The RT-qPCR system used for plasmid
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DNA containing the TCRγ gene cassette derived from UL-1 cells revealed that the system was
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accurate for 10-100,000 copies per reaction and that its sensitivity was 1 cell per 10,000 cells.
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In order to monitor the kinetics of tumour cell numbers in dogs with lymphoma, MRD was
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quantified in the peripheral blood of seven dogs with lymphoma undergoing chemotherapy
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(Yamazaki et al., 2008). Since the lymphoma cells from the seven dogs were shown to be of B
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cell origin, allele-specific oligonucleotide primers and probes were prepared based on the
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sequence of the rearranged IgH gene in each case. In all seven cases, MRD in the peripheral
178
blood was detectable, even in cases of CR. The peripheral blood MRD levels were directly
179
proportional to lymph node size changes evident during remission induction or at relapse. The
180
high instrument sensitivity (1 cell per 10,000 cells) of the RT-qPCR system used in the study
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by Yamazaki et al. (2008) was similar to that of a study by van der Velden et al. (2002) using
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RT-qPCR for detection of MRD in human leukaemia patients. The RT-qPCR system for the
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rearranged IgH/TCR genes was 100 times more sensitive than previous studies using
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conventional PARR with universal primers in dogs with lymphoma (Keller et al., 2004; Lana
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et al., 2006).
186 187
Monitoring minimal residual disease after chemotherapy in dogs with lymphoma
188
Using RT-qPCR for monitoring MRD in canine lymphoma (Yamazaki et al., 2008),
189
a proof-of-concept pilot study was carried out to understand the clinical utility of a multi-drug
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chemotherapy in dogs with lymphoma (Yamazaki et al., 2010). Peripheral blood MRD was
191
monitored in seven dogs with lymphoma, all of which were treated with a CHOP-based
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protocol (UW-25) (Garrett et al., 2002). The sample of seven dogs included one dog at stage
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V (presence of lymphoma cells in peripheral blood) prior to chemotherapy. In this case, the
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clone-specific RT-qPCR revealed a similar number of tumour cells in the peripheral blood to
195
that detected by conventional haematological analysis. Although the other six dogs were in
196
stage III or IV, the clone-specific RT-qPCR assay clearly indicated the presence and quantity
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of tumour cells in the peripheral blood (4.4-140 cells/µL). MRD in peripheral blood gradually
198
decreased after initiation of the UW-25 protocol in all seven dogs, reaching a minimum of
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<0.019-1.0 cells/µL at weeks 4-17 and remaining at these levels until week 25. MRD at weeks
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4, 9, 17 and 25 was significantly lower than the number of neoplastic lymphoid cells
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measured by RT-qPCR at week 1 (before chemotherapy) in the seven dogs.
202 203
The correlation between MRD at the end of chemotherapy and duration of
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remission after chemotherapy was analysed in 17 dogs that completed UW-25. MRD at the
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end of chemotherapy was negatively correlated with duration of remission from the end of
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chemotherapy until relapse. These results suggested that MRD could be used as an objective
207
marker to indicate tumour cell burden in dogs with lymphoma in remission. Furthermore,
208
MRD at the end of chemotherapy could be a prognostic factor predicting duration of
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remission after chemotherapy.
210 211
Prognostic significance of minimal residual disease in the early phase of chemotherapy
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in dogs with lymphoma
213
Although MRD levels after chemotherapy have prognostic significance in dogs
214
with lymphoma (Yamazaki et al., 2010), data at the end of chemotherapy could not be
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obtained in nearly half of the sample population due to the occurrence of progressive disease
216
(PD) during the initial of remission induction therapy (Sorenmo et al., 2010). In order to
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apply the MRD monitoring system to the majority of dogs in this prospective study, Sato et al.
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(2013) examined the prognostic significance of MRD levels in the early phases of a multidrug
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chemotherapy protocol in 36 dogs with multicentric high grade B cell lymphoma. Sequences
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of IgH gene fragments from lymphoma cells were amplified and used to design allele-specific
221
primers and probes for RT-qPCR. The dogs were treated with a modified UW-25 protocol
222
and evaluated for the MRD level at weeks 6 and 11 of UW-25. Of the 31 dogs that remained
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on the protocol by week 11, 14 were MRD negative (less than 10 tumour cells per 105
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peripheral blood mononuclear cells, PBMCs), whereas the other 17 were MRD positive (≥10
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tumour cells per 105 PBMCs). The PFS of dogs with MRD negative status at week 11
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(median 337 days) was significantly longer than that of the MRD positive dogs at the same
227
time point (median 196 days; P = 0.0002). These results highlight the clinical significance of
228
MRD as a prognostic marker in the early phase of chemotherapy.
229 230
Increase in peripheral blood minimal residual disease before clinical relapse in dogs with
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lymphoma that achieved clinical remission following chemotherapy
232
In order to identify the changes in MRD prior to clinical relapse in dogs with
233
lymphoma that had achieved CR following chemotherapy, peripheral blood MRD was
234
monitored by RT-qPCR, which amplified the rearranged IgH gene in 20 dogs with
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multicentric high grade B cell lymphoma (Sato et al., 2011a). MRD measurement and clinical
236
assessment were performed every 2-4 weeks for 28-601 days after completion of
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chemotherapy. An increase in MRD was defined as an increase of more than 0.5, calculated
238
by log10 [copy number of MRD per 105 PBMCs], based on the uncertainty level observed in a
239
canine lymphoma cell line. During the follow-up period, 15 dogs relapsed in 28-320 days
240
(median 120 days) after completion of chemotherapy. An increase in MRD was detected 2
241
weeks or more prior to a relapse event in 14/15 dogs. The time from increased MRD to
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clinical relapse was 0-63 days (median 42 days). In contrast, no increase in MRD was
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detected in five dogs that did not experience clinical relapse. This study indicated that an
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increase in MRD can be detected before clinical relapse in dogs with lymphoma. This leads to
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the consideration of applied early re-induction therapy, based on an increase in MRD before
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clinical relapse (MRD-guided therapy), for the improvement of treatment outcome in canine
247
lymphoma.
248
Cyclophosphamide, doxorubicin, vincristine and prednisolone
249
Evaluation of cytoreductive efficacy of vincristine, cyclophosphamide and doxorubicin
250
in dogs with lymphoma
251
Since a high proportion of dogs with multicentric lymphoma respond well to
252
chemotherapy and achieve CR soon after initiation of chemotherapy, there is a difficulty in
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establishing drug efficacy from a standard clinical evaluation during treatment period. The
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cytoreductive efficacy of vincristine, cyclophosphamide and doxorubicin was evaluated in
255
dogs with lymphoma that received the UW-25 protocol (Garrett et al., 2002) without
256
administration of L-asparaginase at week 1 (because it did not significantly influence
257
treatment outcome; MacDonald et al., 2005; Jeffreys et al., 2005). The number of lymphoma
258
cells in the peripheral blood was measured from diagnosis to week 11 of the modified UW-25
259
in 29 dogs with high grade B cell multicentric lymphoma using clone-specific RT-qPCR to
260
amplify IgH gene fragments (Sato et al., 2011b). The number of lymphoma cells after the first
261
administration of vincristine, cyclophosphamide and doxorubicin in weeks 1-4 was decreased
262
in 29/29 (100%), 15/29 (52%) and 26/27 (96%) dogs, respectively. The cytoreductive efficacy
263
of cyclophosphamide was less than that of vincristine, cyclophosphamide and doxorubicin.
264
Vincristine, cyclophosphamide and doxorubicin administered in weeks 6-9 were effective in
265
5/26 (19%), 5/20 (25%) and 14/19 (74%) dogs, respectively, indicating the sustained
266
cytoreductive efficacy of doxorubicin. Cyclophosphamide non-responders were heavier and
267
exhibited a shorter first remission than cyclophosphamide responders. The study provided
268
several suggestions for the usage of these three chemotherapeutic agents in order to improve
269
treatment efficacy. Vincristine use might be preferred in the early phase because of the
270
decrease in vincristine cytoreductive efficacy evident in the later phase. Cyclophosphamide
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administration can be reconsidered, especially in large dogs (e.g. dose increase or substitution
272
with other agents). Doxorubicin at the dosage used in the current protocol (30 mg/m2 for dogs
273
>10 kg; 1 mg/kg for dogs <10 kg) is highly effective; thus, it is considered to be the main
274
drug in the combination protocol. Findings obtained in this study would be helpful to
275
construct a new or modified chemotherapeutic protocol in order to obtain better treatment
276
outcomes in dogs with lymphoma.
277 278
Quantitative MRD evaluation after or during anti-tumour therapy would provide an
279
objective evaluation comparing the efficacy of different protocols, especially to indicate an
280
advantage of newly introduced modalities, such as high-dose therapy with autologous stem
281
cell transplantation (Frimberger et al., 2006) and immunotherapy with monoclonal antibodies
282
directed to lymphoid tumour cells (Ito et al., 2014). Although clinical parameters, such as OR,
283
PFS and OS, should be defined in clinical trials, MDR monitoring could also be used in
284
combination with clinical response monitoring, since this will help to evaluate its application
285
and give us confidence in the positive utility of the technique.
286 287 288
Clinical usefulness of minimal residual disease monitoring A series of studies indicating the clinical usefulness of MRD monitoring (Yamazaki
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et al., 2008, 2010; Sato et al., 2011a, 2011b, 2013) has the potential to revolutionise the
290
medical control of canine lymphoma. Measurement of MRD levels after chemotherapy can
291
function as an objective parameter for the determination of treatment efficacy. This may allow
292
clinicians to recommend additional treatment plans to animals with relatively high MRD.
293
MRD monitoring results obtained by the newly introduced RT-qPCR technique, in
294
comparison with those obtained by conventional PARR, could be used to evaluate the new
295
protocol. Monitoring of MRD during CR after completion of the chemotherapy would be
296
useful in predicting relapses, leading to the potential application of an early re-induction
297
therapy prior to identifying clinical relapse.
298 299
In the studies of Yamazaki et al. (2008, 2010) and Sato et al. (2011a, 2011b, 2013),
300
peripheral blood samples were used to monitor MRD in dogs with lymphoma. Although there
301
is a possibility of using lymph node aspirates as samples for monitoring MRD in these dogs,
302
this technique is more demanding and requires additional technical expertise. However, in
303
relatively large dogs, mandibular and popliteal lymph nodes are palpable, even in CR, with
304
fresh aspirates from these nodes providing a potentially useful means of evaluating MRD.
305 306
Throughout these studies (Yamazaki et al., 2008, 2010; Sato et al., 2011a, 2011b,
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2013), preparations of allele (clone)-specific oligonucleotides after sequencing the rearranged
308
antigen receptor (IgH or TCR) gene fragments were needed in each dog with lymphoma. This
309
was also the case in recent studies by Gentilini et al. (2010, 2013), who reported successful
310
monitoring of MRD using hairpin-shaped clone-specific primers in dogs with B cell
311
lymphoma. The costs and time required to prepare the allele-specific oligonucleotides can be
312
an obstacle in the application of such sophisticated analytical systems to many canine cases in
313
practice. If more generally applicable procedures using universal primers had reasonable
314
sensitivity in detecting MRD after achieving CR, such techniques may have utility in
315
monitoring dogs with lymphoma under chemotherapy. Currently, preparation of
316
allele-specific oligonucleotides is essential to achieve sufficient sensitivity for quantification
317
of MRD in remission.
318 319
Feline lymphoma is also clinically important due to its high incidence and wide
320
spectrum of clinical manifestations. To aid in the diagnosis of lymphoma, in conjunction with
321
cytological and histological findings, PCR-based clonality assessment via the detection of Ig
322
and TCR gene rearrangements have been developed for use in cats (Moore et al., 2005;
323
Werner et al., 2005). A system for monitoring MRD similarly could be applied to feline
324
lymphomas in the assessment of treatment efficacy and relapse, particularly in nasal and
Page 17 of 26
325
gastrointestinal lymphomas, in which routine clinical examination procedures render
326
evaluation of tumour size difficult.
327 328
Conclusions
329
MRD measurement is a useful prognostic indicator, an objective marker of
330
treatment efficacy and a predictor of clinical relapse in dogs with lymphoma under
331
chemotherapy. Introduction of MRD monitoring will provide a tool to aid in the development
332
of improved treatment strategies for canine lymphoma.
333 334
Conflict of interest statement
335
None of the authors of this paper have a financial or personal relationship with
336
other people or organisations that could inappropriately influence or bias the content of the
337
paper.
338 339
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382
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Keller, R.L., Avery, A.C., Burnett, R.C., Walton, J.A., Olver, C.S., 2004. Detection of
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Ladetto, M., Lobetti-Bodoni, C., Mantoan, B., Ceccarelli, M., Boccomini, C., Genuardi, E.,
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Chiappella, A., Baldini, L., Rossi, G., Pulsoni, A., et al., 2013. Persistence of
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MacDonald, V.S., Thamm, D.H., Kurzman, I.D., Turek, M.M., Vail, D.M., 2005. Does
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Manachai, N., Lacharoje, S., Techangamsuwan, S., Rungsipipat, A., 2014. Detection of
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443 444
Marconato, L., Frayssinet, P., Rouqet, N., Comazzi, S., Leone, V.F., Laganga, P., Rossi, F.,
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Vignoli, M., Pezzoli, L., Aresu, L., 2014. Randomized, placebo-controlled,
446
double-blinded chemoimmunotherapy clinical trial in a pet dog model of diffuse
447
large B-cell lymphoma. Clinical Cancer Research 20, 668-677.
448 449
Miller Jr., W.H., Levine, K., DeBlasio, A., Frankel, S.R., Dmitrovsky, E., Warrell Jr., R.P.,
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1993. Detection of minimal residual disease in acute promyelocytic leukemia by a
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reverse transcription polymerase chain reaction assay for the PML/RAR-α fusion
452
mRNA. Blood 82, 1689-1694.
453 454
Mitterbauer, M., Neumeister. P., Kalhs, P., Brugger, S., Fischer, G., Dieckmann, K., Hoecker,
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P., Hinterberger, W., Linkesch, W., Simonitsch, I., et al., 2001. Long-term clinical
Page 21 of 26
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and molecular remission after allogenic stem cell transplantation (SCT) in patients
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with poor prognosis non-Hodgkin’s lymphoma. Leukemia 15, 635-641.
458 459
Moore, P.F., Woo, J.C., Vernau, W., Kosten, S., Graham, P.S., 2005. Characterization of
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461
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463 464
Myers, N.C. 3rd, Moore, A.S., Rand, W.M., Gliatto, J., Cotter, S.M., 1997. Evaluation of a
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467 468
Pott, C., Brüggemann, M., Ritgen, M., van der Velden, V.H., van Dongen, J.J., Kneba. M.,
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rearrangements and chromosomal translocations as targets for real-time quantitative
471
PCR. Methods in Molecular Biology 971, 175-200.
472 473
Pott, C., Schrader, C., Gesk, S., Harder, L., Tiemann, M., Raff, T., Bruggemann, M., Ritgen,
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M., Gahn, B., Unterhalt, M., et al., 2006. Quantitative assessment of molecular
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remission after high-dose therapy with autologous stem cell transplantation predicts
476
long-term remission in mantle cell lymphoma. Blood 107, 2271-2278.
477 478
Rambaldi, A., Lazzari, M., Manzoni, C., Carlotti, E., Arcaini, L., Baccarani, M., Barbui, T.,
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Bernasconi, C., Dastoli, G., Fuga, G., et al., 2002. Monitoring of minimal residual
480
disease after CHOP and rituximab in previously untreated patients with follicular
481
lymphoma. Blood 99, 856-862.
482 483
Rebhun R.B., Kent, M.S., Borrofka, S.A., Frazier, S., Skorupski, K., Rodriguez, C.O., 2011.
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CHOP chemotherapy for the treatment of canine multicentric T-cell lymphoma.
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Veterinary and Comparative Oncology 9, 38-44.
486 487
Rosolen, A., Perkins, S.L., Pinkerton, C.R., Guillerman, R.P., Sandlund, J.T., Patte, C.,
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Reiter, A., Cairo, M.S., 2015. Revised international pediatric non-Hodgkin
489
lymphoma staging system. Journal of Clinical Oncology 33, 2112-2118.
490
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Sandlund, J.T., Guillerman, R.P., Perkins, S.L., Pinkerton, C.R., Rosolen, A., Patte, C.,
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Reiter, A., Cairo, M.S., 2015. International pediatric non-Hodgkin lymphoma
493
response criteria. Journal of Clinical Oncology 33, 2106-2111.
494 495
Sato, M., Yamazaki, J., Goto-Koshino, Y., Takahashi, M., Fujino, Y., Ohno, K., Tsujimoto, H.,
496
2011a. Increase in minimal residual disease (MRD) in peripheral blood before
497
clinical relapse in dogs with lymphoma that achieved complete remission after
498
chemotherapy. Journal of Veterinary Internal Medicine 25, 292-296.
499 500
Sato, M., Yamazaki, J., Goto-Koshino, Y., Takahashi, M., Fujino, Y., Ohno, K., Tsujimoto, H.,
501
2011b. Evaluation of cytoreductive efficacy of vincristine, cyclophosphamide, and
502
doxorubicin in dogs with lymphoma by measuring the number of neoplastic
503
lymphoid cells with real-time polymerase chain reaction. Journal of Veterinary
504
Internal Medicine 25, 285-291.
505 506
Sato, M., Yamzaki, J., Goto-Koshino, Y., Takahashi, M., Fujino, Y., Ohno, K., Tsujimoto, H.,
507
2013. The prognostic significance of minimal residual disease in the early phases of
508
chemotherapy in dogs with high-grade B-cell lymphoma. The Veterinary Journal 195,
509
319-324.
510 511
Simon, D., Nolte, I., Eberle, N., Abbrederis, N., Killich, M., Hirschberger, J., 2006.
512
Treatment of dogs with lymphoma using a 12-week, maintenance-free combination
513
chemotherapy protocol. Journal of Veterinary Internal Medicine 20, 948-954.
514 515
Sorenmo, K., Overley, B., Krick, E., Ferrara, T., LaBlanc, A., Shofer, F., 2010. Outcome and
516
toxicity associated with a dose-intensified, maintenance-free CHOP-based
517
chemotherapy protocol in canine lymphoma: 130 cases. Veterinary and Comparative
518
Oncology 8, 196-208.
519 520
Tamura, K., Yagihara, H., Isotani, M., Ono, K., Washizu, T., Bonkobara, M., 2006.
521
Development of the polymerase chain reaction assay based on the canine genome
522
database for detection of monoclonality in B cell lymphoma. Veterinary
523
Immunology and Immunopathology 110, 163-167.
524 525
Thilakaratne, D.N., Mayer, M.N., MacDonald, V.S., Jackson, M.L., Trask, B.R., Kidney,
526
B.A., 2010. Clonality and phenotyping of canine lymphomas before chemotherapy
Page 23 of 26
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and during remission using polymerase chain reaction (PCR) on lymph node
528
cytologic smears and peripheral blood. Canadian Veterinary Journal 51, 79-84.
529 530
Uchiyama, M., Maesawa, C., Yashima, A., Tarusawa, M., Satoh, T., Ishida, Y., Ito, S., Murai,
531
K., Enomoto, S., Utsugisawa, T., et al., 2003. Development of consensus
532
fluorogenically labeled probes of the immunoglobulin heavy-chain gene for
533
detecting minimal residual disease in B-cell non-Hodgkin lymphomas. Cancer
534
Science 94, 877-885.
535 536
Vail, D.M., Kisseberth, W.C., Obradovich, J.E., Moore, F.M., London, C.A., MacEwen, E.G.,
537
Ritter, M.A., 1996. Assessment of potential doubling time (Tpot), argyrophilic
538
nucleolar organizer regions (AgNOR), and proliferating cell nuclear antigen (PCNA)
539
as predictors of therapy response in canine non-Hodgkin's lymphoma. Experimental
540
Hematology 24, 807-815.
541 542 543
Valli, V.E., Vernau, W., de Lorimier, L.P., Graham, P.S., Moore, P.F., 2006. Canine indolent nodular lymphoma. Veterinary Pathology 43, 241-256.
544 545
Valli, V.E., San Myint, M., Barthel, A., Bienzle, D., Caswell, J., Colbatzky, F., Durham, A.,
546
Ehrhart, E.J., Johnson, Y., Jones, C., et al., 2011. Classification of canine malignant
547
lymphomas according to the World Health Organization criteria. Veterinary
548
Pathology 48, 198-211.
549 550
van der Velden, V.H., Wijkhuijs, J.M., Jacobs, D.C., van Wering, E.R., van Dongen, J.J.,
551
2002. T cell receptor gamma gene rearrangements as targets for detection of minimal
552
residual disease in acute lymphoblastic leukemia by real-time quantitative PCR
553
analysis. Leukemia 16, 1372-1380.
554 555
Werner, J.A., Woo, J.C., Vernau, W., Graham, P.S., Grahn, R.A., Lyons, L.A., Moore, P.F.,
556
2005. Characterization of feline immunoglobulin heavy chain variable region genes
557
for the molecular diagnosis of B-cell neoplasia. Veterinary Pathology 42, 596-607.
558 559
Yagihara, H., Tamura, K., Isotani, M., Ono, K., Washizu, T., Bonkobara, M., 2007. Genomic
560
organization of the T-cell receptor gamma gene and PCR detection of its clonal
561
rearrangement in canine T-cell lymphoma/leukemia. Veterinary Immunology and
562
Immunopathology 115, 375-382.
563
Page 24 of 26
564
Yamazaki, J., Baba, K., Goto-Koshino, Y., Setoguchi-Mukai, A., Fujino, Y., Ohno, K.,
565
Tsujimoto, H., 2008. Quantitative assessment of minimal residual disease (MRD) in
566
canine lymphoma by using real-time polymerase chain reaction. Veterinary
567
Immunology and Immunopathology 126, 321-331.
568 569
Yamazaki, J., Takahashi, M., Setoguchi, A., Fujino, Y., Ohno, K., Tsujimoto, H., 2010.
570
Monitoring of minimal residual disease (MRD) after multidrug chemotherapy and its
571
correlation to outcome in dogs with lymphoma: A proof-of-concept study. Journal of
572
Veterinary Internal Medicine 24, 897-903.
573 574
Yashima, A., Maesawa, C., Uchiyama, M., Tarusawa, M., Satoh, T., Satoh, M., Enomoto, S.,
575
Sugawara, K., Numaoka, H., Murai, K., Utsugisawa, T., Ishida, Y., Masuda, T., 2003.
576
Quantitative assessment of contaminating tumor cells in autologous peripheral blood
577
stem cells of B-cell non-Hodgkin lymphomas using immunoglobulin heavy chain
578
gene allele-specific oligonucleotide real-time quantitative-polymerase chain reaction.
579
Leukemia Research 27, 925-934.
580 581
Zemann, B.I., Moore, A.S., Rand, W.M, Mason, G., Ruslander, D.M., Frimberger, A.E.,
582
Wood, C.A., L'Heureux, D.A., Gliatto, J., Cotter, S.M., 1998. A combination
583
chemotherapy protocol (VELCAP-L) for dogs with lymphoma. Journal of Veterinary
584
Internal Medicine 12, 465-470.
Page 25 of 26
585
Figure legend
586 587
Fig. 1. Kinetics of tumour cell burdens after anti-tumour therapy. The amount of malignant
588
cells within the body decreases after initiation of chemotherapy and reaches subthreshold
589
detection limits of clinically diagnostic techniques. Such residual malignant cells are
590
considered to be the source of tumour relapses and are known as minimal residual disease
591
(MRD). Clinical remission and clinical relapse are defined as the detection limit of the
592
clinical diagnostic techniques. Molecular remission and molecular relapse are defined by the
593
detection limit of the molecular biology techniques.
Page 26 of 26
S1090-0233(16)30064-8 http://dx.doi.org/doi: 10.1016/j.tvjl.2016.05.012 YTVJL 4821
To appear in:
The Veterinary Journal
Accepted date:
24-5-2016
Please cite this article as: Masahiko Sato, Jumpei Yamazaki, Yuko Goto-Koshino, Asuka Setoguchi, Masashi Takahashi, Kenji Baba, Yasuhito Fujino, Koichi Ohno, Hajime Tsujimoto, Minimal residual disease in canine lymphoma: An objective marker to assess tumour cell burden in remission, The Veterinary Journal (2016), http://dx.doi.org/doi: 10.1016/j.tvjl.2016.05.012. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Review Article
2 3
Minimal residual disease in canine lymphoma: An objective marker to assess tumour
4
cell burden in remission
5 6
Masahiko Sato a, Jumpei Yamazaki b, Yuko Goto-Koshino c, Asuka Setoguchi d, Masashi
7
Takahashi c, Kenji Baba e, Yasuhito Fujino f, Koichi Ohno f, Hajime Tsujimoto c,f,*
8 9
a
Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences,
10
Colorado State University, Fort Collins, CO 80523, USA
11
b
12
University, Sapporo, Hokkaido 060-0818, Japan
13
c
14
University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan
15
d
16
Kagoshima University, Kagoshima, Kagoshima 890-0065, Japan
17
e
18
University, Yamaguchi, Yamaguchi 753-8515, Japan
19
f
20
Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan
Laboratory of Molecular Medicine, Graduate School of Veterinary Medicine, Hokkaido Veterinary Medical Centre, Graduate School of Agricultural and Life Sciences, The Laboratory of Small Animal Internal Medicine, Joint Faculty of Veterinary Medicine, Department of Veterinary Medicine, Joint Faculty of Veterinary Medicine, Yamaguchi Department of Veterinary Internal Medicine, Graduate School of Agricultural and Life
21 22 23 24 25
* Corresponding author: Tel.: +81 3 58415402. E-mail address: [email protected] (H. Tsujimoto). Highlights
26 27
Minimal residual disease (MRD) can be measured in the peripheral blood of dogs with lymphoma (lymphosarcoma).
28
The efficacy of chemotherapy in canine lymphoma can be objectively compared by detection of MRD.
29
The level of MRD is predictive of relapse and prognosis of canine lymphoma.
30
MRD monitoring by real-time quantitative PCR will be of value in evaluating novel
Page 1 of 26
31 32
therapies for canine lymphoma. Abstract
33
Lymphoma is the most common haematopoietic malignancy in dogs. Since a high
34
proportion of dogs with lymphoma achieve remission soon after initiation of chemotherapy,
35
an objective marker assessing treatment efficacy is required. Following clinical remission, the
36
residual population of tumour cells can be referred to as the minimal residual disease (MRD).
37
MRD traditionally has been detected by cytology and flow cytometry; however, if the burden
38
of malignant cells is low, these methods might not be sufficiently sensitive to detect MRD. As
39
an extension of the development of PCR for antigen receptor gene rearrangements (PARR) in
40
dogs, there has been recent progress in the application of real-time quantitative PCR
41
(RT-qPCR) to canine lymphoma. With the RT-qPCR system, a very high sensitivity (1 cell per
42
10,000 cells) has been achieved by preparing allele-specific oligonucleotide primers and
43
probes designed from neoplastic clones of each dog. A series of MRD diagnostics studies
44
employing the RT-qPCR system have revealed its usefulness as a prognostic indicator, an
45
objective marker of treatment efficacy and a predictor of relapse for dogs with lymphoma
46
receiving chemotherapy. Introduction of the MRD monitoring system will provide an
47
innovative scientific tool in the development of superior treatments and monitoring strategies
48
for canine lymphoma.
49 50
Keywords: Canine; Lymphoma; Chemotherapy; Minimal residual disease; Real-time
51
quantitative PCR
52 53 54
Introduction Lymphoma (lymphosarcoma), the most common haematopoietic malignancy in
Page 2 of 26
55
dogs, is characterised by the clonal proliferation of lymphoid cells. In most cases, such as in
56
high grade multicentric B cell lymphoma, the disease is initially responsive to chemotherapy.
57
However, high rates of relapse and mortality are evident in nearly all cases as a result of
58
disease progression.
59 60
Cyclophosphamide, doxorubicin, vincristine and prednisolone (CHOP)-based
61
protocols were introduced in the treatment of canine lymphoma (Greenlee et al., 1990; Keller
62
et al., 1993) as a superior alternative to COP-based protocols (Cotter et al., 1983). The
63
combination of CHOP-based protocols with L-asparaginase as adjunct therapy is referred to
64
as L-CHOP. Overall response (OR) rate, progression-free survival (PFS) and overall survival
65
(OS) in dogs with lymphoma treated with L-CHOP-based protocols (Greenlee et al., 1990;
66
Keller et al., 1993; Vail et al., 1996; Myers et al., 1997; Zemann et al., 1998; Khanna et al.,
67
1998) were 69-87%, 7.3-12.8 months and 10.1-17.0 months, respectively. In these protocols,
68
antineoplastic agents were continuously administered for up to 2-3 years; however,
69
subsequent studies have indicated that similar treatment outcomes were achievable with
70
maintenance-free L-CHOP-based protocols in up to 25 weeks (Garrett et al., 2002;
71
MacDonald et al., 2005; Simon et al., 2006; Hosoya et al., 2007; Burton et al., 2012; Curran
72
and Thamm, 2015). The influence of L-asparaginase on the efficacy of the CHOP/COP
Page 3 of 26
73
protocols was investigated in dogs with multicentric high grade lymphoma in two
74
independent studies, in which outcomes following use of a multi-agent chemotherapy
75
protocol, with or without the addition of L-asparaginase (depending on commercial
76
availability), were compared (Jeffreys et al., 2005; MacDonald et al., 2005). Both of these
77
studies suggested that addition of L-asparaginase to CHOP/COP protocols may not improve
78
treatment outcomes, suggesting that L-asparaginase could be reserved for rescue protocols.
79 80
Recent studies on the clinical application of chemoimmunotherapy (i.e.
81
chemotherapy combined with an autologous tumour cell vaccine) revealed clinical benefits
82
without incidents of increased toxicity in dogs with B cell lymphomas (Aresu et al., 2014;
83
Marconato et al., 2014). Most of the dogs enrolled in these studies had multicentric high
84
grade B cell lymphomas. However, many studies have reported poorer prognoses for high
85
grade T cell lymphomas, indicating the need for alternative protocols. For T cell lymphomas,
86
the use of alkylating agent-based protocols, such as L-asparaginase combined with
87
mechlorethamine, oncovin, procarbazine and prednisone (L-asparaginase-MOPP) was
88
superior to a classic CHOP-based protocol (Brodsky et al., 2009). However, another study
89
reported that the use of a generally applied CHOP-based protocol to dogs with T cell
90
lymphoma produced results comparable to those for B cell lymphomas (Rebhun et al., 2011).
Page 4 of 26
91 92
L-CHOP can induce complete remission (CR) rapidly in a large proportion of dogs
93
with lymphoma; the median time from the first day of L-CHOP to CR was 11 days (Simon et
94
al., 2006). After achieving CR, chemotherapy is usually continued for a certain period, e.g. up
95
to 25 weeks in UW-25 protocol (Garrett et al., 2002). However, most dogs experience relapse
96
after termination of, or during, the protocol. This is indicative of a small, residual, population
97
of malignant tumour cells that persist within the body, referred to as the minimal residual
98
disease (MRD), which are implicated as the source of tumour relapses. In general, following
99
initiation of chemotherapy, the amount of detectable malignant cells within the body
100
decreases to subthreshold detection limits of clinical diagnostic techniques (clinical
101
remission) (Fig. 1). Detection of MRD is facilitated by a sensitive, molecular biological
102
technique that may be subject to, in some cases, further subthreshold decreases to levels
103
below that of its detection limit (molecular remission) following successful remission
104
induction therapy or consolidation therapy. Consequently, the residual malignant cells
105
proliferate after a certain period, resulting in molecular relapse leading to clinical relapse (Fig.
106
1).
107 108
In human haematological malignancies, the quantity of MRD after treatment can be
Page 5 of 26
109
indicative of treatment outcome. High MRD after chemotherapy indicates a poor prognosis
110
for follicular lymphoma (Rambaldi et al., 2002), mantle cell lymphoma (Pott et al., 2006),
111
adult acute lymphoblastic leukaemia (Bruggemann et al., 2006), and acute promyelocytic
112
leukaemia (Miller et al., 1993). Several reports have documented the usefulness of MRD in
113
the molecular assessment of treatment efficacy for diffuse large B cell lymphoma (DLBCL)
114
in human beings (Mitterbauer et al., 2001; Uchiyama et al., 2003; Yashima et al., 2003).
115
DLBCL is the most common histological subtype of canine lymphoma (Valli et al., 2011) and
116
comparative studies between human and canine populations would be possible for this type of
117
lymphoma. MRD diagnostics is of high clinical relevance to human patients affected with B
118
cell non-Hodgkin lymphomas. This may serve as a surrogate parameter in the evaluation of
119
treatment effectiveness and long term prognosis (Ladetto et al., 2013). Subsequently, MRD
120
diagnostics performed by real-time quantitative PCR (RT-qPCR) is now the gold-standard
121
and currently the most sensitive and broadly applied method for the staging, classification and
122
treatment response assessment of human non-Hodgkin lymphoma (Pott et al., 2013; Rosolen
123
et al., 2015; Sandlund et al., 2015).
124 125
The assessment of MRD in human patients with lymphoid malignancies has shown
126
that the prognoses of patients were strongly influenced by the MRD quantities following
Page 6 of 26
127
chemotherapy (or other therapeutic modalities), but were unrelated to the tumour cell burden
128
prior to treatment (Mitterbauer et al., 2001; Rambaldi et al., 2002; Pott et al., 2006; Ladetto et
129
al., 2013). Although available data for canine lymphomas are limited, there are several studies
130
comparing the prognostic values of tumour cell burden amounts before and after
131
chemotherapy. Results of the qualitative status (positive or negative) in the PCR for antigen
132
receptor gene rearrangements (PARR) before chemotherapy were not related to the prognosis
133
of dogs with lymphoma (Lana et al., 2006). Similarly, a correlative, prognostic significance
134
for tumour cell burden prior to the initiation of RT-qPCR-assessed chemotherapy for the
135
immunoglobulin heavy chain (IgH) gene was lacking in dogs with multicentric high grade B
136
cell lymphoma (Sato et al., 2013). However, MRD quantities as assessed by RT-qPCR at
137
weeks 11 and 25 of a standard CHOP protocol (UW-25) (Garrett et al., 2002) were correlated
138
with prognosis in dogs with lymphoma (Yamazaki et al., 2010; Sato et al., 2013). On the basis
139
of these observations, MRD quantity after treatment for a certain period (e.g. 11 weeks or
140
more with the UW-25 protocol) could be used as an objective prognostic marker in canine
141
lymphoma.
142 143
Molecular biological detection of tumour cells in lymph nodes and the peripheral
144
blood of dogs with lymphoid neoplasia has been reported using PARR, resulting in the
Page 7 of 26
145
assessment of clonality and T/B cell lineage of the lymphoid cells (Burnett et al., 2003; Keller
146
et al., 2004; Calzolari et al., 2006; Tamura et al., 2006; Valli et al., 2006; Lana et al., 2006;
147
Yagihara et al., 2007). PARR has also been used for the detection of MRD in dogs with
148
lymphoma that achieved remission after chemotherapy (Thilakaratne et al., 2010; Manachai et
149
al., 2014). Aresu et al. (2014) reported that the combination of PARR and flow cytometry for
150
large CD21+ cells was more sensitive than either technique alone in predicting outcomes of
151
dogs with DLBCL after chemotherapy. All of these studies employed the conventional PCR
152
method using universal primers to detect for the rearrangements of antigen receptor genes.
153
The sensitivity for detecting neoplastic lymphoid cells was shown to be ~1 per 100 (Burnett et
154
al., 2003). Therefore, it is conceivable that tumour cells with clonal rearrangement can
155
become undetectable during remission.
156 157
In a study by Yamazaki et al. (2008), MRD in the peripheral blood was successfully
158
detected and quantified by RT-qPCR using allele-specific primers and probes in most canine
159
lymphoma patients in CR after chemotherapy. Subsequent studies using RT-qPCR procedures
160
enabled the dynamic exploration of tumour cell burden in dogs with lymphoma before, during
161
and after chemotherapy, as well as prior to and during episodes of relapse (Yamazaki et al.,
162
2010; Sato et al., 2011a, b, 2013).
Page 8 of 26
163 164
Quantitative assessment of minimal residual disease in canine lymphoma
165
A quantitative detection system for MRD in canine lymphoma was developed by
166
our group using RT-qPCR (Yamazaki et al., 2008). UL-1, a canine T cell lymphoma-derived
167
cell line, was used to examine the specificity and sensitivity of the assay for detecting MRD.
168
Allele-specific oligonucleotide primers and probes were designed based on the rearranged T
169
cell receptor γ chain (TCRγ) gene sequence of UL-1 cells in conjunction with its downstream
170
sequence obtained from the dog genome database. The RT-qPCR system used for plasmid
171
DNA containing the TCRγ gene cassette derived from UL-1 cells revealed that the system was
172
accurate for 10-100,000 copies per reaction and that its sensitivity was 1 cell per 10,000 cells.
173
In order to monitor the kinetics of tumour cell numbers in dogs with lymphoma, MRD was
174
quantified in the peripheral blood of seven dogs with lymphoma undergoing chemotherapy
175
(Yamazaki et al., 2008). Since the lymphoma cells from the seven dogs were shown to be of B
176
cell origin, allele-specific oligonucleotide primers and probes were prepared based on the
177
sequence of the rearranged IgH gene in each case. In all seven cases, MRD in the peripheral
178
blood was detectable, even in cases of CR. The peripheral blood MRD levels were directly
179
proportional to lymph node size changes evident during remission induction or at relapse. The
180
high instrument sensitivity (1 cell per 10,000 cells) of the RT-qPCR system used in the study
Page 9 of 26
181
by Yamazaki et al. (2008) was similar to that of a study by van der Velden et al. (2002) using
182
RT-qPCR for detection of MRD in human leukaemia patients. The RT-qPCR system for the
183
rearranged IgH/TCR genes was 100 times more sensitive than previous studies using
184
conventional PARR with universal primers in dogs with lymphoma (Keller et al., 2004; Lana
185
et al., 2006).
186 187
Monitoring minimal residual disease after chemotherapy in dogs with lymphoma
188
Using RT-qPCR for monitoring MRD in canine lymphoma (Yamazaki et al., 2008),
189
a proof-of-concept pilot study was carried out to understand the clinical utility of a multi-drug
190
chemotherapy in dogs with lymphoma (Yamazaki et al., 2010). Peripheral blood MRD was
191
monitored in seven dogs with lymphoma, all of which were treated with a CHOP-based
192
protocol (UW-25) (Garrett et al., 2002). The sample of seven dogs included one dog at stage
193
V (presence of lymphoma cells in peripheral blood) prior to chemotherapy. In this case, the
194
clone-specific RT-qPCR revealed a similar number of tumour cells in the peripheral blood to
195
that detected by conventional haematological analysis. Although the other six dogs were in
196
stage III or IV, the clone-specific RT-qPCR assay clearly indicated the presence and quantity
197
of tumour cells in the peripheral blood (4.4-140 cells/µL). MRD in peripheral blood gradually
198
decreased after initiation of the UW-25 protocol in all seven dogs, reaching a minimum of
Page 10 of 26
199
<0.019-1.0 cells/µL at weeks 4-17 and remaining at these levels until week 25. MRD at weeks
200
4, 9, 17 and 25 was significantly lower than the number of neoplastic lymphoid cells
201
measured by RT-qPCR at week 1 (before chemotherapy) in the seven dogs.
202 203
The correlation between MRD at the end of chemotherapy and duration of
204
remission after chemotherapy was analysed in 17 dogs that completed UW-25. MRD at the
205
end of chemotherapy was negatively correlated with duration of remission from the end of
206
chemotherapy until relapse. These results suggested that MRD could be used as an objective
207
marker to indicate tumour cell burden in dogs with lymphoma in remission. Furthermore,
208
MRD at the end of chemotherapy could be a prognostic factor predicting duration of
209
remission after chemotherapy.
210 211
Prognostic significance of minimal residual disease in the early phase of chemotherapy
212
in dogs with lymphoma
213
Although MRD levels after chemotherapy have prognostic significance in dogs
214
with lymphoma (Yamazaki et al., 2010), data at the end of chemotherapy could not be
215
obtained in nearly half of the sample population due to the occurrence of progressive disease
216
(PD) during the initial of remission induction therapy (Sorenmo et al., 2010). In order to
Page 11 of 26
217
apply the MRD monitoring system to the majority of dogs in this prospective study, Sato et al.
218
(2013) examined the prognostic significance of MRD levels in the early phases of a multidrug
219
chemotherapy protocol in 36 dogs with multicentric high grade B cell lymphoma. Sequences
220
of IgH gene fragments from lymphoma cells were amplified and used to design allele-specific
221
primers and probes for RT-qPCR. The dogs were treated with a modified UW-25 protocol
222
and evaluated for the MRD level at weeks 6 and 11 of UW-25. Of the 31 dogs that remained
223
on the protocol by week 11, 14 were MRD negative (less than 10 tumour cells per 105
224
peripheral blood mononuclear cells, PBMCs), whereas the other 17 were MRD positive (≥10
225
tumour cells per 105 PBMCs). The PFS of dogs with MRD negative status at week 11
226
(median 337 days) was significantly longer than that of the MRD positive dogs at the same
227
time point (median 196 days; P = 0.0002). These results highlight the clinical significance of
228
MRD as a prognostic marker in the early phase of chemotherapy.
229 230
Increase in peripheral blood minimal residual disease before clinical relapse in dogs with
231
lymphoma that achieved clinical remission following chemotherapy
232
In order to identify the changes in MRD prior to clinical relapse in dogs with
233
lymphoma that had achieved CR following chemotherapy, peripheral blood MRD was
234
monitored by RT-qPCR, which amplified the rearranged IgH gene in 20 dogs with
Page 12 of 26
235
multicentric high grade B cell lymphoma (Sato et al., 2011a). MRD measurement and clinical
236
assessment were performed every 2-4 weeks for 28-601 days after completion of
237
chemotherapy. An increase in MRD was defined as an increase of more than 0.5, calculated
238
by log10 [copy number of MRD per 105 PBMCs], based on the uncertainty level observed in a
239
canine lymphoma cell line. During the follow-up period, 15 dogs relapsed in 28-320 days
240
(median 120 days) after completion of chemotherapy. An increase in MRD was detected 2
241
weeks or more prior to a relapse event in 14/15 dogs. The time from increased MRD to
242
clinical relapse was 0-63 days (median 42 days). In contrast, no increase in MRD was
243
detected in five dogs that did not experience clinical relapse. This study indicated that an
244
increase in MRD can be detected before clinical relapse in dogs with lymphoma. This leads to
245
the consideration of applied early re-induction therapy, based on an increase in MRD before
246
clinical relapse (MRD-guided therapy), for the improvement of treatment outcome in canine
247
lymphoma.
248
Cyclophosphamide, doxorubicin, vincristine and prednisolone
249
Evaluation of cytoreductive efficacy of vincristine, cyclophosphamide and doxorubicin
250
in dogs with lymphoma
251
Since a high proportion of dogs with multicentric lymphoma respond well to
252
chemotherapy and achieve CR soon after initiation of chemotherapy, there is a difficulty in
Page 13 of 26
253
establishing drug efficacy from a standard clinical evaluation during treatment period. The
254
cytoreductive efficacy of vincristine, cyclophosphamide and doxorubicin was evaluated in
255
dogs with lymphoma that received the UW-25 protocol (Garrett et al., 2002) without
256
administration of L-asparaginase at week 1 (because it did not significantly influence
257
treatment outcome; MacDonald et al., 2005; Jeffreys et al., 2005). The number of lymphoma
258
cells in the peripheral blood was measured from diagnosis to week 11 of the modified UW-25
259
in 29 dogs with high grade B cell multicentric lymphoma using clone-specific RT-qPCR to
260
amplify IgH gene fragments (Sato et al., 2011b). The number of lymphoma cells after the first
261
administration of vincristine, cyclophosphamide and doxorubicin in weeks 1-4 was decreased
262
in 29/29 (100%), 15/29 (52%) and 26/27 (96%) dogs, respectively. The cytoreductive efficacy
263
of cyclophosphamide was less than that of vincristine, cyclophosphamide and doxorubicin.
264
Vincristine, cyclophosphamide and doxorubicin administered in weeks 6-9 were effective in
265
5/26 (19%), 5/20 (25%) and 14/19 (74%) dogs, respectively, indicating the sustained
266
cytoreductive efficacy of doxorubicin. Cyclophosphamide non-responders were heavier and
267
exhibited a shorter first remission than cyclophosphamide responders. The study provided
268
several suggestions for the usage of these three chemotherapeutic agents in order to improve
269
treatment efficacy. Vincristine use might be preferred in the early phase because of the
270
decrease in vincristine cytoreductive efficacy evident in the later phase. Cyclophosphamide
Page 14 of 26
271
administration can be reconsidered, especially in large dogs (e.g. dose increase or substitution
272
with other agents). Doxorubicin at the dosage used in the current protocol (30 mg/m2 for dogs
273
>10 kg; 1 mg/kg for dogs <10 kg) is highly effective; thus, it is considered to be the main
274
drug in the combination protocol. Findings obtained in this study would be helpful to
275
construct a new or modified chemotherapeutic protocol in order to obtain better treatment
276
outcomes in dogs with lymphoma.
277 278
Quantitative MRD evaluation after or during anti-tumour therapy would provide an
279
objective evaluation comparing the efficacy of different protocols, especially to indicate an
280
advantage of newly introduced modalities, such as high-dose therapy with autologous stem
281
cell transplantation (Frimberger et al., 2006) and immunotherapy with monoclonal antibodies
282
directed to lymphoid tumour cells (Ito et al., 2014). Although clinical parameters, such as OR,
283
PFS and OS, should be defined in clinical trials, MDR monitoring could also be used in
284
combination with clinical response monitoring, since this will help to evaluate its application
285
and give us confidence in the positive utility of the technique.
286 287 288
Clinical usefulness of minimal residual disease monitoring A series of studies indicating the clinical usefulness of MRD monitoring (Yamazaki
Page 15 of 26
289
et al., 2008, 2010; Sato et al., 2011a, 2011b, 2013) has the potential to revolutionise the
290
medical control of canine lymphoma. Measurement of MRD levels after chemotherapy can
291
function as an objective parameter for the determination of treatment efficacy. This may allow
292
clinicians to recommend additional treatment plans to animals with relatively high MRD.
293
MRD monitoring results obtained by the newly introduced RT-qPCR technique, in
294
comparison with those obtained by conventional PARR, could be used to evaluate the new
295
protocol. Monitoring of MRD during CR after completion of the chemotherapy would be
296
useful in predicting relapses, leading to the potential application of an early re-induction
297
therapy prior to identifying clinical relapse.
298 299
In the studies of Yamazaki et al. (2008, 2010) and Sato et al. (2011a, 2011b, 2013),
300
peripheral blood samples were used to monitor MRD in dogs with lymphoma. Although there
301
is a possibility of using lymph node aspirates as samples for monitoring MRD in these dogs,
302
this technique is more demanding and requires additional technical expertise. However, in
303
relatively large dogs, mandibular and popliteal lymph nodes are palpable, even in CR, with
304
fresh aspirates from these nodes providing a potentially useful means of evaluating MRD.
305 306
Throughout these studies (Yamazaki et al., 2008, 2010; Sato et al., 2011a, 2011b,
Page 16 of 26
307
2013), preparations of allele (clone)-specific oligonucleotides after sequencing the rearranged
308
antigen receptor (IgH or TCR) gene fragments were needed in each dog with lymphoma. This
309
was also the case in recent studies by Gentilini et al. (2010, 2013), who reported successful
310
monitoring of MRD using hairpin-shaped clone-specific primers in dogs with B cell
311
lymphoma. The costs and time required to prepare the allele-specific oligonucleotides can be
312
an obstacle in the application of such sophisticated analytical systems to many canine cases in
313
practice. If more generally applicable procedures using universal primers had reasonable
314
sensitivity in detecting MRD after achieving CR, such techniques may have utility in
315
monitoring dogs with lymphoma under chemotherapy. Currently, preparation of
316
allele-specific oligonucleotides is essential to achieve sufficient sensitivity for quantification
317
of MRD in remission.
318 319
Feline lymphoma is also clinically important due to its high incidence and wide
320
spectrum of clinical manifestations. To aid in the diagnosis of lymphoma, in conjunction with
321
cytological and histological findings, PCR-based clonality assessment via the detection of Ig
322
and TCR gene rearrangements have been developed for use in cats (Moore et al., 2005;
323
Werner et al., 2005). A system for monitoring MRD similarly could be applied to feline
324
lymphomas in the assessment of treatment efficacy and relapse, particularly in nasal and
Page 17 of 26
325
gastrointestinal lymphomas, in which routine clinical examination procedures render
326
evaluation of tumour size difficult.
327 328
Conclusions
329
MRD measurement is a useful prognostic indicator, an objective marker of
330
treatment efficacy and a predictor of clinical relapse in dogs with lymphoma under
331
chemotherapy. Introduction of MRD monitoring will provide a tool to aid in the development
332
of improved treatment strategies for canine lymphoma.
333 334
Conflict of interest statement
335
None of the authors of this paper have a financial or personal relationship with
336
other people or organisations that could inappropriately influence or bias the content of the
337
paper.
338 339
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Quantitative assessment of contaminating tumor cells in autologous peripheral blood
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585
Figure legend
586 587
Fig. 1. Kinetics of tumour cell burdens after anti-tumour therapy. The amount of malignant
588
cells within the body decreases after initiation of chemotherapy and reaches subthreshold
589
detection limits of clinically diagnostic techniques. Such residual malignant cells are
590
considered to be the source of tumour relapses and are known as minimal residual disease
591
(MRD). Clinical remission and clinical relapse are defined as the detection limit of the
592
clinical diagnostic techniques. Molecular remission and molecular relapse are defined by the
593
detection limit of the molecular biology techniques.
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