British Journal ofHaernatologll, 1995, 90, 113-118 Idiotypic oligonucleotide probes to detect myeloma cells by mRNA in situ hybridization Ross BROWN, XIAO-FENG Luo, JOHN GIBSON, ALEC MORLEY,* PAM SYKES,* MICHAEL BRISCO AND DOUG JOSHUA Institute of Haematology, Royal Prince Alfred Hospital, Sydney, and *Huematology Department, Flinders Medical Centre, Adelaide, Australia Received 16 September 1994; accepted for publication 3 February 1995 Summary. An mRNA in situ hybridization (ISH) method which used non-radioactiveidiotypic oligonucleotideprobes has been used to detect malignant cells in the bone marrow and peripheral blood of patients with multiple myeloma. For each of two patients with multiple myeloma a pair of biotinylated antisense oligonucleotide probes (1 8-22mer) was prepared from non-germline sequences of the rear- ranged immunoglobulin heavy chain (IgH) gene. These oligonucleotide sequences were not homologous with any previously published sequence. The probes from each patient were specific as shown by a failure to hybridize to any cells from six other myeloma patients and four normal indivi- duals. Specific staining of IgH gene mRNA occurred only when the myeloma cells and the sequence of the probe used The diversity of the immunoglobulin repertoire is established during the early stages of B-cell development by a complex series of independent molecular events. Antibody diversity prior to antigen exposure is established by the formation of a vast array of different recombinations of VDJ regions of the immunoglobulin heavy chain (IgH) gene and VJ regions of the light chain gene. Somatic hypermutation of the rearranged immunoglobulin V region genes and their immediate flanking sequences occurs after antigen expo- sure and generates high-aihity antibodies while down- stream switching of the fully assembled V region to a different C region produces a new isotype class (Tonegawa, 1983: French et d. 1989). For patients with Bcell malignancies this series of molecular events provides useful markers of clonality and ontogeny. In acute lymphoblastic leukaemia, for example, clonal succession and oligoclonality have been well demonstrated (Beishuizen et al, 1991; Steenbergen et al, 1993). In multiple myeloma, however, Correspondence: Dr Ross Brown, Haematology Department, Royal Prince Alfred Hospital, Missenden Road, Camperdown, NSW 2050, Australia. @ 1995 Blackwell Science Ltd were from the same patient. Using simultaneous fluorescent immunocytochetnistry it was shown that more than 95% of the ISH-positive cells expressed the malignant light chain in their cytoplasm, ISH positive cells were found in 1-4% of the peripheral blood mononuclear fraction of these two patients. These studies show that idiotypic oligonucleotide IgH gene probes can be used to identify individual cells belonging to the malignant clone and offer the possibility of developing innovative tumour-specific therapeutic procedures using antisense technology for patients with myeloma. Keywords: multiple myeloma, plasma cells, oligonucleotide probes, immunoglobulin variable region. the malignancy develops from B cells in which somatic hypermutation has already occurred, and several groups have now demonstrated that neither the evolution of new IgH gene rearrangements nor ongoing somatic hypermuta- tion occurs during progression of this disease (Ralph et al, 1993: Bakkus et al, 1992).This observation provides us with the opportunity to use non-germline nucleotide sequences of the rearranged IgH gene as patient and tumour-specific markers at any stage of the disease. In a previous study we used PCR to amplie a section of the rearranged IgH gene between the framework 2 and J regions in 21 patients with myeloma (Ralph et ul, 1993). The IgH genes of 10 of these patients were subsequently sequenced and compared with published germline sequences. We have now prepared biotinylated and digoxigenin-labelled anti- sense idiotypic oligonucleotide probes from non-germline IgH gene nucleotide sequences and have performed mRNA in situ hybridization (ISH) using these probes on peripheral blood and bone marrow smears. These idiotypic probes provide a unique means of determining the full spectrum of the cells involved with the malignant clone, characterizing the malignant cells in the peripheral blood and opening new 113
Transcript
British Journal ofHaernatologll, 1995, 90, 113-118
Idiotypic oligonucleotide probes to detect myeloma cells by mRNA in situ hybridization
Ross BROWN, XIAO-FENG Luo, JOHN GIBSON, ALEC MORLEY,* PAM SYKES,* MICHAEL BRISCO AND DOUG JOSHUA Institute of Haematology, Royal Prince Alfred Hospital, Sydney, and *Huematology Department, Flinders Medical Centre, Adelaide, Australia
Received 16 September 1994; accepted for publication 3 February 1995
Summary. An mRNA in situ hybridization (ISH) method which used non-radioactive idiotypic oligonucleotide probes has been used to detect malignant cells in the bone marrow and peripheral blood of patients with multiple myeloma. For each of two patients with multiple myeloma a pair of biotinylated antisense oligonucleotide probes (1 8-22mer) was prepared from non-germline sequences of the rear- ranged immunoglobulin heavy chain (IgH) gene. These oligonucleotide sequences were not homologous with any previously published sequence. The probes from each patient were specific as shown by a failure to hybridize to any cells from six other myeloma patients and four normal indivi- duals. Specific staining of IgH gene mRNA occurred only when the myeloma cells and the sequence of the probe used
The diversity of the immunoglobulin repertoire is established during the early stages of B-cell development by a complex series of independent molecular events. Antibody diversity prior to antigen exposure is established by the formation of a vast array of different recombinations of VDJ regions of the immunoglobulin heavy chain (IgH) gene and VJ regions of the light chain gene. Somatic hypermutation of the rearranged immunoglobulin V region genes and their immediate flanking sequences occurs after antigen expo- sure and generates high-aihity antibodies while down- stream switching of the fully assembled V region to a different C region produces a new isotype class (Tonegawa, 1983: French et d. 1989). For patients with Bcell malignancies this series of molecular events provides useful markers of clonality and ontogeny. In acute lymphoblastic leukaemia, for example, clonal succession and oligoclonality have been well demonstrated (Beishuizen et al, 1991; Steenbergen et al, 1993). In multiple myeloma, however,
Correspondence: Dr Ross Brown, Haematology Department, Royal Prince Alfred Hospital, Missenden Road, Camperdown, NSW 2050, Australia.
@ 1995 Blackwell Science Ltd
were from the same patient. Using simultaneous fluorescent immunocytochetnistry it was shown that more than 95% of the ISH-positive cells expressed the malignant light chain in their cytoplasm, ISH positive cells were found in 1-4% of the peripheral blood mononuclear fraction of these two patients. These studies show that idiotypic oligonucleotide IgH gene probes can be used to identify individual cells belonging to the malignant clone and offer the possibility of developing innovative tumour-specific therapeutic procedures using antisense technology for patients with myeloma.
the malignancy develops from B cells in which somatic hypermutation has already occurred, and several groups have now demonstrated that neither the evolution of new IgH gene rearrangements nor ongoing somatic hypermuta- tion occurs during progression of this disease (Ralph et al, 1993: Bakkus et al, 1992). This observation provides us with the opportunity to use non-germline nucleotide sequences of the rearranged IgH gene as patient and tumour-specific markers at any stage of the disease.
In a previous study we used PCR to amplie a section of the rearranged IgH gene between the framework 2 and J regions in 21 patients with myeloma (Ralph et ul, 1993). The IgH genes of 10 of these patients were subsequently sequenced and compared with published germline sequences. We have now prepared biotinylated and digoxigenin-labelled anti- sense idiotypic oligonucleotide probes from non-germline IgH gene nucleotide sequences and have performed mRNA in situ hybridization (ISH) using these probes on peripheral blood and bone marrow smears. These idiotypic probes provide a unique means of determining the full spectrum of the cells involved with the malignant clone, characterizing the malignant cells in the peripheral blood and opening new
113
114 Ross Brown et a1 avenues to pursue tumour-specific therapy (Dolnick, 1990 Stein & Cohen, 1988).
MATERIALS AND METHODS
Patient selection. For ISH studies, bone marrow cells from patients with myeloma were obtained from the residue of samples aspirated for routine clinical evaluation. Normal bone marrow cells were obtained from donors for dogeneic transplantation after informed consent. The cells were centrifuged on Picoll-hypaqu, washed, and cytospin pre- parations were prepared. Peripheral blood cells were collected after informed consent.
DNA extraction. DNA was prepared by phenol extraction and ethanol precipitation from fixed and stained bone
Germline Pa t i en t 1 Sequence 1 Sequence 2
Germline Pa t ien t 1 Sequence 2
G e r m 1 i n e Pa t ien t 2 Sequence 3
Germline Pa t i en t 2
G e r m 1 i n e Pa t i en t 2 Sequence 4
Germline Pa t ien t 2 Sequence 4
Patient No 1. 3’V to J
marrow cells previously mounted on slides using the method of Briisco et al (1990). After soaking the slide in xylene (3-4d). methanol and water, the cells were scraped from the slide and digested with 80 pg proteinase K in 300 pl NID/PK buffer pH 8-3 (50 mmol/l KC1; 10 mmol/l Tris-HC1; 2.5 mmol/l Mg2CI; 100 mg/l gelatin: 0.45% Nonidet P40: 0.45% Tween 20).
Amplification of lgH genes. A two-round semi-nested PCR with consensus primers to amplify the rearranged IgH gene between the framework 2 (or 3) and J regions was used as previously described (Ralph et al, 1993). The first round used FR2B (or FR3A) and LJH primers and ELJH was used in the second round. When the FR2B primer was used both rounds of PCR consisted of 45 cycles of sequential annealing at 60°C for 30s and denaturation at 94°C for 30s. For the FR3A
A A A . . . . . . . . . . . . . . . . . . . . . . AAATA
Fig 1. Published germline sequences of immunoglobulin heavy chain (IgH) genes are aligned against the IgH gene sequence of two patients with multiple myeloma. Also shown are the sequences from which complementary oligonucleotide probes were prepared. Matched bases are represented by I.’. mismatched are indicated by lower case letters. V, D and J regions are marked above the sequences.
0 1995 Blackwell Science Ltd. British Journal of Haematology 9 0 113-1 18
Myeloma Cells detected by ISH 11 5 mix thawed the annealing mix on contact. Labelling and termination reactions were performed at 21°C. Reaction products were analysed using standard electrophoretic and autoradiography procedures. Automated sequencing was conducted using an Applied Biosystems Taq dye deoxy Terminator Cycle Sequencing Kit (Prober et al. 1987) and an Applied Biosystems 3 73A Automated Sequencer.
mRNA in situ hybridization. DEPC treated and RNase-free solutions and glassware were used throughout. Two antisense oligonucleotide sequences (1 8-24mer) containing predominantly non-germline IgH sequences were chosen for each patient. Each sequence chosen was different from any previously published sequence (GenBank release 79.0). The oligos were either biotinylated during synthesis (Bresatec, Adelaide, Australia) or labelled with digoxigenin-1 1-ddUTP using a DIG oligonucleotide 3’ end labelling kit (Boehringer- Mannheim). Air dried slides were fixed with 3 : 1 ethanol and acetic acid mixture for 30min. Endogenous biotin was blocked by treating with streptavidin. Hybridization was performed at 42°C for 24 h with 50 pl of probe (2 pg) in hybridization buffer containing 50% formamide; 5 x SCC; 25 mg/ml Herring sperm DNA: 10% dextran sulphate (Markovic et al, 1992). Following hybridization, the slides were washed with 2 x SCC and 0.4 x SCC at 42°C and signal was detected using two sequential applications of streptavidin and biotinylated alkaline phosphatase (DAKO ISH detection kit). Digoxigenein labelled probes were
primer the number of cycles was reduced to 30 in the first round and 20 in the second round. DNA from all patients was amplified in duplicate and the products were electro- phoresed on 3.5% agarose gels to check monoclonality.
DNA sequencing. PCR was used to attach M13 universal and reverse sequencing primer sequences onto opposite ends of the amplified IgH gene product. The monoclonal band was cut from a 3.5% agarose gel, soaked in 1 O O p l H20 overnight. 10 pl of this eluate acted as a template for third- round PCR using ELJH-A1 and either FR2-A1 or FR3-A1 as primers. These primers contained USP or RSP sequences with an 11-14 base adaptor sequence that annealed to the 5’ ends of the IgH product. The amplified product was ethanol precipitated and purified by electrophoresis on 1.5% agarose. The band was excised, the gel melted at 65°C in 0.5 mol/l NaCl and DNA was recovered with Tris (10 mmol/ I), EDTA (1 mmol/l) phenol. Two templates were prepared for each patient and each template was sequenced in both orientations. Manual sequencing was performed using a Promega T7 DNA sequencing kit with a modified annealing reaction containing 0.25 pmol template DNA and 10pmol primer and included 10% dimethyl sulphoxide (DMSO) and 0.5% NP-40 (Bachmann et al, 1990; Winship, 1989). The mix was boiled for 5 min, snap frozen in liquid nitrogen and then 5.5 p1 of labelling mix, including Sequnase polymerase, was placed on the side of the tube containing the annealing mix. The tube was centrifuged for 30 s so that the labelling
Fig 2(a). ISH staining of oligonucleotides 1 and 2 on peripheral blood cells of patient 1
Fig 2(b). ISH staining (no positive cells) of oligonucleotides 1 and 2 on peripheral blood cells of patient 2 .
Fig 3. ISH staining of bone marrow cells from patient 1 using oligonucleotides 1 and 2.
0 1995 Blackwell Science Ltd, British IournaI ofHaernatofogy 90: 113-118
Mgeloma Cells detected by ZSH 11 7 mRNA in situ hybridization technique has been used as the detection system. Each unique oligonucleotide probe reacted only with cells of the patient from whom the sequence had previously been determined.
Idiotypic oligonucleotide primers have been used in PCR by several groups to detect minimal disease in patients with acute lymphoblastic leukaemia (Beishuizen et al, 199 1: Steenbergen et al, 1993; Brisco et al, 1993). Although PCR and even Southern blots can demonstrate the presence of circulating myeloma cells (Van Reit et al, 1989: Fend et al. 1993). only in situ hybridization offers the possibility of identifying and phenotyping each individual tumour cell. Witzig et al (1994) quantitated circulating plasma cells by immunofluorescent light chain expression. ISH positivity, as demonstrated in this study, is a more specific way to detect cells belonging to the malignant clone.
We have demonstrated that a significant number of malignant cells (4% in one patient) are present in the peripheral blood of two patients with myeloma in a stable stage of their disease. This has important implications for autologous stem cell harvesters. Both PCR (Henry et al, personal communication) and ISH could be used to quantify the malignant cell content of stem cell harvests and CD34 purified products.
The ability to sequence tumour-specific DNA and then construct unique molecular probes which can identify individual cells belonging to a specific malignant clone has considerable potential for monitoring patients and developing innovative therapy options for patients with multiple myeloma.
detected with an anti-digoxigenin alkaline phosphatase conjugate (Boehringer-Mannheim). Hybridization signals were histochemicaly visualized using S-bromo-4-chloro-3- indolyl phosphate and nitroblue tetrazolium in N,N- dimethylformamide. Slides were counterstained with methyl green for 1 min. Negative control slides were treated with hybridization buffer but no probe.
Fluorescence immunocytochemistry. Bone marrow and peripheral blood slides were stained with FITC-labelled anti kappa, anti lambda and anti human Ig (DAKO) diluted 1 in 50 prior to ISH staining.
RESULTS
The rearranged IgH gene sequences of two patients with myeloma (GenBank accesssion numbers LO6226 and L06227) were aligned against published germline immu- noglobulin sequences to select suitable sequences to be used as idiotypic oligonucleotide probes (Fig 1). At the time of study, both patients were in a stable stage of their disease after chemotherapy, though each had > 10% plasma cells in their bone marrow, Primer sites and VDJ regions are marked above the nucleotide sequences. Antisense oligonucleotide probes containing predominantly non-germline sequences were prepared from the sequence marked.
There was positive staining of IgH gene mRNA in cytospin preparations of 19% of bone marrow and 4% of peripheral blood mononuclear cells from patient 1 using a cocktail of oligonucleotides 1 and 2 and in 1% of peripheral blood mononuclear cells from patient 2 using a cocktail of oligonucleotides 3 and 4. Both biotinylated and digoxi- genin-labelled idiotypic oligonucleotide probes successfully stained IgH gene mRNA from the appropriate patients but not controls. In preliminary studies the intensity of staining with a single digoxigenein probe was barely discernible. In subsequent studies a cocktail of two different biotinylated sequences and sequential application of streptavidin and biotinylated alkaline phosphatase was used to amplify the signal. For each of the probes used, there was specific staining of cells from the same patient (Fig 2a) but cells from other myeloma (n = 6) and normal control samples (n = 4) did not stain (Fig 2b). Simultaneous immunofluorescent staining demonstrated that the ISH positive cells were positive for the same light chain as the M protein.
The positive staining bone marrow cells included cells which were obviously plasma cells, smaller lymphocytoid plasma cells and other cells with lymphoid features (Fig 3). The positive staining cells on peripheral blood slides were probably plasmablastic lymphocytes, though this was difficult to confirm because a Romanowsky stain was not used simultaneously.
DISCUSSION
Idiotypic antisense oligonucleotide probes to patient specific nucleotide sequences of the rearranged IgH gene have been used to identify individual cells of the malignant cell population in bone marrow and peripheral blood samples of patients with multiple myeloma. A non-radioactive
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