Abstract

The majority of BALB/c mice immunized with the BCL1 lymphoma-derived idiotype (Id+) IgM and subsequently challenged with BCL1 tumor cells develop a state of tumor dormancy. The vast majority of dormant lymphoma cells are in cell cycle arrest, but there are also residual replicating cells. In the present studies, we attempted to define features of both the dormant lymphoma cells and the host that lead to escape from dormancy. Escape from dormancy occurs at a steady rate over a 2-year period, suggesting that it is a stochastic process. We found that, in the majority of mice, escape was due to the emergence of genetic variants that were no longer susceptible to the anti-Id–mediated induction of dormancy. Ten percent of these variants were Id; the remainder were Id+ but could grow in the presence of anti-Id antibodies, suggesting that there were mutations in molecules involved in one or more mIg-mediated negative-signaling pathways. In two of five such escapees, alterations in either Syk, HS1, and/or Lyn were observed. In a small percentage of mice, a low titer of circulating anti-Id antibody before tumor challenge correlated with a subsequent, more rapid loss of dormancy.

CANCER DORMANCY is a well-recognized clinical phenomenon in which tumor cells are present but the tumor burden does not increase for long periods of time.1-5 However, tumor cells can regrow many years or even decades later.

We have had a long-term interest in cancer dormancy emanating from studies of a murine lymphoma/leukemia (BCL1 ) in which we encountered dormancy early in our investigations with immunotoxin therapy.6 BCL1 was the first B-cell lymphoma described in mice.7 It arose spontaneously in an elderly BALB/c mouse and is characterized by early splenomegaly and later leukemia. Lymph nodes are not enlarged until late in the course of the disease.8 The tumor cells express IgMλ and IgDλ; the two isotypes share a common idiotype (Id) as defined serologically and by sequence analysis.8,9 

To gain more insight into the mechanisms underlying tumor dormancy, we10-12 and others13,14 have studied BCL1 tumor dormancy in mice immunized with the BCL1 IgM. About 70% of immunized mice do not develop splenomegaly by 60 days11 and this time point (and later) defines dormancy. The dormant state lasts for many months (or years), but dormant tumors can regrow at a later time. The aim of this study was to understand events in both the host and the tumor cells responsible for the outgrowth of dormant lymphoma cells (DLCs) in Id-immunized mice.

MATERIALS AND METHODS

Animals and BCL1 tumors.BALB/c mice were obtained from our University Animal Resources Center (ARC). SCID mice were purchased from the University of Wisconsin and housed and maintained in modified barrier facilities by the ARC. The BCL1 tumor was maintained in vivo by intravenous and intraperitoneal passage in BALB/c mice. (We call these cells wild-type.) The cells bear μλ3 and δλ3 . Five weeks after passage of 5 to 10 × 105 tumor cells, mice were killed and their splenocytes were used as a source of tumor cells. The presence of BCL1 tumor cells in dormant mice was determined by injecting graded numbers of splenocytes from these mice into naive BALB/c mice and/or by FACS analysis of splenocytes.

The assessment of splenomegaly for monitoring tumor growth.To follow the growth of tumor cells over long periods of time, it was necessary to use a noninvasive, quantitative measurement of tumor growth. Previous studies have established that the spleen is the first and primary site of tumor growth in BALB/c mice.8 We, therefore, evaluated splenic enlargement by blinded palpation of a large number of animals to determine the suitability of this method for quantifying tumor.

Determination of splenomegaly by physical palpation.To assign values to the degree of splenomegaly, the ventral surface of the mouse was divided into four equal quadrants progressing from the left rib cage to the right rib cage. Each quadrant was assigned a splenic index with values of 1, 2, 3, and 4; the median line corresponds to the value of 2 (Fig 1). Spleens from normal 12-week-old BALB/c mice have a value of 0.5; ie, the spleen extends halfway between the left rib cage and the first quadrant line that is given the value of 1, a spleen that extends to the median line was given a value of 2, one that extends halfway between the median line and right rib cage was given a value of 3, and one that extends to the right rib cage has a value of 4. If the tip of the spleen is located between two of the primary quadrants, the distance between the lines is estimated in increments of one-eighths. Immunization with the BCL1 Id can result in an increase in spleen size to 1 and occasionally 1.5. Therefore, we selected a spleen index of ≥2 as indicative of tumor growth in BCL1 Id immunized animals. The reproducibility of the spleen palpation was shown by the close correlation of repeated blinded determinations by a single individual using the same animals and the close correlation of independent determinations of splenomegaly by separate individuals. All determinations of splenomegaly were performed in a blinded manner. As shown in Fig 2A, palpation is highly effective at quantifying BCL1 cells in the spleen. In addition, the time of onset of splenomegaly after transferring the BCL1 cells into naive recipients represents an additional means of quantifying the tumor burden (Fig 2B).

Fig. 1.

Determination of spleen indices. The ventral surface of the mouse was divided into four equal quadrants progressing from the left rib cage to the right rib cage. The terminus of each quadrant was assigned a splenic index of 1, 2 (median line of the mouse), 3, and 4 (right rib cage) (see solid lines in figure). Representations of spleens with splenic indices of 1 (black), 2 (dark gray), 3 (light gray), and 4 (white) bordered by black are illustrated. The costal margin is drawn to improve clarity.

Fig. 1.

Determination of spleen indices. The ventral surface of the mouse was divided into four equal quadrants progressing from the left rib cage to the right rib cage. The terminus of each quadrant was assigned a splenic index of 1, 2 (median line of the mouse), 3, and 4 (right rib cage) (see solid lines in figure). Representations of spleens with splenic indices of 1 (black), 2 (dark gray), 3 (light gray), and 4 (white) bordered by black are illustrated. The costal margin is drawn to improve clarity.

Fig. 2.

(A) Splenomegaly, as measured by palpation, is proportional to the number of BCL1 cells in the spleen. Naive BALB/c mice injected with 106 BCL1 cells were palpated and their spleen size was recorded single-blinded. The size was graded as 1 to 4 (spleen index), with ≥2 representing tumor growth (see the Materials and Methods). The animals were killed, the cells were stained, and the number of Id+ cells was determined by FACS analysis. (B) Time of onset of splenomegaly as determined by palpation correlates with the number of BCL1 cells previously injected. Groups of five animals each were injected with graded numbers of BCL1 cells as determined by Id analysis. Splenomegaly was determined by a single observer without knowledge of the treatment protocol. Bars are the SEM.

Fig. 2.

(A) Splenomegaly, as measured by palpation, is proportional to the number of BCL1 cells in the spleen. Naive BALB/c mice injected with 106 BCL1 cells were palpated and their spleen size was recorded single-blinded. The size was graded as 1 to 4 (spleen index), with ≥2 representing tumor growth (see the Materials and Methods). The animals were killed, the cells were stained, and the number of Id+ cells was determined by FACS analysis. (B) Time of onset of splenomegaly as determined by palpation correlates with the number of BCL1 cells previously injected. Groups of five animals each were injected with graded numbers of BCL1 cells as determined by Id analysis. Splenomegaly was determined by a single observer without knowledge of the treatment protocol. Bars are the SEM.

Antibodies.To prepare polyclonal anti-Id, the BCL1 Id was conjugated to keyhole limpet hemocyanin (KLH) and injected into BALB/c mice.11 The total levels of polyclonal mouse IgG anti-BCL1 Id in sera were determined by radioimmunoassay, as previously reported.11 Polyclonal mouse anti-BCL1 Id (MABCL1Id) ascites was produced by intraperitoneal injection of pristane into Id-immune mice followed 1 to 2 weeks later by an intraperitoneal injection of SP2/0 myeloma cells.15,16 6A5 hybridoma cells secreting rat IgG2a anti-BCL1 Id were obtained from Dr Freda Stevenson (Southampton, UK). The C5D5 BCL1 Id+ IgM λ was obtained by fusing BCL1 cells with SP2/0 myeloma cells and purifying the Id+ IgM. Monoclonal B1.1 rat antimouse λ3 (RtAMλ)17 was generated and characterized as described. Normal rat Ig (NRtIg) was purified from pooled rat serum by chromatography on diethyl aminoethyl-Sephadex A-50. Rabbit antimouse μ (RAMμ) and rabbit antimouse Ig (RAMIg) were prepared as described.18 Other antibodies included monoclonal rat antimouse IgM (PharMingen, San Diego, CA),11 mouse antirat λ-1 (PharMingen),12 rabbit anti-HS1 peptide (RbAHS1),19 and monoclonal anti-HS1 (MAHS1).20,21 Rabbit anti-Lyn (RbALyn) sera were produced by immunization with a GST-containing fusion protein incorporating a mouse-Lyn specific sequence located between positions 10 and 66. To prepare rabbit anti-Syk sera (RbASyk), rabbits were immunized with a high-performance liquid chromatography-purified synthetic peptide containing the 28-C-terminal amino acids of the human Syk protein bound to KLH in the presence of carbodimide.

Cytofluorometry.Stained DLCs or wild-type BCL1 cells were analyzed on a FACScan or FACStar Plus equipped with argon ion and helium neon lasers tuned at 488 and 633 nm, respectively (Becton Dickinson, San Jose, CA). Forward or orthogonal light scattering and 3 to 4 fluorochrome emission signals were recorded for each cell. Data were analyzed by Paint-a-Gate software as described.22 This analysis enabled us to perform a multidimensional identification of cells reactive with the antibodies as well as the determination of their relative size (small v large) based on the position of the cells in the correlative display of the forward versus the orthogonal light scattering. The number of BCL1 cells could be measured by determining either the number of Id+ cells or the number of large λ+3 cells in the tumor-bearing mice minus the number of large λ+3 cells in Id-immune non–tumor-bearing mice. The results were similar using these two methods.

Phenotyping.Cells (106) were incubated with either anti-λ or anti-Id antibodies. Binding was detected using a secondary fluorescein isothiocyanate (FITC) mouse antirat κ (MARK) antibody (Becton Dickinson). Alternatively, cells were incubated with a biotinylated anti-λ or anti-Id and incubated with FITC-antimouse IgM and the bound biotinylated antibody was detected with phycoerythrin-streptavidin. Similar results were obtained from staining with anti-Id or anti-λ if the small λ+ cells were excluded. The latter represents about 0.25% of nucleated cells per spleen. Cells were stained at 4°C for 15 minutes. After the final wash, cells were resuspended in 1% paraformaldehyde and maintained at 4°C in the dark before analysis. Control values were subtracted.

Determination of tumor growth in Id-immune mice.Id-immune and control mice were injected with 106 escapee tumor cells or 106 wild-type BCL1 tumor cells as a control and spleens were palpated twice weekly. The day of onset of splenomegaly was recorded for each animal and experimental values were compared with controls by t-test to determine if they were different.11,12 

Immunoblotting.Cells (1 × 107) were washed once in phosphate-buffered saline before lysis in 100 μL lysis buffer for 20 minutes on a shaker at 4°C. After removal of insoluble material at 13,000 RPM for 10 minutes, an equal volume of 2× sodium dodecyl sulfate (SDS) Laemmli sample buffer23 was added to the cell lysate. Protein transfer and immunodetection were performed as described.24 For detection of phosphotyrosil residues, the primary monoclonal antibody (MoAb) was B-PY-20 and the secondary reagent was HRP-SA.

Immune complex kinase assay.Cells (5 × 106) were lysed in 0.5 mL lysis buffer and the kinases were immunoprecipitated by antibodies against Lyn or Syk. The beads were washed twice with kinase buffer (20 mmol/L Tris-HCl, pH 7.5, 100 mmol/L NaCl, 5 mmol/L MnCl2 , 5 mmol/L MgCl2 ) and resuspended in 50 μL of the same buffer before adding 1 μmol/L cold ATP and 10 μCi γ-[32P]ATP (Amersham, Arlington Heights, IL).25 After incubation for 15 minutes at room temperature, the beads were washed five times with washing buffer and the proteins were eluted in 70 μL Laemmli SDS sample buffer. Thirty-five microliters from each sample was electrophoresed on 8% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and the gel was fixed in 15% acetic acid for 30 minutes, washed, and dried. The radioactive bands were visualized and analyzed using a PhosphoroImager System (Molecular Dynamics, Sunnyvale, CA).

RESULTS

Growth of DLCs in Id-immune mice.The kinetics of tumor growth of BCL1 cells in Id-immune and naive BALB/c mice are shown in Fig 3. In about 70% of Id-immune mice, there was no detectable splenomegaly and no increase in BCL1 Id+ cells for 60 days or more.11 In contrast, an increase in both splenomegaly and Id+ cells in naive mice injected with BCL1 cells was detected by 1 month. Hence, 60 days was used as the operational definition of dormancy in these (as in previous) studies.

Fig. 3.

Progressive tumor growth in nonimmune versus Id-immune mice challenged with BCL1 tumor cells. (A) The spleens from naive (○) and Id-immune (•) BALB/c mice injected with 106 BCL1 cells were palpated as described11 on the days indicated. Naive mice have an average spleen size of ∼0.5. Immunization with BCL1 Id results in a spleen size of 1 to 1.5 before BCL1 tumor injection. (B) BCL1 cells (1 × 106) were injected into naive BALB/c mice (○) or into Id-immune BALB/c mice (•). At the times indicated, mice were killed and the number of BCL1 tumor cells was determined by staining with RtABCL1Id for naive animals or RtAMλ for Id-immune animals (endogenous mouse anti-BCL1 Id [MA BCL1 Id] blocks staining with RtABCL1-Id but not the RtAMλ Ab) and analyzed by FACS.

Fig. 3.

Progressive tumor growth in nonimmune versus Id-immune mice challenged with BCL1 tumor cells. (A) The spleens from naive (○) and Id-immune (•) BALB/c mice injected with 106 BCL1 cells were palpated as described11 on the days indicated. Naive mice have an average spleen size of ∼0.5. Immunization with BCL1 Id results in a spleen size of 1 to 1.5 before BCL1 tumor injection. (B) BCL1 cells (1 × 106) were injected into naive BALB/c mice (○) or into Id-immune BALB/c mice (•). At the times indicated, mice were killed and the number of BCL1 tumor cells was determined by staining with RtABCL1Id for naive animals or RtAMλ for Id-immune animals (endogenous mouse anti-BCL1 Id [MA BCL1 Id] blocks staining with RtABCL1-Id but not the RtAMλ Ab) and analyzed by FACS.

Size of the population of DLCs with time after tumor cell challenge.Lack of progressive splenomegaly could be caused by an absence of BCL1 cells in the animal, slow growth of tumor cells due to the ongoing specific immune response, or the presence of a population of tumor cells that is not growing or slowly declining in size. In an attempt to distinguish among these possibilities, dormant Id-immune mice (ie, without splenomegaly) were killed at various times up to 210 days after BCL1 challenge and cells from their spleens were analyzed by flow cytometry for the number of Id+ BCL1 cells. As shown in Fig 4, there was no significant change in the size of the DLC population during the time of observation. Three additional mice were analyzed as late as 450 days. They each had 0.5 to 1 × 106 DLCs in their spleens. These results exclude the explanations that dormancy is due to an absence of tumor cells or to the slow growth of tumor. Rather, a state of dormancy was caused by the failure of the existing DLC population to expand, ie, cells were dividing and dying at the same rate and/or were cell cycle-arrested.

Fig. 4.

The population of DLCs is stable in size. Id-immune mice carrying dormant tumors were killed at various times. The number of large λ+3 cells was determined by FACS analysis. The number in each column indicates the number of mice analyzed for that time frame with the standard deviation indicated by the error bars. The average number of λ+3 cells in Id-immune animals not injected with BCL1 cells (normal B-cell blasts, 0.24 ± 0.09; N = 6) was subtracted from the value obtained for each animal before each group was averaged. The percentage of dormant tumor is the percentage of corrected λ+3 cells in the total splenocyte population for each animal.

Fig. 4.

The population of DLCs is stable in size. Id-immune mice carrying dormant tumors were killed at various times. The number of large λ+3 cells was determined by FACS analysis. The number in each column indicates the number of mice analyzed for that time frame with the standard deviation indicated by the error bars. The average number of λ+3 cells in Id-immune animals not injected with BCL1 cells (normal B-cell blasts, 0.24 ± 0.09; N = 6) was subtracted from the value obtained for each animal before each group was averaged. The percentage of dormant tumor is the percentage of corrected λ+3 cells in the total splenocyte population for each animal.

Malignant potential of DLCs.The next question that arose was whether the DLCs had lost their malignant potential or whether they were fully malignant but maintained in a dormant state. To answer this question, normal mice were injected with various numbers of either DLCs from Id-immune mice or wild-type BCL1 cells from nonimmune mice. Preliminary experiments indicated that cell sorting after staining with anti-λ decreased the tumorigenicity of both wild-type BCL1 cells and the DLCs. Hence, in these experiments, aliquots of the donor tumor cell suspension were assayed for the number of tumor cells present; subsequently, separate unsorted, unstained cells were transferred. In addition, we determined whether cotransfer of B and T cells from Id-immune mice and exposure to anti-Id could influence the behavior of wild-type BCL1 cells in the naive recipient.

Fig. 5.

The malignant potential of DLCs. The percentage of tumor cells was determined by FACS analysis in a population of splenocytes from BCL1 wild-type or DLC-containing spleens. Naive animals were then injected with graded numbers of wild-type BCL1 tumor cells (▵), DLC tumor cells (□), or wild-type BCL1 tumor cells pulsed with 100 μg MαBCL1Id, and co-mixed with 106 splenocytes from BCL1 -Id–immunized mice (○) or wild-type BCL1 tumor cells admixed with a pool of 106 splenocytes from mice containing 0.72% DLCs (•). Cell numbers were 100 (not shown), 30, 10, 6 (not shown), 3, and 1 (not shown). A Breslow-Day test was used to examine the differences between BCL1 and DLC treatment groups across cell number groupings for the occurrence of splenomegaly. Comparisons of time to splenomegaly across treatment groups were provided by numbers of cells entered using a log-rank test (product-limit survival estimates enter into this computation). Multiple comparisons between pairs of treatments were accomplished using a Bonferonni correction to the P values for significant results. Mantel-Haenszel χ2 tests were used to provide an indication of the tendency to increase the occurrence of splenomegaly with increased cell numbers. In comparing BCL1 to DLCs for occurrence of splenomegaly, the Breslow-Day test comparing equal odds ratios of occurrence between the BCL1 and DLCs across cell numbers was not significant (P = .627). The overall odds ratio estimate is 6.103 for likelihood of splenomegaly from BCL1 compared with DLCs. The comparison by log-rank test for time to splenomegaly for mice receiving DLCs versus the other treatment groups was significantly different for cell numbers (P values in parentheses) of 100 (.0001), 30 (.0001), 10 (.0031), and 6 (.0194). No statistical difference was observed when 3 (.351) or 1 (.085) cells were injected. (Analogous results were also seen when the data were analyzed by a standard t-test.) There were no statistical differences in occurrence of splenomegaly or time to splenomegaly among the groups (BCL1 , treated BCL1 , or the BCL1 plus DLCs). Mantel-Haenszel statistics indicate an increased occurrence of splenomegaly for increased cell numbers (P values in parentheses): BCL1 (.001), DLC (.001), and treated BCL1 (.005).

Fig. 5.

The malignant potential of DLCs. The percentage of tumor cells was determined by FACS analysis in a population of splenocytes from BCL1 wild-type or DLC-containing spleens. Naive animals were then injected with graded numbers of wild-type BCL1 tumor cells (▵), DLC tumor cells (□), or wild-type BCL1 tumor cells pulsed with 100 μg MαBCL1Id, and co-mixed with 106 splenocytes from BCL1 -Id–immunized mice (○) or wild-type BCL1 tumor cells admixed with a pool of 106 splenocytes from mice containing 0.72% DLCs (•). Cell numbers were 100 (not shown), 30, 10, 6 (not shown), 3, and 1 (not shown). A Breslow-Day test was used to examine the differences between BCL1 and DLC treatment groups across cell number groupings for the occurrence of splenomegaly. Comparisons of time to splenomegaly across treatment groups were provided by numbers of cells entered using a log-rank test (product-limit survival estimates enter into this computation). Multiple comparisons between pairs of treatments were accomplished using a Bonferonni correction to the P values for significant results. Mantel-Haenszel χ2 tests were used to provide an indication of the tendency to increase the occurrence of splenomegaly with increased cell numbers. In comparing BCL1 to DLCs for occurrence of splenomegaly, the Breslow-Day test comparing equal odds ratios of occurrence between the BCL1 and DLCs across cell numbers was not significant (P = .627). The overall odds ratio estimate is 6.103 for likelihood of splenomegaly from BCL1 compared with DLCs. The comparison by log-rank test for time to splenomegaly for mice receiving DLCs versus the other treatment groups was significantly different for cell numbers (P values in parentheses) of 100 (.0001), 30 (.0001), 10 (.0031), and 6 (.0194). No statistical difference was observed when 3 (.351) or 1 (.085) cells were injected. (Analogous results were also seen when the data were analyzed by a standard t-test.) There were no statistical differences in occurrence of splenomegaly or time to splenomegaly among the groups (BCL1 , treated BCL1 , or the BCL1 plus DLCs). Mantel-Haenszel statistics indicate an increased occurrence of splenomegaly for increased cell numbers (P values in parentheses): BCL1 (.001), DLC (.001), and treated BCL1 (.005).

Figure 5 shows a representative experiment of two performed. There was no significant difference in the number of DLCs compared with wild-type BCL1 cells needed to adoptively transfer progressive tumor growth in recipients.

However, the results did show a significantly longer delay between the regrowth of DLCs as compared with the same number of wild-type BCL1 cells. This could not be accounted for by the transfer of Id-immune B or T cells from the donor spleen because the addition of 99% Id-immune spleen cells from dormant mice to wild-type BCL1 cells did not inhibit tumor growth in adoptive recipients (see Fig 5 legend for statistical analysis).

These results indicate that DLCs do not replicate initially as rapidly as wild-type BCL1 cells but that their replicative capacity is eventually fully restored in an adoptive recipient.

Kinetics of loss of dormancy.We have observed the natural history of dormant BCL1 tumor in 114 mice observed for 610 days after challenge with the BCL1 tumor cells. As shown in Fig 6, there was a steady rate of loss of dormancy over this period of time, suggesting a stochastic process. One likely interpretation is that genetic alterations had taken place in the replicating tumor cells.

Fig. 6.

The loss of dormancy with time after BCL1 challenge. One hundred fourteen dormant mice were examined weekly by palpation for splenic enlargement.11 The straight line was generated by computer analysis of the data; the regression coefficient is .985.

Fig. 6.

The loss of dormancy with time after BCL1 challenge. One hundred fourteen dormant mice were examined weekly by palpation for splenic enlargement.11 The straight line was generated by computer analysis of the data; the regression coefficient is .985.

Expression of mId and mIgM on escapee tumor cells.It might be predicted from studies of others13,14,26-30 that a major mechanism leading to escape of BCL1 cells from dormancy after anti-Id treatment is a loss in the expression of the mId because of the high mutation rate of genes encoding the hypervariable regions of the light and heavy chain. To investigate this, we phenotyped the splenocytes from dormant mice in which tumor had regrown. In such mice, progressive splenomegaly due to regrowth of the BCL1 tumor eventually results in the absorption of all the anti-Id from the sera of these mice. Thus, in the regrowing tumor cells, the Id epitopes are not masked by anti-Id antibodies and escapees can therefore be phenotyped for the expression of Id. An analysis of spleens from 46 animals showed the usual expression of Id and λ in 40 mice; there were 6 animals in which spleen cells were IgM. Splenocytes from these mice were transferred to nonimmune recipients and in 2 of the 6 transfers, Id+ tumor cells grew in the secondary recipient spleens. This suggests that, in these 2 donor mice, anti-Id was either coating the Id+ tumor cells or had downregulated mIgM. Thus, only 4 of 46 mice had lost their Id due to lack of expression of mIgM.

Growth of Id+ escapee tumor cells adoptively transferred Into Id-immune mice.To confirm that Id+ escapees were variants that were resistant to the induction of dormancy by the presence of anti-Id antibodies in secondary recipients, such escapees were injected into Id-immune mice and the mice were followed for tumor growth. Table 1 summarizes the results of these experiments. Of 8 escapees from Id-immune BALB/c mice, 5 showed a significant loss of susceptibility to the induction of dormancy in Id-immune recipients. Dormancy has also been induced in SCID mice passively immunized with rabbit anti-μ and challenged with BCL1 cells.10 Two escapees from such passively immunized SCID mice (nos. 9 and 10) were not susceptible to the induction of dormancy when cells were injected into either Id-immune BALB/c mice or SCID mice passively immunized with rabbit anti-μ.

Table 1.

Transfer of Escapee Tumor

Cells Injected No. of Dormant Mice/Total No. of Recipients (day to splenomegaly ± SD) 
 Nonimmune (control) ID-Immune* SCID + Anti-μ Variant (0-4+) 
BCL1 control 0/14 (28 ± 3) 19/28 (99 ± 47) ND  
Id-immune escapee no. 
0/5 (25 ± 0) 0/11 (47 ± 22) ND +++ 
0/5 (25 ± 0) 4/12 (67 ± 27) ND ++ 
0/5 (30 ± 0) 0/9 (30 ± 0) ND ++++ 
0/5 (47 ± 5) 5/10 (82 ± 43) ND 
2/5 (62 ± 8) 6/10 (79 ± 44) ND 
0/5 (28 ± 0) 1/9 (43 ± 16) ND +++ 
0/4 (29 ± 2) 6/10 (70 ± 33) ND 
0/5 (28 ± 0) 3/10 (44 ± 22) ND ++ 
SCID escapee no. 
0/5 (34 ± 6) 0/9 (36 ± 7) 0/10 (39 ± 2) ++++ 
10 0/5 (28 ± 0) 0/10 (33 ± 4) 0/10 (39 ± 2) ++++ 
Cells Injected No. of Dormant Mice/Total No. of Recipients (day to splenomegaly ± SD) 
 Nonimmune (control) ID-Immune* SCID + Anti-μ Variant (0-4+) 
BCL1 control 0/14 (28 ± 3) 19/28 (99 ± 47) ND  
Id-immune escapee no. 
0/5 (25 ± 0) 0/11 (47 ± 22) ND +++ 
0/5 (25 ± 0) 4/12 (67 ± 27) ND ++ 
0/5 (30 ± 0) 0/9 (30 ± 0) ND ++++ 
0/5 (47 ± 5) 5/10 (82 ± 43) ND 
2/5 (62 ± 8) 6/10 (79 ± 44) ND 
0/5 (28 ± 0) 1/9 (43 ± 16) ND +++ 
0/4 (29 ± 2) 6/10 (70 ± 33) ND 
0/5 (28 ± 0) 3/10 (44 ± 22) ND ++ 
SCID escapee no. 
0/5 (34 ± 6) 0/9 (36 ± 7) 0/10 (39 ± 2) ++++ 
10 0/5 (28 ± 0) 0/10 (33 ± 4) 0/10 (39 ± 2) ++++ 

Abbreviation: ND, not done.

*

The average day to splenomegaly of the escapees from Id-immune mice reinjected into Id-immune animals was compared with the average day to splenomegaly of control BCL1 in Id-immune animals. Five of 8 escapees had a statistically significant decrease in the average day to splenomegaly as determined by the Student's t-test (escapee no. 1 with P = .0013, escapee no. 2 with P = .018, escapee no. 3 with P = .0037, escapee no. 6 with P = .0002, and escapee no. 8 with P = .0001).

Estimation of susceptibility (0-4+) based on both proportion of dormant mice and interval before splenomegaly of challenged Id-immune recipient.

As shown in Table 1, escapees no. 4, 5, and 7 were still susceptible to the induction of dormancy when cells were reinjected into Id-immune mice, suggesting that changes in the host and not in the tumor cells were responsible for escape.

Anti-Id titers in mice with regrowing tumors.It was possible that anti-Id levels and/or cellular immunity were low in escapees no. 4, 5, and 7 and that a poor or waning immune response was responsible for the failure of the 3 mice to maintain the BCL1 cells in a dormant state. Titers of anti-Id could not be determined after growth of the DLCs, because the enlarged tumor cell population adsorbed antibody from the circulation. Therefore, to explore this question, we examined the anti-Id titers in Id-immune mice before challenge with BCL1 cells to determine whether there was a relationship between the levels of IgG anti-Id in response to the immunization regimen and the capacity of the immune host to induce dormancy in the BCL1 population.

As shown in Table 2, the presence of low serum anti-Id titers before BCL1 challenge increased the probability that the tumor would grow rapidly in such mice and that dormancy would not be induced. A higher anti-Id titer correlated with an increase in the proportion of mice that became dormant. The differences between the nondormant and either of the dormant groups is highly significant. In dormant mice observed for the time of onset of splenomegaly (line 3; the other dormant groups were killed for other experimental studies), the duration averaged 132 days, compared with 36 days in the nondormant mice. Thus, the level of anti-Id and, possibly, the level of cellular immunity (which was not assessed) could play a role in the induction and maintenance of dormancy.

Table 2.

Correlation of Initial Serum Level of Mouse Anti-BCL1 Id to Development of Dormancy After BCL1 Challenge

 Serum Anti-BCL1 Id (average μg/mL ± SD) Time of Onset of Splenomegaly (average day ± SD) 
Nondormant animals (N = 15) 70 ± 57 36 ± 15 
Dormant animals (N = 28) 358 ± 546  —  
Subpopulation of dormant animals followed for splenomegaly (N = 11) 269 ± 189 132 ± 74 
 Serum Anti-BCL1 Id (average μg/mL ± SD) Time of Onset of Splenomegaly (average day ± SD) 
Nondormant animals (N = 15) 70 ± 57 36 ± 15 
Dormant animals (N = 28) 358 ± 546  —  
Subpopulation of dormant animals followed for splenomegaly (N = 11) 269 ± 189 132 ± 74 

Forty-three Id-immune animals were bled on day 0, and levels of α Id were determined. BCL1 cells (106) were then injected and the animals were followed for onset of splenomegaly. The differences in average initial serum levels of animals that became dormant versus animals that did not were statistically significant as measured by both the Mann-Whitney U-test (P = .03) and by t-test (P = .02). Because of the necessity of killing dormant animals for analysis, only a subpopulation of dormant animals were followed for the onset of splenomegaly. The average day to splenomegaly for the dormant mice is statistically different by t-test from the average day to splenomegaly for the nondormant animal group (P = .00004). The initial levels of mouse anti-BCL1 Id are also different for these two groups (P = .0008). Serum levels of total anti-BCL1Id IgG were measured by radioimmunoassay as previously described.11 

Analysis of BCL1 escapees.Of the 10 BCL1 escapees described in Table 1, 7 were verified as variants, ie, they would grow when adoptively transferred to Id-immune mice. These included no. 1, 2, 3, 6, 8, 9, and 10. In contrast, escapees no. 4, 5, and 7 were not variants and failed to grow when adoptively transferred into Id-immune mice. All 7 variants examined were Id+, as determined by positive staining with the RAId, and yet were growing in the presence of anti-Id, suggesting that there was a defect in the ability of anti-Id to signal growth arrest. Because these variants were Id+, we hypothesized that the BCL1 cells had become altered in such a way that anti-Id could no longer signal growth arrest. To explore this possibility further, we used Western blots to identify several molecules involved in anti-Ig signaling pathways, ie, Lyn, Syk, and HS1.

Lyn has been implicated in anti-Ig–mediated cell cycle arrest (CCA) of BCL1 cells24 and Syk is involved in the signaling of normal B cells.31 HS1 is a substrate for Lyn21,32 and it has been reported that HS1 is lacking in two variants of a murine B-lymphoma cell line that could not be negatively signaled by anti-μ in vitro.19 IgM-mediated apoptosis was also restored in one of the variants of the murine HS1-deficient B-cell lines by a retroviral expression vector for human HS1.19 To determine whether variants had normal levels of HS1, we used a Western blotting assay that could detect HS1 in BCL1 cells but little in normal spleen cells (Fig 7). BCL1 cells were mixed with normal splenocytes, and the lysates were immunoblotted with RbAHS1.19,20 As shown in Fig 7, the assay could detect HS1 in a mixture of 5% BCL1 cells and 95% normal splenocytes. Using this sensitive assay, lysates from 5 escapees (30% to 80% Id+ cells) and wild-type BCL1 cells (40% to 70% Id+ cells) were immunoblotted with either the RbAHS1 or MAHS1.19-21 Although all lysates were positive in the RbαHS1 blot, one escapee (no. 6) did not blot positively with MAHS1 (Fig 8B). These results suggest that there is a genetic variation in HS1 that affected the epitope detected by the MoAb but not the rabbit antibody. The putative HS1 variant (no. 6) also had no Syk protein detectable by the rabbit antibody (Fig 9).

Fig. 7.

An analysis of HS1 protein in mixtures of BCL1 cells and normal spleen cells. The percentage of BCL1 cells mixed with spleen cells: lane 1, 0%; lane 2, 2%; lane 3, 5%; lane 4, 10%; lane 5, 20%; lane 6, 30%; lane 7, 40%; and lane 8, 50%. The arrow indicates the position of HS1 at 75 kD. The integrated optical densities of the HS1 band are as follows: lane 1, 5.6; lane 2, 5.9; lane 3, 10.8; lane 4, 10.6; lane 5, 18.9; lane 6, 26.2; lane 7, 37.1; and lane 8, 46.2.

Fig. 7.

An analysis of HS1 protein in mixtures of BCL1 cells and normal spleen cells. The percentage of BCL1 cells mixed with spleen cells: lane 1, 0%; lane 2, 2%; lane 3, 5%; lane 4, 10%; lane 5, 20%; lane 6, 30%; lane 7, 40%; and lane 8, 50%. The arrow indicates the position of HS1 at 75 kD. The integrated optical densities of the HS1 band are as follows: lane 1, 5.6; lane 2, 5.9; lane 3, 10.8; lane 4, 10.6; lane 5, 18.9; lane 6, 26.2; lane 7, 37.1; and lane 8, 46.2.

Fig. 8.

Western blot analysis of HS1 in escapees. (A) MoAb AHS1. (B) RbAHS1. Lanes were loaded with equal amounts of cell lysates prepared from the same number of cells. Lane 1, 10% BCL1 cells plus 90% normal splenocytes. Lane 2, wild-type-BCL1 tumor. Lane 3, escapee no. 3. Lane 4, escapee no. 6.

Fig. 8.

Western blot analysis of HS1 in escapees. (A) MoAb AHS1. (B) RbAHS1. Lanes were loaded with equal amounts of cell lysates prepared from the same number of cells. Lane 1, 10% BCL1 cells plus 90% normal splenocytes. Lane 2, wild-type-BCL1 tumor. Lane 3, escapee no. 3. Lane 4, escapee no. 6.

Fig. 9.

Analysis of Lyn and Syk proteins and Lyn kinase in escapees. All lysates were prepared from splenocytes recovered from BCL1 tumor bearing nonimmune mice and BCL1 escapees. The presence of Syk (top) and Lyn (center) were determined by immunoblotting. Lyn tyrosine kinase was immunoprecipitated from separate samples and immune complex kinase assay performed (see the Materials and Methods) (bottom). Lane 1, BCL1 ; lane 2, escapee no. 1; lane 3, escapee no. 3; lane 4, escapee no. 6; lane 5, escapee no. 9; and lane 6, escapee no. 10.

Fig. 9.

Analysis of Lyn and Syk proteins and Lyn kinase in escapees. All lysates were prepared from splenocytes recovered from BCL1 tumor bearing nonimmune mice and BCL1 escapees. The presence of Syk (top) and Lyn (center) were determined by immunoblotting. Lyn tyrosine kinase was immunoprecipitated from separate samples and immune complex kinase assay performed (see the Materials and Methods) (bottom). Lane 1, BCL1 ; lane 2, escapee no. 1; lane 3, escapee no. 3; lane 4, escapee no. 6; lane 5, escapee no. 9; and lane 6, escapee no. 10.

The data in Fig 9 also show that all 5 of the variants tested (no. 1, 3, 6, 9, and 10 in Table 3) had readily detectable Lyn protein, but no. 6 and 9 had markedly reduced Lyn kinase activity as measured by autophosphorylation. Thus, as summarized in Table 3, at least 2 of 5 Id+ escapees examined had detectable alterations in one or more of the three proteins involved in signaling pathways that we studied. The other 3 escapees that were genetic variants could have alterations in other signaling proteins or in Syk kinase activity.

Table 3.

Biochemical Alterations in Genetic Variant Escapees

Animal No. HS1 LYN SYK 
 RbAHS1 MAHS1 Protein Kinase Protein 
− ± − 
± 
10 
Animal No. HS1 LYN SYK 
 RbAHS1 MAHS1 Protein Kinase Protein 
− ± − 
± 
10 

DISCUSSION

In the present study, an immunologic murine model of dormancy was used. Mice were actively or passively immunized so that there were circulating levels of anti-Id or anti-μ antibody. Id+ IgM+, BCL1 cells were then injected into the immunized mice and dormancy was induced in 70% of the recipients. As documented previously,12 dormancy can last for up to 2 years. The major findings to emerge from this study are as follows. (1) The population of DLCs in the spleen was stable for the 210 days of observation. (2) The DLCs were physiologically different from the wild-type DLCs because they grew more slowly for a period of time when transferred to naive recipients. (3) Eventually the DLCs regained most, if not all, of their malignant potential. (4) The kinetics of loss of dormancy occurred at a steady rate during the 2 years of observation. (5) Mice with high levels of serum anti-Id before BCL1 challenge were more likely to become dormant than those with lower levels of anti-Id antibody. (6) About 90% of the escapees were IgM+ and Id+; 10% were IdIgM. Thus, the majority of escapees were Id+ variants with decreased or no susceptibility to anti-Id–mediated induction of dormancy. (7) One of the five Id+ variants tested lacked an HS1 epitope, had no detectable Syk protein, and had markedly reduced Lyn kinase activity. A second Id+ variant had normal levels of HS1, Syk, and Lyn proteins but had markedly reduced Lyn kinase activity.

There is increasing evidence that tumor cells leave the primary site early in the course of the disease and that their presence is not incompatible with long-term survival or cure. However, in the follicular form of non-Hodgkin's lymphoma (NHL), conventional treatment can induce long-term remissions, but DLCs are invariably present and virtually all patients eventually die of the disease.26,33,34 The use of the polymerase chain reaction for lymphomagenic translocations of the VH of the tumor Ig indicate that patients with NHL and several other hematopoietic malignancies have detectable tumor during long-term remission or cure.35-41 For example, in B-cell acute lymphocytic leukemia in childhood, 16 of 17 patients had residual tumor after combination chemotherapy and many carried tumor 6 months to 1 year later and about half of these patients could be cured without further therapy.42 Similar observations have been made in T-cell acute leukemia in children43 and in chronic myelogenous leukemia.44 Even more striking examples of dormancy can be observed in melanoma and breast cancer, where there appears to be a steady rate of recurrence 10 to 20 years after the removal of a primary breast carcinoma and the recurrent tumor frequently grows at a rapid rate.2,3,45 Despite the clinical importance of tumor dormancy, little is known about the changes that take place in the host and/or the tumor cells that allow regrowth (escape) from the dormant state.

A provocative finding to emerge from studies of our model is that, although only a portion of the DLCs are cell cycle arrested,11,12 the population of DLCs is relatively stable for the 7 months of observation. One might have predicted that it would be unlikely that the rate of cell death induced primarily via antibody-mediated apoptosis10 and the rate of replication (a log2 function) would be similar, ie, tumor cells might disappear completely in some animals and grow very rapidly in others. However, there appears to be a balance between the rate of replication and cell death in many of the mice studied. The mechanisms underlying a possible coupling of the antitumor activity of the host with the ability of a subset of tumor cells to replicate are not known. Presumably, there are strong homeostatic mechanisms that keep the proportion of B and T cells commensurate with the host needs, etc.46-48 Perhaps these homeostatic mechanisms together with the immune response are responsible for the stability of the DLC population for many months and/or the CCA and apoptotic pathways are interrelated in some manner. However, regardless of the mechanisms involved, these studies indicate that a tumor cell population was dormant and that neither absence of tumor cells nor very slow growth was responsible for the clinically dormant state. Thus, our operational definition of dormancy is now superseded by formal proof of dormancy.

The present studies indicate that the DLCs eventually express their malignant potential. Thus, there were no significant differences between graded numbers of wild-type and DLCs in transferring progressive tumor growth to nonimmune syngeneic recipients. As few as three DLCs transferred progressive tumor growth to a syngeneic recipient. This result distinguishes the antitumor effect of anti-Id from gene therapy. Anti-Id suppresses the malignant phenotype by signal transduction mechanisms that override the genetic lesions that lead to neoplasia. When the anti-Id is no longer present, the cells can eventually express their malignant phenotype. However, it is provocative that the time at which splenomegaly occurred in these adoptive transfer experiments with DLCs was delayed compared with wild-type BCL1 cells. Thus, at all doses, the DLCs grew more slowly in the syngeneic naive mice than a similar number of wild-type BCL1 cells. This was not due to transfer of Id-immune T or B cells because the addition of such cells to wild-type BCL1 cells had no inhibitory effect. Our tentative conclusion is that there is a transient change in the physiology of these cells induced by their constant interaction with antibody (and Id-specific T cells). The mechanisms underlying this delay in growth are not known. Two possibilities include DNA damage that must be repaired and/or lack of growth factor receptors that must be upregulated.

The steady rate of loss of dormancy over a period of almost 2 years suggests a stochastic process, perhaps involving mutation of a protein in a signaling pathway. We have shown that, in the majority of escapees, alterations in the tumor cells rather than the host were responsible for escape from dormancy. However, we have also shown that changes in the host can probably lead to escape. Thus, we provide evidence to suggest that, in a small percentage of the mice with low anti-Id titers, the tumor regrows. For example, in mice that develop dormancy, the average serum anti-Id concentration before the injection of BCL1 cells was 358 μg/mL, whereas nondormant mice had an average concentration of 70 μg/mL. In addition, the duration of dormancy was higher in mice with higher anti-Id titers. This could be explained by the need of mIg hypercross linking for maximum negative signaling.49 The more effective the antibody level is in reducing the total number of cycling tumor cells via apoptosis and/or cell cycle arrest, the less likely the probability for mutations.

Ten percent of the escapees and their progeny were mIg after transfer to nonimmune mice, suggesting that a mutation had occurred either in the genes encoding the heavy or light chain or one of the proteins that allow surface expression (eg, BIP50). At first glance, these results appear to be different from those reported by Meeker et al26 in which monoclonal anti-Id was used to induce regression of NHL in humans. In their studies, Id variants regrew in many treated patients presumably because of the high rate of mutation in the genes encoding the Ig hypervariable regions, as mentioned above. Our results indicate that, in the presence of polyclonal anti-Id, Id, IgM+ variants did not appear, presumably because it is improbable that several (or more) idiotopes would be altered by mutation in the same cell. An additional important mechanism to explain our data versus those of Meeker et al26 is our recent finding that monoclonal anti-IgM antibodies are frequently ineffective at inducing apoptosis and CCA in a human lymphoma (Burkitt's) cell line.49 However, when three anti-μ MoAbs, recognizing three different epitopes, were used simultaneously in vitro, they were effective at inducing both.49 Hence, for clinical use, it may be important to use a mixture of monoclonal anti-Id antibodies. Indeed, Levy et al27 (personal communication, 1996) have used such a mixture of MoAbs in the treatment of NHL.

In the 90% of escapees that were BCL1 -Id+, other components of the signaling cascade may have undergone genetic variation. Thus, in the limited survey performed here, alterations in Syk, Lyn, and HS1 are suggested by either the loss of an epitope recognized by an MoAb or loss of functional kinase activity. In this regard, prior studies of others have underscored the importance of Lyn, Syk, HS1, and other members of the signaling cascade in conveying signals from mIgM to the nucleus.21,32,51-64 Our own studies using antisense oligonucleotides have implicated Lyn in IgM-mediated CCA.24 Analysis of the DT-40 chicken B-cell Syk cell line provides strong evidence that Syk is essential for IgM-mediated apoptosis.65 Although Lyn, Syk, and HS1 are candidates for mutations, there are many other molecules in the signaling cascade that could also be responsible for loss of susceptibility to the induction of anti-Id–mediated dormancy, eg, PIP2, PLCγ, MAP kinase, BLK, etc.66-75 The molecular basis for these changes is currently under study in cloned cell lines established from these escapees.

With regard to cell signaling, studies by Brown et al34 and Vuist et al76 of NHL patients are particularly pertinent because they have observed a correlation between the capacity of anti-Id to induce phosphorylation in vitro in neoplastic B cells obtained by biopsy and clinical remission in patients.76 This suggests that the signaling capacity of antibody may be essential for effective antitumor activity. This clinical observation is in accord with our conclusions from in vivo and in vitro studies using the BCL1 model in which the critical role of signaling in the induction of dormancy has been proven. Another pertinent observation by this group is that vaccination of NHL patients with the patient's tumor Ig coupled to KLH given in threonyl muramyl dipeptide adjuvant77,78 stimulated an Id-specific humoral or cellular immune response in about half the patients and the majority of such patients do not relapse for periods of up to several years. From the group that did not develop an immune response, the majority have relapsed.

In the experiments described here, the role of Id-specific cellular immunity was not investigated; our prior studies indicate that the major effector pathway in induction of dormancy is mediated by signaling antibody. However, this does not exclude a role for Id-specific T-cell immunity or nonspecific effector functions of the antibodies. With regard to cellular immunity, prior studies have indicated that allogeneic bone marrow transplantation can induce tumor dormancy79-81 and that partial CCA is induced.11,79 Moreover, T cells from Id-immune mice can significantly enhance the induction and maintenance of dormancy induced by passively transferred anti-Id antibody in SCID mice.10 Additionally, Demanet et al82 have shown that both CD4+ and CD8+ cells are required in the successful treatment of BCL1 in vivo with an anti-BCL1Id/anti-CD3 bispecific antibody. Morecki et al83 have presented data consistent with a contribution of cell-mediated immunity in protecting mice immunized with irradiated BCL1 tumor from a subsequent tumor challenge. Hsu et al84 have shown that 3 of 4 patients vaccinated with antigen-pulsed dendritic cells develop cellular responses and undergo tumor regressions. Therefore, it is possible that, when cellular immunity is studied in escapees, a poor Id-specific cellular immune response could also contribute to escape. With regard to effector functions of antibody, eg, ADCC, there is formidable evidence that these functions can contribute in a major way to antibody-induced tumor immunity in general.85 Hence, a dynamic equilibrium may exist between the tumor cells and the several mechanisms by which the immune response inhibits tumor growth and maintains dormancy.

ACKNOWLEDGMENT

We thank Y. Chinn for expert technical assistance and C. Patterson and S. Chadwick for expert secretarial assistance. We also thank J. Bolen for the GST-lyn fusion construct. We are particularly indebted to Dr D. McIntire for his help in statistical analysis.

Supported by National Institutes of Health Grants No. CA-28149 and CA-58321, a grant from The Meadows Foundation, and American Cancer Society Grant No. IM-767.

Address reprint requests to Dr Ellen S. Vitetta, Cancer Immunobiology Center and Department of Microbiology, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd, Dallas, TX 75235.

REFERENCES

REFERENCES
1
Stewart
THM
Hollinshead
AC
Raman
S
Tumor dormancy initiation, maintenance and termination in animals and humans.
Can J Surg
34
1991
321
2
Meltzer
A
Dormancy and breast cancer.
J Surg Oncol
43
1990
181
3
Demicheli R: Tumor dormancy and metastasis development in operable breast cancer: Data from clinical trials, in Yefenof E, Scheuermann RH (eds): Premalignancy and Tumor Dormancy. Georgetown, TX, RG Landes, 1996, p 181
4
Hadfield
G
The dormant cancer cell.
Br Med J
2
1954
607
5
Wheelock
EF
Weinhold
KJ
Levich
J
The tumor dormant state.
Adv Cancer Res
34
1981
107
6
Vitetta
ES
Krolick
KA
Miyama-Inaba
M
Cushley
W
Uhr
JW
Immunotoxins: A new approach to cancer therapy.
Science
219
1983
644
7
Slavin
S
Strober
S
Spontaneous murine B-cell leukaemia.
Nature
272
1978
624
8
Krolick
KA
Isakson
PC
Uhr
JW
Vitetta
ES
BCL1 , a murine model for chronic lymphocytic leukemia: Use of the surface immunoglobulin idiotype for the detection and treatment of tumor.
Immunol Rev
48
1979
81
9
Knapp
MR
Jones
PP
Black
SJ
Vitetta
ES
Slavin
S
Strober
S
Characterization of a spontaneous murine B cell leukemia (BCL1 ). I. Cell surface expression of IgM, IgD, Ia, and FcR.
J Immunol
123
1979
992
10
Racila
E
Scheuermann
RH
Picker
LJ
Yefenof
E
Tucker
T
Chang
W
Marches
R
Street
NE
Vitetta
ES
Uhr
JW
Tumor dormancy and cell signalling. II. Antibody as an agonist in inducing dormancy of a B cell lymphoma in SCID mice.
J Exp Med
181
1995
1539
11
Uhr
JW
Tucker
T
May
RD
Siu
H
Vitetta
ES
Cancer dormancy: Studies of the murine BCL1 lymphoma.
Cancer Res
51
1991
5045S
12
Yefenof
E
Picker
LJ
Scheuermann
RH
Tucker
TF
Vitetta
ES
Uhr
JW
Cancer dormancy: Isolation and characterization of dormant lymphoma cells.
Proc Natl Acad Sci USA
90
1993
1829
13
Stevenson
GA
Stevenson
FK
Prospects for the treatment of B cell tumors using idiotypic vaccination.
Int Rev Immunol
4
1989
271
14
Stevenson
FK
Stevenson
GA
Glennie
MJ
Anti-idiotypic therapy of leukemias and lymphomas.
Chem Immunol
48
1990
126
15
Hoggenraad
NJ
Wraight
CJ
The effect of pristane on ascites tumor formation and monoclonal antibody production.
Methods Enzymol
121
1986
375
16
Lacy
MJ
Voss
EW Jr
A method to induce immune polyclonal ascites fluid in BALB/c mice using Sp2/0-Ag14 cells.
J Immunol Methods
87
1986
169
17
Yagawa
K
Vitetta
ES
Induction of proliferation and differentiation of murine B cells bearing surface Ig lambda by rat monoclonal antibody to lambda chain.
Hybridoma
2
1983
169
18
Yuan
D
Vitetta
ES
Kettman
JR
Cell surface immunoglobulin. XX. Antibody responsiveness of subpopulations of B lymphocytes bearing different isotypes.
J Exp Med
145
1977
1421
19
Fukuda
T
Kitamura
D
Taniuchi
I
Maekawa
Y
Benhamou
LE
Sarthou
P
Watanabe
T
Restoration of surface IgM-mediated apoptosis in an anti-IgM-resistant variant of WEHI-231 lymphoma cells by HS1, a protein-tyrosine kinase substrate.
Proc Natl Acad Sci USA
92
1995
7302
20
Benhamou
LE
Watanabe
T
Kitamura
D
Cazenave
P-A
Sarthou
P
Signaling properties of anti-immunoglobulin — Resistant variants of WEHI-231 B lymphoma cells.
Eur J Immunol
24
1994
1993
21
Yamanashi
Y
Okada
M
Semba
T
Yamori
T
Umemori
H
Tsunasawa
S
Toyoshima
K
Kitamura
D
Watanabe
T
Yamamoto
T
Identification of HS1 protein as a major substrate of protein-tyrosine kinase(s) upon B-cell antigen receptor-mediated signaling.
Proc Natl Acad Sci USA
90
1993
3631
22
Terstappen
LWMM
Mickaels
R
Dost
R
Loken
MR
Increased light scattering resolution facilitates multidimensional flow cytometric analysis.
Cytometry
11
1990
506
23
Sambrook J, Fritsch EF, Mariatis T: Molecular Cloning, A Laboratory Manual. Cold Spring Harbor, NY, Cold Spring Harbor Laboratory, 1989
24
Scheuermann
RH
Racila
E
Tucker
T
Yefenof
E
Street
N
Vitetta
E
Picker
L
Uhr
J
Lyn tyrosine kinase signals cell cycle arrest but not apoptosis in B-lineage lymphoma cells.
Proc Natl Acad Sci USA
91
1994
4048
25
Golden
A
Nemeth
SP
Brugge
JS
Blood platelets express high levels of the pp60c-src-specific tyrosine kinase activity.
Proc Natl Acad Sci USA
83
1986
852
26
Meeker
T
Lowder
J
Cleary
ML
Stewart
S
Warnke
R
Sklar
J
Levy
R
Emergence of idiotype variants during treatment of B-cell lymphoma with anti-idiotype antibodies.
N Engl J Med
312
1985
1658
27
Levy
R
Miller
RA
Therapy of lymphoma directed at idiotypes.
J Natl Cancer Inst
10
1990
61
28
Taya
M
Haimovich
J
Immunotherapy of B lymphoma by anti-idiotype antibodies: Characterization of variant tumor cells appearing a long time after the initial tumor inoculation.
Cancer Immunol Immunother
34
1991
43
29
Roth
MS
Weiner
GJ
Allen
EA
Terry
VH
Harnden
CE
Boehnke
M
Kaminski
MS
Ginsburg
D
Molecular characterization of anti-idiotype antibody-resistant variants of a murine B cell lymphoma.
J Immunol
145
1990
768
30
Weiner
GJ
Kaminski
MS
Anti-idiotypic antibodies recognizing stable epitopes limit the emergence of idiotype variants in a murine B cell lymphoma.
J Immunol
144
1990
2436
31
DeFranco
AL
Transmembrane signaling by antigen receptors of B and T lymphocytes.
Curr Opin Cell Biol
7
1995
163
32
Kitamura
D
Kaneko
H
Taniuchi
I
Akagi
K
Yamamura
K
Watanabe
T
Molecular cloning and characterization of mouse HS1.
Biochem Biophys Res Commun
208
1995
1137
33
Levy
R
Miller
AR
Therapy of lymphoma directed at idiotypes.
Monogr J Natl Cancer Inst
10
1990
61
34
Brown
SL
Miller
RA
Horning
SJ
Czerwinski
D
Hart
SM
McElderry
R
Basham
T
Warnke
RA
Merigan
TC
Levy
R
Treatment of B-cell lymphomas with anti-idiotype antibodies alone and in combination with alpha interferon.
Blood
73
1989
651
35
Sykes
PJ
Neoh
SH
Brisco
MJ
Hughes
E
Condon
J
Morley
AA
Quantitation of targets for PCR by use of limiting dilution.
Biotechniques
13
1992
444
36
Billadeau
D
Blackstadt
M
Greipp
P
Kyle
RA
Oken
MM
Kay
N
Van Ness
B
Analysis of B-lymphoid malignancies using allele-specific polymerase chain reaction: A technique for sequential quantitation of residual disease.
Blood
78
1991
3021
37
Van Dongen
JJM
Breit
TM
Adriaansen
HJ
Keishuizen
A
Hooijkaas
H
Detection of minimal residual disease in acute leukemia by immunological marker analysis and polymerase chain reaction.
Leukemia
6
1992
47
38
Estrov
Z
Ouspenskaia
MV
Felix
EA
McClain
KL
Lee
M
Harris
D
Pinkel
DP
Zipf
TF
Persistence of self-renewing leukemia cell progenitors during remission in children with B-precursor acute lymphoblastic leukemia.
Leukemia
8
1994
46
39
Nizet
Y
Van Daele
S
Lewalle
P
Vaerman
JL
Philippe
M
Vermylen
C
Cornu
G
Ferrant
A
Ferrant
JL
Michaux
JL
Martiat
P
Long-term follow-up of residual disease in acute lymphoblastic leukemia patients in complete remission using clongeneic IgH probes and the polymerase chain reaction.
Blood
82
1993
1618
40
Brisco
MJ
Condon
J
Hughes
E
Neoh
S
Sykes
PJ
Seshadri
R
Toogood
I
Wataers
K
Tauro
G
Ekert
H
Morley
AA
Outcome prediction in childhood acute lymphoblastic leukaemia by molecular quantification of residual disease at the end of induction.
Lancet
343
1994
196
41
Yamada
M
Wasserman
R
Lange
B
Reichard
BA
Womer
RB
Rovera
G
Minimal residual disease in childhood B-lineage lymphoblastic leukemia. Persistence of leukemic cells during the first 18 months of treatment.
N Engl J Med
323
1990
448
42
Biondi
A
Yokota
S
Hansen-Hagge
T
Rossi
V
Giudici
G
Maglia
O
Basso
G
Tell
C
Masera
G
Bartram
C
Minimal residual disease in childhood acute lymphoblastic leukemia: Analysis of patients in continuous complete remission or with consecutive relapse.
Leukemia
6
1992
282
43
Cavé
H
Guidal
C
Rohrlich
P
Delfau
MH
Broyart
A
Lescoeur
B
Rahimy
C
Fenneteau
O
Monplaisir
N
d'Auriol
L
Elion
J
Vilmer
E
Grandchamp
B
Prospective monitoring and quantitation of residual blasts in childhood acute lymphoblastic leukemia by polymerase chain reaction study of δ and γ T-cell receptor genes.
Blood
83
1994
1892
44
Talpaz
M
Estrov
H
Kantarjian
H
Ku
S
Foteh
A
Kurzrock
R
Persistence of dormant leukemic progenitors during interferon-induced remission in chronic myelogenous leukemia.
J Clin Invest
94
1994
1383
45
Henderson JC, Harris JR, Kinne DW, Hellman S: Cancer of the breast, in DeVita VT Jr, Hellman S, Rosenberg SA (eds): Cancer: Principles and Practice of Oncology. Philadelphia, PA, Lippincott, 1989, p 1201
46
Cooke
MP
Heath
AW
Shokat
KM
Zeng
Y
Finkelman
FD
Linsley
PS
Howard
M
Goodnow
CC
Immunoglobulin signal transduction guides the specificity of B cell-T cell interactions and is blocked in tolerant self-reactive B cells.
J Exp Med
179
1994
425
47
Cyster
JG
Hartley
SG
Goodnow
CC
Competition for follicular niches excludes self-reactive cells from the recirculating B-cell repertoire.
Nature
371
1994
389
48
Eris
JM
Basten
A
Brink
R
Doherty
K
Kehry
MR
Hodgkin
PD
Anergic self-reactive B cells present self antigen and respond normally to CD40-dependent T-cell signals but are defective in antigen-receptor-mediated functions.
Proc Natl Acad Sci USA
91
1994
4392
49
Marches
R
Racila
E
Tucker
TF
Picker
L
Mongini
P
Hsueh
R
Vitetta
ES
Scheuermann
RH
Uhr
JW
Tumor dormancy and cell signalling. III: Role of hypercrosslinking of IgM and CD40 on the induction of cell cycle arrest and apoptosis in B lymphoma cells.
Ther Immunol
2
1996
125
50
Knittler
MR
Haas
IG
Interaction of BIP with newly synthesized immunoglobulin light chain molecules: Cycles of sequential binding and release.
EMBO J
11
1992
1573
51
Taniuchi
I
Kitamura
D
Maekawa
Y
Fukuda
T
Kishi
H
Watanabe
T
Antigen-receptor induced clonal expansion and deletion of lymphocytes are impaired in mice lacking HS1 protein, a substrate of the antigen-receptor-coupled tyrosine kinases.
EMBO J
14
1995
3664
52
Desiderio
SV
B-cell activation.
Curr Opin Immunol
4
1992
252
53
Cambier
JC
Bedzyk
W
Campbell
K
Chien
N
Friedrich
J
Harwood
A
Jensen
W
Pleiman
C
Clark
MR
The B cell antigen receptor: Structure and function of primary, secondary, tertiary and quaternary components.
Immunol Rev
132
1993
107
54
Kim
K-M
Alber
G
Weiser
P
Reth
M
Signaling function of the B-cell antigen receptors.
Immunol Rev
132
1993
125
55
Weiss
A
Littman
DR
Signal transduction by lymphocyte antigen receptors.
Cell
76
1994
263
56
Gold
MR
DeFranco
AL
Biochemistry of B lymphocyte activation.
Adv Immunol
55
1994
221
57
Nagai
K
Takata
M
Yamamura
H
Kurosaki
T
Tyrosine phosphorylation of Shc is mediated through Lyn and Syk in B cell receptor signaling.
J Biol Chem
270
1995
6824
58
Burg
DL
Furlong
MT
Harrison
ML
Geahlen
RL
Interactions of Lyn with the antigen receptor during B cell activation.
J Biol Chem
269
1994
28136
59
Aoki
Y
Kim
Y-T
Stillwell
R
Kim
TJ
Pillai
S
The SH2 domains of Src family kinases associate with Syk.
J Biol Chem
270
1995
15658
60
Sidorenko
SP
Law
C-L
Chandran
KA
Clark
EA
Human spleen tyrosine kinase p72Syk associates with the Src-family kinase p53/56Lyn and a 120-kDa phosphoprotein.
Proc Natl Acad Sci USA
92
1995
359
61
Kurosaki
T
Johnson
SA
Pao
L
Sada
K
Yamamura
H
Cambier
JC
Role of the Syk autophosphorylation site and SH2 domains in B cell antigen receptor signaling.
J Exp Med
182
1995
1815
62
Takata
M
Sabe
H
Hata
A
Inazu
T
Homma
Y
Nukada
T
Yamamura
H
Kurosaki
T
Tyrosine kinases Lyn and Syk regulate B cell receptor-coupled Ca2+ mobilization through distinct pathways.
EMBO J
13
1994
1341
63
Kurosaki
T
Takata
M
Yamanashi
U
Inazu
T
Taniguchi
T
Yamamoto
T
Yamamura
H
Syk activation by the Src-family tyrosine kinase in the B cell receptor signaling.
J Exp Med
179
1994
1725
64
Flück
M
Zürcher
G
Andres
A-C
Ziemiecki
A
Molecular characterization of the murine Syk protein tyrosine kinase cDNA, transcripts and protein.
Biochem Biophys Res Commun
213
1995
273
65
Takata
M
Sabe
H
Hata
A
Inazu
T
Homma
Y
Nukada
T
Yamamura
H
Kurosaki
T
Tyrosine kinases Lyn and Syk regulate B cell receptor-coupled Ca++ mobilization through distinct pathways.
EMBO J
13
1994
1341
66
Fischer
G
Kent
SC
Joseph
L
Green
DR
Scott
DW
Lymphoma models for B cell activation and tolerance. X. Anti-mu-mediated growth arrest and apoptosis of murine B cell lymphomas is prevented by the stabilization of myc.
J Exp Med
179
1994
221
67
Lazarus
AH
Kawauchi
K
Rapoport
MJ
Delovitch
TL
Antigen-induced B lymphocyte activation involves the p21ras and ras.GAP signaling pathway.
J Exp Med
178
1993
1765
68
Harwood
AE
Cambier
JC
B cell antigen receptor cross-linking triggers rapid protein kinase C independent activation of p21ras1.
J Immunol
151
1993
4513
69
Joseph
LF
Ezhevsky
S
Scott
DW
Lymphoma models for B-cell activation and tolerance: Anti-immunoglobulin M treatment induces growth arrest by preventing the formation of an active kinase complex which phosphorylates retinoblastoma gene product in G1 .
Cell Growth Differ
6
1995
51
70
Hempel
WM
Schatzman
RC
DeFranco
AL
Tyrosine phosphorylation of phospholipase C-gamma 2 upon cross-linking of membrane Ig on murine B lymphocytes.
J Immunol
148
1992
3021
71
Braun
J
Sha'afi
RI
Unanue
ER
Cross-linking by ligands to surface immunoglobulin triggers mobilization of intracellular 45Ca2+ in B lymphocytes.
J Cell Biol
82
1979
755
72
Ransom
JT
Harris
LK
Cambier
JC
Anti-Ig induces release of inositol 1,4,5-trisphosphate, which mediates mobilization of intracellular Ca++ stores in B lymphocytes.
J Immunol
137
1986
708
73
Wilson
HA
Greenblatt
D
Poenie
M
Finkelman
FD
Tsien
RY
Crosslinkage of B lymphocyte surface immunoglobulin by anti-Ig or antigen induces prolonged oscillation of intracellular ionized calcium.
J Exp Med
166
1987
601
74
Choquet
D
Ku
G
Cassard
S
Malissen
B
Korn
H
Fridman
WH
Bonnerot
C
Different patterns of calcium signaling triggered through two components of the B lymphocyte antigen receptor.
J Biol Chem
269
1994
6491
75
Takata
M
Homma
Y
Kurosaki
T
Requirement of phospholipase C-γ2 activation in surface IgM-induced B cell apoptosis.
J Exp Med
182
1995
907
76
Vuist
WMJ
Levy
R
Maloney
DG
Lymphoma regression induced by monoclonal anti-idiotypic antibodies correlates with their ability to induce Ig signal transduction and is not prevented by tumor expression of high levels of Bcl-2 protein.
Blood
83
1994
899
77
Kwak
LW
Campbell
MJ
Czerwinski
DK
Hart
S
Miller
RA
Levy
R
Induction of immune responses in patients with B-cell lymphoma against the surface immunoglobulin idiotype expressed by their tumors.
N Engl J Med
327
1992
1209
78
Hsu
FJ
Kwak
L
Campbell
M
Liles
T
Czerwinski
D
Hart
S
Syrengelas
A
Miller
R
Levy
R
Clinical trials of idiotype-specific vaccine in B-cell lymphomas.
Ann NY Acad Sci
690
1993
385
79
Siu
H
Vitetta
ES
May
RD
Uhr
JW
Tumor dormancy. I. Regression of BCL1 tumor and induction of a dormant tumor state in mice chimeric at the major histocompatibility complex.
J Immunol
137
1986
1376
80
Weiss
L
Morecki
S
Vitetta
ES
Slavin
S
Suppression and elimination of BCL1 leukemia by allogeneic bone marrow transplantation.
J Immunol
130
1983
2452
81
Weiss
L
Reich
S
Slavin
S
The role of antibodies to IL-2 receptor and Asialo GM1 on graft-versus-leukemia effects induced by bone marrow allografts in murine B cell leukemia.
Bone Marrow Transplant
16
1995
457
82
Demanet
C
Brissinck
J
Leo
O
Moser
M
Thielemans
C
Role of T-cell subsets in the bispecific antibody (anti-idiotype × anti-CD3) treatment of the BCL1 lymphoma.
Cancer Res
54
1994
2973
83
Morecki
S
Pugatsch
T
Levi
S
Moshel
Y
Slavin
S
Tumor-cell vaccination induces tumor dormancy in a murine model of B-cell leukemia/lymphoma (BCL1 ).
Int J Cancer
65
1996
204
84
Hsu
FJ
Benike
C
Fagnoni
F
Liles
TM
Czerwinski
D
Taidi
B
Engleman
EG
Levy
R
Vaccination of patients with B-cell lymphoma using autologous antigen-pulsed dendritic cells.
Nat Med
2
1996
52
85
Vitetta
ES
Uhr
JW
Monoclonal antibodies as agonists: An expanded role for their use in cancer therapy.
Cancer Res
54
1994
5301