Cancer Therapy: Preclinical
`
`Comparative Impact of Trastuzumab and Cyclophosphamide
`
`on HER-2—Positive Human Breast Cancer Xenografts
`
`Giulio Francia,1'2 Shan Man,"2 Chyan-Jang Lee,3 Christina R. Lee,"2
`Ping Xu,"2 Miriam E. Mossoba,2 Urban Emmenegger,1'2
`Jeffrey A. Medin,2'3 and Robert S. Kerbel1'2
`
`Abstract
`
`Purpose: Metronomic chemotherapy is a minimally toxic and frequently effective new
`treatment strategy that is beginning to show promising phase II clinical trial results,
`particularly for metastatic breast cancer when combined with various molecularly tar-
`geted antitumor agents. Here, we assessed a treatment strategy that uses trastuzumab
`plus daily oral metronomic cyclophosphamide on metastatic Her-2—positive human
`breast cancer models.
`
`Experimental Design: Treatments were initiated on orthotopic transplanted primary
`tumors as well as established visceral metastatic disease of two independent Her-2—
`positive breast cancer models, both independently derived from the human MDA-
`MB—231 breast cancer cell line. Outcome was assessed by noninvasive measurements
`of tumor cell—secreted human choriogonadotropin in the urine as a surrogate marker of
`relative tumor burden, or by whole body bioluminescent imaging, in addition to pro—
`longation of survival.
`Results: Orthotopic primary tumors responded to trastuzumab monotherapy with sig-
`nificant growth delays, whereas minimal antitumor effect was observed when mice
`with metastatic disease were treated. Nevertheless, trastuzumab showed a benefit in
`this latter setting when combined with metronomic low-dose cyclophosphamide as
`assessed by prolongation of survival. This benefit was similar to trastuzumab plus
`maximum tolerated dose cyclophosphamide, but was associated with lesser toxicity.
`Conclusions: Trastuzumab combined with metronomic cyclophosphamide may be an
`effective long-term maintenance strategy for the treatment of Her—2—positive metastatic
`breast cancer. (Clin Cancer Res 2009;15(20):6358—66)
`
`One significant advance in breast cancer medical oncology
`over the last decade has been the development and approval
`of drugs that target the erbB-2/Her-2 (Her-2) oncogene (1, 2).
`These drugs include trastuzumab (Herceptin), the humanized
`
`Authors‘ Affiliations: 1Molecular and Cellular Biology Research, Sunnybrook
`Health Sciences Centre, 2Department of Medical Biophysics, and 3Division of
`Stem Cell and Developmental Biology, Ontario Cancer Institute/University
`Health Network, University of Toronto, Toronto, Ontario, Canada
`Received 4/13/09; revised 7/7/09; accepted 7/9/09; published OnlineFirst
`10/13/09.
`Grant support: NIH, the National Cancer Institute of Canada, and Canadian
`Institutes for Health Research (RS. Kerbel). RS. Kerbel holds a Tier | Canada
`Research Chair in Tumor Biology, Angiogenesis and Antiangiogenic Therapy.
`U. Emmenegger was funded by OICR (Clinician—Scientist || Investigator
`Award). Trastuzumab was a generous gift from Genentech.
`The costs of publication of this article were defrayed in part by the payment
`of page charges. This article must therefore be hereby marked advertisement
`in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
`Requests for reprints: Robert S. Kerbel or Giulio Francia, Molecular and
`Cellular Biology Research, Sunnybrook Health Science Centre, 8-217
`Research Building, 2075 Bayview Avenue, Toronto, Ontario, Canada M4N
`3M5. Phone: 416-480-5711; Fax; 416-480-5884; E—mail: robert.kerbel@sri.
`utoronto.ca.
`© 2009 American Association for Cancer Research.
`doi:1o.1158/1078-0432.CCR-09-0931
`
`monoclonal antibody (2—5), and lapatinib (Tykerb), a small
`molecule receptor tyrosine kinase inhibitor that targets both
`the epidermal growth factor receptor (erbB-l) and Her-2 (6).
`Trastuzumab has shown benefit in a number of randomized
`
`clinical trials involving breast cancer patients whose tumors
`overexpress Her—2, in both the metastatic and adjuvant treat-
`ment settings, especially the latter (2, 7, 8). With respect to
`treatment of metastatic disease, the clinical results have high-
`lighted the importance of both intrinsic and acquired resis-
`tance, because more than half of breast cancer patients whose
`tumors overexpress Her—2 do not respond to trastuzumab, and
`among those that do, acquired resistance to the drug inevitably
`develops, generally within 1 year of treatment initiation (2, 9).
`Moreover, trastuzumab monotherapy for the treatment of ad-
`vanced metastatic disease in patients is associated with mini-
`mal, if any, activity; its benefit is derived by integration with
`chemotherapy. In contrast, trastuzumab monotherapy is effec-
`tive in preclinical models, but these invariably involve treat—
`ment of localized transplanted primary tumors, not Visceral
`metastatic disease.
`
`Recently, we have developed models of metastatic disease
`using either the parental MDA-MB—231 human breast cancer
`cell line (10) or a Her-2+ variant of MDA-MB—231 called H2N
`(11, 12). These models are associated with extensive visceral
`
`Genentech 2080 - Celltrion v. Genentech - |PR2017-01122
`
`Clin Cancer Res 2009;15l20) October 15, 2009
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`6358
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`

`

`Metronomic Therapy for HER-2+ Metastases
`
`LM2-4H2N (Her—2 pos-
`Spontaneous (orthotopic) metastasis assays.
`itive) and met2 (Her-2 positive) cells were injected into the mammary
`fat pad of female C817 SCID mice. LM2-4H2N and met2 tumors were
`measured using calipers and removed when they reached an average
`size of 500 mm3. Mice were monitored for body weight, B—hCG urine
`levels, or luciferase expression. End points were determined according
`to institutional guidelines; mice were sacrificed when cachexia (>15%
`body weight loss) was observed, or when mice showed evidence of
`lymph node metastases, or when they exhibited moribund symptoms
`(lethargy and/or reduced mobility). Statistical analysis (log-rank test for
`survival or ANOVA with Newman—Keuls comparison test for weight
`loss) was done using Prism software (GraphPad).
`Trastuzumab administration. Treatments for primary tumors were
`initiated when the average tumor size was ~250 mm3. Treatment of
`metastatic disease was initiated 3 wk after resection of the primary tu-
`mor. This time point was empirically determined as optimal in view of
`the sporadic rate of appearance of spontaneous metastases (data not
`shown). lf treatment was initiated beyond 3 wk after surgery (when
`up to 50% of the mice would show detectable hCG/luciferin lumines-
`cence), we observed that mice with the most advanced disease would
`
`
`
`
`Ld CTX+Trastuzumab
`—-- MTD C1X+Trastuzumab
`<~*-- Control
`
` -
`
`
`
`
`
`
`
`Translational Relevance
`
`
`
`The Her-2—targeting antibody, trastuzumab, in
`combination with chemotherapy has proved effec-
`tive against Her-2—overexpressing breast cancers in
`the clinic, both in the metastatic as well as in the
`adjuvant setting. Here, we used models of preclinical
`Her-2—positive metastatic breast cancer to test the
`efficacy of trastuzumab combined with one of two
`forms of administration of chemotherapy (using
`cyclophosphamide): either pulsatile maximum toler-
`ated dose or a daily "low—dose" extended metro-
`nomic delivery. Combinations of trastuzumab with
`either mode of chemotherapy administration proved
`effective in suppressing the progression of Her—
`positive metastatic disease. Furthermore, the combi-
`nation involving metronomic chemotherapy did not
`exhibit the severe toxicity associated with the maxi-
`mum tolerated dose regimen. These results suggest
`that trastuzumab plus metronomic chemotherapy
`may be an effective long-term treatment regimen
`for metastatic Her-2—positive breast cancer.
`
`metastases that can become established in the lungs, liver, and
`lymph nodes of most mice within one or several months of re-
`section of the primary tumor (10, 11).
`Given the development of these metastatic models of Her-
`2—positive breast cancer, we decided to initiate a preclinical
`analysis of trastuzumab plus chemotherapy using either a con—
`ventional—type maximum tolerated dose (MTD) chemotherapy
`regimen or continuous low-dose metronomic chemotherapy,
`using the same drug, cyclophosphamide (CTX). Our results,
`which constitute one of the first reports of experimental therapy
`of metastatic Her—Z—positive xenografts, illustrate the possible
`benefits of using trastuzumab in combination with metronom—
`ic chemotherapy for metastatic breast cancer, for which prelim-
`inary clinical evidence is also beginning to emerge (13).
`
`Materials and Methods
`
`Cell lines. The parent MDA-MB-231 human breast cancer cell line
`(14) was used to derive the erbBZ—transduced 231-H2N (12), the meta-
`static variants met2 (11), and LM2-4H2N (erbBZtransduced LM2-4
`cells; ref. 10). LM2»4 cells are highly metastatic from a primary ortho-
`topic transplant, after surgical resection of the tumor, as previously de-
`scribed (10). The LM2-4 cells were cotransfected with the firefly
`luciferase vector pCL3 (Promega Corporation) and pSVZneo, and
`selected using C418, from which a luciferase—expressing clone was
`isolated; these cells were then used to generate the LM2-4H2N
`(erbB2—transduced LM2-4 cells) as previously described (12). The
`met2 cells were obtained as described in the Results section.
`fl—hCG measurements.
`is—hCC in the mouse urine was measured as
`previously detailed (11), and normalized to urine creatinine levels (us-
`ing QuantiChrom TM Creatinine assay kit from BioAssay Systems) as
`described by Shih et al. (15).
`Female 4-wk-old CB17 severe com-
`Orthotopic tumor implantation.
`bined immunodeficient (SCID) mice were purchased from Charles
`River Canada (Saint-Constant) and allowed to acdimatize for 2 wk MDA-
`MB-231 cell variants were harvested by trypsin treatment, washed thrice
`in ice-cold PBS, and resuspended in serum—free DMEM. Cells (2 x 10") were
`injected in 50-pL volumes into the inguinal mammary fat pad.
`
`1800
`1 600
`
`3:3"aoS060
`
`800
`600
`400
`
`100
`120
`140
`160
`”406080444404444
`
`180 200
`
`
`(mm3)
`
`Tumorvolume
`Days
`(g)
`
`MouseWeight
`
`204060
`
`120 140 160.180 200
`100
`80
`444
`
`
`Days
`
`
`Impact of concurrent combination of trastuzumab-chemotherapy
`Fig. 1.
`(CTX) therapy regimens on orthotopic primary H2N tumors in SCID mice.
`When tumors reached 250 mm3, mice were treated with saline (control,
`n = 3) or metronomic low—dose (Ld) CTX plus trastuzumab (Ld CTX +
`trastuzumab, n = 8), or with a MTD CTX plus trastuzumab (MTD CTX+
`trastuzumab, where MTD CTX is 210 mg/kg every 21 d, n = 5). Top, tumor
`volume; bottom, mouse weights. Arrows, each MTD cycle (i.e., every 21 d).
`*, one mouse in each group had to be sacrificed at the indicated point.
`Trastuzumab was given twice weekly at 20 mg/kg i.p., and metronomic
`low-dose CTX was ~20 mg/kg/d.
`
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`
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`

`Fig. 2. Schematic of met2 metastatic
`tumor therapy experiment. Human
`breast cancer MDA-MBZ31 cells were
`modified to express Her—2 and secrete
`hCG, and subsequently selected for
`high metastatic potential through two
`rounds of in vivo selection, as
`described previously (10). The resulting
`met2 metastatic variant was generated,
`and subsequently injected into female
`SCID mice. The growth of this tumor
`can be monitored via hCG levels in the
`mouse urine. Mice were then used to
`
`assess the impact of therapies on the
`growth of primary tumors and, in
`parallel, in mice with metastatic
`disease.
`
`Cancer Therapy: Preclinical
`
`MDA—MB-231 —>H2N—» H2N.hCG 4‘
`Her-2 transduction
`hCG transfection
`‘ '1' °.
`(du Manoir et al 2006)
`
`.
`go
`Two rounds of
`in vivo selection
`(Francia 3‘ 3' 2008)
`
`_
`
`_
`1
`H21\.hCG.met21mfp
`. l
`:- 07/ —’
`a...“
`i
`
`| Primal
`’1'
`.1
`1 a
`d“
`
`tumor (500mm3)
`.
`. —’
`surglcal resection
`
`-
`
`.
`
`"
`
`_
`
`'
`
`o;
`
`P11ma1
`
`tumot (250n1m3)
`
`Urine hCG analysis
`
`-/-trastuzumab ip
`_/_ LDM CTX
`-/— MTD CTX
`
`
`
`WU
`
`----- Ld CTX
`~_, Trastuzumab
`—— Ld CTX+Trastuzumab -—~—- control
`C!O
`
`.
`-/—trastuzumab 1p
`’/' LDM CTX
`
`
`
`Caliper measurements,
`Urine hCG analysis
`PRIMARY TUMOR
`
`
`
`
`
`hCG(mlUImgCreatlnlne)
`
`rine hCG analysis
`Days
`
`20
`
`40
`
`60
`
`80 100 120 140 160 180 200
`
`2D
`
`40
`
`60
`
`80 100 120 140 160 180 200
`Days
`
`—-— MTD CTX+Trastuzurmb --—- Control
`- MTD
`, Ld CTX+Trastuzun‘ab ~4~ Ld CTX
`.. , Trastuzumab
`
`
`
`MouseWeight(9)
`
`
`
`MouseWeight(9)
`
`25.5
`
`
`
`MouseWeight(g)
`
`20406080100120140160180200
`
`'
`
`20 406080100120140150180200
`AAAAAAAA
`
`. 20406080100120140160180200
`
`Fig. 3. A, effect on tumor volume (top) and hCG levels (bottom) of the different therapies on met2 orthotopically implanted primary tumors. When tumors
`reached 250 mm3, mice were treated with saline (control, n = 5), or trastuzumab alone (n = 6), or low-dose metronomic CTX alone (Ld CTX, n = 6) or the
`combination of trastuzumab plus low-dose metronomic CTX (Ld CTX+ trastuzumab, n = 6). The curves show that, similar to H2N tumors (see Fig. 1), the met2
`line gave rise to primary tumors that are highly responsive to the low-dose metronomic CTX + trastuzumab combination therapy. The urine hCG values
`(corresponding to pooled urine hCG for each group, normalized to urine Creatinine levels) were found to be concordant with tumor volume measurements.
`8, treatment of met2 metastases and assessment of mouse weights. Orthotopically implanted met2 tumors were surgically removed, and 3 wk later, various
`therapies were initiated. Mice were treated with MTD CTX alone (MTD, r1 = 6) or low—dose CTX alone (n = 6) or control saline (n = 6), or trastuzumab
`alone (n = 6). In addition, other mice were given trastuzumab plus metronomic CTX (n = 12), or trastuzumab plus MTD CTX (n = 12; arrows, MTD dosing).
`Weight loss on day 115 for MTD therapies was significantly different (P< 0.05) versus the other regimens. Trastuzumab was given twice weekly at 20 mg/kg
`i.p., and low—dose metronomic CTX alone was ~20 mg/kg/d.
`
`Clin Cancer Res 2009;15(20) October 15, 2009
`
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`

`Metronomic Therapy for HER-2+ Metastases
`
`have to be sacrificed before the end of the 5th week, i.e., before any
`treatment could start to have an impact. Trastuzumab was given twice
`weekly at 20 mg/kg i.p. (12).
`M'ID and metronomic C’IX administration. CI'X treatment was ini-
`tiated concomitant with trastuzumab therapy. Metronomic CTX was ad-
`ministered at 20 mg/kg (cumulative dose, 420 mg/kg/Zl (1) via the
`drinking water (16). A conventional MTD CTX cycle was given as
`70 mg/kg i.p. on day 1, then again on days 3 and 5; mice were then
`allowed a break period to recover until day 21 when a subsequent
`MTD CTX cycle would begin again (cumulative dose, 210 mg/kg/21 d).
`Bioluminescent (luciferin) imaging. Mice were injected (i.p.) with
`150 mg/kg of luciferin (Caliper Life Sciences) made up in PBS, and
`10 min, later they were anaesthetized and imaged for 1 to 60 3. Imaging
`was done using a IVISZOO Xenogen system (Xenogen) following the
`manufacturer's recommendations. Luminescence was detected via the
`IVIS camera and analyzed using the Living Image software (Xenogen).
`
`Results
`
`Impact of trastuzumab plus metronomic or MTD C’IX on ortho-
`topic H2N tumors.
`Previously, we described the combination
`of trastuzumab plus metronomic CTX as an effective treatment
`for primary H2N tumors when compared with trastuzumab
`plus MTD CTX treatment (12). However, in that study, a
`Kaplan-Meier survival analysis showed that the principal reason
`for the difference was the extreme toxicity that developed fol-
`lowing a third round of MTD CI'X (12). Thus, MTD CI'X plus
`trastuzumab was not a less effective antitumor therapy, but
`rather one that eventually became too toxic (12). Therefore,
`to compare over a long-term period metronomic-based sched-
`ule with a more conventional-type regimen (i.e., several near
`MTD rounds of chemotherapy), we chose an empirical 30% re-
`duction ”MTD" CTX group. We reasoned that this would com-
`pensate for the multiple MTD CTX rounds (as opposed to the
`one or two rounds commonly used in most preclinical studies)
`
`in our regimen. Thus, instead of 300 mg/kg (12), we chose 210
`mg/kg of CTX for each round of MTD. H2N tumors were grown
`orthotopically and treated with saline (control) or with one of
`two combinations of CTX plus trastuzumab. One combination
`involved low-dose metronomic CTX at a daily dose of 20 mg/kg
`administered in the drinking water, plus trastuzumab. The
`other combination was near MTD CI'X (210 mg/kg/Zl-day cy-
`cle) plus trastuzumab. Figure 1 shows that the tumor response
`was virtually identical when comparing the two combination
`therapies. However, the MTD-based regimen did show toxicity,
`particularly after the fifth MTD close (around days 100-110).
`Around day 192, as both therapies were beginning to fail, we
`noticed a drop in weight in some of the mice of both groups
`that did not seem to follow any particular CTX administration.
`Autopsy on these mice revealed the presence of metastases to the
`lungs, bone marrow, and brain (data not shown). Thus, long-
`term treatment of trastuzumab plus CI‘X is effective, but distant
`metastases eventually develop, which seem resistant to the same
`therapy that continues to inhibit primary tumor growth.
`Efficacy of trastuzumab-based chemotherapy regimens on spon-
`taneous metastatic disease. The aforementioned results raise
`
`the question of whether our previous analysis (12) overesti-
`mated the benefits of our combination therapies, because they
`were only tested on single, localized primary tumors, and not
`on metastatic disease. Furthermore, because it is extremely rare
`for H2N tumors to metastasize (12), it was not an appropriate
`model to address this question. Indeed, this probably explains
`why it took over 192 days for even a small fraction of the mice
`to show evidence of H2N metastatic disease.
`
`We therefore ”tagged” the H2N line with hCG, to monitor
`growth of metastatic disease by measuring hCG levels in the
`mouse urine (11, 15), and then used two rounds of in viva
`selection of H2N.hCG cells, as detailed elsewhere (11). This
`selection protocol, which took ~12 months, resulted in the
`
`N
`
`
`hCG(mlUlmg
`
` Creatlnlne) aQ hCGlmlU/mgCreatinine)
`
`_. o
`
`
`
`
`
`"' Control —— Ld CTX+Trastuzumab -— MTD CTX+Trasmzumab
`
`
`D
`"~— Contml
`... v Trasluzumah
`
`_... Ld CTX
`— MTD CTXsTrastuaJmab
`«~- Ld crx 7+7 Trastuzumab
`----- MTD crx
`
`r - Ld CTX+Ttasluzurer "w MTD (31X
`
`
`
`g.-
`
`
`
`-o
`
`NO
`
`
`Percentsurvival
`
`
`
`
`
` o
`
`
`AmonOOO
`
`NO
`
`Fig. 3. Continued. C, top, hCG curves of met2 metastases treated with the regimens indicated. Note at the start oftherapy (day 24), the low hCG levels made it
`difficult to accurately normalize the groups. As a consequence, some groups (e.g., low-dose metronomic CTX alone, see days 25-50) subsequently turned out
`to have relatively low hCG levels. Nonetheless, sequential hCG measurements showed controls and the monotherapies to rapidly increase after a lag
`phase (particularly long for the low-dose metronomic CTX group). In contrast, mice treated with the combination therapies did not show increases in
`hCG readings for 3 mo after treatment was initiated. The combination of MTD CTX + trastuzumab showed a decrease in hCG levels suggesting this approach
`to be the most effective therapy. Low—dose metronomic CTX + trastuzumab showed unchanged hCG levels for the first 3 mo of treatment, followed by
`an increase thereafter. Boxed graph, the same data with expanded Y-axis and only showing the hCG curves for the combination therapies for ease of
`comparison. Bottom, graph showing the same data on the full range of detected hCG values (i.e., 0600), indicating that at the time of sacrifice monotherapies
`had 100-fold higher hCG burden than the ongoing combination therapies. Note that the data are of pooled urine hCG, normalized to creatinine levels.
`D, corresponding survival curve of therapy experiment for met2 metastases (see C) showing the impact of MTD CTX+ trastuzumab (P= 0.03) in this
`model, relative to the other regimens tested. MTD CTX is 210 mg/kg every 21 d. Trastuzumab was given twice weekly at 20 mg/kg i.p., and Ld CTX was
`~20 mg/kg/d.
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`Cancer Therapy: Preclinical
`
`Table 1. MDA-M3231 generated Her-positive Variants
`
`Variant line
`
`Details
`
`Her-2 transduced (retrovirus)/YFP Luciferase transfected
`
`H2N
`met2 (H2NhCGmet2)
`LM2-4H2N (LM2-4H2Nluc+)
`
`Her-2 transduced (retrovirus)/YFP
`Her-2 transduced (retrovirus)/YFP hCG transfected
`
`Metastatic potential
`
`Reference
`
`Low
`High
`Very high
`
`du Manoir et al. 2006
`Francia et al. 2008
`Munoz et al. 2006
`
`establishment of the metastatic cell line called met2 (11). Next,
`having developed the model, we repeated the experiment
`described in Fig. 1—using the met2 line, i.e., we assessed its
`response to therapy as a primary tumor. This was undertaken
`to confirm that the met2 line is responsive to the combination
`therapies. In parallel, and at the same time using the same
`batch of met2 cells, a spontaneous metastasis model was eval-
`uated to test the effect of the therapies on metastatic disease.
`An outline of this experimental plan is shown schematically
`in Fig. 2. For the primary tumors, we noted the expected re-
`sponse pattern (Fig. 3A) as was originally observed with the pa-
`rental H2N (Fig. 1). Thus met2 cells gave rise to primary tumors
`that were highly responsive to trastuzumab plus low-dose met-
`ronomic CTX, a finding independently confirmed by monitor-
`ing hCG urine levels (Fig. 3A). Regression of established tumors
`was induced, followed by 120 days of suppressed growth dur—
`ing continuous therapy. Regrowth (relapse) became apparent
`around day 170.
`In the parallel experiment involving metastatic disease, ther-
`apy was initiated 3 weeks after surgery. Mouse weights were
`then monitored (Fig. 38). For the control groups, we observed
`a slow but steady increase in average mouse weight. In contrast,
`MTD CTX caused cycles of weight loss that coincided with
`rounds of MTD dosing. Interestingly, and consistent with
`previous studies (12), in between MTD rounds the mice main-
`tained a steady average weight (around 20 grams; Fig. SB). The
`steady weight increase seen in the control group was also ob—
`served in the trastuzumab monotherapy groups, but this in-
`crease was reduced in the trastuzumab plus low-dose CTX
`group. Thus, although the trastuzumab plus low-dose CTX reg-
`imen did not result in any significant toxicity, the length (i.e.,
`>200 days) of this experiment allowed us to appreciate that this
`regimen may impair aging-related increase (albeit modest) in
`body weight.
`Although the mice were monitored to evaluate the therapies
`by a traditional survival curve, we could also obtain interim da—
`ta on the relative metastatic burden in each treatment group
`(Fig. BC) by monitoring urine hCG levels. These measurements
`indicated that both controls and MTD CTX monotherapy were
`ineffective at preventing a surge in hCG levels. The same appar-
`ent lack of activity was observed with trastuzumab monother-
`apy, which is consistent with earlier observations using this
`model (11). In contrast, the combination of MTD CTX plus
`trastuzumab caused hCG levels to drop to barely detectable le—
`vels. Metronomic CTX plus trastuzumab also had a suppressive
`impact on hCG levels, although the kinetics of the hCG sup-
`pression were less dramatic. Therefore, trastuzumab can be
`effectively combined with either mode of CTX administration
`(i.e., MTD or metronomic) to treat Her—2-positive breast cancer
`metastases. However, the results also indicate that trastuzumab
`plus MTD CTX is the most effective treatment, at least initially
`(in the debulking of metastatic disease, as assessed by hCG
`
`levels). On the other hand, the trastuzumab plus low—dose
`CTX combination was not as toxic as the MTD CTX combina-
`
`tion treatment (Fig. SB). Thus, determining the preferred treat-
`ment option between these two regimens could depend on the
`relative importance of antitumor efficacy versus acceptable le-
`vels of toxicity. This can be assessed in the resulting survival
`curve for this experiment (Fig. 3D). The data showed that both
`combination regimens were effective in prolonging survival,
`and that the superior survival curve was actually obtained with
`the trastuzumab plus MTD CTX regimen (consistent with the
`hCG data; Fig. 3C). However, it should be stressed that 2 of
`12 mice in this group had to be sacrificed (between days 190
`and 220) due to extreme weight loss, and that at autopsy, no
`evidence of metastatic disease was found. Thus, these mice
`mOSt likely succumbed to toxicity caused by the treatment. Im-
`portantly, we never observed any such toxicity in the trastuzu—
`mab plus low-dose CTX group in the ZOO-day therapy period.
`Evaluation ofan independent model of metastasis. To exclude
`the possibility that the above observations were unique to the
`met2 model, we developed an independent spontaneously me-
`tastatic and Her-2-positive breast cancer model. The LM2-4 line
`was previously generated as a highly metastatic variant of the
`MDA—231 human breast cancer cell line, following in viva selec-
`tion (10), and later transfected with luciferase to generate the
`luc14+line (Table 1). Subsequently, the luc14+ line was trans-
`duced with H2N/YFP, resulting in the generation of the W2-
`4luc+H2N cell line (Fig. 4) that has stable and high Her—2
`expression and expresses luciferase. This tumor line was
`implanted orthotopically into female SCID mice, and 3 weeks
`after surgery, treatments were initiated. The survival curve
`(Fig. 5A) shows that the monotherapies were less effective than
`the combination regimens. Trastuzumab plus MTD CTX proved
`equivalent to trastuzumab plus low-dose CTX. All mice that
`showed detectable luciferase luminescence after surgery
`(Fig. SB) eventually succumbed to metastatic disease. Indeed,
`in this model, we did not observe a debulking of metastatic
`disease, as was observed in Fig. 3C with hCG levels for the
`groups treated with trastuzumab plus MTD CTX.
`Analysis of mouse weights (Fig. 5C) showed that controls,
`metronomic CTX, and trastuzumab monotherapies exhibited
`an aging-related increase in weight. In contrast, MID CTX and
`trastuzumab plus MTD CTX led to cycles of weight-loss that
`correlated with the timing of the MTD dosing (particularly at
`days 100 and 140 after surgery). Thus, in this model, trastuzu-
`mab plus low—dose CTX is superior to trastuzumab plus MTD
`CTX in that equivalent survival curves were generated, yet the
`metronomic—based regimen lacked the repeating cycles of
`weight loss. We conclude that both are valid combination treat-
`ment regimens, and that evaluating the most effective regimens
`depends on the balance of the need to target the tumor popula-
`tion with the desire of achieving such effects with minimal asso-
`ciated toxicity.
`
`Clin Cancer Res 2009;15(20) October 15, 2009
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`6362
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`

`

`Discussion
`
`This study represents the latest in a series of investigations,
`including from our laboratory, evaluating therapeutic outcomes
`after therapy is initiated in mice with established visceral me-
`tastatic disease, and comparing the results with those ob-
`tained using the more traditional primary tumor models,
`including orthotopic transplants (10, 11, 17—19). This partic-
`ular study represents the first detailed preclinical study of
`Her—2—positive spontaneous metastatic breast cancer (14),
`of which we are aware, which uses mice with established me-
`tastatic disease. Previous studies have used Her—2—transfected
`
`MDA-MB-231 cells injected into the arterial circulation as a
`means of generating ”artificial” metastases, e.g., brain metas-
`tases, as a model for therapy of Her-2 metastatic breast cancer
`using drugs such as lapatinib (20). These studies involved ini-
`tiation of therapy within one or a few days of tumor cell in-
`jection and thus did not involve macroscopic established
`metastasis. In our studies with orthotopically implanted
`and intact primary H2N tumors, we observed that such ”pri-
`mary” tumors would eventually start to relapse on treatment
`using the combination of trastuzumab plus metronomic CTX;
`in some cases, this was accompanied by the development of
`metastatic disease. However the minimal metastatic capacity
`of the H2N tumor model made it initially difficult to ascer—
`tain whether this result was due to the ineffectiveness of the
`
`therapy against late-developing metastatic disease or simply
`to metastases that had also developed resistance to the treat—
`ment. We therefore developed the metastatic met2 model
`(11), with the hCG ”tag" allowing us to generate a ”metasta-
`ses”-growth curve (by plotting the relative weekly pooled
`urine hCG levels).
`We have used the met2 model to test the effectiveness of tras-
`
`tuzumab plus CTX regimens on established spontaneous meta-
`static disease after primary tumor resection. Therapies were also
`tested, in parallel, using the intact primary orthotopic tumor
`model. This is a fundamental aspect of our study, because,
`as discussed above, the majority of preclinical therapies re-
`ported in the literature are routinely assessed using only
`primary tumor models, either ectopic or orthotopic. Further-
`more, in the few cases where therapies were initiated against
`
`Metronomic Therapy for HER-2+ Metastases
`
`spontaneous metastatic disease, there was no (control) equiv-
`alent test on a primary tumor using the same tumor cell po-
`pulations. We found trastuzumab combinations (i.e., with
`either MTD or metronomic CTX) to show superimposable tu-
`mor regressions followed by growth delays. Yet hCG analysis
`of metastatic growth (which is another fundamental aspect of
`our study) showed that the impact of these therapies was not
`equivalent when applied to the metastatic setting. This result
`indicates that growth behavior and kinetics at a secondary
`(metastatic) site are unique and distinct phenomena from
`those at the primary site.
`Of considerable interest, especially from a clinical perspec-
`tive, is the contrast in the results we observed with the drug
`combinations tested to those obtained against orthotopically
`implanted primary tumors. Thus, we observed less impres-
`sive treatment benefits when treating established metastatic
`disease. We would argue that the experiments described
`here, although more time consuming, may be necessary to
`avoid or minimize the over interpretations that could be
`drawn from results obtained based only on treating primary
`tumors. In this respect, we note that the hCG methodology
`permitted an evaluation of the effectiveness of the different
`regimens on metastatic disease within 50 days from the start
`of treatment.
`We also monitored the relative toxicity of long—term therapies
`particularly in view of the overt toxicity associated with MTD
`protocols. This also applies in the clinical setting, as dose—
`limiting toxicities of chemotherapeutic agents are sometimes
`implicated in the deaths of a small percentage of patients in
`clinical trials (21). Indeed, although in one of our metastatic
`models (met2) we observed that trastuzumab plus MTD che-
`motherapy was most effective in increasing survival, this
`was accompanied by a 16% rate of death of mice that showed
`no evidence of metastases. These mice seem to have succumbed
`
`to the overt toxicity of the long-term MTD regimen. In contrast,
`no such severe toxicity was observed in the trastuzumab
`plus low-dose CTX group. These results suggest that after an
`initial MTD-based treatment regimen, it may be beneficial to
`switch to a minimally toxic maintenance metronomic—type
`regimen—as we and others have already suggested for CTX
`(22) or vinblastine regimens (23—25). Thus, an interesting
`
`Fig. 4. Schematic of LM2—4H2N therapy
`experiment. The human breast cancer
`cell line MDA—M3231 was selected
`in vivo for high metastatic potential (10)
`and thereafter transfected to express
`luciferase, and then transduced to
`express Her-2. The resulting LM2—4H2N
`line was injected orthotopically into
`female SCID mice. A spontaneous
`metastasis assay was carried out, with
`therapies beginning 3 wk after resection
`of the primary tumors.
`
`MDA-MB-231
`
`In vivo
`selection
`(Munoz et al 2006)
`LM2-4 metastatic variant
`|uc+
`Her-2 transduction (H2N)
`
`1
`LM2-4/lUC+/H2N
`
`i
`
`l
`
`+/-trastuzumab ip
`+/- LDM CTX
`+/- MTD CTX
`
`i Primagl tumor (500mm3)
`
`3
`
`d
`
`
`Luciferase imaging and survival analysis
`
`www.aacrjourna|s.org