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Advances in Brief |
Departments of Pathology [W. F. S., S. D., H. T. Y.], Medicine [M. D. V., R. O., P. K., F. M.], Surgery [R. L. S., H. M.], Radiology [A.Y. K.], and Radiation Oncology [S. C. F.], and Kaplan Comprehensive Cancer Center, New York University Medical Center, New York, New York 10016
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ABSTRACT |
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 Top ABSTRACT IntroductionPatients and MethodsResultsDiscussionREFERENCES |
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Introduction |
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TopABSTRACTIntroductionPatients and MethodsResultsDiscussionREFERENCES |
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The two main reasons supporting the treatment of breast cancer with neoadjuvant (rather than conventional postoperative) chemotherapy are: (a) to predict patient outcome based on the observed response to treatment; and (b) to reduce the size of the primary tumor and thus facilitate surgical management (1, 2, 3, 4) . Clinical trials have indicated that the extent of response of the primary tumor to neoadjuvant chemotherapy correlates with disease-free and overall survival (1 , 2 , 5, 6, 7) . On the basis of the clinicopathological status of patients at the time of surgery (after neoadjuvant chemotherapy; Ref. 8 ), a minority (3–12%) of patients obtain complete pathological response, but these patients have the longest survival (1 , 2 , 5 , 7) . Another minority of patients (12–25%) who obtained minimal or no pathological response (<50% tumor diameter reduction by clinical measurement) fare significantly worse, whereas the majority (70–80%) of women with a partial response to treatment have an intermediate outcome (1 , 2) . Indeed, the outcomes for women with a partial response to treatment are probably variable, and these differences may be difficult to predict based solely on clinicopathological response. If the extent of tumor reduction at the completion of neoadjuvant treatment predicts for patient survival, then early prediction of tumor response to the neoadjuvant chemotherapy could yield useful information. Early identification of women who are expected to have no response or an incomplete response could enable their selection as candidates for modification or intensification of their neoadjuvant treatment, possibly including the selection of specific additional agents to augment the efficacy of cancer cell killing (4 , 9 , 10) .
An important challenge is to accurately and noninvasively assess the extent of cancer that is present before and after treatment, particularly if tumor response is to be used as an end point for clinical studies. Of note, approximately 1 of 4 women who have a clinical complete response (no palpable tumor) actually have a pathological complete response (3) . Conventional breast imaging offers an accessible and promising approach. Studies of breast cancers, with or without neoadjuvant chemotherapy, have also shown that ultrasound measurements predict pathological tumor size more accurately than mammographic or clinical measurements (11, 12, 13) , although this conclusion is not unanimous (14) . Ultrasound measurements in three planes allow for approximation of tumor volume before and after treatment, and therefore a change in volume can be determined as an objective parameter of response. The advantage of this approach is quantification of tumor response that can be compared with potential predictors of outcome.
Paclitaxel is a taxane with activity against breast cancer. In vitro treatment of cells with paclitaxel rapidly induces the accumulation of cells in G2-M phases of the cell cycle (after polymerization of microtubules) and leads to apoptosis in susceptible cells (15, 16, 17) . In paclitaxel-treated MCF-7 breast cancer cells, apoptotic bodies are seen by light microscopy within 24 h of treatment and peak at 48–72 h (18 , 19) . In a mouse breast cancer model (an inoculated transplantable, spontaneous, mammary carcinoma in C3Hf/Kam mice), accumulation of mitotic figures is seen with light microscopy at 9 h, and apoptotic indices peak at 18–24 h after a single dose (60 mg/kg) of paclitaxel (20) . Both mitotic and apoptotic microscopic indices return to pretreatment levels by 4 days in this model (20) . The peak apoptotic index and the pretreatment apoptotic index correlated with murine tumor reduction by paclitaxel therapy (21) . These preclinical data support our hypothesis that early cellular responses after the first dose of neoadjuvant paclitaxel chemotherapy for breast cancer predict for the extent of tumor reduction.
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Patients and Methods |
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TopABSTRACTIntroductionPatients and MethodsResultsDiscussionREFERENCES |
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Women who opted for neoadjuvant paclitaxel therapy were also invited to have serial FNAs to assess cellular responses to the first dose of paclitaxel. In this protocol, a baseline FNA was performed before the core biopsy (prior to chemotherapy) and at 24, 48, 72, and 96 h after the first paclitaxel infusion began. All samples were from the primary breast tumor, not from palpable lymph nodes. At each time point, a single-pass FNA was performed using a 25-gauge needle, and the cellular sample was divided onto seven glass slides using a spreader slide. The spreader slide was stained with Diff-quik (Allegiance, McGaw Park, IL) for an immediate microscopic interpretation of the specimen adequacy. The first glass slide was immediately fixed in 95% ethanol and then stained with H&E. H&E stain, rather than in situ terminal transferase UTP nick end labeling, was used to identify apoptotic cells because there is excellent cytological detail, close inter-observer consistency, and less artifact and loss of specificity using H&E stain (22, 23, 24) . We abandoned the terminal transferase UTP nick end labeling (TUNEL) assay to identify apoptotic cells in our samples because the majority of cells had positive staining (even in nontreated control samples), possibly because of DNA stand breaks from smearing, drying, and/or formalin fixation (23 , 24) . Microscopic counts of the number of identifiable mitotic figures or apoptotic bodies in one thousand cancer cells (x400, x600; Olympus BH2 microscope) were recorded as percentages (indices). The index at each time point was then divided by the pretreatment baseline index (0 h) and expressed as a ratio. The ratio indicates the proportion of change of the index compared with the baseline index and corrects for variability in baseline indices from different patients’ tumors. The sum of these relative changes in apoptotic or mitotic index (each compared with baseline) was calculated to assess the cumulative response for the first 4 days of treatment.
Radiological and clinical measurements of the primary tumor were
performed prior to the first dose of paclitaxel and after the fourth
cycle of paclitaxel (prior to surgery). Clinical measurements were made
of the maximal palpable tumor diameter. Mammographic and ultrasound
measurements of the tumor dimensions were made in three dimensions
[antero-posterior (AP), transverse (T), and
sagittal (S)]. The tumor volume was calculated as
/6 x (AP x T x S)
(Ref. 25
). A radiologist reviewed the mammograms and
ultrasound images from each patient and independently decided which
modality most accurately demonstrated the tumor mass for measurements
of both the pre- and posttreatment tumor volumes. After surgery, the
pathological specimen was thoroughly sampled and photographed by the
investigators, with measurement and extensive histological sampling of
the tumor bed. Preoperative clinical, mammography, and ultrasound
measures of greatest residual tumor diameter were compared with the
greatest pathological tumor diameter using Pearson’s correlation
coefficient (r) to measure their linear relationship.
We introduced a HCF to approximate the microscopic proportion (0–1) of carcinoma cells within recognizable tumor in the histological sections from the pretreatment core biopsy and the posttreatment tumor bed. This HCF corrects for extensive areas of fibrosis or necrosis that would be identified as tumor using radiological studies. The histologically corrected radiological tumor volumes pre- and posttreatment were called "radiological cancer volume." Each cancer volume was calculated by multiplying the radiological tumor volume with the respective HCF. The proportion of residual cancer after treatment was then calculated as a ratio: radiological cancer volume after treatment divided by radiological cancer volume before treatment.
Quantification of cancer response offers a potential advantage for comparison of response with potential predictors of outcome in small clinical study populations. The same radiological modality was used to measure the tumor volume before and after treatment; therefore, inherent errors of the radiological measurement should be similar and may therefore cancel out when the before and after treatment measurements are expressed as a ratio. We believe that this approach is better than a comparison of two measurements that were derived using different methods, e.g., clinical pretreatment and pathological posttreatment measurements. We compared the observed cellular responses (G2-M arrest and apoptosis) with the proportion of residual cancer after treatment using Pearson’s correlation coefficient (r). The scatter plots from these pilot data indicated a relationship between cellular response and tumor reduction. We assumed a linear relationship only for simplicity, recognizing that the number of patients in this pilot study are too few for a sophisticated statistical analysis of variables.
A Macintosh G3 computer (Apple Computer, Cupertino, CA) was used with Deltagraph Professional v2.0.3 (SPSS, Inc., Chicago, IL) and Statview v5 (SAS Institute, Inc., Cary, NC) software to produce time response curves, scatter plots, and statistical analyses. Regional heterogeneity of apoptotic and mitotic indices within a tumor mass was evaluated in a separate control group of untreated resected breast cancers. Samples were obtained from up to 10 different sites within the primary tumor using the FNA and sample preparation methods described above. The coefficient of variation (SD/mean) was then calculated for apoptotic index and mitotic index. Ideally, we would also have evaluated the temporal heterogeneity of our measured indices from serial daily FNAs of the tumors in a control group of untreated women, but we believed that was not clinically reasonable. Temporal heterogeneity could be studied in an appropriate animal model (20 , 21) .
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Results |
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TopABSTRACTIntroductionPatients and MethodsResultsDiscussionREFERENCES |
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showed average coefficients of variation of 0.25 for apoptotic index
and 0.46 for mitotic index. For most of the 11 treated women, there was
a 3.0–6.0-fold increase in apoptotic activity (relative to the
pretreatment baseline measurement) at some time during the first 4 days
after the first dose of neoadjuvant paclitaxel treatment (Fig. 1
a). There was also a 5.0–50.0-fold relative increase in
mitotic index (relative to the pretreatment baseline measurement) at
some time during the first 4 days after the first dose of neoadjuvant
paclitaxel treatment (Fig. 1
b). Therefore, the apoptotic and
mitotic arrest responses were clearly in excess of the expected range
from regional heterogeneity within the tumor.
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a).
Noticeably, the apoptotic response was sustained for 2 patients (nos.
10 and 11), who had a complete pathological response or a single
microscopic residual tumor focus (Figs. 1
a and
2; Table 2
). An apoptotic response either did not occur, or was not sustained
beyond day 1, in 3 patients whose cancer volume did not decrease after
treatment (patients 1, 2, and 9; Fig. 1
a; Table 2
). The
profiles of mitotic response were specific for each patient but did not
appear to relate to the measured tumor response to treatment (Fig. 1
b).
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). Therefore, radiological measurements were used for the subsequent
analysis of tumor volume response to treatment. A radiologist
independently selected ultrasound in 9 patients and mammography in 2
patients as the better imaging technique from which to accurately
measure the tumor volume. The clinicopathological status of response to
neoadjuvant treatment can be compared with these radiological
measurements of tumor volume change in Table 2
. These data
suggest that the clinicopathological status assessment may sometimes
overestimate the extent of tumor reduction from neoadjuvant
chemotherapy (patient 9, Table 2
; Fig. 4
). Radiological volume measurement alone would have underestimated a
complete pathological response for patient 11 (Fig. 2
; Table 2
). As
described in "Patients and Methods," we introduced a HCF to rectify
any problem of areas of mass not containing cancer. Sometimes, there
was more extensive fibrosis after treatment, as demonstrated by
differing HCF values in posttreatment specimens from patients 4, 5, 6,
7, and 11 (Table 2)
. Occasionally, there was a greater proportion of
the mass that contained carcinoma after treatment (patient 2). A HCF
was not obtainable after paclitaxel therapy for patient 3, because she
continued with Adriamycin-based chemotherapy before surgery (Table 2)
.
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a). The relative change in
apoptotic index (compared with the pretreatment index) at each day
after the first dose did inversely correlate with the proportion of
residual cancer volume [r = -0.51 (day 1), -0.86
(day 2), -0.88 (day 3), and -0.78 (day 4)] (Fig. 5)
. The cumulative apoptotic response (sum of relative changes in
apoptotic index for days 1–4 after the first dose) showed an even
stronger inverse correlation (r = -0.97) with the
proportion of residual cancer volume (Fig. 6)
. There was no correlation between the cumulative mitotic response and
the proportion of residual cancer after the neoadjuvant therapy
(r = -0.18) or the cumulative apoptotic response
(r = 0.25). We note that our sample size (11 patients)
is too small for detailed statistical analysis. Linear correlation
coefficients were used to simply describe our observations from the
pilot data.
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Discussion |
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TopABSTRACTIntroductionPatients and MethodsResultsDiscussionREFERENCES |
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Tumor imaging with measurements in three dimensions, combined with correction for the histopathological proportion of malignant cells within the mass, enabled us to quantify the primary cancer response to neoadjuvant treatment. We observed good correlations between the radiological measurements of greatest tumor diameter immediately prior to surgery and the macroscopic pathological diameter of the tumor bed at resection, which are consistent with most published studies of untreated resected breast cancers (11, 12, 13) . We note that a quantitative measure of tumor reduction could enable more precise measurement of the treatment response in the majority (70–80%) of patients who have an intermediate (partial) response to neoadjuvant therapy, rather than grouping all of them together (1 , 2) . Furthermore, if meaningful and predictive information about the early cellular responses to treatment can be obtained using FNA, it could benefit those patients who will have an incomplete (partial) response. Early recognition of such patients might enable a specific early intervention during their treatment to increase the cell killing (by targeting the likely mechanism of impaired chemotherapeutic efficacy) and so improve the benefit from neoadjuvant treatment.
Our limited data did not identify a specific day from which tumor
samples should ideally be obtained, because there was variable timing
of the response profiles of apoptosis and mitotic accumulation (Figs. 1
and 5)
. Therefore, we could not accurately predict the time to capture
cells at the exact peak of cellular response. However, our results
suggest that a sustained apoptotic response to treatment is
important for greater cancer volume reduction. Further studies might
better define the optimal number and timing of samples. Our data from
patients, and other published data from mice, show that the apoptotic
response to a single dose of paclitaxel lasts for 
4 days (20
, 21)
. This theoretically favors a weekly schedule for neoadjuvant
paclitaxel, rather than less frequent dosing intervals of 2–3 weeks. A
confirmatory study could be undertaken during a weekly neoadjuvant
paclitaxel regimen to assess whether the lower paclitaxel dose in a
weekly regimen induces similar apoptotic responses.
The cumulative apoptotic response during the first 96 h after the
first dose of paclitaxel had an almost linear relationship to the
extent of tumor reduction (Fig. 4)
. This was despite the observed
variability in timing of the apoptotic response in individual patients.
Perhaps different waves of apoptosis occur during the first 4
days as different molecular pathways are activated and lead to
activation of caspases and apoptosis at different rates (26
, 27)
. For example, one measure of short-term cell death
(viability at 24 h) after chemotherapy in vitro does
not always predict a longer-term anticancer effect (colony formation at
8 days; Ref. 28
). The cumulative apoptotic response (from
daily measurements) may correct for the variability in rates of
induction of apoptosis by different mechanisms.
It is intriguing to consider whether FNA could be used to study the role of drug resistance mechanisms, and/or inhibition of apoptosis, in breast cancers treated with neoadjuvant chemotherapy. Although the potential mechanisms of resistance to chemotherapy-induced cell killing are myriad (4 , 28) , the induction of a multiple drug resistance gene (MDR-1) product has been implicated as a cause of resistance to neoadjuvant chemotherapy for breast cancer (29, 30, 31, 32) . Also, blunted and truncated apoptotic responses to neoadjuvant paclitaxel in nonresponsive breast cancers may result from an inability of the cancer cells to efficiently enter and complete the apoptotic pathways that should be induced by paclitaxel (4 , 27 , 33, 34, 35, 36, 37, 38) . There is also evidence that extensive apoptosis from taxanes can decrease the intravascular and interstitial pressure in solid tumors, and that may improve drug delivery in subsequent chemotherapy cycles, hence compounding the effectiveness of treatment (39) . Knowledge of likely mechanisms of resistance to chemotherapy-induced apoptosis for an individual patient should provide an opportunity for early and targeted intervention to enhance the extent of cell killing, tumor reduction, and survival benefit (9 , 10 , 40, 41, 42, 43, 44, 45, 46, 47) .
Larger clinical trials (with statistical power) and more sophisticated molecular analyses of the responses of cancer cells are needed to validate these pilot data. The similarities between our results from patients in a clinical trial and the published preclinical data provide a foundation for more detailed studies of the molecular and cellular responses to paclitaxel chemotherapy in murine models and clinically (20 , 21) . We have demonstrated in this study that serial FNAs are a minimally invasive adjunct to therapeutic clinical trials with a real potential to obtain samples that can improve our knowledge of breast cancer cellular responses to chemotherapy in vivo. This may ultimately allow us to individually tailor each woman’s neoadjuvant treatment protocol.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by National Cancer Institute Grant R21
CA66229 pilot study funds (to W. F. S.), a New York State Health
Science Research Board Department of Health EMPIRE award (to
W. F. S.), an American Cancer Society grant (to M. D. V.), and a
California Breast Cancer Research Program grant (to S. C. F.).
Presented in part at the American Association for Cancer Research
special meeting "Molecular Determinants of Sensitivity to Anti-Tumor
Agents," Canada, March 1999. 
2 To whom requests for reprints should be
addressed, at Department of Pathology, Box 85, University of Texas
M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX
77030. Phone: (713) 792-7962; Fax: (713) 745-8610; E-mail: fsymmans{at}mdanderson.org
3 The abbreviations used are: FNA, fine-needle
aspiration; HCF, histopathological correlation factor.
Received 5/18/00; revised 10/ 3/00; accepted 10/13/00.
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TopABSTRACTIntroductionPatients and MethodsResultsDiscussionREFERENCES |
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