Download Immune Changes in Patients with Locally Advanced Breast Cancer

Immune Changes in Patients with Locally Advanced
Breast Cancer Receiving Neoadjuvant Chemotherapy
CAB‹O⁄LU N1, MÜSLÜMANO⁄LU M2, TÜTÜNCÜ Y3, AD‹N-C‹NAR S4, GÜREL N5, K‹RAN B5,
IGC‹ A2, ÖZMEN V2, DA⁄O⁄LU T2, AYDINER A2, DEN‹Z G4
1Department
of General Surgery, Haseki Research Hospital, Istanbul, Turkey
University, Istanbul Faculty of Medicine, Departments of General Surgery,
3Department of Internal Medicine, Division of Diabetes, 4Institute of Experimental Medicine (DETAE), Department of
Immunology, 5Department of Microbiology, Virology and Basic Immunology Division, Istanbul, Turkey
1Istanbul
ABSTRACT
Object: The effect of neoadjuvant chemotherapy on cellular immunity was investigated in patients with locally advanced
breast cancer by evaluating the changes in peripheral blood lymphocyte (PBL) subpopulations and natural killer cell activity (NKA) after chemotherapy.
Materials and Methods: Blood samples were obtained from 13 advanced breast cancer patients before and after 3 or 4
cycles of the combination chemotherapy 5-fluorouracil, epirubicin, and cyclophosphamide. PBLs including CD3+, CD4+,
CD8+, CD19+, CD25+, CD45RA+, CD45R0+, CD56+ and γδ-T cells were evaluated by flow-cytometric analyses by using specific monoclonal antibodies. NKA was assessed by anti-candidal indexes.
Results: After chemotherapy, the CD19+ B and CD45RA+ naive T-lymphocyte percentages and the CD4/CD8 ratio were
decreased (p<0.001, p=0.06, p=00.04, respectively), while the CD8+ cytotoxic-supressor T cells were increased (p<0.001). In
subgroup analysis, the anti-candidal indexes were found to be decreased (p=0.04), while the CD56+ (p=0.005), and CD45RO+
(p=0.05) memory T-cell percentages were increased in the responsive-group (n=7) after chemotherapy. However, the anti-candidal indexes (p=0.01) were observed to be increased, and the CD25+ activated T-cell percentages (p=0.004) were decreased
in the chemotherapy non-responsive group (n=6) unlike the other groups. No statistically significant changes were observed in
CD3+, CD4+, and γδ-T cell percentages after chemotherapy in the whole group and subgroup analyses.
Conclusion: Our results suggest that the anthracycline-based chemotherapy regimen induced increases in CD45RO+ memory T-cell percentages may enhance the efficacy of chemoimmunotherapy trials in chemotherapy-responsive patients.
Detailed phenotypical and functional characterization of intratumoral lymphocytes warrants further investigation in terms of
contrary findings on PBL percentages and NK cell function after chemotherapy.
Note: Part of this study was presented in ASCO 1998.
Turk J Immunol, 2008; 13: 15-20
Key Word: Cellular immunity, lymphocytes, peripheral blood, natural killer cells, lymphocyte subpopulations, natural killer
cell activity, locally advanced breast cancer, neoadjuvant chemotherapy
Received: 04.04.2008
Revised: 22.09.2008
Accepted: 16.10.2008
been suggested that natural killer (NK) cells may provide a first line defense against newly developed
malignancies as immunological effector cells1-4. The
activity of NK cells is mediated primarily by large granuler lymphocytes, which are capable of lysing tumor
cells without prior sensitization5.
INTRODUCTION
are many host factors that influence the
T here
growth and the metastatic potential of tumors.
Among these factors, the immune system may play an
important role in the control of cancer spread. It has
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TURK J IMMUNOL
Vol.13, No.1, 2008
breast ultrasound. A neoadjuvant (preoperative)
chemotherapy was planned for those patients who
were considered to have locally advanced breast
cancer according to the TNM staging (T2N1-N3, or
T3 or T4, and any N)14. Fine needle aspiration or trucut needle biopsy or incisional biopsy were performed for histopathological diagnosis. Further
staging work-up included a chest X-ray, a bone scan
and abdominal ultrasound or thorax & abdominal CT
for detection any sistemic metastases.
All patients received 3 or 4 cycles of the combination chemotherapy, 5-fluorouracil (500 mg/m2),
epirubicin (80 mg/m2), and cyclophosphamide (500
mg/m2) (FEC) every 3 weeks as primary chemotherapy before surgery. The response to neoadjuvant
chemotherapy was evaluated every 2 cycles by
physical examination, and by ultrasonography or by
mammography along with physical exam once the
neoadjuvant chemotherapy was completed. Clinical
responses were based on the primary tumor and
axillary lymph node response and were categorized
as follows: complete response (CR) = total resolution
of the breast mass and axillary adenopathy on physical and radiographic examination; partial response
(PR) = 50% or greater diminution of bidimensional
tumor or axillary lymph nodes; stable disease (SD) =
no more than 25% increase or decrease in tumor size
or no change in lymph node; progressive disease
(PD) = more than 25% increase in tumor. Blood samples in heparinized tubes (5-10 cc) were obtained
from 13 consecutive locally advanced breast cancer
patients before and after 3 or 4 cycles of FEC.
A modified radical mastectomy was performed
after the neoadjuvant chemotherapy. The
histopathologic type of tumor, surgical margin
involvement and axillary involvement was detected
in the specimen. Hormone receptors including
estrogen and progesterone receptors were studied
in the biopsy specimen which was performed before
the neoadjuvant chemotherapy. The pathologic
response was also evaluated in the mastectomy
specimen and absence of invasive cancer was considered a pathologic complete response.
Patients completed their chemotherapy regimen
following surgery by additional 3 or 4 cycles of FEC,
or by Taxotere alone (75 mg/m2) if the chemotherapy
response was considered as no response or progression. After completion of chemotherapy, patients had
The other pathway of the immune system to
eradicate the cancer cells is by recognising cancer
cells by activating lymphocytes and driving the cancer cell to becoming susceptible to kill6. Numerous
studies have demonstrated that both CD8 and CD4
subsets of lymphocytes must be activated to elucidate a tumoricidal effect. Impaired cell-mediated
immunity is a poor prognostic factor in addition to
the presence of uncontrolled cancer cell growth and
metastases7. Furthermore, some studies showed
elevated levels of tumor antigen-specific autoantibodies in cancer patients8,9. Other reports demonstrated strong infiltration of primary tumors and
draining lymph nodes by tumor-specific cytotoxic T
lymphocytes10. Mccoy et al11 also demonstrated that
cell-mediated immunity to tumor-associated antigens is a stronger prognostic factor than stage,
grade, and lymph node status.
The most widely used anticancer agents for
breast cancer are anthracyclines including doxorubicin and epirubicin that were shown to improve survival in patients in adjuvant setting12. Both of these
drugs exert antitumor effects through interference
with DNA synthesis and function, especially during
the S and G2 phases of the cell cycle. Furthermore,
they also cause cytocidal activity by intercalation
between nucleotide base pairs resulting in inhibition
of nucleic acid (DNA and RNA) and in DNA cleavage
by topoisomerase II13. In general, administration of
cytotoxic chemotherapy is characterized by bone
marrow supression that results in reduced numbers
of peripheral blood immune effector cells including
neutropenia. These effects are mostly reversible,
and do not lead to permanent alterations in immune
function.
Due to the conflicting results in the literature, the
present study investigated the effect of the neoadjuvant combination chemotherapy regimen including
epirubicin on cellular immunity in patients with locally advanced breast cancer by evaluating the
differential changes in peripheral blood lymphocyte
(PBL) subpopulations and natural killer cell activity
(NKA) after chemotherapy.
MATERIALS AND METHODS
Patients and Study design
Patients were evaluated by physical exam and
radiologic imaging including mammography and
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Vol.13, No.1, 2008
TURK J IMMUNOL
radiation treatment including 50 Gy to the thorax and
to the lymphatic basins (axilla, supraclavicular nodes
and internal mammarial chain). Radiotherapy was
administered as 20 fractions over a 4 week periods.
Tamoxifen (20 mg/day) was given to estrogen receptor positive patients for five years. Patients were followed up by the surgeon and oncologists periodically after completion of their radiotherapy.
Analyses of Peripheral Blood
Lymphocyte Subsets
Blood samples for analysis of the PBL subsets
and NKA were drawn before and 2 weeks after the
completion 3 cycles neoadjuvant chemotherapy.
Samples from each patient were processed immediately for the phenotypic analysis and NKA.
Phenotypic analyses were performed using a
FACScan flow cytometer (Becton Dickinson, USA)
equipped with a 15-mW air-cooled argon-ion laser.
The percentages of peripheral blood lymphocyte
subsets including CD3+, CD4+, CD8+, CD19+,
CD25+, CD45RA+, CD45R0+, CD56+, γδ-T cells were
measured by using specific monoclonal antibodies.
FACScan analyses were performed by 3 investigators (S.A., N.G. and B.K.) who were blinded to clinical data and NKA results.
NK Cytotoxic Cell Activity
NK cytotoxicity was assessed as the anticandidal
colorimetric index, which was found to be a simple
method to assess NKA15,16. Ten-milliliter blood samples were taken in standard tubes with heparin and
then centrifuged by Ficoll Isopaque. Lymphocyte layers were diluted with RPMI-1640, and living cells
stained with trypan blue were counted.
By using lymphocytes as effector cells and
Candida (Candida stellatoidea) as target cells, cells
were mixed in the ratios of 1/5 or 1/25 target:effector cells. As the control, 0.025 ml Candida solution in
phosphate-buffered saline, neutral pH, was inoculated into blood agar. After 48 h, colonies were counted in triplicate and the anti-candidal index was calculated: anti-candidal index (%) = 1 - experimental
colony unit/control colony unit x 100.
Statistical Analyses
Statistical tests were performed by using SPSS
11.00 for Windows statistical software (SPSS Inc,
Chicago, IL). Comparison between the means of the
two groups were made by using the paired
Student's t test. All p values were two-sided in tests
and p values <0.05 were considered significant.
RESULTS
The median age was 43 (range, 24-75).
According to the TNM staging classification, 12
patients were clinically T4 N0-N2 as stage IIIB and
one patient was T2N2 as stage IIIA without any sistemic metastases before neoadjuvant chemotherapy. Partial response was obtained in 7 patients
whereas no significant response to chemotherapy
was observed in 6 patients who therefore were considered as non-responsive patients. When all the
patients’ immunological results before and after
chemotherapy were analyzed (Table 1), the CD8+
cytotoxic-suppressor T cell percentages were found
to be increased following chemotherapy (p<0.001).
Furthermore, the CD4/CD8 ratio (p=0.04), and the
CD19+ B lymphocyte (p<0.001), and CD45RA+ nonactivated T cell percentages were decreased after
chemotherapy even though the decrease in
CD45RA+ population did not reach the statistical
significance (p= 0.06).
In subgroup analyses (Table 1), in the chemotherapy-responsive group, the CD8+ cytotoxic-supressor
T cell percentages were increased (p<0.001), whereas the CD4/CD8 ratio (p<0.04), and the CD19+
B lymphocyte
percentages (p<0.001) were
decreased similar to the whole group analyses.
Interestingly, however, CD56+ NK cells (p=0.005),
and CD45RO+ memory T-cells (p=0.05) were found
to be increased, whereas the NKA (p=0.04) were
decreased in this group after chemotherapy.
In the chemotherapy nonresponsive group
(Table 1), the CD8+ cytotoxic-supressor T cell perTable 1. Monoclonal antibodies that were used in
this study
17
Monoclonal
Peripheral Blood
Antibodies
Lymphocyte Subpopulations
CD3
Total matureT cells
CD4
T-helper cells
CD8
Supressor-cytotoxic T-cells
CD25
Activated T-cells (IL-2 receptor)
CD19
Total B-lymphocytes
CD56
Natural Killer Cells
CD45RA
Non-activated T cells
CD45RO
Memory T-cells
T-cell γδ
γδ-T lymphocytes
TURK J IMMUNOL
Vol.13, No.1, 2008
centages were increased (p=0.03), whereas the
CD19+ B lymphocyte percentages (p=0.04) were
decreased similar to the whole group and to the
chemotherapy-responsive
group
analyses.
Nevertherless, unlike the other groups, the CD25+
activated T-cell percentages (p=0.004) were
decreased, and the NKA (p=0.01) was found to be
increased in this group analyses after chemotherapy. However, no statistically significant changes
were observed in other PBL subpopulation percentages including CD3+, CD4+, and γδ-T cell populations in the whole group and subgroup analyses
after chemotherapy.
chemotherapy responsive-group whereas the NKA
was observed to be increased in the chemotherapy
non-responsive group following chemotherapy.
In concordance with previous studies,
decreased CD19+ B-lymphocyte percentages were
obtained following chemotherapy in patients with
breast cancer17,19. Furthermore, similar to our results,
Murta et al17 observed increased levels of CD8+
cytotoxic-supressor T cells in patients after FEC regimen as neoadjuvant chemotherapy. In subgroup
analyses according to the response to chemotherapy however, they also observed an increased ratio
CD4/CD8 ratio in chemotherapy-responsive patients
compared to nonresponders unlike our findings.
It has been recently suggested that chemotherapy can impair cell function through the destruction
of bone marrow environment. Solomayer et al20
reported reduced numbers of activated NK and NKT cells 24 months after surgery and adjuvant
chemotherapy in breast cancer patients indicating a
long-lasting negative effect of chemotherapy on the
bone marrow immune system. However, the mechanisms are not well understood. Another study
demonstrated that the number of NK cells (CD16+
and CD56+) in cancer patients decreased after
chemotherapy regimen including bleomycin, etoposide and cisplatin21. On the contrary, FEC regimen
caused an increase in the percentages of NK cells
and T-cytotoxic cells suggesting that different
chemotherapy regimens may have different effects
on immune cells.
DISCUSSION
It is well known that chemotherapy induces bone
marrow suppression that may cause myelosupression and leucopenia. We therefore investigated in
this study whether the FEC regimen which is the
most common chemotherapy regimen used in
breast cancer could effect the natural killer cell activity and peripheral blood lymphocyte subsets in
patients. We briefly found that even though there
were decreases in the CD19+ B-lymphocyte and
CD45RA+ naive T-lymphocyte percentages and the
CD4/CD8 ratio and increases in the CD8+ cytotoxicsuppressor T cells, the NKA did not show any
change after chemotherapy. In the subgroup analyses, the CD45RO+ memory T-cell percentages were
interestingly found to be increased in the
Table 2. Changes in Peripheral Blood Lymphocyte Subpopulation Ratios and Natural Killer Cell Activity in
Patients with Locally Advanced Breast Cancer after Chemotherapy (mean±SD)
All patients (n=13)
CD3
CD4
Before CT
69.4±9.0
5.4±10
After CT
73.4±7.0
P-value
0.2
CD8
CD4/CD8
CD19
CD25
CD56
CD45RA CD45RO
γδ-T
cell
NKA
27.9±8.7 1.3±0.4
9.3±3.7
6.1±3.1 20.4±13.6 46.6±7.6 45.6±6.1
5.8±4.7 34.8±13.3
32.3±12.5 37.7±7.6 0.9±0.5
2.4±1.5
4.1±3.0
25.2±9.5
36±6.7
48.4±21.1
12±15.8 32.5±10.3
<0.001
0.3
0.1
0.06
0.7
45±8.7
48.7±7.6
0.6
<0.001
0.04
0.3
0.7
CT-responsive (n=7)
Before CT
69.8±8.6
After CT
73±6.6
P-value
0.5
37.8±9.7
27.6±9.6 1.5±0.4
29.9±15.8 37.4±9.1 0.9±0.6
0.4
0.01
0.05
9.9±3.1 6.75±4.5 17.7±12.1
1.8±1.3 6.75±1.7 24.7±9.5 32.3±3.8 63.7±10.3
0.001
0.999
0.005
8.6±4.6
5.3±1.0
24.3±16
0.2
0.05
4±1.0
40.6±12.4
5.7±2.1
27.5±9.5
0.4
0.04
8.5±7.8
25.5±5.1
CT-nonresponsive (n=6)
Before CT
69±10.5
32±10.2
28.4±8.3 1.04±0.2
After CT
74±8.2
35.8±5.3
38±5.8 0.97±0.2
P-value
0.2
0.3
0.03
0.6
47.8±7.9 43.2±4.5
3.2±1.5 0.51±0.6 25.8±10.7 38.8±7.5
0.04
18
0.004
0.8
0.3
37±20.3
0.6
21.5±26.2 46.9±10.7
0.5
0.01
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TURK J IMMUNOL
Furthermore,
previous studies showed a
decreased activity of NK cells during chemotherapy
including regimens with antracyclines, fluorouracil or
cyclophosphamide or melphalan with methotrexate
in patients with breast cancer even though they did
not observe any change in NK cell percentages22,27.
In our study, no change was seen in NK cell percentages or NKA after FEC chemotherapy. Interestingly
however, in the subgroup analyses, there was an
increase of NK cell percentages and a decrease in
NKA in chemotherapy-responsive patients whereas
an increased NKA was observed in chemotherapynonresponsive patients. The discrepancies in the
findings of our study and other reports might be due
to the different techniques used to assess NK cell
activity since we used Candida cells as target cells
for NK cells as opposed to K562 human erythroleukemia cell line used in the majority of other
studies22,24,26.
Brittenden et al28 similarly reported that NK or lymphokine-activated killer cell activity was significantly
suppressed in locally advanced breast cancer
patients receiving CHOP chemotherapy that could be
enhanced by L-arginine supplementation. However,
optimal levels of antibody dependent cellular cytotoxicity of CD16+ NK cells could be obtained in patients
with breast cancer against MCFHER2/neu in the presence of Herceptin, a humanized recombinant monoclonal antibody recognizing HER2/neu antigen in
breast cancer cells, after chemotherapy completion29.
All these results suggest that chemotherapy induced
changes in natural immunity could be modulated by
immunotherapy strategies to enhance the antitumoral
response in patients.
Furthermore, we demonstrated increased percentages of CD45+ memory T-cells and CD8+ T lymphocytes in chemotherapy-responsive patients after
chemotherapy. Eralp et al30 similarly found antigenspecific CD8+ T lymphocytes were induced by
doxorubicin or paclitaxel chemotherapy and vaccination with gene vaccine to HER2/neu in mice that were
injected with an aggressive breast tumor cell line that
expresses HER2/neu. In this study, neither vaccination nor chemotherapy alone significantly reduced the
tumor growth in mice, whereas chemotherapy followed by vaccination interestingly significantly inhibited the tumor growth. Similarly, effective antitumoral
response could be obtained in mice inoculated with
E0771 breast medullar adenocarcinoma cells by a
combination chemoimmunotherapy consisting of a
single dose of doxorubicin injection followed by daily
injections of IL-231. This combination therapy, not
chemotherapy or immunotherapy alone caused a
tumor-free long-term survival of 40% of mice and the
majority of the surviving mice had specific memory
cells for E0771 cells. The increased CD45RO+ memory T- cell percentages might contribute to the
chemoimmunotherapy trials in breast cancer patients
concomitant with chemotherapy or following
chemotherapy.
CONCLUSION
Our results suggest that the anthracycline-based
chemotherapy regimen-induced increases in memory T-cells in chemotherapy-responsive patients may
further enhance the efficacy of chemoimmunotherapy and/or vaccination trials in patients32. Detailed
phenotypical and functional characterization of intratumoral lymphocytes along with peripheral blood
lymphocytes warrants further investigation to design
a realistic treatment option regarding chemoimmunotherapy trials in terms of contrary findings on
PBL percentages and NK cell function33.
CORRESPONDENCE
Neslihan Cabioglu
Zeytinlik mah. Odabafl› sok. No: 47/10,
34710 Bak›rköy, ‹stanbul
Phone: 00 90 532 505 77 24
E-mail: [email protected]
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