ANTICANCER RESEARCH 28: 1797-1804 (2008)
`
`Changes in Tumour Biological Markers during
`Primary Systemic Chemotherapy (PST)
`
`HANS NEUBAUERl, CHRISTIAN GALLl, ULRICH VOGELZ, RENE H0RNUNG1,
`DIETHELM WALLWIENER1,ERICH SOLOMAYER1 and TANJA FEHM1
`
`[Department of Gynaecology and Obstetrics, Calwerstr. 7, 72076 Tubingen;
`2Department of Pathology, University of Tubingen, Liebermeisterstr. 8, 72076 Tubingen Germany,
`
`Abstract. Background: The influence of primary systemic
`therapy (PST) on the expression of relevant therapeutic
`markers is still under investigation. Patients and Methods:
`Corresponding “baseline” biopsies and post—chemotherapy
`surgical specimens from 87 patients treated with neoadjuvant
`anthracycline— or taxane—based chemotherapy were analysed
`for the expression of the oestrogen receptor (ER),
`the
`progesterone receptor (PR), the B—cell lymphoma protein 2
`(Bcl—Z), the v—erb—b2 erythroblastic leukemia viral oncogene
`homolog 2 (HerZ/neu),
`the tumour protein p53 and the
`proliferation—related Ki—67 antigen. Results: The pathological
`response rate was 70% . Twenty—three tumours (26%)
`changed hormone receptor classification after chemotherapy
`( 7, ER; 16 PR). A significant change was also observed for
`Her2/neu status. Eleven tumours which were positive prior
`to PST down—regulated HerZ/neu after chemotherapy. The
`median Ki—67 index decreased from 30% before to 13%
`after treatment (p<0.0]). Minor changes were observed in
`the expression of Bcl—2 and p53 ( 9% ). Only the reduction of
`Ki—67 was associated with pathological response to PST.
`Conclusion: HerZ/neu status as well as ER and PR status
`
`should be re—evaluated on post—chemotherapy surgical
`specimens since changes can be observed.
`
`Primary systemic therapy (PST) is the standard treatment for
`locally advanced breast cancer. The major aim of systemic
`therapy in these patients is to facilitate breast conserving
`therapy and to eradicate distant micrometastatic disease. In
`recent years, PST has also been offered to patients with
`smaller tumours who were expected to receive post—operative
`systemic therapy (1). PST is as effective as post—operative
`chemotherapy in these patients, but offers the possibility of
`
`Correspondence to: Tanja Fehm, MD, Department of Gynaecology
`and Obstetrics, Tubingen, Calwerstr. 7, 72076 Tubingen, Germany.
`Tel: +49 7071 2980782, e—mail: tanja.fehm@med.uni—tuebingen.de
`
`Key Words: Breast cancer, oestrogen receptor, progesterone receptor,
`PST.
`
`in vivo chemosensitivity testing (2—4). Moreover, based on
`the pathological
`response to chemotherapy prognostic
`information can be obtained. Patients with complete
`remission of the primary tumour have a better clinical
`outcome compared to those with partial remission or non—
`responders (5, 6).
`Additionally, micrometastasis and the dissemination of
`tumour cells into the body’s circulation which takes place
`at early stages of the tumour may be treated at the earliest
`possible moment
`thereby improving prognosis. Besides
`these clinical aspects PST provides an ideal model
`to
`evaluate the role of biological markers as predictive and
`prognostic
`factors. Many retrospective
`studies have
`identified patterns of biomarker expression before or after
`chemotherapy which have predictive
`or prognostic
`significance in relation to different clinical end—points.
`Current clinical assessment assumes that the response of a
`tumour mass in total is representative of all the tumour cells
`and that tumours are of clonal or oligoclonal origin. However,
`breast cancer is characterized by its cellular heterogeneity.
`Therefore, PST might lead to an in vivo selection of a fraction
`of the tumour cells with different expression levels of tumour
`biological markers and a different phenotype compared to the
`pre—treatment tumour. Prior to PST tumour biological factors,
`such as Her2/neu, oestrogen receptor (ER) and progesterone
`receptor (PR) status, are routinely determined to inform post—
`operative, adjuvant treatment decisions regarding trastuzumab
`and endocrine therapy. However, if residual tumour cells do
`express different levels of tumour biological markers after
`PST these should be reassessed after PST to make sure that
`
`post— surgery treatment is tailored to the residual tumour cells.
`In order to get a better insight into breast cancer response
`to chemotherapy this study evaluated changes in ER and PR,
`Her2/neu, p53, Bcl—2, and Ki—67 before and after PST.
`
`Patients and Methods
`
`Patients. Eighty seven patients with invasive primary breast
`carcinoma (postchemotherapy pathologic (yp)T0—T4, ypNO—NZ,
`grade I—III) which have underwent PST at the Department of
`Gynaecology and Obstetrics, University Hospital, Tuebingen,
`
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`
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`
`ANTICANCER RESEARCH 28: 1797-1804 (2008)
`
`Table 1. Antibodies.
`
`
`
`
`
` Antibodies Antigen Dilution Cut- off
`
`
`
`NCL—ER-6F111
`NCL—PGR-3121
`A 04852
`
`hER
`hPR
`Her2/neu
`
`1:100
`1:300
`1:2000
`
`>10%
`>10%
`Score 2+ / 3+
`
`>15%
`1:100
`p53
`DO-71
`> 10%
`1:200
`bcl- 2
`clone 1242
`
`
`
`1:200Ki-67MIB-1,M 72402 >30%
`
`1Novocastra, New Castle, UK; 2Dako, Glostrup, Denmark.
`
`Germany, from January 2002 until January 2005 were included in
`this study. All the patients underwent diagnostic core biopsy of the
`breast
`tumour to confirm invasive cancer before commencing
`treatment. All specimens were obtained after written informed
`consent and collected using a protocol approved by the local ethics
`committee (AZ 266/98).
`
`Chemotherapy schedules and surgery. The patients received six
`cycles of either anthracycline (n=60) or taxane (n=27) based
`regimen administered at 21—day intervals. Surgery was performed
`approximately 1 month after the final cycle of chemotherapy. The
`patients who had no remaining invasive cancer in the breast and
`who were lymph node negative were considered to have a
`pathological complete response (CR).
`
`Response assessment. The response of the tumours to the neoadjuvant
`chemotherapy was evaluated pathologically by clas sifying the regressive
`changes using a semiquantitative scoring system from 0 to 4 (0=no
`effect, 1=resorption and tumour sclerosis, 2=minimal residual invasive
`tumour [<0 .5 cm], 3=residual non—invasive tumour only, 4=no tumour
`detectable) according to the tumour regression grading described by
`Sinn et al. (7). A consultant pathologist (U. Vogel) blinded to clinical
`outcome reviewed all paired biopsy and surgical specimens.
`
`Immunohistochemical technique. The immunohistochemical (IHC)
`analysis was performed on both cut core biopsies and surgical
`resection specimens for each patient. The tissue had been fixed in
`4.5% buffered formalin (pH 7.0) and embedded in paraffin. The
`IHC was performed on 3 to 5 pm thick sections mounted on poly—
`L—lysine slides using a commercially available ABC kit (Vectastain,
`Vector Laboratories, Burlingame, CA, USA). The primary
`antibodies were diluted in Tris—HCl (pH 7.5) and applied according
`to the manufacturer’s instruction as listed in Table I. DAB (3,3’
`diaminobenzidine) was used as chromogen. Finally, the slides were
`counterstained with Mayer’s hematoxylin for 10 sec and mounted
`for examination. For assessment of the proliferation index (Ki—67) ,
`p53 expression, ER and PR status, the percentage of cells with
`nuclear reactivity was recorded (8). For Bcl—2 protein expression,
`the percentage of cells with strong cytoplasmic or perinuclear
`expression was scored. For Her2/neu expression, only membranous
`staining was evaluated. Her2/neu expression was then scored semi—
`quantitatively using the 0—3+ score (0: no staining or membrane
`staining in <10% of tumour cells, 1+: >10% of tumour cells with
`weakly positive incomplete membrane staining, 2+: >10% of
`tumour cells with weak to moderate staining of the entire
`membrane, 3+: >10% of tumour cells with strong staining of the
`entire membrane). The cut—offs for positivity are listed in Table I.
`
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`
`Table 11. Basic characteristics of patients after primary systemic
`therapy.
`
` N (%)
`
`87 (100)
`
`Total
`Menopausal status
`Pre
`Post
`Tumour size
`ypTo
`ypTl
`ypT2-4
`Nodal status
`ypN neg
`ypN pos
`Grading
`I-II
`III
`missing
`Histology
`Ductal
`Lobular
`Others
`Primary systemic therapy
`Anthracycline based
`Taxane based
`Therapy response
`1 (1)
`CR
`60 (69)
`PR
`24 (28)
`SD
`
`PD 2 (2)
`
`38 (44)
`49 (56)
`
`1 (1)
`35 (41)
`50 (58)
`
`37 (43)
`50 (57)
`
`50 (58)
`33 (38)
`4 (5)
`
`62 (71)
`16 (19)
`9 (10)
`
`60 (69)
`27 (31)
`
`ypT: post-chemotherapy pathologic T classification, ypN: post-
`chemotherapy pathologic N classification, CR: complete remission, PR:
`partial remission, SD: stable disease, PD: progressive disease.
`
`Statistical methods. The statistical analysis was carried out using SPSS
`(version 11.5; SPSS, Chigaco, IL, USA). The associations between
`ordinal variables were assessed using Chi—sqare analyses or the Fisher
`Exact Test in the case of 2 ’ 2 variables. The analyses involving Ki—67
`as a continuous variable were investigated using ANOVA.
`
`Results
`
`Clinical characteristics. The clinical data are presented in Table
`11. After PST 41% of the patients had ypTl and 58% had
`ypT2—T4 tumours with grade I—11 in 58% and III in 38% of the
`cases. Positive lymph nodes were seen in 57% of the patients.
`The predominant tumour type was invasive ductal carcinoma
`(71%) followed by lobular carcinoma in 19% of the cases.
`
`Response to treatment. Out of the 87 patients, treated with
`neoadjuvant chemotherapy, one patient showed complete
`remission (CR). Partial remission (PR) was seen in 60 patients
`and stable disease (SD) or progression of disease (PD) was
`observed in 24 patients and five patients, respectively. The
`pathological response rate was 70% (Table II).
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`Neubauer et al: Changes in Tumour Markers during Primary Systemic Chemotherapy
`
`Table III. Tumour biological factors before and after primary systemic
`therapy.
`
`Table IV. Treatment related changes of ER and PR.
`
`PR status
`
`
`
` Marker N Ncgineg Posipos Negipos Posineg Change
`
`
`
`
`
`ER
`
`PR
`
`87
`
`87
`
`24 (28%) 56 (64%)
`
`3 (3%)
`
`4 (5%)
`
`7 (8%)
`
`36 (41%) 35 (40%)
`
`3 (3%)
`
`13 (15%)
`
`16 (18%)
`
`Her2/neu 86
`
`46 (53%) 27 (31%)
`
`2 (2%)
`
`11 (13%)
`
`13 (15%)
`
`neg % neg neg % pos pos % neg pos % pos Total
`
`ER status
`
`22
`
`24
`0
`2
`0
`neg % neg
`3
`1
`0
`1
`neg % pos
`pos % neg
`0
`3
`0
`4
`pos % pos
`12
`2
`8
`34
`56
`
`
`8 (13%)
`8 (13%)
`-
`38 (61%) 16 (26%)
`62
`p53
`2 (3%) 22 (35%) 24 (38%)
`25 (40%) 14 (22%)
`63
`Ki—67
`36 3 13 35Total 87
`Bcl-2
`62
`12 (19%) 42 (68%)
`2 (3%)
`6 (10%)
`8 (13%)
`
`
`
`
`
`
`
`
`
`
`
`Expression of biological markers. The expression of the
`different
`tumour markers determined in the diagnostic
`“baseline” biopsies
`and
`post—chemotherapy
`surgical
`specimens and the changes of tumour biological factor
`expression during treatment are shown in Table III.
`The expression of ER was assessed in all 87 pre— and post—
`chemotherapy sample pairs and remained the same in 80 of
`them. Three initially ER negative tumours were ER positive
`and four initially ER positive tumours were negative after
`chemotherapy. The difference in ER expression level before
`and after chemotherapy exposure was not statistically
`significant. The expression of PR was also determined in all
`87 pre— and post—chemotherapy sample pairs and no change in
`expression was observed in 71. A switch from PR negative
`before to PR positive post—chemotherapy was detected in 3%
`and from PR positive before to negative after chemotherapy
`in 15% .
`
`Her2/neu expression remained unchanged in 73 out of 86
`tested pre— and post—chemotherapy sample pairs. In two
`patients, Her2/neu expression switched from negative before
`to positive after PST while in 11 cases it changed from
`positive to negative.
`Expression of p53 and bcl—2 remained unchanged in 87%
`of the 62 pre— and post—chemotherapy sample pairs which
`could be determined. The difference in p53 or bcl—2
`expression level before and after PST was not statistically
`significant. Ki—67 expression remained unchanged in 62%
`of the 63 cases determined. In 3% the expression switched
`from negative to positive while in 35% the Ki—67 count was
`positive before treatment and negative after PST. The mean
`proliferation fraction was 30% before PST and 13% after
`chemotherapy (p<0 .001,
`two—sided
`t—test
`for paired
`samples). Only the reduction of Ki—67 was associated with
`pathological response to PST.
`
`tumour biological markers.
`Correlation of difi‘erent
`Significant correlations of the expression were obtained for
`ER and PR (p<0.01, Table IV) and for PR and Bel—2
`(p<0.01, data not shown). For ER and PR, 19 tumours
`
`changed the expression of either or both receptors (22% ).
`Ten of them did not change the expression of ER, but
`switched from PR positive to PR negative after PST (53% ).
`Additionally, three tumours regulated both, ER and PR,
`down after PST (16%).
`
`Discussion
`
`Significant effects on the expression of tumour biological
`markers by primary chemotherapy are controversely
`discussed with some groups reporting no changes (9—14)
`and others observing changes
`(15—20, Table V) of
`expression. Rody et al.
`(15) obtained a switch in
`expression for ER, PR, or Her2/neu from positive to
`negative in 45.7% of cases and vice versa in 22.7%
`following neoadjuvant chemotherapy. In a study performed
`by Piper et al. (16) of those patients who did not achieve
`a pCR, a change in tumor markers was seen in 25.7% of
`patients. ER changed in 33% and PR in 42% . Her2
`changed in 25% of the patients. Burcombe et al. (17)
`report that 9% of the tumours changed hormone receptor
`classification after neoadjuvant chemotherapy (3% ER,
`6% PR); HER—2 staining changed in nine cases. Median
`Ki—67 index was 24.9% before and 18.1% after treatment.
`
`In another study a significant decrease in the ER levels by
`24% in patients responding to anthracycline—based PST
`was detected (18). Further, a significant change in
`hormonal
`receptor
`content
`after
`pre—operative
`chemotherapy was observed in a total of 33% of patients
`(19). ER changed in 17% , PR in 22% , and both ER and
`PR in 6% . However, ER and PR status did not appear to
`predict or correlate with response to chemotherapy (19).
`Finally, a significant down—regulation of ER (14%) and
`PR (52%) was also identified using hormone receptor IHC
`in breast cancer patients receiving different regimens of
`PST (20). Seven (50%) of these patients were pre—
`menopausal suggesting that PST may have exerted an
`endocrine effect by rendering women post—menopausal
`after chemotherapy. Their observations might also explain
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`ANTICANCER RESEARCH 28: 1797-1804 (2008)
`
`Table V. Overview ofpublished response rates and changes of tumour biolagical factors during treatment.
`
`Reference
`
`Year
`
`No. of patients
`
`Treatment
`
`
`Change of tumour biological marker
`
`ER
`PR
`Her2
`Ki- 67
`
`
`(15)
`(16)
`
`(17)
`(18)
`(19)
`
`2006
`2004
`
`2005
`1997
`1996
`
`70
`43
`
`118
`29
`21
`
`AT
`A,AT
`
`A, MMM
`A, CMF
`A, CMF
`
`H 45.7% a’d, 22.7% axe %
`e 25.7%a %
`42%
`33 %
`6%
`3%
`n.c.
`24%c, s
`n.d.
`F 33% a, 6%bfi
`17%
`22%
`
`(20)
`2003
`191
`A, CMF, T
`14%d
`52%d
`n.d.
`n.d.
`
`n.d.
`n.d.
`
`24.9% a 18.1%
`15% #11%*
`
`25 %
`8%
`n.d.
`n.d.
`
`CMF:
`regimen, MMM: methotrexate/mitoxantrone/mitomycin,
`taxane- containing
`T:
`regimen,
`anthracycline- containing
`A:
`cyclophosphamide/methotrexate/5-fluorouracil, *=not significant, s=significant. a) change of at least one marker, b) change of both markers, c)
`change of mean in responders, d) change from positive to negative; e) change from negative to positive. n.c.= no change, n.d.:not determined.
`
`our result that most tumours down—regulating PR were ER
`positive (n28) as this might indicate a change in ER
`signaling capacity. An attractive hypothesis to explain the
`progression to steroid independence is that the tumour
`acquires the ability to constitutively express autocrine
`growth factors. In MCF—7 cells it has been observed that
`overexpression of fibroblast growth factor (FGF) induced
`an
`oestrogen—independent
`phenotype, which
`acted
`downstream of the ER as the ER level was not changed in
`these cells (21). The predictive value of PR has long been
`attributed to the dependence of PR expression on ER
`activity, with the absence of PR reflecting a nonfunctional
`ER. Two neoadjuvant studies confirmed the observation
`that PR negative tumours respond less well to hormonal
`therapy than PR positive tumours (22, 23). It might
`therefore well be that neoadjuvant chemotherapy is
`selecting for residual PR negative tumour cells or tumour
`cells that are able to alter their PR expression resulting in
`recurrences less susceptible to hormonal therapy. These
`data along with our results indicate that post—operative
`marker studies should be performed given the possibility
`of a change in status.
`rapidly
`A majority of studies have confirmed that
`proliferating tumours confer a poor prognosis (24—33). A
`reduction in Ki—67 index has been demonstrated after
`
`neoadjuvant chemotherapy (10, 17, 34), tamoxifen therapy
`(35) , and chemoendocrine therapy (36, 37). Our observation
`that the median Ki—67 index dropped from 30% to 13%
`post—PST was
`in line with them and indicated that
`chemotherapy was exerting an anti—proliferative effect on
`the tumours. This suggests that probably a proliferating state
`renders tumour cells more sensitive to chemotherapy since
`structural damage during DNA synthesis
`induced by
`anthracycline exposure decreases the viability of newly
`formed cancer cells (10, 38—44).
`
`The reduction of Ki—67 labelling in residual tissue at
`the end of chemotherapy, raises the question of whether
`the decrease in proliferation resulted from down—
`regulation in the entire cell population by a triggered
`‘switching off’ of proliferative regulators, or reflected
`selection of residual, less proliferative cells that were
`intrinsically less sensitive to chemotherapy and were
`preserved throughout treatment.
`The difference in p53 expression level before and after
`chemotherapy exposure was not statistically significant in
`our study. Similarly, several neoadjuvant studies have
`failed to detect a predictive value of p53 staining with
`regards to chemoresponsiveness in breast carcinomas (45—
`48). However, evidence from in vitro (49) and animal
`studies (50) has shown that defective p53 was associated
`with resistance to chemotherapy. Furthermore, loss of p53
`function correlated with multidrug resistance in many
`tumour
`types and specific p53 mutations have been
`associated with
`resistance
`to doxorubicin in
`the
`
`neoadjuvant setting (51, 52).
`The situation of Bcl—2 expression in breast cancer is not
`clear—cut with some studies indicating that Bel—2 has no
`predictive value in terms of chemoresponse (18, 41, 53, 54).
`These data support our results that the difference in Bel—2
`expression level before and after chemotherapy exposure
`was not statistically significant. In contrast, in a very small
`series of women with advanced breast cancer, Bel—2
`negativity was associated with an increased response rate to
`neoadjuvant chemotherapy (47). Interestingly, in our study
`Bel—2 expression correlated with the expression of PR as has
`been observed by others before in breast cancer (55) and
`endometrial cancer (56).
`The transmembrane receptor Her2/neu is overexpressed in
`about 25% of breast tumours (57) and is associated with poor
`outcome (58), and relative sensitivity to anthracycline
`
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`Neubauer et al: Changes in Tumour Markers during Primary Systemic Chemotherapy
`
`regimens (59, 60). In our study 13% of the tumours switched
`from Her2/neu positive to Her2/neu negative after PST.
`However, it could not be distinguished if the receptor was
`actively down—regulated in theses cases or if neoadjuvant
`treatment was selecting for Her2/neu negative tumour cells.
`Taucher et al. analyzing an anthracycline/taxane—based
`neoadjuvant therapy regime found no significant change of
`Her2/neu expression (61). Thus, at present,
`the data in
`neoadjuvant studies are conflicting with respect to Her2/neu
`status and response to anthracyclines, but based on our results
`we recommend that the Her2/neu score is re—evaluated in post—
`treatment tissue.
`
`A possible reason for the observed status variation may
`be reflected by either sampling error within heterogeneous
`tumours or the immunostaining of core biopsies. Our
`sample cohort did not include non—neoadjuvant therapy
`control patients for comparison. It has been reported that
`ER/PR status
`changed
`in
`5—6%
`of
`neoadjuvant
`chemotherapy and control groups due to tissue sampling
`(16, 62). If 5—6% is deducted from the 26% total ER/PR
`changes, approximately 20% of the hormone receptor
`changes would still have been caused by neoadjuvant
`treatment. Also, in a previously reported series of 236
`patients treated without
`intervening chemotherapy the
`hormone receptor status was highly representative of the
`entire resected tumour (20). This result suggests that
`sampling error did not account for the observed hormone
`receptor “down—regulation” seen in some cases.
`
`A second cause of variation might be technical as a recent
`study has shown a discordance rate in hormone receptor
`(HR) status of 9% between core biopsy and surgical
`specimens due to fixation or technical artefacts of IHC (63,
`64). However, such discordance of HR status was very low
`(3%) in a previously reported control group (65). Thus
`although some of the discordance observed in our series
`might have been caused by technical caveats the published
`data suggest that such differences are rare and have minor
`clinical significance.
`We therefore conclude that the changes in tumour
`marker expression observed in our study were changes
`induced by the treatment itself. Patients treated with
`adjuvant hormonal therapy are traditionally selected by
`an assessment of their HR status since HR—positive status
`predicts
`response
`to
`adjuvant hormonal
`therapy.
`Therefore, if PST changes the phenotype of the residual
`tumour cells, post—operative, adjuvant treatment decisions
`regarding e.g. trastuzumab and endocrine therapy might
`be optimized by re—evaluating the expression level of
`Her2/neu and ER on post—surgical tumour tissues. This
`view is supported by the observation that residual disease
`after PST, rather than parameters evaluated on the initial
`tumour biopsy, should be considered for patient prognosis
`(66). Survival after PST was related to the HR status of
`
`the residual disease with a high discordance in the HR
`status between the initial biopsy and the remaining
`tumour at surgery.
`
`Conclusion
`
`Her2/neu status as well as ER and PR status should be re—
`
`evaluated on post—chemotherapy surgical specimens since
`changes can be observed. The clinical relevance of these changes
`to adjuvant endocrine therapy or trastuzumab requires further
`long term follow—up and until such data becomes available,
`caution should be exercised when basing adjuvant therapy
`regimens on pre—operative tumour marker studies alone.
`
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