Expression of LRP and MDR1 in locally advanced breast cancer predicts axillary node invasion at the time of rescue mastectomy after induction chemotherapy

Abstract

Axillary node status after induction chemotherapy for locally advanced breast cancer has been shown on multivariate analysis to be an independent predictor of relapse. However, it has been postulated that responders to induction chemotherapy with a clinically negative axilla could be spared the burden of lymphadenectomy, because most of them will not show histological nodal invasion. P-glycoprotein expression in the rescue mastectomy specimen has finally been identified as a significant predictor of patient survival. We studied the expression of the genes encoding multidrug resistance associated protein (MDR1) and lung cancer associated resistance protein (LRP) in formalin-fixed, paraffin-embedded tumor samples from 52 patients treated for locally advanced breast cancer by means of induction chemotherapy followed by rescue mastectomy. P-glycoprotein expression was assessed by means of immunohistochemistry before treatment in 23 cases, and by means of reverse-transcriptase-mediated polymerase chain reaction (RT-PCR) after treatment in 46 (6 failed). LRP expression was detected by means of immunohistochemistry, with the LRP-56 monoclonal antibody, in 31 cases before treatment. Immunohistochemistry for detecting the expression of c-erb-B2, p53, Ki67, estrogen receptor and progesterone receptor are routinely performed in our laboratory in every case, and the results obtained were included in the study. All patients had received between two and six cycles of standard 5-fluorouracil, doxorubicin and cyclophosphamide (FAC) chemotherapy, with two exceptions [one patient received four cycles of a docetaxel-adriamycin combination, and the other four cycles of standard cyclophosphamide-methotrexate-5-fluorouracil (CMF) polychemotherapy]. Response was assessed in accordance with the Response Evaluation Criteria In Solid Tumors (RECIST). By these, 2 patients achieved a complete clinical response, 37 a partial response, and the remaining 13 showed stable disease. This makes a total clinical response rate of 75.0%. None achieved a complete pathological response. < 0.05). Finally, in a logistic regression multivariate model, histology other than ductal, a Ki67 labeling index of at least 20% and the combination of LRP and MDR1 positivity emerged as independent predictors of axillary node invasion at the time of rescue mastectomy. The expression of different genes involved in resistance to chemotherapy, both before and after treatment with neoadjuvant, is associated with the presence of axillary node invasion at rescue surgery in locally advanced breast cancer. This might reflect the presence of intrinsically resistant clones before any form of therapy, which persist after it, and could be helpful both for prognosis and for the choice of individual treatment.

Background:

Axillary node status after induction chemotherapy for locally advanced breast cancer has been shown on multivariate analysis to be an independent predictor of relapse. However, it has been postulated that responders to induction chemotherapy with a clinically negative axilla could be spared the burden of lymphadenectomy, because most of them will not show histological nodal invasion. P-glycoprotein expression in the rescue mastectomy specimen has finally been identified as a significant predictor of patient survival.

Methods:

We studied the expression of the genes encoding multidrug resistance associated protein (MDR1) and lung cancer associated resistance protein (LRP) in formalin-fixed, paraffin-embedded tumor samples from 52 patients treated for locally advanced breast cancer by means of induction chemotherapy followed by rescue mastectomy. P-glycoprotein expression was assessed by means of immunohistochemistry before treatment in 23 cases, and by means of reverse-transcriptase-mediated polymerase chain reaction (RT-PCR) after treatment in 46 (6 failed). LRP expression was detected by means of immunohistochemistry, with the LRP-56 monoclonal antibody, in 31 cases before treatment. Immunohistochemistry for detecting the expression of c-erb-B2, p53, Ki67, estrogen receptor and progesterone receptor are routinely performed in our laboratory in every case, and the results obtained were included in the study. All patients had received between two and six cycles of standard 5-fluorouracil, doxorubicin and cyclophosphamide (FAC) chemotherapy, with two exceptions [one patient received four cycles of a docetaxel-adriamycin combination, and the other four cycles of standard cyclophosphamide-methotrexate-5-fluorouracil (CMF) polychemotherapy]. Response was assessed in accordance with the Response Evaluation Criteria In Solid Tumors (RECIST). By these, 2 patients achieved a complete clinical response, 37 a partial response, and the remaining 13 showed stable disease. This makes a total clinical response rate of 75.0%. None achieved a complete pathological response.

Results:

< 0.05). Finally, in a logistic regression multivariate model, histology other than ductal, a Ki67 labeling index of at least 20% and the combination of LRP and MDR1 positivity emerged as independent predictors of axillary node invasion at the time of rescue mastectomy.

Conclusion:

The expression of different genes involved in resistance to chemotherapy, both before and after treatment with neoadjuvant, is associated with the presence of axillary node invasion at rescue surgery in locally advanced breast cancer. This might reflect the presence of intrinsically resistant clones before any form of therapy, which persist after it, and could be helpful both for prognosis and for the choice of individual treatment.

Introduction

].

] that P-glycoprotein expression could indicate the presence of multidrug-resistant clones, from which tumor recurrence and dissemination might arise.

] have clearly shown that LRP is involved in resistance to adriamycin, and would therefore be a good candidate for research in association with this tumor for the above-mentioned reasons.

We have studied the expression of MDR1 and LRP both before and after neoajuvant chemotherapy in the present series of patients. The results have been correlated with the response to treatment, the presence of invaded axillary nodes, and also with that of other conventional prognostic markers (hormone receptors, Ki67 labeling index, mutant p53 expression, and c-erb-B2 overexpression).

Immunohistochemistry

], which takes into account both the strength of the staining reaction and the proportion of reactive tumor cells. After application of the scale, tumors were considered positive for MDR1 when there was distinct membrane staining (besides the ubiquitous and difficult to interpret cytoplasmic staining, which was more or less present in all specimens) in more than 10% of the tumor cells, and for LRP when more than 20% of the tumor cells showed specific staining against a negative background.

] and routinely employed at our laboratory.

Reverse-transcriptase-mediated polymerase chain reaction (RT-PCR)

for 15 min. Afterwards, the upper phase was transferred to a new tube, avoiding the interphase, and precipitated in an equal volume of propan-2-ol, then pelleted, washed and dried.

) were added, along with 1 μl of RNase-free bovine pancreatic DNase I (40 U/μl; Boehringer Mannheim, Germany). This mixture was incubated at room temperature for 30 min and stopped by the addition of 0.1 vol. of 20 mM EDTA.

, 5'-GAG CAT ACA TAT GTT CAA ACT TC-3') and β-globin or GADPH (commonly used primers as an internal amplification control). RT-PCR was performed in a total volume of 25 μl. After incubation at 50°C for 20 min, the three-step PCR was performed for 35 cycles in a Perkin-Elmer 9700 thermal cycler: 30 s denaturation at 94°C, followed by annealing for 20 s at 52°C and finally extension for 30 s at 72°C.

and control amplified product and run in a 3% (w/v) agarose gel at a constant 70 V in Tris-borate-EDTA buffer for 45 min. The size of amplified bands was 168 base pairs (bp) for mdr1 and 130-400 for GADPH and β-globin, depending on the primers used.

).

Statistics

). Because MDR1 post-chemotherapy (RT-PCR) and LRP were interrelated (all MDR1-positive cases were also LRP-positive), both variables were subsequently combined into into a single one with three different categories: both negative, MDR1-negative/LRP-positive and both positive. Information about this new variable was absent for 22 women. Given the relatively small sample size of the study, these missing values were treated as a separate category, which allowed us to include them in the multivariate analysis.

).

Results

MDR1 expression was measured by means of RT-PCR in rescue mastectomy specimens from patients previously subjected to induction chemotherapy for locally advanced breast cancer. MDR1-mRNA was successfully extracted from 51/52 tumor paraffin blocks, and was effectively retrotranscribed and amplified in 46/51 instances. Additionally, MDR1 expression before chemotherapeutic treatment was assessed by means of immunohistochemistry in 23 cases. LRP expression before chemotherapy was studied accordingly in 31 cases.

), which makes the detection of reactive tumor cells very easy. No association of LRP expression and the subsequent response to therapy could be discerned (although this observation is of little value, given the low sample number and the fact that most tumors showed some response).

). The logistic regression model is much more stringent, because it has to accommodate all data and because missing data are included in it as a distinct category, thus diluting the statistical power of some correlations, which nevertheless do exist if analyzed individually (as in this case).

= 0.052).

= 0.028), and thus (with very much care) was not attributable to pure chance.

).

= 0.052). In fact, 18/22 positive cases (81.8%) showed nodal invasion, against only 13/24 (54.2%) negative ones.

).

Discussion

] addressed the possible significance of the MDR1 gene in breast cancer through a large meta-analysis, and cautiously stated in their conclusions that 'we found no evidence to support the assumption that MDR1/gp170 expression has no role in breast cancer' and, a little more daringly, that 'while the precise role of MDR1/gp170 in breast cancer remains to be established definitively, it seems likely that, in tumors where expression is detectable, this expression contributes to the multidrug-resistant phenotype'. Our present findings seem only to corroborate this statement.

], and we must humbly admit that their methodological criticisms largely apply to them (although also to most, if not all, other studies considered). This, however, speaks in favor of the plausible role of the MDR1 gene in the development of resistance to chemotherapy in breast cancer, which persisted throughout the meta-analysis, in spite of the many justified criticisms of the studies included in it.

]. Support for the notion that there is indeed some relationship between oncogenic activation including c-erb-B2 expression is provided by experimental evidence that the transfection of c-erb-B2 and c-Ha-ras to human breast epithelial cells that initially do not express the MDR1 gene confers on them the full MDR phenotype. So, MDR1 expression could indirectly represent just an increased malignancy of the tumors, and nodal invasion would simply reflect their increased aggressiveness, and not the existence of chemotherapy-resistant tumor cell clones.

However, the multivariate analysis of the present study shows that LRP and MDR1 expression is significantly associated with nodal metastasis independently of a high proliferation rate, which was another of the significant (and independent) predictors identified here. In addition, MDR1 and c-erb-B2 were not significantly coexpressed this time, which seems to contradict our previous reports; however, this might be attributable to several causes, among which are the different subsets of tumors studied in them (only T4b tumors in one, which are a minority in this study, and preselected, highly aggressive operable tumors in the other), the use of different tumor material, at least in the first study (frozen tumors), and the use of different antibodies (c219 and NCL-pGlyP). In the present study, the relationship of nodal invasion to the expression of MDR1 and LRP is therefore clearcut, and clearly outweighs an eventual association with c-erb-B2 expression or high proliferation. This might explain axillary node invasion purely as a function of tumor aggressiveness via oncogenic activation, with MDR1 and LRP expression being only a byproduct of it.

], and if breast cancer patients die from their disease, they do so precisely because of distant metatstases. From our results, finally, it seems evident once more that resistance to chemotherapy is a multifactorial phenomenon, where not just one but many genes are involved.

Abbreviations

CMF = cyclophosphamide–methotrexate–5-fluorouracil; RT–PCR = reverse-transcriptase-mediated polymerase chain reaction.

Acknowledgements

This research was financed by grant FIS 99/0621 from Fondo de Investigación Sanitaria, Spain.

Figures and Tables

Ethidium-bromide-stained agarose gel after RT-PCR amplification of MDR1 mRNA. The MDR1-expressing tumors are demonstrated by the presence of a 168 base-pair band, above the β-globin positive control (lanes 3, 4, 7, 8, 11, 13, 15, 16, 18, 20 and 22).

Strong reactivity with the LRP-56 monoclonal antibody of breast cancer tumor cells, as opposed to the inert stromal background. Streptavidin-biotin-peroxidase staining. Magnification ×400.

Clinical features of tumors, immunohistochemistry of tru-cut core biopsies before chemotherapy, and reverse-transcriptase-mediated polymerase chain reaction (RT-PCR) after chemotherapy

Responses: 1, positive, 0, negative. Positivity levels: MDR1 (c494), only membrane staining in more than 10% of tumor cells; LRP, more than 20% reactive cells (for details, see the text). CCR, complete clinical response; PR, partial response; SD, stable disease; F, 5-fluorouracyl; A, doxorubicin (adriamycin); C, cyclophosphamide; M, methotrexate; undiff, undifferentiated; ND, not done; Ttere, taxotere.

Univariate analysis of prognostic factors related to axillary lymph node invasion

CI, confidence interval; ER, estrogen receptor; PR, progesterone receptor; RT-PCR, reverse-transcriptase-mediated polymerase chain reaction.

Multivariate analysis of prognostic factors related to axillary lymph node invasion

Keywords

• axillary nodes
• chemotherapy
• LRP
• multidrug resistant
• neoadjuvant