REVIEWS Biomarkers of residual disease after neoadjuvant therapy for breast cancer Frederique Penault-Llorca1,2 and Nina Radosevic-Robin1,2
Abstract | Nowadays, the decision of which adjuvant treatment should be given to patients with residual breast cancer after neoadjuvant therapy is based on the initial, pretreatment breast cancer molecular subtype and on the estimated residual tumour burden after neoadjuvant therapy. Substantial biological differences exist, however, between treatment-naive breast cancer and the residual tissue that remains after neoadjuvant therapy. In addition, the evaluation of relapse risk in patients is subject to a lack of uniformity in pathological qualification and quantification of remnant breast cancer following neoadjuvant treatment. In this Review, we present the recent recommendations for standardized evaluation of response to neoadjuvant therapy in patients with breast cancer, followed by a comprehensive overview of the pathobiological features of the residual disease after neoadjuvant therapy, which could serve as prognostic biomarkers or guide the choice of targeted adjuvant approaches. These biomarker candidates are at different stages of development, but some already have demonstrated superior prognostic value compared with biomarkers derived from pretreatment breast-cancer characteristics. The evidence presented herein indicates that further research on the biology of breast cancer that persists after neoadjuvant therapy is necessary to improve the management of this disease.
Department of Pathology, Jean Perrin Comprehensive Cancer Centre, 58 rue Montalembert, BP392, 63011 Clermont-Ferrand, France. 2 ERTICa Research Group, EA4677 University of Auvergne, 49 Boulevard Francois Mitterrand, 63000 Clermont-Ferrand, France. 1
Correspondence to F.P.‑L. frederique.penault-llorca@ cjp.fr doi:10.1038/nrclinonc.2016.1 Published online 9 Feb 2016
Breast cancer is a heterogeneous disease encompassing more than 20 histopathological subtypes and, at least, four relevant molecular subgroups (luminal A and B, HER2‑enriched, and basal-like)1,2. The molecular sub‑ groups, also known as the intrinsic subtypes of breast cancer, were defined by gene-expression profiles, and have distinct clinical features, metastatic behaviour and prognosis. At present, gene arrays are not suitable for application to routine clinical practice and, there‑ fore, a simplified surrogate molecular classification based on immunohistochemical markers was developed, resulting in four categories of breast cancer: luminal A, luminal B, triple-negative (TNBC) and HER2‑enriched (HER2‑positive/non-luminal breast cancer)3. Even after the adoption of these subtypes, intertumour and intra tumour breast-cancer heterogeneity remain the princi‑ pal causes of the marked differences observed in patient responses to therapy and their prognosis4. Neoadjuvant therapy (NAT) has become part of the standard-of‑care treatment of patients with locally advanced breast cancer. Besides reducing tumour burden and thus permitting conservative breast surgery, NAT provides a unique opportunity to evaluate the response of patients with breast cancer to different treatments, which is assessable by non-invasive and invasive methods after completion of treatment. The mainstay of the response
evaluation is the pathological examination of the breast tissue and lymph nodes that are surgically removed after NAT. The findings of numerous studies have demon‑ strated that the degree of reduction in tumour burden after NAT influences disease-free survival (DFS) and overall survival. Indeed, in a pooled analysis of neo‑ adjuvant trials in >12,000 patients with breast cancer published in 2014, patients who achieved a pathological complete response (pCR), defined as the histologically confirmed total absence of invasive cancer cells in the breast and axillary nodes, had a significantly improved overall survival compared with patients who had resid‑ ual breast tumour (HR 0.36; 95% CI: 0.31–0.42)5. Among patients who achieved a pCR, the most prominent reduc‑ tion of cancer-related death risk was observed in patients with TNBC, HER2-positive/hormone receptor (HR)negative breast cancer treated by trastuzumab as well as those not treated with trastuzumab and with high-grade HR-positive/HER2-negative breast cancer (risk reduction of 84% (95% CI 75–89%), 92% (95% CI 78–97%), 71% (95% CI 50–83%), and 71% (95% CI 35–87%), respec‑ tively)6. Finally, in 2014, the FDA recommended pCR rate as an acceptable end point for accelerated approval of new agents for NAT in patients with high-risk early stage breast cancer7,8. The final full approval of such agents remains, however, dependent on demonstration of
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REVIEWS The comparison of breast cancer burden and patho biology before and after treatment is an indispensable • Neoadjuvant therapy (NAT) provides a unique opportunity to assess the response part of response evaluation, which necessitates that a of patients with breast cancer to different treatments proper clinical work‑up is performed before therapy; • Standards for pathological examination need to be standardized in order to enable a pretherapy core-needle biopsy is required to enable the reproducible evaluation of the residual disease that persists after NAT diagnosis of invasive cancer, to determine the tumour • Residual disease remaining after NAT is different from treatment-naive breast cancer type, grade and cellularity, and to classify the tumour into • ‘Classic’ histopathological parameters, such as ypTNM, grade, mitotic index, and one of the four immunohistochemistry ( IHC)-based hormone-receptor, HER2 and Ki67 status, provide valuable prognostic and predictive intrinsic molecular subtypes13. information when assessed in residual breast cancer tissue after NAT Communication between pathologists and the multi‑ • Genomic and proteomic markers of residual breast cancer are currently under disciplinary clinical team is mandatory for the optimal development, and might inform patient stratification for adjuvant treatment handling of post-therapy surgical specimens. Indeed, • Immune markers are among the most-promising biomarkers in the post NAT setting, major obstacles can arise from a lack of pathologist– in which extensive tumour infiltration by lymphocytes indicates a good prognosis, clinician communication (BOX 1). Clinical examination, irrespective of residual tumour size together with mammography and ultrasonography assessment, can reveal several patterns of response to therapy, such as a complete response, concentric tumour an improvement in event-free survival. These new rules shrinkage, or scattered foci of residual tumour14–16. PET highlight the importance of defining standard operating and MRI-based assessments might eventually supplement procedures for the pathological evaluation of a response treatment-response evaluation, but these modalities are to therapy in patients with breast cancer, in particular not currently used routinely or recommended for assess‑ with regards to reporting the results of clinical trials. ing response to therapy. Several studies have demon‑ Besides the degree of tumour-burden reduction after strated promising results after 18F-flurodeoxyglucose NAT, emerging evidence indicates that the pathobiologi‑ (FDG)–PET is used in early (after two cycles of ther‑ cal features of post-therapy breast cancer residual dis‑ apy) response prediction17–19, and/or in the assessment ease are major determinants of patient outcome. These of breast-cancer response after a full course of NAT20. characteristics can differ from the baseline, pretreatment Ongoing validation studies of PET and MRI use for features because resistant cancer-cell subclones can be monitoring patient responses are expected to provide selected for by therapeutic agents; although these sub‑ more conclusive results in the coming years. clones might have been undetectable before therapy, The post-NAT breast-tissue specimens should be they might predominate in the residual tumour popula‑ oriented by the surgeon following surgical removal. The tion after NAT9–11. The decision-making process for the gross pathological examination is the most-challenging choice of adjuvant treatment is, therefore, transitioning part of post-NAT disease assessment because treatment- from the use of traditional approaches based on the induced changes to tissue characteristics can make assessment of pre-NAT tumour parameters to being localizing the tumour bed difficult. At the beginning of guided by the features of the residual disease after NAT. the gross examination, the post-therapy breast-tumour At present, prognostic biomarkers based on post- specimen is measured and inked using multiple colours NAT breast-cancer characteristics remain scarce and for margin assessment, and is then cut into 3–5 mm pCR can be considered as the only currently validated thick slices. If the residual tumour is macroscopically biomarker of survival. New techniques to assess gene- visible, the pathologist examines the slices and performs expression and protein-expression, however, have started gross evaluation of the tumour size and margin status. to reveal potential biomarkers in the breast-cancer tissue Insertion of metallic clips into the tumour bed before remaining after NAT that should be of clinical relevance therapy is strongly recommended, because the location if their effectiveness is validated in large studies. of the clips can guide the surgical excision and the gross In this Review, we first discuss how pathological pathological examination in the event of a substantial or response of patients with breast cancer to NAT can be complete pathological response to NAT13. The sampling evaluated. We then provide a comprehensive overview of the tumour bed must be systematic and exhaustive to of putative biomarkers, based on the characteristics of ensure the correct histopathological status is reported. In post-NAT breast-cancer tissue, which could serve as indi‑ the case of a clinically nonpalpable breast mass (clinical cators of a patient’s long-term prognosis and thus help complete response), at least 10–15 tumour bed samples personalize adjuvant treatment of patients with breast are typically taken and subsequently examined for the cancer. presence of microscopic residual disease. Small breast- resection specimens (4–5 cm longest dimension, either Evaluating response to NAT length or width) should be submitted for pathological A great diversity of systems have been developed to clas‑ evaluation in their entirety12. The surgically removed sify the pathological response of patients with breast can‑ axillary nodes are processed the same way as is per‑ cer to NAT. Given the lack of consensus on this issue, an formed in patients who do not receive NAT and instead international multidisciplinary working group has pro‑ undergo upfront resection, with complete inclusion of duced guidelines covering various aspects of post-NAT each node in the pathological assessment after slicing — breast-cancer evaluation, which are summarized in this except for the obviously metastatic ones, for which one section (BOXES 1–4) and have been reviewed elsewhere12,13. slice per node is assessed. Key points
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REVIEWS After the sections are fixed in formalin and embed‑ ded in paraffin (FFPE), those with suspected areas of residual tumour and/or involved margins are evaluated microscopically using haematoxylin and eosin (H&E) staining. The microscopic evaluation assesses tumour size (ypT), type and grade, presence of emboli and of carcinoma in situ, and lymph-node (ypN) and surgical- margin status12. The residual-tumour size is then evalu‑ ated following the American Joint Committee on Cancer (AJCC) rule21 by measuring the largest contiguous focus of invasive cancer in the breast. The accuracy of this assessment can be impaired by the pattern of response to therapy; in the case of multifocal disease (multiple independent tumours), only the biggest lesion is used for the ypT determination21. The histological subtype is usu‑ ally not affected by NAT, except for some tumours that acquire a single cell-differentiation pattern that mimicks the features of lobular carcinoma22. In addition, cytotoxic therapy can induce microscopic changes, such as cel‑ lular and nuclear pleomorphism, multinucleation, and hyperchromasia; therefore, the assessment of post-NAT tumour grade is only possible if pretreatment biopsy samples are available for comparison. Tumour hetero‑ geneity might be revealed after therapy, as reflected by Box 1 | Common pitfalls in gross evaluation of post-NAT breast specimens Tumour-related • Not knowing that a neoadjuvant treatment has been administered • Not knowing where the tumour is supposed to be • Lack of proper tumour-bed definition/examination (palpation, confrontation with specimen radiography, clip localization, extensive sampling of at least 10–15 slices) Lymph-node-related • Lack of information derived from sentinel lymph node biopsy before NAT (risk of understaging, impossibility to determine residual cancer burden) • Post-treatment lymph-node atrophy (treatment-induced lymph-node thinning) NAT, neoadjuvant therapy.
Box 2 | Common pitfalls in microscopic evaluation of post-NAT breast specimens Tumour-related • Difficulty distinguishing ductal carcinoma in situ from invasive carcinoma • The presence of scattered single cancer cells (can be missed in the absence of extensive sampling) • Challenges in recognizing differences between single cancer cells and tissue macrophages/histiocytes (requires the use of immunohistochemistry in difficult cases) • Use of lymphovascular invasion as the sole evidence of residual cancer (no pathological complete response) • Difficulty distinguishing scarring from needle biopsy from the fibrotic tumour bed • Possible overestimation of tumour-bed cellularity Lymph-node-related • Issues relating to atrophy of lymph nodes (such as differentiating between therapeutically-induced fibrosis and atrophied lymph nodes) • Difficulty recognizing the difference between single cancer cells and tissue macrophages/histiocytes • Challenges in distinguishing foreign-body granuloma (due to prior clip placement) from apocrine carcinoma or treatment-altered carcinoma NAT, neoadjuvant therapy.
different degrees of response within the breast mass, and should be reported. Mitotic count is frequently modi‑ fied by therapy and must be reported separately from tumour grade. The cellularity of the residual tumour is a major parameter to assess after NAT. Sometimes, despite the absence of tumour shrinkage, a clear decrease in cellular‑ ity is observed, underscoring the effect of therapy. Thus, cellularity evaluation is included in several systems of grading the breast-cancer response to therapy23–25. The presence or absence of residual post-NAT ductal carcinoma in situ (DCIS) also needs to be reported, and is included in some of the published classification sys‑ tems25–27. Similarly, the presence of lymphovascular inva‑ sion (LVI) must be stated, because cancer emboli have been demonstrated to be more resistant to treatment than cancer cells outside of vascular spaces28. A criti cal situation is residual tumour characterized by the presence of LVI in the absence of other microscopically detectable invasive tumour cells. In such cases, the BIGNABCG group clearly stipulates “residual LVI should not be classed as pCR” (REF. 13). The therapeutic effect of NAT can be microscopically observed as residual, invasive or in situ breast cells that demonstrate characteristics associated with cell necrosis and/or apoptosis, multinucleation and pleomorphism; in the stroma, the therapeutic effect is reflected by the presence of fibrosis, sclerosis, necrosis, haemosiderin deposits, and microcalcifications, and infiltration by foamy macrophages, neutrophils or lymphocytes. Margin assessment can be impaired in the presence of a good clinical and macroscopic response to NAT, or if a scattered-foci pattern of histological response is observed. The correlation of pathology and radiology findings is important for a correct locoregional man‑ agement of such residual tumours, particularly when the scattered foci are suspected to be close to, or at the surgical margins. The post-NAT pathological examination of lymph nodes presents numerous obstacles and, for this reason, particular rules must be followed (BOXES 2,3). The pathol‑ ogist reports the number of involved nodes, the size of the largest metastatic deposit, and the signs of thera‑ peutic effects. The inclusion of certain elements in the pathology report of patients with persistent post-NAT residual disease is recommended (BOX 4).
Biomarkers of residual disease Residual tumour burden Multiple classification systems have been proposed for grading the pathological response to therapy in patients with breast cancer (TABLE 1), with substantial hetero geneity both in the definition of pCR5,23,26–36 and in the recommendations for quantification of the residual tumour23,29,36. This heterogeneity is an obstacle that lim‑ its comparisons of data from different clinical trials. In this subsection we reflect on what should be considered a pCR of patients with breast cancer to therapy and, fur‑ thermore, we present the most important current sys‑ tems for quantification of residual-tumour-burden and the prognostic information they provide.
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REVIEWS Box 3 | Lymph-node evaluation in the neoadjuvant setting13 Before treatment • Clinical assessment: ultrasonography is recommended • In patients with suspicious lymph nodes, fine-needle aspiration or core biopsy should be performed • Sentinel-lymph-node biopsy precludes a correct assessment of nodal residual disease and the calculation of residual cancer burden and, thus, should be avoided in clinical trials After treatment • Immunohistochemistry and additional sectioning levels are not routinely required, but can help clarify identity of suspicious cells • In the absence of tumour cells, fibrosis, macrophages, tumour-negative lymph nodes should be reported • In the absence of tumour cells but presence of fibrosis, effects of chemotherapy and any presence of ‘sterilized’ nodes should be reported • If residual disease is observed in the lymph node, tumour-positive nodes should be reported and the size of the entire area infiltrated by tumour cells should be measured, even if few cells are present • In the neoadjuvant setting, in the presence of micrometastatic disease or single tumour cells, report positive nodes (ypN1 or 2)
Box 4 | Elements of the pathology report for post-NAT residual breast cancer13 Breast Gross description: • Tumour bed size Microscopic description: • Residual tumour size according to the American Joint Committee on Cancer staging21 • In the case of multifocal residual disease: -- Measurement of two dimensions of largest cross-section of residual tumour bed (in case the entire area is involved); if tumour cells are present within a sole tumour bed, the largest diameter is used for ypT -- Measurement of the largest tumour residue diameter (in the case of multiple tumours present on pretreatment imaging or tumour foci separated by normal breast tissue) for ypT • Residual tumour cellularity • Percentage of ductal carcinoma in situ Lymph-node status • Number of involved nodes and size of largest metastatic deposit NAT, neoadjuvant therapy.
Pathological complete response. A FDA proposal, pub‑ lished in 2014 (REF. 8), defined a pCR as ypT0/Tis ypN0 disease (absence of invasive cancer in the breast and axillary nodes), a definition we agree with. The FDA definition of pCR highlights the total microscopic dis‑ appearance of invasive disease but allows the persistence of an in situ component7. The influence of the post-NAT in situ carcinoma on disease outcome has been analysed in several large studies. For example, von Minckwitz et al.37 reported that patients with residual DCIS had a higher relapse rate than patients without an in situ com‑ ponent. By contrast, a meta-analysis of 12 neoadjuvant randomized trials (CTNeoBC) demonstrated similar event-free survival and overall survival rates regard‑ less of the presence of residual DCIS5. Concordantly, we and others have shown that the distinction between
post-NAT ypT0 and ypTis has no prognostic rele‑ vance38–40; therefore, we endorse ypT0/Tis ypN0 as the correct definition of pCR, which has already been included in other published guidelines41,42. Pathological partial response. The dichotomization of the response to NAT into pCR and non-pCR does not account for the prognostic significance of various forms and extents of post-therapy residual disease, such as scat‑ tered residual tumour cells, a residue of only a few hun‑ dreds of cells or a large residual tumour. Furthermore, pCR confers a survival advantage, but this criteria is far from being a perfect and universal prognostic indica‑ tor. Moreover, controversy exists on whether the sur‑ vival advantage associated with achievement of a pCR is limited to some breast cancer intrinsic subtypes43 or is independent of subtype classification44. Indeed, findings of various trials have shown that patients with luminal breast cancer who do not have a pCR nevertheless had a favourable prognosis5,37,45, which indicates that sub‑ groups of patients with an incomplete pathological response to NAT might have different prognoses that can be revealed through a more-detailed analysis of the residual tumour tissues. Symmans and colleagues25, from the MD Anderson Center, have developed a system to quantitate residual disease, termed the ‘Residual Cancer Burden’ (RCB) index. The RCB index is calculated as a continuous parameter combining six variables, all obtained by microscopic evaluation of the resection specimen: two dimensions of the post-treatment breast-tumour bed, its cellularity, percentage of carcinoma in situ, number of metastatic nodes, and the diameter of the largest nodal metastatic lesion25. The RCB index enables the classi‑ fication of residual disease into four categories: RCB‑0 (pCR), RCB‑I (minimal residual disease), RCB‑II (mod‑ erate residual disease) and RCB-III (extensive residual disease). The overall probability of relapse at 5 years was found to be similar for subgroups of patients with RCB‑0 and RCB‑I (5.4% and 2.4%, respectively), whereas the risk of relapse was significantly higher for RCB-III (53.6%). Thus, this system classifies patients who have benefited from therapy as RCB‑0 or RCB‑I. Among patients with disease classified as RCB-III after NAT, all of those who did not receive adjuvant hormone therapy experienced distant relapses within 3 years, and even 40% of the patients who received such therapy relapsed within 5 years25. In a study in which investigators evalu ated 100 patients with breast cancer, pathology slides and reports were independently reviewed by five patholo‑ gists, and the results demonstrated that the RCB index is a highly reproducible and accurate tool to predict distant recurrence-free survival (RFS) and overall sur‑ vival46. Accordingly, several prospective clinical studies (NSABP, CALGB, ABSCSG, GEICAM, ACOSOG and I-SPY)46 have included RCB as a primary or secondary end point of patient response to chemotherapy. Our group has proposed another system of residual disease quantification that takes into account the tumour size, Scarf–Bloom–Richardson (SBR) grade47 and the number of involved nodes. This parameter, named
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REVIEWS Table 1 | Classifications of breast cancer pathological response to neoadjuvant therapy Name
Grades defined
Histology
GEPARDO30
4 (pCR)
No detectable residual tumour cells
3
DCIS only
2
Minimal invasive residual tumour (90% reduction in tumour cellularity; only small clusters or individual tumour cells remaining
5 (pCR)
No invasive tumour cells (DCIS allowed)
0
No response; almost no change in tumour cells
1a
Mild response; mild changes in tumour cells everywhere / marked changes in 1/3 but
Abstract | Nowadays, the decision of which adjuvant treatment should be given to patients with residual breast cancer after neoadjuvant therapy is based on the initial, pretreatment breast cancer molecular subtype and on the estimated residual tumour burden after neoadjuvant therapy. Substantial biological differences exist, however, between treatment-naive breast cancer and the residual tissue that remains after neoadjuvant therapy. In addition, the evaluation of relapse risk in patients is subject to a lack of uniformity in pathological qualification and quantification of remnant breast cancer following neoadjuvant treatment. In this Review, we present the recent recommendations for standardized evaluation of response to neoadjuvant therapy in patients with breast cancer, followed by a comprehensive overview of the pathobiological features of the residual disease after neoadjuvant therapy, which could serve as prognostic biomarkers or guide the choice of targeted adjuvant approaches. These biomarker candidates are at different stages of development, but some already have demonstrated superior prognostic value compared with biomarkers derived from pretreatment breast-cancer characteristics. The evidence presented herein indicates that further research on the biology of breast cancer that persists after neoadjuvant therapy is necessary to improve the management of this disease.
Department of Pathology, Jean Perrin Comprehensive Cancer Centre, 58 rue Montalembert, BP392, 63011 Clermont-Ferrand, France. 2 ERTICa Research Group, EA4677 University of Auvergne, 49 Boulevard Francois Mitterrand, 63000 Clermont-Ferrand, France. 1
Correspondence to F.P.‑L. frederique.penault-llorca@ cjp.fr doi:10.1038/nrclinonc.2016.1 Published online 9 Feb 2016
Breast cancer is a heterogeneous disease encompassing more than 20 histopathological subtypes and, at least, four relevant molecular subgroups (luminal A and B, HER2‑enriched, and basal-like)1,2. The molecular sub‑ groups, also known as the intrinsic subtypes of breast cancer, were defined by gene-expression profiles, and have distinct clinical features, metastatic behaviour and prognosis. At present, gene arrays are not suitable for application to routine clinical practice and, there‑ fore, a simplified surrogate molecular classification based on immunohistochemical markers was developed, resulting in four categories of breast cancer: luminal A, luminal B, triple-negative (TNBC) and HER2‑enriched (HER2‑positive/non-luminal breast cancer)3. Even after the adoption of these subtypes, intertumour and intra tumour breast-cancer heterogeneity remain the princi‑ pal causes of the marked differences observed in patient responses to therapy and their prognosis4. Neoadjuvant therapy (NAT) has become part of the standard-of‑care treatment of patients with locally advanced breast cancer. Besides reducing tumour burden and thus permitting conservative breast surgery, NAT provides a unique opportunity to evaluate the response of patients with breast cancer to different treatments, which is assessable by non-invasive and invasive methods after completion of treatment. The mainstay of the response
evaluation is the pathological examination of the breast tissue and lymph nodes that are surgically removed after NAT. The findings of numerous studies have demon‑ strated that the degree of reduction in tumour burden after NAT influences disease-free survival (DFS) and overall survival. Indeed, in a pooled analysis of neo‑ adjuvant trials in >12,000 patients with breast cancer published in 2014, patients who achieved a pathological complete response (pCR), defined as the histologically confirmed total absence of invasive cancer cells in the breast and axillary nodes, had a significantly improved overall survival compared with patients who had resid‑ ual breast tumour (HR 0.36; 95% CI: 0.31–0.42)5. Among patients who achieved a pCR, the most prominent reduc‑ tion of cancer-related death risk was observed in patients with TNBC, HER2-positive/hormone receptor (HR)negative breast cancer treated by trastuzumab as well as those not treated with trastuzumab and with high-grade HR-positive/HER2-negative breast cancer (risk reduction of 84% (95% CI 75–89%), 92% (95% CI 78–97%), 71% (95% CI 50–83%), and 71% (95% CI 35–87%), respec‑ tively)6. Finally, in 2014, the FDA recommended pCR rate as an acceptable end point for accelerated approval of new agents for NAT in patients with high-risk early stage breast cancer7,8. The final full approval of such agents remains, however, dependent on demonstration of
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REVIEWS The comparison of breast cancer burden and patho biology before and after treatment is an indispensable • Neoadjuvant therapy (NAT) provides a unique opportunity to assess the response part of response evaluation, which necessitates that a of patients with breast cancer to different treatments proper clinical work‑up is performed before therapy; • Standards for pathological examination need to be standardized in order to enable a pretherapy core-needle biopsy is required to enable the reproducible evaluation of the residual disease that persists after NAT diagnosis of invasive cancer, to determine the tumour • Residual disease remaining after NAT is different from treatment-naive breast cancer type, grade and cellularity, and to classify the tumour into • ‘Classic’ histopathological parameters, such as ypTNM, grade, mitotic index, and one of the four immunohistochemistry ( IHC)-based hormone-receptor, HER2 and Ki67 status, provide valuable prognostic and predictive intrinsic molecular subtypes13. information when assessed in residual breast cancer tissue after NAT Communication between pathologists and the multi‑ • Genomic and proteomic markers of residual breast cancer are currently under disciplinary clinical team is mandatory for the optimal development, and might inform patient stratification for adjuvant treatment handling of post-therapy surgical specimens. Indeed, • Immune markers are among the most-promising biomarkers in the post NAT setting, major obstacles can arise from a lack of pathologist– in which extensive tumour infiltration by lymphocytes indicates a good prognosis, clinician communication (BOX 1). Clinical examination, irrespective of residual tumour size together with mammography and ultrasonography assessment, can reveal several patterns of response to therapy, such as a complete response, concentric tumour an improvement in event-free survival. These new rules shrinkage, or scattered foci of residual tumour14–16. PET highlight the importance of defining standard operating and MRI-based assessments might eventually supplement procedures for the pathological evaluation of a response treatment-response evaluation, but these modalities are to therapy in patients with breast cancer, in particular not currently used routinely or recommended for assess‑ with regards to reporting the results of clinical trials. ing response to therapy. Several studies have demon‑ Besides the degree of tumour-burden reduction after strated promising results after 18F-flurodeoxyglucose NAT, emerging evidence indicates that the pathobiologi‑ (FDG)–PET is used in early (after two cycles of ther‑ cal features of post-therapy breast cancer residual dis‑ apy) response prediction17–19, and/or in the assessment ease are major determinants of patient outcome. These of breast-cancer response after a full course of NAT20. characteristics can differ from the baseline, pretreatment Ongoing validation studies of PET and MRI use for features because resistant cancer-cell subclones can be monitoring patient responses are expected to provide selected for by therapeutic agents; although these sub‑ more conclusive results in the coming years. clones might have been undetectable before therapy, The post-NAT breast-tissue specimens should be they might predominate in the residual tumour popula‑ oriented by the surgeon following surgical removal. The tion after NAT9–11. The decision-making process for the gross pathological examination is the most-challenging choice of adjuvant treatment is, therefore, transitioning part of post-NAT disease assessment because treatment- from the use of traditional approaches based on the induced changes to tissue characteristics can make assessment of pre-NAT tumour parameters to being localizing the tumour bed difficult. At the beginning of guided by the features of the residual disease after NAT. the gross examination, the post-therapy breast-tumour At present, prognostic biomarkers based on post- specimen is measured and inked using multiple colours NAT breast-cancer characteristics remain scarce and for margin assessment, and is then cut into 3–5 mm pCR can be considered as the only currently validated thick slices. If the residual tumour is macroscopically biomarker of survival. New techniques to assess gene- visible, the pathologist examines the slices and performs expression and protein-expression, however, have started gross evaluation of the tumour size and margin status. to reveal potential biomarkers in the breast-cancer tissue Insertion of metallic clips into the tumour bed before remaining after NAT that should be of clinical relevance therapy is strongly recommended, because the location if their effectiveness is validated in large studies. of the clips can guide the surgical excision and the gross In this Review, we first discuss how pathological pathological examination in the event of a substantial or response of patients with breast cancer to NAT can be complete pathological response to NAT13. The sampling evaluated. We then provide a comprehensive overview of the tumour bed must be systematic and exhaustive to of putative biomarkers, based on the characteristics of ensure the correct histopathological status is reported. In post-NAT breast-cancer tissue, which could serve as indi‑ the case of a clinically nonpalpable breast mass (clinical cators of a patient’s long-term prognosis and thus help complete response), at least 10–15 tumour bed samples personalize adjuvant treatment of patients with breast are typically taken and subsequently examined for the cancer. presence of microscopic residual disease. Small breast- resection specimens (4–5 cm longest dimension, either Evaluating response to NAT length or width) should be submitted for pathological A great diversity of systems have been developed to clas‑ evaluation in their entirety12. The surgically removed sify the pathological response of patients with breast can‑ axillary nodes are processed the same way as is per‑ cer to NAT. Given the lack of consensus on this issue, an formed in patients who do not receive NAT and instead international multidisciplinary working group has pro‑ undergo upfront resection, with complete inclusion of duced guidelines covering various aspects of post-NAT each node in the pathological assessment after slicing — breast-cancer evaluation, which are summarized in this except for the obviously metastatic ones, for which one section (BOXES 1–4) and have been reviewed elsewhere12,13. slice per node is assessed. Key points
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REVIEWS After the sections are fixed in formalin and embed‑ ded in paraffin (FFPE), those with suspected areas of residual tumour and/or involved margins are evaluated microscopically using haematoxylin and eosin (H&E) staining. The microscopic evaluation assesses tumour size (ypT), type and grade, presence of emboli and of carcinoma in situ, and lymph-node (ypN) and surgical- margin status12. The residual-tumour size is then evalu‑ ated following the American Joint Committee on Cancer (AJCC) rule21 by measuring the largest contiguous focus of invasive cancer in the breast. The accuracy of this assessment can be impaired by the pattern of response to therapy; in the case of multifocal disease (multiple independent tumours), only the biggest lesion is used for the ypT determination21. The histological subtype is usu‑ ally not affected by NAT, except for some tumours that acquire a single cell-differentiation pattern that mimicks the features of lobular carcinoma22. In addition, cytotoxic therapy can induce microscopic changes, such as cel‑ lular and nuclear pleomorphism, multinucleation, and hyperchromasia; therefore, the assessment of post-NAT tumour grade is only possible if pretreatment biopsy samples are available for comparison. Tumour hetero‑ geneity might be revealed after therapy, as reflected by Box 1 | Common pitfalls in gross evaluation of post-NAT breast specimens Tumour-related • Not knowing that a neoadjuvant treatment has been administered • Not knowing where the tumour is supposed to be • Lack of proper tumour-bed definition/examination (palpation, confrontation with specimen radiography, clip localization, extensive sampling of at least 10–15 slices) Lymph-node-related • Lack of information derived from sentinel lymph node biopsy before NAT (risk of understaging, impossibility to determine residual cancer burden) • Post-treatment lymph-node atrophy (treatment-induced lymph-node thinning) NAT, neoadjuvant therapy.
Box 2 | Common pitfalls in microscopic evaluation of post-NAT breast specimens Tumour-related • Difficulty distinguishing ductal carcinoma in situ from invasive carcinoma • The presence of scattered single cancer cells (can be missed in the absence of extensive sampling) • Challenges in recognizing differences between single cancer cells and tissue macrophages/histiocytes (requires the use of immunohistochemistry in difficult cases) • Use of lymphovascular invasion as the sole evidence of residual cancer (no pathological complete response) • Difficulty distinguishing scarring from needle biopsy from the fibrotic tumour bed • Possible overestimation of tumour-bed cellularity Lymph-node-related • Issues relating to atrophy of lymph nodes (such as differentiating between therapeutically-induced fibrosis and atrophied lymph nodes) • Difficulty recognizing the difference between single cancer cells and tissue macrophages/histiocytes • Challenges in distinguishing foreign-body granuloma (due to prior clip placement) from apocrine carcinoma or treatment-altered carcinoma NAT, neoadjuvant therapy.
different degrees of response within the breast mass, and should be reported. Mitotic count is frequently modi‑ fied by therapy and must be reported separately from tumour grade. The cellularity of the residual tumour is a major parameter to assess after NAT. Sometimes, despite the absence of tumour shrinkage, a clear decrease in cellular‑ ity is observed, underscoring the effect of therapy. Thus, cellularity evaluation is included in several systems of grading the breast-cancer response to therapy23–25. The presence or absence of residual post-NAT ductal carcinoma in situ (DCIS) also needs to be reported, and is included in some of the published classification sys‑ tems25–27. Similarly, the presence of lymphovascular inva‑ sion (LVI) must be stated, because cancer emboli have been demonstrated to be more resistant to treatment than cancer cells outside of vascular spaces28. A criti cal situation is residual tumour characterized by the presence of LVI in the absence of other microscopically detectable invasive tumour cells. In such cases, the BIGNABCG group clearly stipulates “residual LVI should not be classed as pCR” (REF. 13). The therapeutic effect of NAT can be microscopically observed as residual, invasive or in situ breast cells that demonstrate characteristics associated with cell necrosis and/or apoptosis, multinucleation and pleomorphism; in the stroma, the therapeutic effect is reflected by the presence of fibrosis, sclerosis, necrosis, haemosiderin deposits, and microcalcifications, and infiltration by foamy macrophages, neutrophils or lymphocytes. Margin assessment can be impaired in the presence of a good clinical and macroscopic response to NAT, or if a scattered-foci pattern of histological response is observed. The correlation of pathology and radiology findings is important for a correct locoregional man‑ agement of such residual tumours, particularly when the scattered foci are suspected to be close to, or at the surgical margins. The post-NAT pathological examination of lymph nodes presents numerous obstacles and, for this reason, particular rules must be followed (BOXES 2,3). The pathol‑ ogist reports the number of involved nodes, the size of the largest metastatic deposit, and the signs of thera‑ peutic effects. The inclusion of certain elements in the pathology report of patients with persistent post-NAT residual disease is recommended (BOX 4).
Biomarkers of residual disease Residual tumour burden Multiple classification systems have been proposed for grading the pathological response to therapy in patients with breast cancer (TABLE 1), with substantial hetero geneity both in the definition of pCR5,23,26–36 and in the recommendations for quantification of the residual tumour23,29,36. This heterogeneity is an obstacle that lim‑ its comparisons of data from different clinical trials. In this subsection we reflect on what should be considered a pCR of patients with breast cancer to therapy and, fur‑ thermore, we present the most important current sys‑ tems for quantification of residual-tumour-burden and the prognostic information they provide.
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REVIEWS Box 3 | Lymph-node evaluation in the neoadjuvant setting13 Before treatment • Clinical assessment: ultrasonography is recommended • In patients with suspicious lymph nodes, fine-needle aspiration or core biopsy should be performed • Sentinel-lymph-node biopsy precludes a correct assessment of nodal residual disease and the calculation of residual cancer burden and, thus, should be avoided in clinical trials After treatment • Immunohistochemistry and additional sectioning levels are not routinely required, but can help clarify identity of suspicious cells • In the absence of tumour cells, fibrosis, macrophages, tumour-negative lymph nodes should be reported • In the absence of tumour cells but presence of fibrosis, effects of chemotherapy and any presence of ‘sterilized’ nodes should be reported • If residual disease is observed in the lymph node, tumour-positive nodes should be reported and the size of the entire area infiltrated by tumour cells should be measured, even if few cells are present • In the neoadjuvant setting, in the presence of micrometastatic disease or single tumour cells, report positive nodes (ypN1 or 2)
Box 4 | Elements of the pathology report for post-NAT residual breast cancer13 Breast Gross description: • Tumour bed size Microscopic description: • Residual tumour size according to the American Joint Committee on Cancer staging21 • In the case of multifocal residual disease: -- Measurement of two dimensions of largest cross-section of residual tumour bed (in case the entire area is involved); if tumour cells are present within a sole tumour bed, the largest diameter is used for ypT -- Measurement of the largest tumour residue diameter (in the case of multiple tumours present on pretreatment imaging or tumour foci separated by normal breast tissue) for ypT • Residual tumour cellularity • Percentage of ductal carcinoma in situ Lymph-node status • Number of involved nodes and size of largest metastatic deposit NAT, neoadjuvant therapy.
Pathological complete response. A FDA proposal, pub‑ lished in 2014 (REF. 8), defined a pCR as ypT0/Tis ypN0 disease (absence of invasive cancer in the breast and axillary nodes), a definition we agree with. The FDA definition of pCR highlights the total microscopic dis‑ appearance of invasive disease but allows the persistence of an in situ component7. The influence of the post-NAT in situ carcinoma on disease outcome has been analysed in several large studies. For example, von Minckwitz et al.37 reported that patients with residual DCIS had a higher relapse rate than patients without an in situ com‑ ponent. By contrast, a meta-analysis of 12 neoadjuvant randomized trials (CTNeoBC) demonstrated similar event-free survival and overall survival rates regard‑ less of the presence of residual DCIS5. Concordantly, we and others have shown that the distinction between
post-NAT ypT0 and ypTis has no prognostic rele‑ vance38–40; therefore, we endorse ypT0/Tis ypN0 as the correct definition of pCR, which has already been included in other published guidelines41,42. Pathological partial response. The dichotomization of the response to NAT into pCR and non-pCR does not account for the prognostic significance of various forms and extents of post-therapy residual disease, such as scat‑ tered residual tumour cells, a residue of only a few hun‑ dreds of cells or a large residual tumour. Furthermore, pCR confers a survival advantage, but this criteria is far from being a perfect and universal prognostic indica‑ tor. Moreover, controversy exists on whether the sur‑ vival advantage associated with achievement of a pCR is limited to some breast cancer intrinsic subtypes43 or is independent of subtype classification44. Indeed, findings of various trials have shown that patients with luminal breast cancer who do not have a pCR nevertheless had a favourable prognosis5,37,45, which indicates that sub‑ groups of patients with an incomplete pathological response to NAT might have different prognoses that can be revealed through a more-detailed analysis of the residual tumour tissues. Symmans and colleagues25, from the MD Anderson Center, have developed a system to quantitate residual disease, termed the ‘Residual Cancer Burden’ (RCB) index. The RCB index is calculated as a continuous parameter combining six variables, all obtained by microscopic evaluation of the resection specimen: two dimensions of the post-treatment breast-tumour bed, its cellularity, percentage of carcinoma in situ, number of metastatic nodes, and the diameter of the largest nodal metastatic lesion25. The RCB index enables the classi‑ fication of residual disease into four categories: RCB‑0 (pCR), RCB‑I (minimal residual disease), RCB‑II (mod‑ erate residual disease) and RCB-III (extensive residual disease). The overall probability of relapse at 5 years was found to be similar for subgroups of patients with RCB‑0 and RCB‑I (5.4% and 2.4%, respectively), whereas the risk of relapse was significantly higher for RCB-III (53.6%). Thus, this system classifies patients who have benefited from therapy as RCB‑0 or RCB‑I. Among patients with disease classified as RCB-III after NAT, all of those who did not receive adjuvant hormone therapy experienced distant relapses within 3 years, and even 40% of the patients who received such therapy relapsed within 5 years25. In a study in which investigators evalu ated 100 patients with breast cancer, pathology slides and reports were independently reviewed by five patholo‑ gists, and the results demonstrated that the RCB index is a highly reproducible and accurate tool to predict distant recurrence-free survival (RFS) and overall sur‑ vival46. Accordingly, several prospective clinical studies (NSABP, CALGB, ABSCSG, GEICAM, ACOSOG and I-SPY)46 have included RCB as a primary or secondary end point of patient response to chemotherapy. Our group has proposed another system of residual disease quantification that takes into account the tumour size, Scarf–Bloom–Richardson (SBR) grade47 and the number of involved nodes. This parameter, named
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REVIEWS Table 1 | Classifications of breast cancer pathological response to neoadjuvant therapy Name
Grades defined
Histology
GEPARDO30
4 (pCR)
No detectable residual tumour cells
3
DCIS only
2
Minimal invasive residual tumour (90% reduction in tumour cellularity; only small clusters or individual tumour cells remaining
5 (pCR)
No invasive tumour cells (DCIS allowed)
0
No response; almost no change in tumour cells
1a
Mild response; mild changes in tumour cells everywhere / marked changes in 1/3 but
