Pathology of Breast Carcinomas After Neoadjuvant Chemotherapy: An Overview With Recommendations on Specimen Processing and Reporting Open Access

Neoadjuvant therapy (NAT), also termed primary or preoperative therapy, refers to the treatment of patients with systemic agents before definitive surgical removal of a carcinoma. It was originally used as a standard treatment for inflammatory and inoperable locally advanced breast cancers.1,2 Diagnosis was typically made by fine-needle aspiration. Therefore, pathologic information about the carcinoma (eg, type and grade) and information for staging (eg, size and lymph node status) was limited. The combination of image-guided core needle biopsy, sentinel lymph node biopsy (or fine-needle aspiration of a palpable or suspicious node), and new breast imaging modalities (such as magnetic resonance imaging) have substantially increased the ability to accurately classify and stage carcinomas before surgical excision. Thus, increasing numbers of women with earlier-stage operable breast cancers are now being treated with chemotherapy or hormonal agents before surgery.3–5 Recently, a detailed review on “Preoperative Therapy in Invasive Breast Cancer: Reviewing the State of the Science and Exploring New Research Directions” was conducted at the 2007 National Cancer Institute State of the Science meeting. The materials presented at this meeting are publicly available at http://ctep.cancer.gov/bcmeeting.

Published clinical trials have shown that systemic therapy before or after surgery gives identical results for locoregional control and metastasis-free survival.2,3,6 However, the following are several major advantages of neoadjuvant therapy.

1. The efficacy of systemic therapy can be assessed in vivo. The response, or nonresponse, of cancers can be determined in individual patients. In all studies, compared to poor responders, patients who achieve a good clinical response have improved long-term disease-free survival and overall survival, indicating that clinical response can be used as an early surrogate of outcome. Patients who achieve a pathologic complete response (pCR), that is, no residual invasive carcinoma in the breast or lymph nodes, have an excellent prognosis.2,7–15 Information about response can also be used to modify or change treatment for patients whose carcinomas show little or no change after initial treatment.

2. Tumor response is a short-term endpoint for clinical trials. After a breast carcinoma is surgically removed, the next endpoint for a clinical trial is recurrence or death. However, these events may not occur for many years or even decades. In addition, if a patient does well, it cannot be determined if this outcome is due to the effectiveness of the systemic treatment or because the carcinoma was completely removed by surgery. Tumor response provides important clinical information within months, which can allow more rapid assessment of new treatments with fewer patients.

3. There is increased eligibility for breast conservation. In most studies of NAT, most carcinomas are reduced in size, and some women are able to conserve their breast instead of requiring mastectomy.16 

4. Research linking tumor response to tissue samples is facilitated. In many trials, tissue samples are collected before, during, and after treatment. Tumor samples paired with information about tumor response are powerful research tools for identifying techniques to predict response, as well as for understanding how and why tumors respond to therapy. Such studies have identified gene expression profiles associated with tumor response.17–20 

Here we describe the (1) pathologic changes in breast cancers after treatment, (2) systems for evaluating response to treatment, and (3) recommendations for pathologic examination and reporting.

Although there are many different combinations of agents used for NAT, typical changes are seen in most carcinomas with any type of treatment. The following changes are commonly observed after neoadjuvant therapy.

Neoadjuvant therapy reduces the size of the primary tumor for most patients. Size can usually be determined by palpation in patients with a minimal treatment response. However, tumors that have undergone a marked response are more difficult to palpate. The reason for the change in palpability is secondary to marked softening of the tumor stroma, the quality of the desmoplastic response, and changes in cellularity. In addition, a decrease in blood flow, as frequently detected by magnetic resonance imaging, could contribute to a change in the firmness of the carcinoma. In cases of a marked or complete response, it may not be possible to identify the original tumor site (tumor bed) by gross examination.

Carcinomas typically become less cellular after treatment, even when there is not a marked decrease in size. Loss of cellularity correlates with clinical response and prognosis.10,21 For tumors that remain as a contiguous mass, cellularity can be estimated over the entire carcinoma. Estimates of cellularity are more difficult when there has been a marked response, because islands of highly cellular carcinoma may be interspersed within a large, difficult-to-delineate tumor bed. Since carcinomas before treatment also vary greatly in cellularity, a change in cellularity is easier to determine if the pretreatment carcinoma is available for comparison.

Most carcinomas do not change in appearance after treatment, except for the loss of cellularity. However, some tumors may appear to be higher grade, and in rare instances may be of lower grade because of the cytomorphologic changes seen in the residual tumor cells from treatment effect.2,22,23 A change in tumor grade can only be assessed by comparing the posttreatment tumor to the pretreatment biopsy before attributing the cellular pleomorphism to treatment effect. The cytologic effects of treatment resulting in a change in the grade of cancers have not been clearly correlated with clinical outcome, and it is not yet known if this will be an important prognostic factor. For unknown reasons, in some tumors, the in situ carcinoma and tumor emboli in vascular spaces (lymphovascular invasion or LVI) are relatively resistant to treatment when compared to carcinoma invading the stroma.22 

For a detailed description of the treatment-induced changes, see the “Microscopic Appearance of Tumor Bed” in the section titled, “Pathologic Examination After Neoadjuvant Therapy.”

In general, tumor markers remain the same before and after treatment.24 In rare instances of discrepancies, laboratory error in testing, interpretation of the stains, tumor sampling (eg, there may be little tumor to evaluate either before or after treatment) and tumor heterogeneity must be considered. However, in some cases, a change in tumor marker expression may result directly from specific types of neoadjuvant therapy. For example, progesterone receptor is frequently lost after treatment with aromatase inhibitors, but not with tamoxifen.25 HER2/neu expression rarely changes after chemotherapy,26 but may be diminished in a subset of carcinomas after treatment with trastuzumab (Herceptin; Genentech, South San Francisco, Calif).27 It is unknown if this change is due to downregulation or selection of tumor cells not expressing HER2/neu. In a recent study by Tacca et al,28 98 (23%) of 420 patients who received NAT had a change in hormone receptor status on repeat immunohistochemical studies. In this study, 61 of 145 patients (42%) who were initially receptor negative became receptor positive. The hormone receptor–positive switch was significantly correlated with better overall survival in these patients, compared with patients with unchanged hormone receptor–negative tumors.

Changes in Ki-67 (MIB-1) have been suggested as a means to measure response to therapy, particularly with hormonal therapy where inhibition of proliferation is the primary goal, though hormonal agents have the capability of promoting cell death. Complete pathologic responses are uncommon after such treatment and changes in proliferation have not yet been linked to survival after hormonal treatment.25 

Lymph node status is the most important prognostic factor in patients who receive neoadjuvant therapy. Evaluation of treatment response in lymph nodes is more complicated and may not be possible in certain patients, as the only involved nodes might have been removed at the time of sentinel node biopsy before NAT. Thus, it is preferable to evaluate clinically palpable or sonographically abnormal nodes with fine-needle aspiration rather than with biopsy if NAT is planned. Patients who have negative sentinel node biopsy before receiving NAT usually do not undergo axillary dissection (ie, they are assumed to be node negative).

The response in the breast and in the lymph node is generally similar. Patients with complete response in both breast and axillary lymph nodes have significantly improved overall and disease-free survival.9 In fact, the prognostic significance of a pathologic complete response in the breast is marginal or absent in models that control for pathologic responses in lymph nodes.8,9,14,29 

Not all of a patient's lymph node metastases will respond equally to chemotherapy. Some nodal metastases with good response may not leave any evidence of prior tumor involvement or may show fibrous scarring with little or no residual carcinoma, whereas other nodes may have large metastases after treatment.

The significance of the size of a metastatic deposit in a lymph node depends on whether or not the patient has been treated. In the National Surgical Adjuvant Breast and Bowel Project (NSABP) B-18, at 9 years of follow-up, patients with negative nodes or micrometastases, who were not treated with chemotherapy before surgery, had identical survival, whereas those patients with macrometastases had a significantly worse survival.2 However, after NAT, survival of patients with minimetastases (<1.0 mm) and micrometastases (<2.0 mm) in lymph nodes was similar to that of patients with macrometastases and significantly worse than that of patients with negative nodes.2 Thus, the prognostic significance of small lymph node metastases after neoadjuvant therapy, particularly in a lymph node with prior greater involvement, is different when these same findings are present in the node of a patient who has not received NAT.2,30 Micrometastases in lymph nodes in patients who receive NAT probably represent macrometastases that have partially responded to chemotherapy.

The same prognostic factors evaluated for untreated carcinomas are important for carcinomas after treatment, but may be modified by changes caused by the treatment. Several studies have attempted to provide criteria for pathologic response after treatment (Table 1). Although the criteria for classifying pathologic response have not been standardized, in most studies the degree of response to treatment has been shown to correlate with survival (Table 2). In general, all of the systems recognize a category of pathologic complete response (pCR) and a category of little or no response. The number of categories of partial response in different systems ranges from 1 to 4, or response may be expressed by a continuous variable.13 In most systems, a pCR requires the absence of invasive carcinoma in the breast. Residual ductal carcinoma in situ may be present, as this finding should not alter survival.31 In systems that include lymph nodes, the nodes must also be free of carcinoma for a pCR. The issue of appropriate classification of “isolated tumor cells” as “node negative” or “node positive” after treatment has not been specifically addressed by any study.

Below are listed some of the systems with a brief description on the classification and categories of responses.

Carcinomas are assigned a posttreatment T and N category indicated by the prefix “y.” The posttreatment AJCC (American Joint Committee on Cancer) stage retains prognostic information but relies predominantly on information about tumor size and lymph node status, which can be difficult to evaluate after NAT.12 Carey et al12 defined the T category for AJCC staging system as follows: “When tumor shrinkage from chemotherapy resulted in nests of residual tumors, these tumors were categorized by the distance over which the nests extended unless there were clearly defined multicentric tumor foci that could be distinguished pathologically.” Therefore, this system equates large contiguous carcinomas with microscopic foci scattered in a tumor bed. This system does not include changes in cellularity or lymphovascular invasion (LVI). The posttreatment “y” AJCC stage, by itself, does not give an indication of the response to treatment.

This was a system used for one of the largest studies comparing neoadjuvant to adjuvant therapy.2 Three categories of responses were recognized with only 1 category of partial response. Partial response was defined as presence of sparse invasive tumor. Metastases to lymph nodes were analyzed separately. The study was correlated with overall survival and disease-free survival.

Response was divided into 5 grades based on a comparison of tumor cellularity before and after treatment and was correlated with disease-free and overall survival.10 This study showed that a grade-4 response (almost a pCR) had a worse prognosis than a pCR (grade 5), providing evidence that this type of response should be kept as a separate group. However, this system does not include the response in lymph nodes or the presence of LVI. Thus, it is possible that the patients with a grade 4 response who did poorly either had lymph node metastases or had LVI.

This system classified the treatment response of 45 women with inflammatory carcinoma into 4 groups with 1 category of partial response.7 The authors separated cases with residual ductal carcinoma in situ from cases with no residual carcinoma. In most systems, these are both grouped together as pCR. In this system, partial response category was combined with the no response category for outcome analysis.

This system classified treatment response in 36 patients for both the primary carcinoma and the lymph nodes.8 The category of pCR (called T-A) includes a “near total therapeutic effect,” implying that small foci of invasive carcinoma may be present. There are 2 categories of partial response. This system does not include LVI. The study showed that patients with T-A had a better survival at 5 years than the other 3 categories of patients, but did not show differences in the categories of partial response.

This system was developed to calculate residual cancer burden (RCB) in 382 patients in 2 different treatment cohorts for prediction of distant relapse–free survival.15 This system uses residual invasive carcinoma cellularity distributed over the tumor bed, the number of lymph nodes with metastases, and the size of the largest metastasis combined mathematically into a continuous index to define 4 categories of RCB (RCB-0 through RCB-III). Although the system requires the use of a formula, a Web-based calculation script is freely available to calculate the scores (http://www.mdanderson.org/breastcancer_RCB).

In addition to typical neoadjuvant therapy (NAT) in which chemotherapy is completed before surgery, some protocols may involve short-term therapy (eg, weeks instead of months) between diagnosis and definitive surgery. Alternatively, therapies based on NAT protocols using only hormonal agents may need to be of longer duration to develop a maximal response. Different classification schemes for tumor response will be developed as treatments are used in different ways.

Tumor response can be evaluated clinically by palpation or by breast imaging, and these are useful techniques to monitor response during therapy. However, clinical and radiologic assessments of response often underestimate or overestimate the amount of residual carcinoma present. Thus, pathologic examination of the excised tumor bed is the gold standard and is essential for identifying the group of patients (typically 15% to 28%) with a pathologic complete response to treatment, as well as the other 60% to 70% of patients who have a partial response to treatment.2,7–14 

Typically, the pretreatment specimen will be a core needle biopsy. Because 15% to 28% of patients will have no residual tumor after NAT, it is important to have an adequate pretreatment sample in which an unequivocal diagnosis of invasive carcinoma is established and evaluation of hormone receptors and HER2/neu status is completed before treatment. Predictors of a pCR include high histologic grade, nonlobular histologic type, estrogen receptor negativity, extensive tumor necrosis, and HER2/ neu overexpression (for patients who would receive treatment targeting this antigen).32 It is therefore helpful to include histologic type, grade, minimal size, presence of tumor necrosis, and LVI to enable comparison of these features to the posttreatment carcinoma.

It is important for the pathologist to be aware of the use of neoadjuvant therapy before examining a pathologic specimen. Unfortunately, this history is not always provided. Important clues are a long interval between a prior core needle biopsy and a subsequent excision (typically months) and/or a specimen without an obvious biopsy cavity or a clearly palpable carcinoma. The following clinical information should be provided to the pathologist.

1. Presentation of the lesion before treatment (palpable mass or radiologic lesion, skin changes such as edema, erythema, fixation to chest wall)

2. Size of the invasive carcinoma before treatment

3. Prior diagnostic procedure: core needle biopsy or incisional biopsy—ideally, this specimen should be available for comparison with the posttreatment carcinoma

4. Presence of a clip and/or calcifications in the tumor

5. Prior evaluation of lymph nodes (fine-needle aspiration, core biopsy, or sentinel lymph node biopsy) and the results

6. Type of neoadjuvant therapy

7. Clinical/radiologic response of the carcinoma to the treatment (complete, partial, minimal)

It is imperative to identify the original tumor (tumor bed) to determine whether or not the patient has had a pCR and to allow localization for breast preservation. If the patient had a complete response clinically and radiologically, it is unlikely a gross lesion will be present. However, microscopic examination in such cases may reveal substantial residual tumor. Therefore, for patients who receive NAT, a clip should be placed during a diagnostic core needle biopsy or at the first few cycles of therapy, ideally before the tumor is too soft or unrecognizable on imaging studies. If a clip is not placed, it may be impossible to reliably identify the tumor bed. When calcifications are associated with the carcinoma, they usually remain after treatment and can be detected on a specimen radiograph. Ideally, candidates who qualify for breast-conserving surgery after NAT should undergo wire/needle localized excisional biopsy. It is prudent to obtain a specimen radiograph demonstrating a clip or calcifications to document that the tumor bed was excised. Lacking these, one must rely on the surgeon and/or microscopic detection of the tumor bed to ensure that the prior tumor site was removed, especially when no gross lesion is visible. For mastectomy specimens without a radiologically identifiable lesion, a detailed description of the pretreatment tumor location (eg, quadrant, distance from nipple) and sutures placed by the surgeon are extremely helpful in finding the tumor bed.

Grossly, the tumor bed may have the appearance of a subtle irregular area of rubbery fibrous tissue (Figure 1). Residual tumor may be recognized as fleshy nodules within the tumor bed (Figure 2). It is important to document the size of the grossly visible tumor bed and any recognizable residual tumor. In some cases, the tumor bed may not be recognizable grossly. For patients who undergo breast-conserving surgery, the specimens should be differentially inked to assess margins in the event residual tumor is found on microscopic examination.

The degree to which a residual tumor bed should be sampled has not been established. For initial sampling, 1 block per centimeter of pretreatment tumor size is reasonable. If residual tumor is found, additional sampling is not necessary. If no tumor is found, it is not yet known how much additional sampling is needed. However, if the residual tumor bed is small, the entire area should be submitted for pathologic analysis. If the residual tumor is still large (>5 cm), then one should consider examining at least 5 representative sections from the largest cross-sectional area. The Web site that offers the residual cancer burden calculator (http://www.mdanderson.org/breastcancer_RCB) has some recommendations on tumor sampling.

The presence of a tumor bed must be confirmed microscopically when residual carcinoma is not present. Microscopically, the tumor bed is characterized by an area of hyalinized vascular stroma with stromal edema and fibroelastosis, often without the presence of normal glandular breast ducts and lobules (Figure 3, A). The stroma is often infiltrated by foamy histiocytes, sometimes forming large sheets, aggregates of lymphocytes, and hemosiderin pigment (Figure 3, B and C). Areas of tumor necrosis may leave nodules of histiocytes and cholesterol clefts (Figure 3, D).

The cytologic features of most carcinomas do not change after treatment except for the decrease in cellularity. However, some carcinomas (Figure 4, A) show changes indicative of treatment effect (Figure 4, B). These changes include distortion of glandular architecture, enlarged tumor cells due to increased cytoplasm, cytoplasmic vacuolization and eosinophilic change, pleomorphic and bizarre nuclei, and decreased mitotic activity (Figure 4, B, inset). Residual tumor cells are often distributed either singly or in clusters; in the latter case, the borders are typically well defined and the cells tend to shrink away from the stroma (Figure 4, B). This feature should not be misinterpreted as lymphovascular invasion. In cases of near complete pathologic response, scattered single degenerated tumor cells may show multinucleation, hyperchromasia, and nuclear smudging, making them difficult to detect on routine hematoxylin-eosin stain (Figure 5, A and B). In difficult cases, immunohistochemical stains to distinguish between epithelial cells (cytokeratins AE1/ AE3 or cytokeratin 7) and histiocytes (CD68 or CD163) are helpful in identifying the residual tumor cells, as well as in the evaluation of surgical margins. Residual ductal carcinoma in situ usually does not show morphologic alteration after treatment.

Cytotoxic treatment effect also occurs in the nontumor–bearing breast parenchyma in the form of moderate to marked sclerosis of basement membranes of the ductal and acinar components of the terminal duct–lobular unit. Scattered epithelial cells in this unit may show cytologic and nuclear enlargement (Figure 6), which should not be confused with residual in situ carcinoma.

Margins can be more difficult to evaluate after neoadjuvant therapy (NAT). It is preferable to excise the entire tumor bed with a rim of normal tissue to ensure there is no residual carcinoma. However, the extent of the tumor bed can be difficult to determine on imaging by the radiologist, grossly by the surgeon, and macroscopically by the pathologist when there has been a pronounced response to treatment. The significance of tumor bed at the margin is unclear in patients with a pCR. In cases with scattered residual foci of invasive carcinoma or ductal carcinoma in situ throughout a tumor bed, tumor bed changes at the margin may be predictive of the possibility of residual carcinoma in the breast.

The axillary tail should be carefully searched for lymph nodes and all nodes thinly sectioned and completely submitted. In general, after NAT, lymph nodes are difficult to recognize because of atrophy and fibrosis. When it is difficult to locate lymph nodes, fibrotic areas in the axillary fat and tissue around the vessels should be submitted, which may reveal small atrophic nodes on microscopic examination.

Patients who have a positive lymph node by fine-needle aspiration (or core needle biopsy) before neoadjuvant therapy usually undergo completion axillary dissection at the time of primary tumor resection. This approach helps to stratify patients into 3 groups, namely those with (1) positive nodes, with or without evidence of disease regression (partial or no response); (2) negative nodes, with evidence of treatment-induced change but no viable tumor (complete response); and (3) negative nodes without treatment effect (complete response). If a positive lymph node was removed before therapy, and the lymph nodes after therapy are not involved by metastases, response to therapy cannot be evaluated with certainty.

Lymph node metastases that show complete response to treatment are often replaced by hyaline stromal scars, mucin pools, or aggregates of histiocytes without any viable tumor cells (Figure 7, A and B). Complete pathologic response to prior metastatic involvement in some cases cannot be determined with certainty because metastasis can resolve without a scar or may leave small fibrous scars. However, it is unusual to see large fibrous scars in lymph nodes of patients who undergo surgery first.33 Therefore, the presence of a large scar in a lymph node without tumor cells most likely is indicative of a complete response to therapy.

Partial response in lymph nodes is characterized by isolated or clusters of tumor cells surrounded by thin or thick hyaline stromal fibrosis (Figure 8, A and B). Immunohistochemical stains (cytokeratins) can be used to identify tumor cells that are difficult to characterize on routine hematoxylin-eosin stain. Patients who have residual metastatic tumor with evidence of treatment effect have better disease-free survival and lower relapse rates than patients who have positive nodes without evidence of such changes.34 Therefore, it is important to make notation of treatment effect in lymph nodes.

Pathology reports on treated tumors should include the following information.

1. Presence and size of tumor bed: important for documentation, especially in cases with pathologic complete response

2. Size and extent of residual tumor

     Two-dimensional measurements of the largest area of invasive cancer

     Number of foci or number of blocks with foci of invasion

3. Average cancer cellularity of the residual tumor bed (see Table 1 for Miller-Payne grading system, which requires evaluation of the change in cellularity and for RCB system, which has examples and guidelines to assess residual cellularity)

4. Appearance of the residual tumor and grade, if applicable: compare to pretreatment carcinoma, if possible

5. Viability (necrosis, mitotic figures); proliferation index by MIB-1 (Ki-67) may be requested for some protocols

6. Lymphovascular invasion

7. Presence and extent of ductal carcinoma in situ (percentage of in situ component when using the RCB system)

8. Margins with respect to tumor bed, invasive, and in situ carcinoma

9. A comment on the overall response to treatment

1. Number of lymph nodes

2. Number of lymph nodes with metastases

3. Size of the largest metastasis

4. Presence of extranodal extension (measurement of largest extent of extranodal extension may be requested by some radiation oncologists)

5. Number of metastases with evidence of treatment response

6. Number of lymph nodes with evidence of treatment response but without tumor cells (ie, fibrosis, necrosis, aggregates of histiocytes)

1. By AJCC staging, pT category and pN category assigned a prefix “y” (“p” refers to pathologic classification)

2. Response category according to 1 of the classification systems as used by specific institutions or for clinical protocols

Neoadjuvant therapy is being offered more commonly to patients with earlier-stage breast cancer and is likely to become the standard of care for patients receiving systemic therapy. Because clinical and radiologic responses do not correlate well with residual tumor after treatment, pathologic evaluation of tumor response is the gold standard. Pathologists have played, and will continue to play, an important role in providing this information to optimize the knowledge gained by this approach to breast cancer therapy. The role of pathologists is vital in standardizing the existing classification schemes and in developing new schemes for ongoing trials.