reviews Annals of Oncology 26: 1280–1291, 2015

doi:10.1093/annonc/mdv161
Published online 27 May 2015

Recommendations for standardized pathological

characterization of residual disease for neoadjuvant
clinical trials of breast cancer by the BIG-NABCG
collaboration
V. Bossuyt1*, E. Provenzano2, W. F. Symmans3, J. C. Boughey4, C. Coles5, G. Curigliano6,
J. M. Dixon7, L. J. Esserman8, G. Fastner9, T. Kuehn10, F. Peintinger11,12, G. von Minckwitz13,
J. White14, W. Yang15, S. Badve16, C. Denkert17, G. MacGrogan18, F. Penault-Llorca19,
G. Viale20 & D. Cameron21 of the Breast International Group-North American Breast Cancer Group
(BIG-NABCG) collaboration
1
Department of Pathology, Yale University, New Haven, USA; 2Department of Oncology, University of Cambridge, Addenbrooke’s Hospital, Cambridge, UK; 3Department
of Pathology, The University of Texas M.D. Anderson Cancer Center, Houston; 4Division of Subspecialty General Surgery, Mayo Clinic, Rochester, USA; 5Oncology Centre,
Cambridge University Hospitals National Health Services Foundation Trust, Cambridge, UK; 6Division of Early Drug Development for Innovative Therapies, European
Institute of Oncology, Milan, Italy; 7Edinburgh Breast Unit, Western General Hospital, Edinburgh, UK; 8Carol Franc Buck Breast Care Center, University of California,
San Francisco, USA; 9Department of Radiotherapy and Radiation Oncology, Landeskrankenhaus, Paracelsus Medical University, Salzburg, Austria; 10Department of
Gynecology and Obstetrics, Interdisciplinary Breast Center, Klinikum Esslingen, Essligen, Germany; 11Institute of Pathology, Medical University of Graz, Graz; 12Breast
Center Salzburg, Paracelsus Medical University, University Hospital Salzburg, Salzburg, Austria; 13German Breast Group, Neu-Isenburg, and University Women’s Hospital,
Frankfurt, Germany; 14Department of Radiation Oncology, Ohio State University Comprehensive Cancer Center, Columbus; 15Department of Diagnostic Radiology, The
University of Texas M.D. Anderson Cancer Center, Houston; 16Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis,
USA; 17Institute of Pathology, Charité Hospital, Campus Mitte, Berlin, Germany; 18Department of Biopathology, Institut Bergonié, Bordeaux; 19Centre Jean Perrin,
Clermont-Ferrand, and Université d’Auvergne, France; 20Department of Pathology, European Institute of Oncology and University of Milan, Milan, Italy; 21Edinburgh Cancer
reviews

Research UK Centre, The University of Edinburgh, Edinburgh, UK

Received 12 March 2015; accepted 28 March 2015

Neoadjuvant systemic therapy (NAST) provides the unique opportunity to assess response to treatment after months
rather than years of follow-up. However, significant variability exists in methods of pathologic assessment of response to
NAST, and thus its interpretation for subsequent clinical decisions. Our international multidisciplinary working group was
convened by the Breast International Group-North American Breast Cancer Group (BIG-NABCG) collaboration and
tasked to recommend practical methods for standardized evaluation of the post-NAST surgical breast cancer specimen
for clinical trials that promote accurate and reliable designation of pathologic complete response ( pCR) and meaningful
characterization of residual disease. Recommendations include multidisciplinary communication; clinical marking of the
tumor site (clips); and radiologic, photographic, or pictorial imaging of the sliced specimen, to map the tissue sections
and reconcile macroscopic and microscopic findings. The information required to define pCR (ypT0/is ypN0 or ypT0 yp
N0), residual ypT and ypN stage using the current AJCC/UICC system, and the Residual Cancer Burden system were
recommended for quantification of residual disease in clinical trials.
Key words: breast cancer, neoadjuvant systemic therapy, residual disease, pathologic complete response, pathologic
assessment, response evaluation

introduction breast cancer subset [2, 3]. The US Food and Drug Administra-
tion (FDA) has recommended pathologic complete response
Response to neoadjuvant systemic therapy (NAST) is an excel- ( pCR) as an end point for accelerated approval of new agents
lent indicator of outcome [1], especially when evaluated by for neoadjuvant treatment of high-risk early-stage breast cancer,
and recently approved pertuzumab based on the increase in
pCR rate [4–6]. An FDA meta-analysis failed to show signifi-
*Correspondence to: Dr Veerle Bossuyt, Department of Pathology, Yale University, PO
Box 208023, 310 Cedar Street, LH108, New Haven, CT 06520-8023, USA. Tel: +1-203- cant improvement in event-free or overall survival related to
785-5793; Fax: +1-203-785-3583; E-mail: [email protected] improved pCR rates in most included trials [1]. Therefore,

© The Author 2015. Published by Oxford University Press on behalf of the European Society for Medical Oncology.
All rights reserved. For permissions, please email: [email protected].
Annals of Oncology reviews
accelerated approval can be based on an improved pCR rate; but approach based on personal experience and our review of the
improved event-free survival remains the end point for full SOP’s of major NAST breast cancer trials.
approval. This new regulatory pathway emphasizes the import-
ance of standardized, reproducible methods of pathology evalu- recommendations
ation and reporting for neoadjuvant clinical trials. To use pCR While the basic principles are similar in neoadjuvant and adju-
to demonstrate treatment efficacy of novel therapies, we must vant situations, NAST specimens are generally more challenging.
have a standard definition and approach to pCR assessment. Therefore, multidisciplinary communication is essential before,
Moreover, with data emerging on regional recurrence risk based during, and after NAST. These recommendations are intended
on response in the breast and lymph nodes, decisions about sub- for clinical trials; but they can be optionally incorporated into
sequent therapy might be based on pathologic assessment of routine practice because, in our opinion, standardization is most
response [7]. effective when uniformly applied.
Post-NAST changes are complex. Several reviews of the differ-
ent classification systems for post-NAST specimens are available
initial diagnosis
[8–11]. Careful, systematic evaluation of the post-NAST speci-
men in the context of clinical and imaging findings is required A percutaneous image-guided core-needle biopsy (CNB) is
for accurate diagnosis. Individual pathologists’ experience with strongly recommended. The CNB must be adequate for an un-
NAST specimens and standardization appears to affect results. equivocal diagnosis of invasion.
For example in a 2004 study [12], using the Chevallier system Pretreatment tumor size (T stage) is based on imaging and
[13], pCR rates dropped—from 16% and 10% for arms A and B, physical examination. Histologic type, tumor grade, estrogen
respectively, to 8% and 6%—from local to central pathology receptor (ER), progesterone receptor (PR), HER2, and other
review. Similarly, in the I-SPY 1 trial, pCR rates fell by almost parameters used to determine neoadjuvant treatment should be
10% after training in the Residual Cancer Burden (RCB) system, evaluated on the pretreatment CNB.
which requires a standardized approach for pathologic evalu- To ensure accurate diagnosis and ancillary tests, an adequate
ation to map gross and microscopic findings (L. J. Esserman, number of sufficiently thick, nonfragmented cores obtained
personal communication). A standard approach to post-NAST with an appropriate-gauge needle are needed. Samples obtained
pathologic assessment of breast cancer would improve compari- from different parts of the tumor, for example by angling the
sons between clinical trials, enable accumulation of more robust needle, may be helpful.
evidence in controversial areas of practice such as specimen Ideally, baseline CNBs for research are obtained at the time of
handling, and better serve each patient. diagnostic biopsy. A separate research biopsy is an alternative.
Clinical trials often incorporate research biopsies at additional
time points (e.g. after the first cycle or mid-course). We endorse
the recommendations from a previous BIG-NABCG working
methods group addressing this topic [15].
To build consensus on a more standard characterization of residual However, removing too much tumor for diagnosis by oversam-
disease in breast cancer neoadjuvant trials, the BIG-NABCG pling of tumor with wide-gauge needles interferes with response
collaboration convened an international working group of path- assessment.
ologists, radiologists, surgeons, gynecologists, and medical and We strongly recommend clip placement at the time of diagnos-
radiation oncologists. Members were selected via BIG-NABCG tic or research biopsy [16]. If, after the first or subsequent cycle(s)
leadership and working group co-chair nomination, as well as re- of chemotherapy, the decrease in tumor volume suggests a pos-
ferred by sites involved in neoadjuvant trials. The working group sible complete response and a clip was not placed previously, it is
reviewed standard operating procedures (SOPs) for pathologic as- imperative to place a clip, even if mastectomy is planned. After
sessment from 28 major NAST breast cancer trials and 25 trial completion of NAST, it may be difficult to identify the correct
sites, finding a variety of approaches (supplementary Material S1, area in the breast or to ensure that the appropriate area was
available at Annals of Oncology online). Moreover, sites submit- excised, if no clip was placed.
ting SOPs noted a need for a standard. The working group devel-
oped practical recommendations for the post-NAST pathologic evaluation of the axilla pre-NAST
assessment of breast cancer in neoadjuvant clinical trials. Systemic or local treatment decisions may be based on axillary
Detailed recommendations for pathologists, including more status at presentation (pre-NAST). Pre-NAST sentinel lymph
in-depth discussion of their evidence basis, are published in our node biopsy (SLNB) is not recommended because assessment of
pathology-focused paper [14]. This paper summarizes the nodal response in the axilla, a very important determinant of sur-
recommendations for pathologic assessment for a multidiscip- vival post-NAST, is unreliable after excision of a positive node.
linary audience, addresses the prerequisites for accurate patho- Furthermore, this invalidates the RCB score and the ypN stage,
logic assessment, and explains how a standardized approach and potentially compromises comparisons of pCR results across
would benefit the entire medical team. different studies. This position should be balanced against the
The majority of available evidence pertains to neoadjuvant accuracy of SLNB post-NAST [17]. Post-NAST SLNB is strongly
chemotherapy. However, we did not identify existing data or con- recommended.
ceptual reasons to limit this standardization effort to neoadjuvant So, to obtain maximum information about the axillary status
chemotherapy only. When evidence was found lacking, the pre-NAST for systemic or local treatment decisions, routine
recommendations represent a consensus opinion for a pragmatic ultrasound of the regional nodal basins is strongly encouraged.

Volume 26 | No. 7 | July 2015 doi:10.1093/annonc/mdv161 | 

reviews Annals of Oncology

Diagnosis of clinically or radiologically abnormal lymph nodes sample (Table 1). Furthermore, images of the sliced resection
by fine-needle aspiration or CNB [18] is strongly recommended specimen (with a scale for measurement) are useful as maps on
before NAST. Clip placement into the biopsied node may which to annotate the tissue sections that correspond to the
improve the accuracy of post-NAST SLNB. However, in clinical- different slides. This greatly helps the pathologist to reconstruct
ly node-negative patients, it may be that pretherapeutic sentinel the extent and location of disease after reviewing the slides
lymph node status may determine systemic or local treatment in under the microscope. This technique is critical for more
some cases. standardized and accurate staging of the residual tumor and
calculation of RCB, and generally requires fewer tissue blocks to
preoperative staging and surgery post-NAST be processed, less time, and less expense.
Preoperative imaging should be appropriate for the clinical stage Figure 1 summarizes possible patterns of tumor response in
at presentation, and is important to document the clinical the breast and associated sampling problems affecting determin-
extent of residual disease. ation of extent and cellularity of residual disease. Because of these
Surgical resection volume is based on preoperative imaging. issues, we recommend systematic sampling with mapping of the
All detectable residual disease should be removed by the surgery specimen as described below and further detailed in our path-
with clear margins [19–21]. In cases of complete radiologic re- ology-focused paper [14]. This pragmatic approach is a consensus
sponse, the center of the tumor bed should be removed, includ- based on personal experience and our review of the SOPs of
ing any radiologic clips. Pre- or intraoperative localization major NAST breast cancer trials.
techniques and orientation of the specimen by the surgeon are If the resection specimen is small (e.g. <30 g), it would be
imperative. In addition, marking the tumor bed with clips at the reasonable to process all of the excised tissue for microscopic
time of surgery is encouraged [22]. evaluation. However, description or preferably an image of the
sliced specimen should still be used to map the location of each
essential information accompanying the post-NAST tissue section. Using the techniques described below, it is often
surgical specimen still possible to collect research samples.
Table 1 lists the clinical information that should be available Information on pre-NAST tumor size and location is critical.
to the pathologist for optimal evaluation of the post-NAST Systematic sampling should include macroscopically visible
specimen. The supplementary Material S2, available at Annals of tumor/tumor bed and immediately adjacent tissue, to represent
Oncology online, includes a suggested template requisition form the area suspected of involvement by carcinoma before treatment
to send with the post-NAST specimen. At an absolute minimum, (area of interest, AI). Extent of sampling is thus determined by
the specimen must be clearly marked as post-NAST and the the pretreatment size in addition to macroscopic pathologic eval-
pre-NAST location and size of the tumor must be indicated. uation, supplemented by any specimen radiography. Sometimes,
additional sampling may be needed after reviewing the initial sec-
evaluation of the post-NAST surgical specimen tions under the microscope.
Pathologic evaluation of the post-NAST specimen must ensure Accurate description or diagrams (maps, ideally drawn on
that the surgery is adequate (identify tumor bed, assess margins), digital photographs or radiographs) must be used to reconstruct
evaluate prognostic factors (document pCR or confirm size/extent the specimen after microscopic evaluation for accurate measure-
of residual tumor, and allow microscopic estimate of residual ments of extent of residual disease and estimates of cellularity, as
tumor cellularity), and permit collection of research samples. well as to ensure adequacy of sampling (Figure 2). A cutoff of an
entire cross section of the AI per 1 cm of pretreatment size or, for
recommended data in the pathology report. Table 2 summarizes
very large tumors, five representative blocks of a cross section of
the recommended elements not always routinely included in the
AI per 1–2 cm of pretreatment size, with a maximum of ∼25
adjuvant setting but recommended in the pathology report of
blocks of AI, should be sufficient to confidently document pCR in
the post-NAST specimen. We strongly recommend that the
most cases, provided a tumor bed or clip is identified.
manner of specimen processing and reporting allow for tumor
staging and calculation of the RCB score [25, 26], as described
collection of tissue samples for research purposes. We generally
below. In addition, we also encourage reporting using another
recommend that dedicated research samples (to be frozen or
system (e.g. Chevallier, Sataloff ) [13, 27] when it is preferred
otherwise prepared in a nonclinical manner) only be collected if
locally. Particularly, whenever the Miller–Payne or Pinder
there is grossly obvious residual invasive cancer. Such sites are the
systems [10, 28] are likely to be used, we recommend reporting
most likely to contain diagnostically expendable tumor tissue of
the cellularity of the pre-NAST CNB. The US National Cancer
sufficient cellularity for research use. One can thin a section and
Institute’s Breast Oncology Local Disease (BOLD) Task Force
submit the trim for research. Another practical approach is to
has also recommended standardized data elements for collection
obtain small cylinders of tissue for research from the slices with a
in preoperative breast cancer clinical trials [29].
punch biopsy tool. Where the research tissue was collected can so
extent of sampling. Accurate, reproducible documentation of be readily identified on the histology slides. A previous inter-
pathologic response to NAST requires adequate sampling of the national working group has addressed the collection of biospeci-
correct area of the breast. Overly exhaustive sampling and mens from NAST breast cancer clinical trials in detail [15].
histologic evaluation of the entire tumor bed are not required
and not as efficient or informative as informed mapping of the tumor grade and type. Histologic tumor type can be more
specimen. Clinical and imaging information, as well as marking difficult to ascertain after NAST. NAST can cause nuclear
of the tumor site, are critical in the selection of the areas to hyperchromasia and pleomorphism and can alter the mitotic

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Annals of Oncology reviews
Table 1. Essential information to be provided to the pathologist with the surgical specimen removed after neoadjuvant systemic therapy
Essential and critical information to be provided or made available to the pathologist
This information is very important to maintain a high quality of histopathological evaluation and to minimize turnaround time. A suggested requisition
form is provided in the supplementary Material S2, available at Annals of Oncology online.
Comment

1 Clearly marked as post-NAST specimen. In daily clinical routine, this information is often not passed along to the pathologist.
The pathologist should be informed of any previous therapy (hormonal therapy,
chemotherapy, radiation therapy, and/or other therapy) for the cancer.
2 Is this part of a clinical trial? Important to follow trial protocol.
Does the trial protocol recommend a grading system for Pathologist needs to know this information in advance of the surgery in order to follow
response? protocol for description, processing, and reporting of the specimen.
If this is a drug registration trial, the pathologist should be blinded to the treatment arm,
or arrange for independent blinded secondary review of the case by another colleague.
3 Results of previous core biopsies, especially if they were Core biopsy results: histologic type, grade, ER/PR, HER2 (and Ki67).
carried out in another hospital. Lab reference number.
Ideally, slides should be available for review.
4 Pretreatment lymph node status and method of This information is essential for a correct nodal status.
assessment. If nodal status was assessed by sentinel lymph node biopsy or percutaneous biopsy (core-
needle biopsy or fine-needle aspiration biopsy) before neoadjuvant treatment, what
were the results (number of nodes sampled, number of positive nodes, size of largest
metastasis)? Was a clip placed in the sampled lymph node?
5 Clinical tumor size(s) before and after chemotherapy. This information is important (i) to estimate the response to chemotherapy (=differences
The information is best given as size in cm or mm, rather in tumor sizes) and (ii) to select the sampling area.
than clinical tumor stage. If a large pretherapeutic tumor has been diagnosed, the pathologist will perform a more
Different imaging modalities may provide different sizes. extensive sampling to rule out multifocal residual disease.
If the clinical response is suggestive of a complete response, the pathologist will also
perform a more extensive sampling.
In contrast, if the clinical evaluation suggests no response, the histopathological
turnaround time can be reduced, as the extensive sampling might not be necessary.
For multifocal tumors, size of each tumor should be given.
Imaging modality (mammography/US/MRI) and the chemotherapy cycle number at post-
treatment imaging are informative, as are patterns of response (e.g. scattered versus
concentric shrinking).
6 Location of the tumor/tumor bed/residual tumor after This information is important in particular for large resection specimens.
chemotherapy. Detailed description of the location with, for example for mastectomy specimens, ‘o’clock
The information is best given in a scheme/drawing. radius’ and distance from nipple is more helpful than just a quadrant.
Procedure used for marking pretreatment tumor location should be noted. Location
ideally marked/bracketed with clip (or ink) before treatment.
Information on presence of calcifications should be provided because associated
calcifications could be used to localize a lesion.
The surgeon can mark the location with a stitch on the specimen.
Specimen radiography can help localize lesions, clips, and calcifications.
For multifocal tumors, location of each tumor should be given.
7 Information on close (e.g. <5 mm) margins based on This is particularly relevant for large specimens. Close (e.g. <5 mm) margins need a more
intraoperative findings/specimen radiography. extensive sampling.
8 Clinical and radiologic response to treatment in the axilla. Clinical exam is sensitive to disease >1 cm in size.
US is currently the imaging modality of choice for assessment of response in the axilla
pre- and post-treatment and has the additional advantage of guiding percutaneous
biopsy and clip placement to identify specific nodes for response evaluation at final
histopathological evaluation.

ER/PR, estrogen receptor, progesterone receptor; US, ultrasound; MRI, magnetic resonance imaging.

rate [10]; however, histologic grade should be compared with difficult to see grossly. The residual carcinoma may be present as
the pretreatment biopsy before assuming that findings are small foci scattered over a (ill-defined) tumor bed [31].
treatment-related. The most recent (7th edition) [23] American Joint Committee
on Cancer (AJCC)/Union for International Cancer Control
tumor extent and cellularity. Tumor size/extent is often difficult (UICC) recommendation to measure the largest contiguous
to assess after NAST. Residual tumor is often softer and more focus excluding areas with intervening fibrosis may result in a

Volume 26 | No. 7 | July 2015 doi:10.1093/annonc/mdv161 | 

reviews Annals of Oncology

Table 2. Elements not always routinely included in the adjuvant setting but recommended in the pathology report of the post-NAST specimena
Report the elements as for any other type of specimen, plus the following:
Comment

1. Size (A) Two dimensions of largest cross section of entire area involved by In the opinion of the working group, the
(possibly scattered) residual invasive tumor foci (=largest distance largest dimension in (A) (longest blue
between invasive tumor cell foci) arrow), together with tumor cellularity, is
and likely a better indicator of response than
(B) Extent of largest contiguous focus of invasive carcinoma as recommended by measurement (B) [19, 24].
AJCC 7th edition [23] The report should clearly state how the size
was determined and which dimension was
Largest dimension of tumor bed
used for staging, especially in cases with
scattered residual disease, where there is
(A) Two dimensions of largest possible interobserver variability due to
cross section of entire area
involved by scattered residual differences in guidelines regarding how size
tumor foci should be measured.
(A) is needed to calculate the Residual Cancer
Burden (RCB) score.

(B) Extent of largest contiguous focus

2. Cellularity – Qualitative statement Assessment of average cancer cellularity
– Largest cross section of residual tumor bed represented in blocks: … across the largest cross section of the
(e.g. ‘G through F’) residual tumor bed (that contains residual
– Compare with pretreatment cellularity if available (Miller–Payne or cancer) is needed to calculate the Residual
Pinder Systems) Cancer Burden (RCB) score.
3. Tumor bed – Identified or not
– Presence of tumor bed at margin
4. Lymph node – Size of largest metastasis The largest distance between tumor cell foci
metastasis including intervening areas of fibrosis.
Size of largest metastasis is needed to
calculate the Residual Cancer Burden
(RCB) score.
5. Treatment – Presence of treatment effect in the breast
effect – Number of lymph nodes with possible treatment effect

a
This table discusses only those elements specific to NAST that may not be routinely included in pathology reports for non-NAST specimens. A complete
list of elements recommended in the pathology report of the post-NAST specimen can be found in our pathology-focused paper [14]. Information about
size, cellularity, and lymph node metastasis is required for quantification of residual disease.

systematic artificial down-staging of tumors with a scattered re- to only assess the most cellular areas of the tumor. Even pretreat-
sponse pattern if scattered tumor nests were part of a single tumor ment cellularity is often heterogeneous, with pretreatment CNB
mass before treatment. There are currently no data linking this only partly representing the tumor. Similarly, changes in tumor
tumor measurement to survival outcome. Note that prior publica- cellularity induced by NAST are commonly heterogeneous
tions concerning prognosis of yp-TNM staging used the earlier (Figure 1). Systematic sampling as described above is therefore
(6th edition) [32] AJCC staging system, which considered the needed to accurately assess cellularity.
largest extent of residual cancer allowing for intervening fibrosis Although it ignores pretreatment cellularity, the RCB system
[24, 33]. To consider tumors multifocal, they should be separated offers several advantages in addressing this heterogeneity. The
by abundant normal breast or adipose tissue and should be mea- RCB system standardizes sampling of specimens and interprets
sured independently and documented. In this situation, dimen- the average invasive cancer cellularity (by area) across the entire
sions from the largest tumor deposit should be used for AJCC residual tumor bed. The residual tumor bed area is initially deter-
staging, with ‘m’ indicating the presence of multiple tumors. mined from the macroscopic evaluation combined with any spe-
Generally, tumor cellularity decreases with tumor size; but this cimen radiography, and revised after the corresponding tissue
is quite variable and so their combined results are more inform- sections from that area have been studied under the microscope.
ative [19]. In some cases, tumor size may not decrease, but over- Calibrating the observer’s eyes to the online cellularity standard
all cellularity may decrease markedly (Figure 1). Comparison of provided on the RCB website can be helpful [25]. The images in
pre- and post-treatment cellularity is the key element of several the publication for the Miller–Payne score are also helpful [28].
systems for classifying residual disease, including the Miller– Assessment of RCB is quite reproducible [34]. This system
Payne and Pinder systems [10, 28]. These systems, however, do helps to standardize gross and microscopic methods, and is
not state how to deal with heterogeneity, and it can be tempting more efficiently utilized in a prospective manner, rather than by

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Annals of Oncology reviews
A Apparent complete response on gross examination

b Tumor bed grossly visible Tumor bed indistinct

Specimen
Clip No residual
a disease grossly
or microscopically
Pre-treatment
tumor

c
Microscopic
tumor Residual disease
on microscopic
examination only

Patterns of partial response (Residual disease)

B
Decrease in cellularity Concentric shrinking
size unchanged Cellularity similar a b
Specimen

Pre-treatment
tumor
Heterogeneous decrease in cellularity c e
Size changed or unchanged
d
Pre-treatment Microscopic
tumor tumor

Tumor bed Grossly visible

tumor

C Problems with random sampling in heterogeneous response

Varied cellularity Large areas without residual disease (scatter pattern)
1 2 1 2 1 2 1 2

Slices Slices

Black blocks: almost no response Black blocks: residual disease

Blue blocks: significant response Blue blocks: complete response

Figure 1. Patterns of response in the breast and problems related to sampling for histologic evaluation: schematic overview with gross and microscopic illustra-
tions. Photos courtesy of Veerle Bossuyt. (A) In some cases with complete response, a residual tumor bed is visible. In others, the tumor bed is indistinct and
sampling of the correct area can only be confirmed by thorough clinical and imaging correlation and identification of a clip. Often, residual microscopic disease
is identified when there is no residual tumor grossly. (a) Gross photograph of tumor bed (arrow). (b) Low-power hematoxylin and eosin (H&E) slide of this
tumor bed. No residual tumor is identified. (c) High-power of H&E slide of the tumor bed from a different patient with rare residual invasive carcinoma cells
(small arrows). (B) A partial response ranges from a decrease in cellularity with unchanged tumor size to concentric tumor shrinking with unchanged tumor
cellularity. Often the decrease in cellularity is heterogeneous and residual disease extends beyond the grossly visible tumor bed. (a) Gross photograph of tumor
bed with residual tumor (arrow). (b) H&E slides (low and high power) of different patient with residual invasive carcinoma concentrated in a nodule with high
cellularity within the tumor bed (concentric shrinking). (c) Gross photograph of most common pattern of residual disease with scattered residual tumor across
a fibrous tumor bed. (d,e) Medium power of H&E slides from two different blocks of the tumor bed (black and blue boxes) illustrating that cellularity often
varies greatly from block to block. (C) When decrease in cellularity is heterogeneous, random sampling can lead to very different estimates of cellularity. When
the decrease in cellularity is so heterogeneous that there are apparent areas with complete response (no residual disease) and apparent multiple foci of residual
tumor (scatter pattern), there are interobserver variability and inconsistencies among guidelines in size measurements and when to consider multiple foci. For
example, for AJCC staging, the largest contiguous focus of invasive carcinoma should be measured [23]. Intervening areas of fibrosis are specifically excluded,
whereas other systems include these areas [24–26, 32, 33]. Moreover, there can be interobserver variability in how much fibrosis to allow within this largest con-
tiguous focus.

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reviews Annals of Oncology

A Grouped Scattered
C

10%

20%

30%

40%

B D15 D20

D25

D2
0

A: 3.6 mm

D15

D25

Figure 2. Example of mapping of a post-NAST lumpectomy specimen. The specimen is serially sliced and radiographed (A). A diagram allows the pathologist
to correlate the microscopic findings with the gross findings and the specimen radiograph and to reconstruct the location of the microscopic residual disease in
the specimen for size measurements (B) [30]. The cellularity is assessed across the largest cross section of residual microscopic disease and compared with a
computer-generated standard to improve reproducibility (C) [25, 30]. (The average cellularity in this example is ∼30%.). Adapted by permission from the
American Association for Cancer Research: Symmans WF. “Pathologic Evaluation After Neoadjuvant Chemotherapy: Standardizing Management of the
Surgical Specimen and Assessing Response to Neoadjuvant Therapies: The Promises and Challenges of Pathologic Complete Response.” Regulatory Science
and Policy Session, 8 April 2013. Washington, DC: American Association for Cancer Research (AACR) Annual Meeting 2013. http://webcast.aacr.org/console/
player/20130?mediaType=audio& Source: see ref. [30].

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Annals of Oncology reviews
retrospective review. As a first step, we advocate reporting the In current practice, the choice of adjuvant therapy is dictated
2D size of the largest distance between residual tumor cell nests by the results at primary diagnosis. However, patients with re-
in a cross section of the entire area involved by residual tumor. sidual disease that originally had negative receptor status can be
The relevant sections should be recorded in the pathology report. re-tested to re-evaluate for eligibility for a targeted adjuvant
Thus, the benefit of uniform sampling is achieved and RCB as- treatment. Re-assessment of hormone receptor (HR) and HER2
sessment, including average cellularity of the tumor bed area, can in all cases with residual disease can be considered in clinical
be carried out upon local or central review, as preferred. trials to gather high-quality data to clarify these issues. Otherwise,
we recommend repeat testing only in circumstances where the
margins. Assessment of margins may be less reliable post- clinical course or pathologic findings suggest repeat testing may
NAST in cases with scattered response. Tumor bed extending to yield a different result that would change treatment. If pre- and
the margins should be documented. post-NAST results are discrepant, retesting of the pretreatment
biopsy should also be considered.
evaluation of the axilla post-NAST Ki67 expression correlates with long-term outcome, whether
Post-NAST lymph node status is an important determinant of sur- natural prognosis or after endocrine [53] or chemo-endocrine
vival, regardless of response within the breast [7, 35–41]. The accur- therapy [54, 55]. Despite concerns about the analytical reprodu-
acy of SLNB after NAST is an important clinical research topic, cibility of Ki67 measurements [56–58], the test is used at many
especially for patients presenting with positive lymph nodes [17, 42]. institutions for basic risk assessment to tailor adjuvant therapy
Pathologic evaluation is the same as for non-NAST speci- based on markedly low or high values that more reliably classify
mens, although lymph nodes may be more difficult to identify risk, and is a component of several multivariate prediction
grossly. All surgically removed lymph nodes should be entirely models in the post-NAST setting—e.g. the preoperative endo-
submitted for histologic evaluation, sectioned at 2-mm intervals. crine prognostic index (PEPI) and the residual proliferative
Additional levels and immunohistochemistry (IHC) are not cancer burden (RPCB) [53, 59].
routinely required. The number of positive lymph nodes, size of
the largest metastasis, and presence of micrometastasis and iso-
pathologic complete response
lated tumor cells (ITCs) are predictors of worse survival and
should be recorded [40, 43]. When ITCs ( pN0i+) are present in To date, a variety of definitions of pCR have been used in neoad-
the lymph nodes, this is not considered pCR. Molecular assays juvant clinical trials in breast cancer, impeding cross-trial inter-
(e.g. OSNA) to evaluate sentinel lymph node (SLN) are not pretation of data [5, 14]. In its guidance on the potential use of
usually calibrated to detect ITCs [44] and are therefore not pCR to accelerate drug approval, the US FDA defines pCR as
recommended post-NAST [45]. either ypT0/isypN0 or ypT0ypN0 [1, 5]. Indeed, there are excel-
The presence of treatment effect in the lymph nodes may lent data and a strong consensus to include absence of disease in
provide additional prognostic information and should be recorded both the breast and the lymph nodes in a standard definition of
[46]; however, this can be difficult to discern. Small fibrous scars pCR [1, 7, 35–41].
suggestive of prior lymph node involvement or treatment effect Whereas it is clear that patients with residual carcinoma in
can also be seen in patients without treatment [47]. NAST effect the lymph nodes only (ypT0/is ypN+) have a considerably infer-
cannot always reliably be distinguished from previous biopsy site ior prognosis [1, 7, 35–41], the significance of residual in situ
changes. Furthermore, granulomas can form around radiologic carcinoma [ductal carcinoma in situ (DCIS) alone] is not entire-
clips within lymph nodes and previously involved lymph nodes ly clear. Both ypT0/is ypN0 and ypT0 ypN0 have comparable
may look completely normal after NAST. The pathology report survival and are correlated with improved survival [1]. In a
should state if a clip is identified and specify the histologic findings pooled analysis from the German Breast Group, absence of
(involvement, possible treatment effect, or biopsy site changes) in DCIS in addition to the absence of invasive carcinoma (ypT0
the lymph node with the clip. ypN0) identified a smaller group of patients with the best prog-
nosis [3]. However, in patients treated at MD Anderson Cancer
Center, there was no difference in survival between patients with
HR, HER2, and Ki67 post-NAST ypT0 ypN0 and ypTis ypN0 [60]. Future studies should pro-
Receptor status can differ between pre-NAST and post-NAST spectively select either ypT0/is ypN0 or ypT0 ypN0 as a primary
tumor samples. Two meta-analyses report discordant results of end point and state which definition is used; we would recom-
13% and 18% for ER, 32% and 26% for PR, and 9% and 6% mend also reporting the other as a secondary/exploratory end
for HER2, respectively, before and after chemotherapy [48, 49]. point. Pathologists should report DCIS, remembering to note if it
Trastuzumab may increase the rate of negative conversion is absent, so the data needed for further examination of outcomes
for HER2 [50, 51]. Reasons for this discordance include tech- associated with these two pCR definitions can be gathered.
nical failure, intratumoral heterogeneity of marker expression, Table 3 summarizes our recommendations for the assessment
and changes induced by therapy. However, ER, PR, and HER2 of pCR. IHC is not required but may be helpful to visualize
assays are not 100% accurate and reproducible—i.e. repeating tumor cells when hematoxylin and eosin (H&E) staining is in-
the assays will inevitably lead to some discordant results [52]. conclusive, or as part of an SOP for SLN evaluation. If residual
The reported rates of discordant results should be interpreted tumor cells are present, they should be considered in the same
in the context of the expected discordance rate from tech- manner whether identified on H&E or IHC. The companion
nical variability in repeated measurements (∼10% for a 95% pathology paper discusses occasionally controversial elements
accurate test.) in detail [14].

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reviews Annals of Oncology

Table 3. Requirements for accurate and reliable histologic assessment of pathologic complete response (pCR)

Assessment of pathologic complete response (pCR)

pCR = No residual invasive carcinoma in the breast and in all sampled lymph nodes
(ypT0/is ypN0 or ypT0 ypN0) [1, 5]
Requires adequate sampling of the correct area of the breast:
– Correlate area to sample with clinical and imaging findings (pretreatment tumor size and location)
– Identify clip, if present/tumor bed
– Document the (largest) cross section(s) of pretreatment area of involvement with a map of the tissue blocks. (For initially large tumors, 5 representative
blocks per 1–2 cm of pretreatment size with maximum of ∼25 blocks should be sufficient.)a
Immunohistochemistry is not routinely required but may be helpful.
All surgically removed lymph nodes must be entirely submitted for histologic evaluation, sectioned at 2-mm intervals. (Additional levels and
immunohistochemistry are not routinely required.)
Occasionally controversial elements:
pCR NOT pCR Insufficient Comment
evidence
Ductal carcinoma in situ (DCIS) x x pCR definitions vary [1, 3, 60]; adding pT0 or pTis clarifies the pCR definition
Lobular carcinoma in situ x
Lymphovascular invasion (LVI) x x Very rarely a problem for designation as pCR or not because significant
LVI-only residual disease without residual disease in the lymph nodes is
extremely rare.
Micro- and macrometastasis in lymph x Residual disease in the lymph nodes confers a worse prognosis irrespective of
node(s) (pN1mic and above) the presence of disease in the breast [7, 35–41].
Isolated tumor cells in lymph node(s) x The significance of micrometastases and isolated tumor cells is different in the
(pN0i+) neoadjuvant setting than in the adjuvant setting [43].

a
The FDA has recommended a minimum of one block per cm of pretreatment tumor size or at least 10 blocks in total, whichever is greater [5].

residual disease into four classes (0, I, II, and III) [26]. The quantitative RCB
Using pCR as the only indicator of response to NAST underesti- score can be incorporated into a multivariate model. RCB has
mates the clinical benefit a patient receives in terms of event-free been validated in several independent cohorts, and is prognostic
survival. Simulations show that measures of residual disease can at 5 years and beyond 10 years overall and in phenotypic sub-
improve the power of neoadjuvant clinical trials and will improve groups [26, 62]. A prescriptive protocol for pathologists is
estimates of survival benefits [61]. There is great interest to gain available at http://www3.mdanderson.org/app/medcalc/index.cfm?
further prognostic information from the extent of residual disease pagename=jsconvert3 [25].
through evaluation of, e.g. yp-stage, RCB, and PEPI, and also
from the biology of residual disease, e.g. using Ki67 and multi-
gene assays in residual disease [26, 53, 59, 62, 63]. conclusions
Different classification systems can yield different estimates of We propose a standardized evaluation of the post-NAST surgi-
future risk [41]. Systems that combine clinical, pathologic, and cal specimen in breast cancer neoadjuvant clinical trials that can
biomarker information pre- and post-NAST, thus incorporating be optionally incorporated into routine practice and promotes
information about pretreatment tumor burden, residual disease, accuracy and reproducibility of response assessment across insti-
and biology, will likely be the most useful [62]. For example, tutions. Rather than exhaustive sampling, thorough sampling in
the clinical–pathologic stage-estrogen/grade (CPS-EG) combines the areas of the specimen identified by informed mapping,
pathologic stage post-NAST with clinical stage pre-NAST, taking into account clinical and imaging information, is needed.
nuclear grade, and ER status, and has been independently vali- The standard proposed also allows collection of research tissue
dated to identify patients with residual disease post-NAST who and better serves the study of response to NAST. pCR and RCB
have a high risk of relapse [33]. Particularly in HR + HER2− have robust long-term prognostic data for breast cancer overall
breast cancer, pretreatment variables are prognostic beyond re- and within phenotypic subsets. The AJCC/UICC yp-staging
sidual disease measures [62]. system is internationally endorsed.
At present, we recommend both the RCB system and the Clearly identifying resection specimens as post-NAST is
current AJCC/UICC staging system to quantify residual disease essential. We recommend that the post-NAST pathology evalu-
in neoadjuvant trials. The RCB score incorporates pCR as a ation and pathology report include:
score of zero and combines findings in the primary tumor bed
(size and average cellularity of largest cross section of residual 1. The information needed to determine pCR versus residual
tumor bed) and the regional lymph nodes (number of and size disease, using either of the definitions proposed by the FDA
of largest metastases) to quantify increasing amounts of residual meta-analysis [1] (ypT0/is ypN0 or ypT0 ypN0).
disease as an increasing continuous RCB score that is subdivided 2. AJCC/UICC ypT and ypN stage.

 | Bossuyt et al. Volume 26 | No. 7 | July 2015

Annals of Oncology reviews
3. More detailed quantification of residual disease. Ideally, the Hospital, Breast Health Center, Rhode Island; Jeremy Thomas
RCB system and/or any other classification system that is of Western General Hospital, NHS Lothian, Edinburgh, UK;
locally preferred or required for a clinical trial protocol relevant Gary Unzeitig of Laredo Breast Care, Texas; Jelle Wesseling of
to the patient is included in the report. When RCB is not Netherlands Cancer Institute; and Marc Wilt of Centre Paul
reported, we advocate reporting the 2D size of the largest dis- Strauss, Strasbourg, France.
tance between residual tumor cell nests in a cross section of the We also thank Rebecca Enos of the EMMES Corporation for
entire area involved by residual tumor and identifying the rele- information gathering and for coordination and administrative
vant sections in the pathology report with at least a qualitative support of the BIG-NABCG Residual Disease Characterization
assessment of cellularity across this entire area. Working Group. We also thank the Breast Cancer Research
Foundation (BCRF) for its support of the BIG-NABCG collab-
We hope that direct, prospective comparisons of different oration, including the BIG-NABCG meeting where this working
classification systems will provide greater clarity for pathologic group was proposed.
reporting of residual disease.
Finally, the pathologic assessment of residual disease forms an
important component of multivariate approaches that combine
pre- and post-treatment burden of disease and biological charac- disclosure
teristics to better define prognosis after NAST. WFS filed Residual Cancer Burden (RCB) as intellectual
property (Nuvera Biosciences), patenting the RCB equation.
(The RCB calculator is freely available on the worldwide web.)
acknowledgements WFS reports current stock in Nuvera Biosciences and past stock
We thank the BIG-NABCG leadership: Nancy E. Davidson, in Amgen.
from University of Pittsburgh Cancer Institute and UPMC GMG reports grants and personal fees from Roche, and
Cancer Center, Pittsburgh, Pennsylvania. Martine Piccart, of personal fees from Sanofi Aventis, outside the submitted work.
Institut Jules Bordet, Université Libre de Bruxelles, Brussels, All remaining authors have declared no conflict of interest.
Belgium. Larry Norton, Memorial Sloan-Kettering Cancer
Center, New York, New York.
We also thank the following for providing their input: Helena references
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Annals of Oncology 26: 1291–1299, 2015

doi:10.1093/annonc/mdv022
Published online 20 January 2015

Genetics of breast cancer: a topic in evolution

S. Shiovitz1,2* & L. A. Korde1,2,3
1
Divison of Medical Oncology, University of Washington, Seattle; Divisions of 2Clinical Research; 3Public Health Sciences,
Fred Hutchinson Cancer Research Center, Seattle, USA

Received 21 January 2014; revised 2 December 2014; accepted 31 December 2014

A hereditary predisposition to breast cancer significantly influences screening and follow-up recommendations for
high-risk women. However, in patients with a suggestive personal and/or family history, a specific predisposing gene is
identified in <30% of cases. Up to 25% of hereditary cases are due to a mutation in one of the few identified rare, but
highly penetrant genes (BRCA1, BRCA2, PTEN, TP53, CDH1, and STK11), which confer up to an 80% lifetime risk of
breast cancer. An additional 2%–3% of cases are due to a mutation in a rare, moderate-penetrance gene (e.g. CHEK2,
BRIP1, ATM, and PALB2), each associated with a twofold increase in risk. Prediction models suggest that there are un-
likely to be additional yet to be identified high-penetrance genes. Investigation of common, low-penetrance alleles contrib-
uting to risk in a polygenic fashion has yielded a small number of suggestive single-nucleotide polymorphisms (SNPs), but
the contributive risk of an individual SNP is quite small. Mutation testing is currently recommended for individual genes in
the appropriate clinical setting where there is a high index of suspicion for a specific mutated gene or syndrome. Next-
generation sequencing offers a new venue for risk assessment. At the present time, there are clear clinical guidelines
for individuals with a mutation in a high-penetrance gene. Otherwise, standard models are used to predict an individual’s
lifetime risk by clinical and family history rather than genomic information.
Key words: breast cancer, family history, genetics, screening, multiplex gene panels, BRCA

introduction were 464 000 cases of female breast cancer and 131 000 deaths in
Europe in 2012 [1]. The American Cancer Society estimates
Breast cancer is the most common malignancy in women in that, in the United States, there were ∼232 000 new breast
Europe and the United States and second leading cause of cancer cases (of which 2000 were male breast cancer) and
cancer-related death. A recent publication estimated that there 40 000 deaths in 2013 [2]. There is no single definition of
‘familial’ breast cancer, but generally accepted criteria include:
*Correspondence to: Dr Stacey Shiovitz, Seattle Cancer Care Alliance, 825 Eastlake Ave (i) at least three breast and/or ovarian cancer cases in a family;
E., G4830, Seattle, WA 98109, USA. Tel: +1 206-288-6658; Email: [email protected]

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