The clinical assessment of nodal status (as in TNM classification) is unreliable.^2,³ Palpable nodes may be enlarged because they show benign reactive changes whilst nodes bearing tumour deposits can be impalpable. Although axillary sonography is a moderately sensitive and fairly specific technique in the diagnosis of axillary metastatic involvement,⁴ histological examination of the lymph nodes from the axilla should be carried out in all patients with primary operable invasive breast cancer. It is well known that patients who have histologically confirmed lymph node involvement have a significantly poorer prognosis than those without nodal metastases. The 10-year survival is reduced from 85% for patients with no nodal involvement to 40% for those with involvement of multiple nodes. Prognosis worsens the greater the number of nodes involved, and the level of nodal involvement can provide useful information; metastasis to the higher level nodes (level II or III) in the axilla and particularly those at the apex (level III) carries a worse prognosis (Fig. 2.1).

Figure 2.1 Overall survival for patients in the NTPBCS according to lymph node stage (P < 0.0001).

Studies show that overall survival is decreased as the number of nodes that are involved increases.5–7

Optimal management of the axilla is discussed in Chapter 9.

Sentinel node biopsy or blue dye directed axillary sampling has been shown to provide accurate prognostic information and has an extremely low rate of lymphoedema.² Of 1275 patients undergoing axillary sampling, only 0.04% suffered symptomatic complaints of arm swelling and lymphoedema.

A refinement of lymph node sampling is provided by the technique of sentinel lymph node (SLN) biopsy. There have been several studies to examine the validity of this technique. The ALMANAC trial provides level I evidence for UK-based practice.³

From a pathological perspective, there are unresolved questions about how to optimally assess the sentinel node. Studies have shown that the mean number of sentinel nodes is close to 3. As the number of nodes removed during SLN biopsy is fewer, pathologists are now challenged to process these few nodes optimally. Several methods have been studied: routine paraffin histology; intraoperative frozen section; immunohistochemistry and more intensive methods such as serial sectioning and molecular methods including polymerase chain reaction (PCR) and reverse transcription–PCR (RT–PCR).

At present there is no consensus for the optimal handling of the SLN in the laboratory. Most centres have developed their own ‘in-house’ protocols, which are invariably tied to the institution’s research ambitions. This ad hoc approach can influence interpretation of results, as the amount of material assessed between centres is not uniform. In a number of large studies, using different histopathological techniques, the SLN false-negative rate (defined as how often the SLN is negative for malignancy when cancer is present in the rest of the axilla) varies between 0% and 11%.

In Nottingham the MRC ALMANAC protocol is employed to assess all sentinel node biopsies. These nodes are cut into slices about 3 mm thick, taken perpendicular to the long axis of the node to maximise the assessment of the marginal sinus, with one node per cassette. The majority of nodes can be completely embedded in one cassette. Larger nodes have alternate slices embedded and may require more than one cassette. Large, obviously involved nodes have one section taken.

Intraoperative frozen sections or imprint cytology of axillary lymph nodes have been advocated by some authors. Conventional frozen sections have a high false-negative rate of between 10% and 30%. More intensive intraoperative assessment with serial sections and immunohistochemistry has been described,⁸ but is time consuming and labour intensive. Frozen section is best applied to selected cases; for example, if the node is macroscopically abnormal and this is confirmed histologically to be metastatic carcinoma, further axillary surgery can be performed immediately. Some studies have found low false-negative rates of 2–3% with intraoperative imprint cytology,⁹ but not all have been able to achieve this level of accuracy. Lymph node status can be assessed peroperatively using ultrasound combined with FNA or core biopsy, and this reduces the need for preoperative frozen section and imprint cytology to assess axillary nodes intraoperatively.

RT–PCR is more sensitive than immunohistochemistry at detecting metastatic tumour in axillary nodes. Two types of methods have been used to detect tumour cells with molecular techniques. First, a genetic defect such as chromosomal rearrangement or mutation can be used. The problem with this is that no single genetic defect is seen in all breast carcinomas. The second method is to use a molecular marker that is present in tumour cells but not in the adjacent tissue. To identify a marker that has this specificity and is expressed in the majority of tumours is difficult. It may be necessary to use a panel of markers. A major problem with PCR is the potentially high false-positive rate due to the sensitivity of the method and potential for contamination. In addition, it is not possible with PCR to determine whether the DNA comes from cells that are viable. An advantage of haematoxylin/eosin sections and immunohistochemistry is that the morphology of the cells can be examined and malignancy confirmed. A major unresolved question is whether carcinoma detected only by PCR or RT–PCR has the same prognostic significance as haematoxylin/eosin positivity.

The detection rate of micrometastases in axillary lymph nodes has been reported to range from 9% to 46%.¹⁰ Studies have used serial sectioning with or without immunohistochemical stains to detect micrometastatic foci and these methods have increased detection rates.

The European Working Group for Breast Screening Pathology ¹¹ has formulated working guidelines for the assessment and pathological work-up of SLNs in breast cancer. From a literature review available at that time, the committee concluded that it was not possible to determine the significance of micrometastasis or isolated tumour cells. They noted that approximately 18% of cases are associated with other nodal (non-sentinel node) metastases. False-negative rates are most often determined via immunohistochemistry (IHC). However, at present it is recommended that an intensive work-up is not justified on a population level. The committee did suggest multilevel assessment and, where resources permit, intraoperative assessment, although the results of Z0011 (see Chapter 7) suggest that some node-positive patients may not require axillary dissection and this brings the whole issue of intraoperative assessment into question. The current view is that IHC should not be used routinely to assess axillary lymph nodes.

Tumour size

Tumour size is one of the most powerful predictors of prognostic factor in breast cancer.^12,¹³ The frequency of nodal metastases in patients with tumours < 10 mm is 10–20%,¹³ and node-negative patients with tumours < 10 mm have a 10-year disease-free survival rate of greater than 90%.¹⁴ Tumour size is a time-dependent prognostic factor that depends on the period between tumour development and detection, and on the balance between tumour cell proliferation and death (tumour growth rate). It is well known that the association between increasing tumour size and increasing number of positive lymph nodes and worse outcome is highly statistically significant.¹³ Therefore, the main aim of population screening with mammography is to detect smaller tumours that are likely to have a better outcome than those that present symptomatically (of a larger size). Patients with smaller tumours have a better long-term survival than those with larger tumours (Fig. 2.2). Estimation of tumour size has assumed particular importance since the introduction of population screening. In most studies the frequency of axillary lymph node metastasis in small < 15 mm (so-called minimally invasive carcinoma (MIC)) invasive carcinomas is 15–20%,^15–17 compared with over 40% in tumours measuring 15 mm or more.¹⁸ During the prevalent round of breast screening even more favourable results are obtained, with the frequency of axillary lymph node metastasis ranging from 0% to 15%.^19–22 The Nottingham Tenovus Primary Breast Cancer Study (NTPBCS) has generated data that suggest the cut-off point of 10 mm is not the best discriminator for MIC. Life-table analysis of survival curves found no difference between tumours measuring up to 9 mm and those measuring 10–14 mm. This indicates that < 15 mm may be a more realistic watershed in defining small, invasive, carcinomas of good prognosis. It is clear that pathological tumour size is a valuable prognostic factor and it has become an important quality assurance measure for breast screening.^21,^23–26 It is also used in part to judge the ability of radiologists to detect small impalpable invasive carcinomas.

Figure 2.2 Overall survival according to size.

Differentiation

Modern pathologists have recognised that invasive carcinomas can be divided according to their degree of differentiation. There are two ways to achieve this: (i) by allocating a histological type according to the architectural pattern of the tumour; (ii) by assigning a grade of differentiation based on semiquantitative evaluation of structural characteristics.

Certain histological types of invasive carcinoma carry a favourable prognosis. Tubular, mucinous, invasive cribriform, medullary and tubulolobular types, together with rare tumour types such as adenoid cystic carcinoma, adenomyoepithelioma and low-grade and squamous carcinoma, have all been reported to have a more favourable outcome than invasive carcinoma of no special type (ductal NST). Undoubtedly, assessment of histological type provides prognostic information in breast cancer. However, this effect is relatively small in multivariate analysis ²⁷ when compared with the prognostic value of histological grade; histological type may prove to be more useful in increasing our understanding of the biology of breast cancers.²⁸

Invasive lobular carcinoma (ILC) is of particular importance. It comprises approximately 5–15% of breast cancers and appears to have a distinct biology. It is less common than invasive carcinoma of no special type, also called invasive ductal carcinoma (IDC).

Recently, Rakha et al.²⁹ examined a large group of 5680 breast tumours (415 patients (8%) with pure ILC and 2901 (55.7%) with IDC (no special type)) and demonstrated that, compared to IDC, patients with ILC tended to be older and have tumours that were more frequently of lower grade (typically grade 2; 84%), hormone-receptor positive (86% compared to 61% in IDC), of larger size and with absence of vascular invasion. More patients with ILC compared with IDC were placed in the good Nottingham Prognostic Index group (40% compared to 21% in IDC). ILC showed indolent but progressive behavioural characteristics with nearly linear survival curves that crossed those of IDC after approximately 10 years of follow-up, thus eventually exhibiting a worse long-term outcome. Interestingly, ILC showed a better response to adjuvant hormonal therapy (HT), with improvement in survival in patients who received HT compared with matched patients with IDC.

Tumour grade

The Nottingham method (Elston and Ellis) is a modification and improvement of previously existing morphological grading systems and is able to provide greater objectivity of grade.³⁰

In brief, assessment of grade considers three tumour characteristics: tubule formation, nuclear pleomorphism and mitotic counts. A numerical scoring system of 1–3 is used for each factor individually. The three scores are added together to produce scores of 3–9 on which an overall tumour grade is assigned:

• Grade 1 – well differentiated = score 3–5 points.

• Grade 2 – moderately differentiated = score 6–7 points.

• Grade 3 – poorly differentiated = score 8–9 points.

It should be noted that grade is valuable irrespective of histological type.

There is a highly significant correlation between tumour grade with long-term prognosis (Fig. 2.3); patients with grade 1 tumours have a 93% chance of surviving 10 years after diagnosis, whereas in patients with grade 3 tumours this is reduced to 70%. It has now been shown conclusively that the Nottingham method, with its more objective criteria, has excellent reproducibility when used by experienced pathologists.

Figure 2.3 Overall survival according to grade.

Interestingly, grade is not included in the recent revision of the TNM staging system of breast cancer as its value is questioned in certain settings such as lobular type.

The value of grade was addressed by Rakha et al.³¹ in a large study of grade and overall survival in a large and well-characterised consecutive series of operable breast cancer (n = 2219 cases), with a long-term follow-up (median 111 months) using the Nottingham histological grading system. Histological grade was strongly associated with both breast cancer-specific survival (BCSS) and disease-free survival (DFS) in the whole series as well as in different subgroups based on tumour size (pT1a, pT1b, pT1c and pT2) and lymph node (LN) stages (pN0 and pN1 and pN2). The authors were able to demonstrate differences in survival between different individual grades (1, 2 and 3). In multivariate analyses histological grade was an independent predictor of both BCSS and DFS.

Additionally, the usefulness of routinely assessing grade in invasive lobular cancer was examined by this group in 4987 patients,³² of whom 517 were pure ILC cases. The majority of ILC was of classical type or mixed lobular variants (89%). Most ILC cases were moderately differentiated (grade 2) tumours (76%), while a small proportion of tumours were either grade 1 or 3 tumours (12% each). There were significant associations between histological grade and other clinicopathological variables of poor prognosis such as larger tumour size, positive lymph node status, vascular invasion, oestrogen receptor and androgen receptor negativity, and p53 positivity. Multivariate analyses showed that histological grade was an independent predictor of BCSS and disease-free interval.

Vascular invasion

The presence of tumour emboli in vascular and lymphatic spaces has emerged as an important prognostic factor. Several studies have now shown that the presence of vascular invasion correlates closely with local and regional lymph node involvement.^33,³⁴ It has been suggested that it can provide prognostic information as powerful as lymph node stage. Reproducibility is the limiting factor in its widespread adoption and routine clinical assessment. In a Nottingham study of assessing vascular invasion, the issue of reproducibility was addressed specifically. Of 1704 cases, a subset of 400 cases was examined by two or more pathologists. There was a 77% overall inter-observer agreement on histological features and an 85.8% overall agreement in the classification of vascular invasion. Other studies have reported a similarly high concurrence between pathologists.^35,³⁶ The assessment of vascular invasion is subjective but there is good evidence that a high rate of concurrence can be obtained as long as strict criteria are used. Lymphatic and vascular invasion is considered to be a valuable surrogate for lymph node stage in cases where nodes have not been removed for examination. In patients whose axillary nodes are tumour free on histological examination there is a correlation between the presence of lymphovascular invasion (LVI) and early recurrence.

Recently, Lee et al.³⁷ assessed the prognostic value of LVI in a group of 2760 patients with node-negative breast cancer with long-term follow-up (median 13 years). This study demonstrated a strong association between poor histological grade and younger age with LVI-positive cancers. LVI was prognostically significant and was independent of grade, size and type for overall survival (see Fig. 2.4).

Figure 2.4 LVI and breast cancer-specific survival.

Molecular/predictive factors

Despite the overall association between molecular markers with prognosis and outcome, they are limited in their ability to capture the nuances of the complex cascade of events that drive the clinical behaviour of breast cancer. Morphological factors are unable to predict response to systemic treatments, and tumours of apparently homogeneous morphological characteristics still vary in response to therapy and have distinct outcomes. The use of strategies ranging from hormone therapy to chemotherapy and recently to novel receptor-directed therapies and vaccines relies on the expression of predictive factors by the cancer, either individually (e.g. oestrogen receptors (ERs), progesterone receptors (PRs), human epidermal growth factor receptor (HER-2) and epidermal growth factor receptor (EGFR)) or globally (e.g. high-throughput gene expression assays). These molecular markers are used not only to guide treatments but are also being used to monitor response and detect relapse. It is envisaged that molecular predictive markers may form the basis of tailoring an individual’s adjuvant therapy based on the genetic fingerprint of their cancer.

Recently, Tsutsui et al.⁴⁷ reported on a large series of 1029 patients (with adjuvant therapy) and found EGFR to be of independent prognostic value irrespective of nodal status. In contrast, studies by Rampaul et al.⁴⁸ and Ferrero et al.⁴⁹ (without adjuvant therapy) showed EGFR to be of no prognostic value (these latter series also incorporated grade and had longer follow-up).

The predictive value of this marker for hormone resistance or responsiveness is better defined. EGFR tumours are considered more resistant to endocrine therapy, and there exists an inverse relationship with EGFR-negative cancers (they are more likely ER positive), being more often sensitive to endocrine manipulation. Though some studies have been able to demonstrate that EGFR status is a predictor of tamoxifen failure and even response rates,⁵⁰ there are conflicting data from well-designed level II studies⁵¹ that have shown no value of EGFR in predicting the efficacy of tamoxifen in high-risk postmenopausal women.

Perhaps the most exciting use of EGFR status and indeed the driving impetus for clinicians and scientist to measure it accurately is its putative role in defining those who should respond to novel EGFR-directed therapies.

HER-2

HER-2 is an important target in the development of a variety of new cancer therapies, which include monoclonal antibody (mAb)-based therapy, small-molecule drugs directed at the internal tyrosine kinase portion of the HER-2 oncoprotein, and vaccines. The most widely known HER-2-directed therapy is trastuzumab (Herceptin; Genentech, South San Francisco, CA, USA). Trastuzumab is a humanised recombinant mAb that specifically targets the HER-2 extracellular domain. There are a variety of techniques available to determine HER-2 status in breast cancer, some of which are employed for research purposes only. In diagnostic pathology laboratories HER-2 status is assessed routinely either by IHC, which assesses expression of the HER-2 oncoprotein, and fluorescence in situ hybridisation (FISH), which measures the number of HER2 gene copies per chromosome 17 or gene amplification. Modifications of ISH using colorimetric detection are being developed, including chromogenic in situ hybridisation (CISH) and silver-enhanced in situ hybridisation (SISH).

Methods of HER-2 testing: Current guidelines for HER-2 testing ⁵² specify the methods that are suitable to detect either the HER-2 protein (by IHC) or gene amplification (using FISH or other in situ methods). Guidelines stress the need for stringent, reproducible and consistent criteria for testing.

Immunohistochemistry for HER-2 testing: Among the methods in use for determining HER-2 status, IHC is the most widely used. In studies employing various commercially available antibodies, a wide variety of sensitivity and specificity in fixed paraffin-embedded tissues is seen.^53,⁵⁴ Antigen retrieval techniques are currently not standardised and they introduce the potential for false-positive staining. Nonetheless, IHC possesses many advantages to support its widespread adoption: (i) it allows for the preservation of tissue architecture and so can be used to identify local areas of overexpression within a heterogeneous sample, and can distinguish between HER-2 positivity in in-situ and invasive cancer; (ii) it is applicable to routine patient samples, facilitating use as a diagnostic test, and this allows prospective and retrospective research studies of HER-2 status to be undertaken.

Two Food and Drug Administration (FDA)-approved IHC tests for determining HER-2 status are available: HercepTest (DAKO, Carpeteria, CA, USA), based upon a polyclonal antibody; and CB11 (Pathway, Ventana Medical Systems, Tucson, AZ, USA), based upon a monoclonal antibody. The National Comprehensive Cancer Network guidelines ⁵⁵ classify an IHC score of 0 or 1 + as representing HER-2-negative status, 3 + as positive, while 2 + is equivocal. Positive staining is defined as strong, continuous membranous expression of HER-2 in at least 10% of tumour cells. However, a joint report from the American Society of Clinical Oncology (ASCO) and the College of American Pathologists (CAP)⁵² specified a threshold of > 30% strong circumferential membrane staining for a positive result. If both uniformity and a homogeneous, dark circumferential pattern are seen, resultant cases are likely to be amplified by FISH as well as positive for HER-2 protein expression. The equivocal range for IHC (score 2 +), which may include up to 23% of samples, is defined as complete membrane staining that is either non-uniform or weak in intensity but with obvious circumferential distribution in at least 10% of cells. Equivocal or inconclusive results should be tested by FISH. Consistent with previous guidelines, a negative HER-2 test is defined as either an IHC result of 0 or 1 + for cellular membrane protein expression (no staining or weak, incomplete membrane staining in any proportion of tumour cells), though up to 5% of 1 + on IHC may be FISH positive.

In situ hybridisation for HER-2 testing: FISH and CISH measure directly the number of HER2

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