SPECIAL FEATURE Recent advances in sentinel node biopsy for breast cancer

One-step nucleic acid amplification assay (OSNA) for sentinellymph node biopsy

Yasuhiro Tamaki

Received: 19 June 2012 / Accepted: 27 June 2012

� The Japanese Breast Cancer Society 2012

Abstract The one-step nucleic acid amplification assay

(OSNA) has been developed for the intraoperative rapid

detection of sentinel lymph node metastasis. Pooled analysis

of recent studies comparing OSNA with pathology indicated

that OSNA is as accurate as pathology (96.3 % concordance

rate) and is useful for making the decision to omit axillary

dissection for OSNA-negative patients (97.4 % negative

predictive value). The advantage of OSNA over pathology is

that the former allows the semiquantitative evaluation of

total tumor volume in the node when a whole node is

examined. OSNA is expected to be a powerful tool for the

estimation of risk of non-sentinel lymph node metastasis and

also patient prognosis, though further studies about this issue

with larger numbers of patients is needed.

Keywords Breast � Cancer � Sentinel � OSNA

Introduction

Sentinel lymph node biopsy (SLNB) has been performed as a

standard surgical procedure for early breast cancer. Axillary

dissection can be omitted for patients with negative sentinel

lymph nodes (SLNs) to avoid postoperative complications,

though it is considered necessary for those with positive SLNs

to achieve local control and to accurately assess the stage of

the disease. Therefore, accurate and rapid assessment of SLN

metastasis during operation is important for making the

decision to perform or omit axillary completion. For this

purpose, a rapid molecular detection system targeting mRNA

of cytokeratin 19 (CK19)—the one-step nucleic acid ampli-

fication assay (OSNA)—was developed by Sysmex (Kobe,

Japan), and first reported by Tsujimoto et al. [1]. OSNA

showed equal accuracy to pathological examination for the

detection of lymph node metastasis, and is expected be an

alternative method to pathology for SLNB detecting micro-

metastasis (MIC) with higher sensitivity and less labor.

On the other hand, management of patients with positive

SLNs has been changing over the last year. The newest version

of the National Cancer Comprehensive Network Guidelines

for breast cancer recommends to consider no further surgery

for the axilla of a patient meeting all of the following criteria,

i.e., who has T1 or T2 tumor with 1 or 2 positive SLNs,

undergoes breast-conserving therapy and whole breast radi-

ation therapy, and received no neoadjuvant chemotherapy,

based on the results of the American College of Surgeons

Oncology Group (ACOSOG) Z0011 trial reported by Giuli-

ano et al. in 2011 [2]. Furthermore, detailed analysis of SLNs

obtained in the former Z0010 trial showed that occult

metastases in SLNs detected by immunohistochemistry (IHC)

had no impact on overall survival of women receiving breast-

conserving surgery and SLNB [3]. These findings indicated

that intraoperative examination for metastasis, especially

MIC, in SLNs is not needed for patients with 1 or 2 SLNs.

In these circumstances, one should consider how and why to

use OSNA in clinical situations. In this paper, the reliability and

utility of OSNAfor SLNexaminationare reviewed, and its future

perspectives in light of recent studies of SLNB are discussed.

Materials and methods

A search for literature relevant to OSNA was performed

using a computerized search engine PubMed with keywords

Y. Tamaki (&)

Department of Breast and Endocrine Surgery,

Osaka Medical Center for Cancer and Cardiovascular Diseases,

1-3-3, Nakamichi, Higashinari-ku, Osaka 537-8511, Japan

e-mail: tamaki-ya@mc.pref.osaka.jp

123

Breast Cancer

DOI 10.1007/s12282-012-0390-x

‘‘OSNA’’ and ‘‘Breast’’. Twenty-one original (2 were non-

English) and one review articles were found. There were

nine comparison studies of OSNA with pathology, and

seven observational studies for its clinical use in SLNB.

Reliability of OSNA compared with pathology

Between 2008 and 2012, there are nine studies comparing

OSNA with pathological examination (Table 1). Dissected

axillary lymph nodes were used in three studies [4–6], and

SLNs used in six [7–12]. Lymph nodes were sliced into 4

pieces of 1- or 2-mm thickness (a, b, c, d). Two slices (a, c)

were examined immediately or after storage in liquid

nitrogen at -80 �C. The other two slices (b, d) were fixed

and embedded for pathological examination. Multi-step

sections were examined with hematoxylin and eosin (HE)

staining basically, and additional analysis using IHC was

performed in several studies. Judgment of positive or

negative metastasis status by OSNA was done according to

the cutoff level indicated by Tsujimoto, i.e., negative for a

case with less than 2.5 9 102 CK19 mRNA copies/

lL, ? positive with at least 2.5 9 102 CK19 mRNA cop-

ies/lL and less than 5 9 103 CK19 mRNA copies/lL, and

?? positive with at least 5 9 103 CK19 mRNA copies/lL

[1].

The results of the studies are shown in Table 1. The

sensitivity, specificity, and accuracy of OSNA for the

detection of metastasis in lymph nodes ranged from 75.0 to

98.1, 91.7 to 98.4, and 91.8 to 96.3 %, respectively, and

OSNA is statistically judged as being reliable for the

pathological examination. From the pooled analysis of the

nine studies, the corresponding values are 87.9, 94.8, and

93.6 % (Table 2), and these values may become higher

because those in each study were higher after further

analysis for discordant cases. However, there still were a

few discordant cases. Because different samples from one

node are examined by means of each method, discordant

results caused by tissue allocation bias are inevitable.

When OSNA is used for SLNB, local recurrence caused by

false negative assessment must be a significant problem.

From this point of view, the high negative predictive value

of OSNA (97.4 %) can be an assurance for making the

decision to omit axillary dissection. From the results of

these comparison studies, the cutoff values of CK19

mRNA copy numbers are considered appropriate, and

OSNA can be an alternative to pathology for SLN

examination.

Assay time

The time required for the assay is also an important factor

for the intraoperative immediate examination for SLNs.

Amplification of CK19 mRNA in the assay takes only

16 min. However, it takes additional time to homogenize

each lymph node and put each sample into a test tube.

Therefore, the total assay time depends on the number of

lymph nodes. Several reports showed it was 32–33 min for

one node, and 37–40 min for 2 nodes [7–9, 11]. The

average time for OSNA in SLNB was reported as 36 min

[12].

Table 1 Comparison studies of the OSNA assay with pathological examination

Author Subjects Sensitivitya (%) Specificitya (%) Accordancea (%) NPVa (%) PPVa (%) Ref no.

Visser 346 ALNs 95.3 94.7 94.8 98.9 80.3 [4]

Schem 343 ALNs 98.1 91.7 91.8 99.1 80.0 [5]

Tamaki 450 ALNs 87.5 94.1 92.9 97.2 76.1 [6]

Feldman 1,044 SLNs 77.5 95.8 93.4 96.6 73.8 [7]

Khaddage 46 Pts

80 SLNs

80.0

88.2

97.2

98.4

93.5

96.3

94.6

96.9

88.9

93.8

[8]

Snookb 194 Pts

395 SLNs

89.8

91.7

94.5

96.9

93.4

95.9

96.5

98.1

84.6

86.8

[9]

Le Frere-Belda 233 Pts

503 SLNs

76.8

80.9

88.0

93.9

86.3

92.2

94.9

97.2

58.9

65.4

[10]

Bernet 181 SLNs 89.2 95.8 94.5 97.2 84.6 [11]

Sagara 61 SLNs 75.0 98.0 93.4 94.1 90.0 [12]

Pts patients, ALNs axillary lymph nodes, SLNs sentinel lymph nodes, NPV negative predictive value, PPV positive predictive value,

Ref no. reference numbera Values were calculated on the basis of the original data reported, if they were not shown in the original papersb Data shown were calculated after discordant case analysis and the exclusion of samples affected by tissue allocation bias

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Clinical use of OSNA for sentinel lymph node biopsy

How to examine SLNs using OSNA in the clinical setting

is another important issue, and still controversial. Theo-

retically, a whole-node examination using OSNA will be

the best, because it provides a complete assessment of

tumor volume in the node. This is a great advantage of

OSNA over pathology; however, pathology is still a gold

standard at present. Furthermore, very rare cases do not

express CK19 mRNA, and there might also be some

mechanical or human error in OSNA, which may account

for loss of data resulting in failure of risk assessment of the

patient. It may be recommended to examine at least one

slice of each SLN by pathology for a while.

There are seven reports showing the data of OSNA used

for SLNB in the clinical setting (Table 3). In four reports,

almost all parts of the SLN were examined by means of

OSNA except for a central 1-mm slice, which was sampled

for pathological assessment [8, 13–15]. A whole-node

examination by means of OSNA was performed in

remaining three [12, 16, 17]. Generally, OSNA detected

greater numbers of patients with positive SLNs than

pathology. In patients who underwent axillary dissection,

10–22 % of patient with positive SLNs were assessed as

OSNA ? and 34–54 % of those with OSNA ?? SLNs had

non-SLN metastases. Previous reports examining SLNs by

means of pathology with HE and/or IHC showed similar

results, i.e., 20–23 % of patients with MIC in SLNs and

45–50 % of those with macrometastasis in SLNs had non-

SLN metastases [18, 19]. Thus, the assessment of SLN

metastasis by means of OSNA can be a promising tool for

the prediction of non-SLN metastasis.

Discussion

A lot of comparison studies revealed that OSNA is as

reliable as pathology, and the observational studies indi-

cated that OSNA can be an alternative method to pathology

for the examination of SLNs as described. However, there

still exist some controversies to discuss. One of those is the

false negative assessment by OSNA caused by low

expression of CK19. The incidence of low expression of

CK19 protein in ductal and lobular breast cancer was

reported as about 1.2 %, and significantly associated with

the triple-negative subtype [20, 21]. However, the inci-

dence of discordant cases between OSNA and pathology

caused by low expression of CK19 was as low as 0.5 %

[6, 9], and this difference may caused by the difference of

expression of protein and mRNA of CK19. Recent thera-

peutic guidelines for breast cancer recommend chemo-

therapy as an adjuvant treatment for the majority of

patients with triple-negative subtype of invasive cancer.

Therefore, the clinical effect of this false negative may

become minimal. However, to reduce the risk of underes-

timation of the stage of disease, the use of touch print

cytology or HE staining for a central slice together with

OSNA, or CK19 immunostaining for needle biopsy spec-

imens obtained before surgery is recommended [22].

The clinical significance of MIC detected by OSNA is

also a controversial issue. A lot of papers discussed the

association between MIC in axillary lymph nodes and

clinical outcomes. Some showed that MIC is associated

with poorer prognosis [23–25], and others advocated that

Table 2 Pooled analysis per node of 9 studies comparing OSNA and

pathology

Pathologically

positive

Pathologically

negative

Total

OSNA ? and

??

516 146 662

OSNA - 71 2,668 2,739

Total 587 2,814 3,401

Sensitivity = 87.9 %, specificity = 94.8 %, accuracy = 93.6 %, neg-

ative predictive value = 97.4 %, positive predictive value = 77.9 %

Table 3 Clinical studies of routine use of OSNA for sentinel lymph node biopsy

Author No. of Pt Material Detection rate (%) Non-SNL metastases (%) Ref no.

Pathology OSNA Pathology OSNA OSNA ? OSNA ??

Khaddage 197 1-mm slicea Remainder 12.7 21.3 10.3 54.0 [8]

Tamaki 417 1-mm slicea Remainder 15.8 22.5 17.6 44.0 [13]

Godey 258 1-mm slicea Remainder 15.5 26.4 10.8 34.4 [14]

Vogue 55 1-mm slicea Remainder 10.9 52.7 – – [15]

Osako 531 (618 HCP) Whole node 17.6 22.8 – – [16]

Sagara 248 NT Whole node – 20.2 – – [12]

Castellano 110 (169 HCP) Whole node 27.2 29.0 22 42 [17]

No. of Pt number of patients, HCP historical control patients, NT not tested, Ref no. reference numbera A central 1-mm-thick slice of each sentinel lymph node was examined by pathology

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there is no relationship between them [3, 26, 27]. Weaver

et al. [28] performed further analysis for negative SLNs

obtained in the National Surgical Adjuvant Breast and

Bowel Project (NSABP) trial B-32, and reported that occult

metastases were significantly associated with both disease-

free and overall survival of the patients. However, they

concluded that additional evaluation with IHC for negative

SLNs to find occult metastasis is not needed, because the

magnitude of the difference in outcome was so small.

Although MIC may be actually associated with patient

prognosis, adjuvant systemic therapy and/or breast irradi-

ation may give some effect on the outcome of patients.

Thus, de Boer reported that adjuvant systemic therapy

could improve the 5-year disease-free survival of nearly

10 % of the patients with MIC [24]. It is important to select

appropriate adjuvant systemic therapy for each patient on

the basis of an accurate assessment of lymph node metas-

tasis. In this context, OSNA is suitable for SLN evaluation

because of its accuracy and easy operation. However, the

current assessment of OSNA for lymph node metastasis is

based on that of pathology, i.e., OSNA ? corresponds to

MIC and OSNA ?? to macrometastasis. Direct compari-

son between CK19 mRNA copy numbers and patient

prognosis is necessary to confirm the relationship between

tumor volume in the axillary node and survival of patients.

Whether or not to perform axillary dissection for

patients with SLNs assessed as positive by means of OSNA

becomes another issue for discussion. More than 10 % of

patients with SLNs assessed as ? positive by OSNA and

more than 30 % of those assessed as ?? positive were

revealed to have non-sentinel node metastasis, and axillary

dissection has been recommended for such patients.

Actually, Pepels et al. [29] warn that no additional axillary

treatment in patients with SLN micrometastases is associ-

ated with an increased 5-year regional recurrence rate. On

the other hand, the ACOSOG Z0011 trial showed that there

was no significant difference of both local recurrence rate

and overall survival between patients with one or two

positive SLNs who underwent additional axillary dissec-

tion and those who received no axillary surgery [3, 30].

This means that intraoperative assessment of SLNs itself is

no longer needed for patients who have only one or two

SLNs and undergo breast-conserving surgery with post-

operative radiation therapy. However, it is unknown whe-

ther axillary dissection can be omitted for patients with

positive SLNs who undergo mastectomy. Further studies

with greater numbers of patients are necessary to find the

answer to this question. The essential issue is accurate

assessment of metastasis for limited numbers of SLNs,

regardless of the timing of the examination of SLNs, to

choose appropriate adjuvant treatments, and OSNA is

useful for this purpose.

Application of OSNA for patients treated with neoad-

juvant chemotherapy is the next issue to be studied. There

are no published data examining the usefulness and accu-

racy of OSNA compared with pathology for patients with

neoadjuvant chemotherapy at present, except for one report

of a study using OSNA for such patients [31]. After neo-

adjuvant chemotherapy, lymph nodes often show severe

fibrotic change containing scattered tumor cells, which

need IHC to be confirmed. OSNA is expected to evaluate

accurate tumor volume remaining after chemotherapy

without regard to fibrotic change of the tissue.

The one-step nucleic acid amplification assay is a useful

and reliable tool to evaluate lymph node metastasis with

equal accuracy to conventional pathological examination,

and is expected to have an advantage over pathology in the

semiquantitative assessment of tumor burden when a whole

lymph node is examined. However, future studies are

needed to confirm the association among tumor volume in

SLNs assessed by OSNA, non-SLN metastasis, and patient

prognosis.

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