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Implementation of molecular-based intraoperative sentinel node analysis: practical, logistical and controversial issues.

Introduction

Sentinel node biopsy Sentinel node biopsy
A newer procedure performed in order to determine whether breast cancer has spread to auxiliary (underarm) lymph nodes. A blue radioactive tracer and/or blue dye is injected into the area of the breast tumor.
 is the established standard for staging the axilla axilla /ax·il·la/ (ak-sil´ah) pl. axil´lae   [L.] the armpit.ax´illary

ax·il·la
n. pl. ax·il·lae
See armpit.
 in clinically node-negative breast cancer [1]. It can be carried out in all patients who have undergone preoperative pre·op·er·a·tive
adj.
Preceding a surgical operation.



preoperative

preceding an operation.


preoperative care
the preparation of a patient before operation.
 assessment showing no evidence of axillary ax·il·lar·y
n.
Relating to the axilla.


Axillary
Located in or near the armpit.

Mentioned in: Mastectomy


axillary

of or pertaining to the armpit.
 disease on imaging, or needle biopsy

The ability to analyse the sentinel node status during the operation allows the patient to have a therapeutic axillary clearance at the same time if malignancy is detected within the node. This potentially avoids a delayed second hospital admission, anaesthetic and operation. Between 25% and 30% of patients undergoing sentinel node biopsy have a positive biopsy [2,3].

Intraoperative techniques for assessing sentinel nodes date back over a decade. Today's established techniques include cytological cytological, cytologic

pertaining to cytology.


cytological examination
examination of material for purposes of cytology. Carried out on cerebrospinal fluid, joint fluid, aspirates of body cavities and cystic lesions.
, histological and, most recently, molecular-based analyses. A review of the different techniques has reported differences in sensitivity and specificity for frozen-section and imprint cytology [4,5], with each having specific advantages and disadvantages (Figure 1). Molecular techniques have the potential to achieve greater sensitivity, in part due to the reduction in sampling error associated with a less thorough examination of a node [6].

We have been carrying out sentinel node biopsy in our unit since 1999 (ALMANAC Trial) and since 2004 as standard treatment. We introduced the GeneSearch [TM] Breast Lymph Node Assay (BLNA BLNA Bilateral Lightwave Network Analyzer
BLNA Busy Line No Answer
) (Veridex, Warren, New Jersey, USA), the first commercially available qRT-PCR (quantitative reverse transcriptase- polymerase chain reaction) assay for intraoperative assessment of sentinel lymph node Sentinel lymph node
The first lymph node to receive lymph fluid from a tumor. If the sentinel node is cancer-free, then it is likely that the cancerous cells have not metastasized.

Mentioned in: Vulvar Cancer
 material, into our breast unit in November 2007. The kit uses two gene markers, mammoglobin (MGB MGB Mini-Gastric Bypass
MGB Minor Groove Binder (molecular biology)
MGB Manual Gearbox
MGB Matthew Good Band
MGB May God Bless
MGB Medial Geniculate Body
MGB Medium Girder Bridge
MGB Motor Gun Boat
MGB Microsoft Global Briefing
) 1, a breast-specific marker, and cytokeratin (CK) 19, an epithelial marker. This FDA-approved/CE-marked test detects mRNA from the marker genes that are expressed in breast and epithelial cells but not in lymphoid lymphoid /lym·phoid/ (lim´foid) resembling or pertaining to lymph or tissue of the lymphoid system.

lym·phoid
adj.
Of or relating to lymph or the lymphatic tissue where lymphocytes are formed.
 cells. The other commercially available test. One Step Nucleic Acid Amplification (OSNA OSNA Ornithological Societies of North America
OSNA Oregon Student Nurses Association
OSNA Open Service Network Architecture
) (Sysmex, Kobe, Japan) works in a similar fashion but only looks at one gene marker: CK19.

Business case

Before any new intervention or procedure is carried out in a trust, internal governance approval has to be sought and a business case made that evaluates the pros and cons. Costs have to be taken into account for equipment, additional personnel required and theatre utility, and these balanced against the additional costs associated with a second hospital admission for a delayed axillary clearance. On a broader scale, the additional clinical benefits being provided to a local population should be considered. The current tariff structure of remuneration makes intraoperative testing non-profitable from an individual trust viewpoint, but it becomes cost neutral, or offers minor savings to the health economy, by reducing the additional costs associated with a second hospital admission [7].

Validation, training and learning curve

Our biomedical bi·o·med·i·cal
adj.
1. Of or relating to biomedicine.

2. Of, relating to, or involving biological, medical, and physical sciences.
 scientists had to undergo formal training to carry out the technique. This training was provided by Johnson & Johnson, the parent company of Veridex, as a 3-day course in Strasbourg. Newer members of the team can now be trained in house by those carrying out the procedure regularly. There was a short learning curve, with 98% concordance being reached after 50 cases, and we carried out 125 cases in our unit before we went live, performing immediate axillary clearance when the intraoperative testing was positive. This enabled us to fully validate our intraoperative test results with conventional histological analysis and look at any safety aspects. Since introduction of the procedure in the unit, the time taken from removing the sentinel node to having a definitive positive or negative result has been reduced from 60 minutes to 30 minutes. Positive results can be obtained even earlier if there is early detection of enough positive material.

Equipment required

The hardware costs for intraoperative molecular-based testing vary from approximately 30,000 [pounds sterling] to 60,000 [pounds sterling], depending on which method of testing is adopted. The equipment includes basic standard laboratory consumables, reagents, homogeniser, weighing scales, printer, freezer and a cell cycler. Although not essential, the equipment and laboratory will ideally be set up in close vicinity to the operating theatre. This facilitates cross-specialty learning from the theatre staff, surgeons and biomedical scientists and, in our unit, has definitely helped in implementing the new technique.

Methodology

Patient consent is obtained preoperatively from all patients undergoing sentinel node biopsy so that material can be used and stored as required in the future. The sentinel nodes are identified using dual localisation (programming) localisation - (l10n) Adapting a product to meet the language, cultural and other requirements of a specific target market "locale".

Localisation includes the translation of the user interface, on-line help and documentation, and ensuring the images and
 with a blue dye and radioactive marker. The nodes are then harvested, and each node removed can start being processed without any need to wait for all sentinel nodes to be retrieved. The fat surrounding the node is removed by either the surgeon or biomedical scientist. The nodes are sectioned at 2-mm intervals, and alternate slices analysed using the BLNA. Remaining slices of node are sent for histology, including CK19 immunohistochemistry at multiple levels. For molecular analysis, the samples are weighed, homogenised and RNA RNA: see nucleic acid.
RNA
 in full ribonucleic acid

One of the two main types of nucleic acid (the other being DNA), which functions in cellular protein synthesis in all living cells and replaces DNA as the carrier of genetic
 eluted. The PCR PCR polymerase chain reaction.

PCR
abbr.
polymerase chain reaction


Polymerase chain reaction (PCR) 
 mix is then prepared with the primers and probes and finally the RNA is added before being placed in the cell cycler. The product of the RT-PCR reaction is quantified by fluorescence and recorded in graph and numerical form on a computer screen. Positive and negative controls are used in all PCR runs to ensure quality control issues are met anc to assess for any contamination. Final results of each run for each node are classified as positive (micrometastases/ macrometastases), negative--or invalid, if the controls fail.

Patient counselling/leaflets

Patients who undergo immediate axillary clearance will have an axillary drain placed at the time of surgery. When the patient recovers from their anaesthetic, they are immediately aware that they have had more extensive surgery than they had been expecting, with the implication that their cancer is no longer confined to the breast. Although it has not yet been formally evaluated, this can potentially lead to increased anxiety--both from a prognostic viewpoint and because of the increased likelihood of additional adjuvant adjuvant /ad·ju·vant/ (aj?dbobr-vant) (a-joo´vant)
1. assisting or aiding.

2. a substance that aids another, such as an auxiliary remedy.

3.
 therapies in the form of chemotherapy or radiotherapy. Patients should therefore be given enough information prior to the surgery to understand the implications of intraoperative testing. We have designed and distributed written patient information leaflets to all patients undergoing intraoperative testing as an additional resource. Patients are also made fully aware of the possibility of discordance discordance /dis·cor·dance/ (dis-kord´ans) the occurrence of a given trait in only one member of a twin pair.discor´dant

dis·cor·dance
n.
 between the intraoperative test result and final histology, and the implications of this.

Discordant patient results

A prospective database has to be set up and maintained in order to enable the analysis of discordant results. Our previously published results have shown overall concordance of 95% between histological analysis, including micrometastases, and molecular analysis (sensitivity 96%, specificity 95%) [7). If there is discordance between the intraoperative test and conventional histology, this is likely to be due to sampling error with each evaluating alternate slices of the node. As the node is sectioned in 2-mm slices, it is likely that any discordant result is based on tissue less than 2 mm thick and therefore a positive result on molecular analysis with no histological evidence of metastases Metastasis (plural, metastases)
A tumor growth or deposit that has spread via lymph or blood to an area of the body remote from the primary tumor.

Mentioned in: Malignant Melanoma
 could be reasonably treated as micrometastatic disease. Where there is discordance, there is no way of knowing whether the test has failed (although the positive and negative controls confirm the internal validity of each test run) and has produced a false-negative or a false-positive result, or whether the finding is due to sampling error. We have so far resisted testing the entire node using the molecular-based assay but this may be something that will require further exploration in the future.

Planning lists

Predicting and stratifying which patients are more likely to have positive sentinel nodes, based on preoperative tumour grade, size and presence of lymphovascular invasion, can be helpful when planning a list. A list can potentially over-run if two or three patients undergoing sentinel node biopsy in a full day's operating session end up having an immediate axillary clearance--or under-run if all sentinel nodes are negative. Flexible working patterns of theatre staff can be useful if there are regular overruns. Sentinel nodes are harvested prior to any surgery on the breast which allows evaluation of the sentinel nodes in time for the surgeon to carry out appropriate surgery to the breast. We have found that this does not increase the theatre time if the sentinel node is negative, and that 40 minutes' additional theatre time is required per axillary clearance [7]. Patients having sentinel node biopsy without axillary clearance are often fit to be discharged on the day of their surgery. Those having additional immediate axillary clearance are likely to remain in hospital for an extra day or two.

Issues around micrometastases

There is outcome controversy as to the best practice when dealing with micrometastases in the sentinel node and there are ongoing prospective studies evaluating their prognostic significance. However, studies have shown an incidence of 10-15% of finding further involvement of non-sentinel nodes when axillary clearance is subsequently carried out [8]. The current American Society of Clinical Oncology (ASCO ASCO American Society of Clinical Oncology
ASCO Association of Schools and Colleges of Optometry (since 1941; Rockville, Maryland)
ASCO Australian Standard Classification of Occupations
ASCO Automatic Switch Company
) guidelines state that all patients who have sentinel node micrometastases should undergo axillary clearance. The software initially provided with the Veridex kit was unable to distinguish whether a positive result was indicative of micrometastases or macro metastases. Modification to the software and interpretation of the analyses now allows this distinction.

Veridex withdrawal

In December 2009, Johnson & Johnson indicated that it would stop the manufacturing of the BLNA kits and would be withdrawing its product support in August 2010. The reason for this was not publicly stated but was most likely the result of poor uptake in the USA. This led to our unit looking at alternative methods for carrying out the intraoperative analysis. In collaboration with the pathology department in a separate NHS trust, a new assay, Metasin, was developed and validated. Metasin was based on the technique of qRT-PCR along similar lines to Veridex [9]. This was validated using BLNA kits in parallel, as controls. The data we have analysed so far has indicated that the assay is as robust and safe as Veridex, and significantly cheaper.

Discussion

Intraoperative analysis of sentinel nodes can be carried out by a number of different techniques. This allows patients to undergo a single operation to treat and stage the breast cancer. There is widespread variation in practice in the UK, with many units not actually carrying out any form of intraoperative testing, but there is a trend towards using molecular-based assays as the technique of choice, with increasing evidence of its greater sensitivity. It is likely to become the gold standard for analysis over conventional histological and cytological techniques. Automation and molecular whole-node analysis may reduce the intense workload of the histopathologist but this may have to be tempered with the loss of some prognostic information and also the potential loss of long-term archiving of paraffin blocks. However the choice of best technique of intraoperative nodal assessment for an individual unit is likely to be determined by cost, expertise and local availability of service.

Acknowledgements

The authors would like to thank: I Cree, Consultant Pathologist, M Jeffrey, Consultant Pathologist, A McDowell, Biomedical Scientist, and G Gabriel, Biomedical Scientist, of Queen Alexandra Hospital, Portsmouth, who have all been instrumental in the facilitation of intraoperative sentinel node testing in our unit.

References

[1.] National Institute for Health and Clinical Excellence. Guideline 80: Early and locally advanced breast cancer. Diagnosis and treatment. National Institute for Health and Clinical Excellence, 2009. Available at: www.nice.org.uk/nicemedia/pdf/ CG80NICEGuideline.pdf (last accessed November 2010).

[2.] Turner R, Ollila D, Krasne D and Giuliano A. Histopathologic validation of the sentinel lymph node hypothesis for breast carcinoma. Ann Surg. 1997, 226, 271-278.

[3.] Mansel R, Fallowfield, L, Kissin M eta/. Randomised Adj. 1. randomised - set up or distributed in a deliberately random way
randomized

irregular - contrary to rule or accepted order or general practice; "irregular hiring practices"
 multicentre trial of sentinel node biopsy versus standard axillary treatment in operable operable /op·er·a·ble/ (op´er-ah-b'l) subject to being operated upon with a reasonable degree of safety; appropriate for surgical removal.

op·er·a·ble
adj.
 breast cancer: the ALMANAC Trial. I Natl Cancer Inst, 2006, 98, 599-609.

[4.] Tew K, Irwig L Matthews Aef al. Meta-analysis of sentinel node imprint cytology in breast cancer. Br J Surg, 2005, 92, 10681080

[5.] Aihara T, Munakata S, Morino H and Takatsuka Y. Comparison of frozen section and touch imprint cytology for evaluation of sentinel lymph node metastasis metastasis /me·tas·ta·sis/ (me-tas´tah-sis) pl. metas´tases  
1. transfer of disease from one organ or part of the body to another not directly connected with it, due either to transfer of pathogenic microorganisms or to
 in breast cancer. Ann Surg Oncol, 2004, 11, 747- 750.

[6.] Layfield D, Agrawal A, Roche H and Cutress R. Intraoperative assessment of sentinel lymph nodes in breast cancer. Br J Surg, 2010. September [epub ahead of print], doi: 10.1002/bjs.7229.

[7.] Cutress R, McDowell A, Gabriel F ef al. Observational and cost analysis of the implementation of breast cancer sentinel node intra-operative molecular diagnosis. J Clin Pathol, 2010, 6, 522529

[8.] Cserni G, Gregori D, Merletti F et al. Meta-analysis of non-sentinel node metastases associated with micrometastatic sentinel nodes in breast cancer. Br J Surg, 2004, 91, 1245-1252.

[9.] Al-Ramadhani S, Balaraman P, Mascall A et al. Validation of metasin: a novel real-time PCR assay for metastatic Metastatic
The term used to describe a secondary cancer, or one that has spread from one area of the body to another.

Mentioned in: Coagulation Disorders


metastatic

pertaining to or of the nature of a metastasis.
 carcinoma. Poster presentation (P10) at Pathological Society of Great Britain and Ireland Great Britain and Ireland are the two largest islands in the British Isles. A former state, the United Kingdom of Great Britain and Ireland, was composed of the political union of the two. , Winter meeting, 2010. Available at: www.pathsoc org/files/meetings/winter2010/05.01.106552ProgMAINv10(web). pdf (last accessed October 2010).

Avi Agrawal, Martin Wise and Constantinos Yiangou

Department of Breast Surgery, Portsmouth Hospitals NHS Trust, Hampshire, UK

Correspondence to: Avi Agrawal

Breast Unit, Queen Alexandra Hospital

Southwick Hill Road, Cosham, Portsmouth

Hampshire P06 3LY, UK

(email: avi.agrawal@porthosp.nhs NHS
abbr.
National Health Service


NHS (in Britain) National Health Service
.uk)
Figure 1: Advantages and disadvantages of various techniques of
intraoperative sentinel lymph node assessment

Technique          Advantages                  Disadvantages

Frozen-section     Very established technique  Expensive
-76% sensitivity   Equipment widely available  Loss of tissue
-99% specificity                               Labour-intensive
                                               Operator-dependent
                                               Histopathologist
                                                 and technician
                                                 required

Imprint/scrape     Low preparation time        Cytologist required
  cytology
-63% sensitivity   Low cost                    Higher false-negative
-99% specificity   No loss of tissue             results in micro
                                                 metastatic and
                                                 lobular carcinoma

Molecular-         Reduced sample error        High initial capital
  based assay                                    outlay
-96% sensitivity   Trained technician          Requires specific
                     only required               training/learning
                                                 curve
-95% specificity   Greater volume analysis     Unable to assess
                     of lymph node               extranodal/
                                                 extracapsular spread
                   No intra-observer           No archival paraffin
                     variation                   section material
                                                 available
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Title Annotation:Feature Article
Author:Agrawal, Avi; Wise, Martin; Yiangou, Constantinos
Publication:Advances in Breast Cancer
Article Type:Disease/Disorder overview
Date:Dec 1, 2010
Words:2302
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