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Novel Intraoperative Molecular Test for Sentinel Lymph Node Metastases in Patients With Early-Stage Breast Cancer

Thomas B. Julian, Peter Blumencranz, Kenneth Deck, Pat Whitworth, Donald A. Berry, Scott M. Berry, Anne Rosenberg, Anees B. Chagpar, Douglas Reintgen, Peter Beitsch, Rache Simmons, Sukamal Saha, Eleftherios P. Mamounas, Armando Giuliano

From the Department of Human Oncology, Allegheny General Hospital/Allegheny Cancer Center, Pittsburgh; Department of Surgery, Jefferson University Hospital, Philadelphia, PA; Department of Surgery, Morton Plant Mease Healthcare, Clearwater; Department of Surgery, Lakeland Regional Medical Center, Lakeland, FL; Department of Surgery, South Orange County Surgical Medical Group, Laguna Hills; Department of Surgery, John Wayne Cancer Institute, Santa Monica, CA; Nashville Breast Center, Nashville, TN; Berry Consultants LLC, Houston; Department of Surgery, Dallas Surgical Group, Dallas, TX; Department of Surgery, University of Louisville, Louisville, KY; Department of Surgery, Weill-Cornell Breast Center, New York, NY; Department of Surgery, McLaren Regional Medical Center, Flint, MI; and Department of Surgery, Aultman Hospital, Canton, OH

Corresponding author: Thomas B. Julian, MD, Allegheny Breast Care Center, Allegheny General Hospital, 320 E North Ave, Pittsburgh, PA 15212; e-mail: tjulian{at}wpahs.org

	ABSTRACT

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Purpose An accurate, intraoperative sentinel lymph node (SLN) test could decrease delayed axillary dissections. Molecular tests may be more sensitive than current intraoperative tests but historically have not been rapid enough and have not been properly validated. We present the results from a large, prospective evaluation of the first rapid molecular SLN test, the Breast Lymph Node (BLN) Assay.

Methods A beta trial (n = 304) to determine the threshold levels of mammaglobin and cytokeratin 19 correlating with metastasis greater than 0.2 mm and a validation trial (n = 416) to validate the threshold cutoffs were conducted. Alternating portions from each SLN were processed for histology and the BLN Assay.

Results BLN Assay performance against extensive permanent-section histology verified by central pathology review was similar to that expected of standard permanent-section histology: sensitivity, 87.6%; specificity, 94.2%; positive predictive value, 86.2%; and negative predictive value (NPV), 94.9%. In 319 patients with both frozen-section hematoxylin and eosin results and BLN Assay results, the BLN Assay had higher sensitivity (95.6%) and NPV (98.2%) than frozen section (sensitivity, 85.6%; NPV, 94.5%). The assay can be performed in approximately 36 to 46 minutes for one to three nodes.

Conclusion The BLN Assay allows a rapid evaluation of 50% of each SLN. Comparison with permanent-section histology on adjacent node pieces evaluated by expert pathologists indicated that the BLN Assay was more sensitive than current intraoperative techniques while maintaining high specificity. These data indicate that the assay may be clinically useful for intraoperative or postoperative axillary lymph node dissection decisions.

	INTRODUCTION

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Sentinel lymph node biopsy (SLNB) provides accurate axillary staging while sparing node-negative patients the morbidity associated with axillary lymph node dissection (ALND).^1-5 Current intraoperative sentinel lymph node (SLN) tests have reportedly high false-negative rates,^6,7 leading to second operations when metastases are identified postoperatively by permanent-section hematoxylin and eosin (HE) or immunohistochemistry (IHC). This generates additional cost, time, and patient anxiety. Furthermore, the second operation may be difficult to perform because of edematous healing and difficult identification of anatomic landmarks.⁸

A rapid molecular test has been developed to reduce second-surgery ALNDs. The GeneSearch Breast Lymph Node (BLN) Assay (Veridex LLC, Raritan, NJ) is a real-time reverse-transcriptase polymerase chain reaction (RT-PCR) assay for detection of nodal metastases greater than 0.2 mm.^9-14 Results from two consecutive, independent, prospective, blinded clinical trials of the BLN Assay are presented here.

	METHODS

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The BLN Assay evaluation consisted of (1) a beta (cutoff) trial (n = 304 patients) to determine the threshold between normal and cancer-related levels of the markers mammaglobin (MG) and cytokeratin-19 (CK-19), and (2) a validation trial (n = 416 patients) to verify the chosen assay threshold cutoffs. Both trials were completed between July 2004 and December 2005 (United States Food and Drug Administration Pre-IDE I040002).

Male or female patients 18 years of age with a diagnosis of invasive adenocarcinoma of the breast and scheduled for an SLNB were considered for inclusion in this study. The study protocol was approved by the institutional review boards at the 12 participating United States sites, and patients gave written informed consent. Trained site personnel performed the BLN Assay, and results were blinded to all site personnel and central pathologists.

Node Processing
SLNs were identified and dissected according to the standard procedures of each site. Each node was sectioned along the short axis into multiple sections (1.5 to 3 mm thick), with alternating sections prepared for histology or the BLN Assay.

Tissue sections reserved for histology were processed for intraoperative cytopathology and/or frozen section, and for permanent-section HE (site slides) according to the standard procedures of each site. Patient management was based on the analysis of these slides. Additional HE slides (three levels, approximately 150 µm apart; central slides) were prepared and sent to the central study pathologists for evaluation. Sections were also stained for IHC, either on-site or by an outside laboratory (Albany Medical College, Department of Pathology, Albany, New York), and evaluated by either the site pathologist or the central pathologists.

Histologic Evaluation
Any histologic method has sampling limitations, thus there is no gold standard against which to evaluate new tests but only an existing standard. For these studies, additional node sampling by HE and central review of slides were adopted to optimize histologic assessment. A node was considered positive for metastasis when a focus greater than 0.2 mm was confirmed on the same slides by two independent pathologists. Two or three central pathologists independently evaluated each HE and IHC central slide, and at least one central pathologist had to confirm positivity on site slides. When either the final site slide result and/or the final central slide result revealed metastases, the overall histology result (OHR) for the node was positive.

BLN Assay Testing
The BLN Assay measures the expression of breast or epithelial cell-specific MG and CK-19 mRNA, respectively. Elevated levels of these analyte markers indicates the presence of metastases greater than 0.2 mm. An internal control marker, porphobilinogen deaminase, is also included in this PCR reaction.

Alternating sections from the same node were combined and immediately processed as fresh tissue as per the BLN Assay instructions for use.

BLN Assay Result Interpretation
All BLN Assay raw cycle threshold (Ct) data for the markers were provided to the sponsor (Veridex LLC) for analysis. If either or both CK-19 and MG are positive, the assay result is positive. Cutoff values were chosen based on the beta study data set of 304 patients. First, the two markers’ cutoffs were examined to find all combinations that resulted in an assay specificity of 95%, then sensitivity was maximized.

Additional Molecular Testing
Residual nodal homogenate or RNA extract from samples with discordant results between OHR and the BLN Assay were tested with independent molecular markers for nodal metastases.^15,16 This testing can provide a valuable indication of the percentage of disagreement in results likely caused by the assay and histology evaluating different parts of the node. For this confirmatory test, the cutoff values for its four markers (PIP, PDEF, B305D, and B726) were predetermined to achieve 100% specificity with a lower 95% CI of no less than 95%.^14,16 The results of this confirmatory test were not used in the calculations of BLN Assay performance. Technicians were blinded to previous results.

Analysis
The primary analysis was the overall performance of the BLN Assay (positive, negative, or invalid result) compared on a patient level with permanent-section histology (OHR; negative, or positive with metastases > 0.2 mm) in adjacent node tissue. This evaluation allows only a relative (v histology) estimate of the Assay's true performance, because different tissue was tested by the two methods, and histologic sampling has practical limitations.

The validation study design was Bayesian, which incorporates preplanned interim analyses and offers the potential benefit of ending a study earlier if predetermined success or futility criteria are met. The predetermined decision criteria for the validation study were as follows:

• When the posterior probabilities are larger than 0.985 that the assay sensitivity is at least 0.70 and that the specificity is at least 0.90, then the trial is stopped and considered a success.
  
• When the predictive probability of a successful trial given 700 patients is smaller than 0.05, the trial is stopped for futility.
  
• When neither of the above criteria are met, then the trial continues up to 700 patients, with preplanned analysis of every additional 50 patients.
  

Type I error was controlled at 0.05. The first analysis was to take place when at least 200 patients had complete data. Because of batched reviews of site slides by the central pathologists, 412 patients ’ data were available for the first analysis, at which time the assay performance met the criteria for trial success.

Patients with invalid BLN Assay results were considered assay negative for performance calculations and were included in the primary analysis because, in clinical use, invalid results would not provide conclusive evidence of metastases. Second-surgery ALND would be required if nodal metastases were found later by histology. Invalid results can arise from inappropriate sampling (eg, all adipose tissue or no nodal tissue) or processing, operator error, or equipment malfunction.

Bayesian statistical study design and analysis were performed by Berry Consultants, LLC (Houston, TX). Other statistical analyses and all data validation were performed by the sponsor.

	RESULTS

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Final Statistical Results
Final analysis included 416 total patients (four patients did not have full data sets for the first interim analysis), with an observed sensitivity of 0.876 (95% credible interval, 0.811 to 0.928) and an observed specificity of 0.942 (95% credible interval, 0.913 to 0.966).

Patients
Patient demographics and cancer history are provided in Table 1 for the beta and validation studies. Using the predetermined cutoffs, 421 validation study patients had final BLN Assay results of positive (29.5%), negative (62.5%), or invalid (8.1%). Many of the invalid results were a result of inexperience with the assay or with any molecular techniques for many operators. From reviewing the errors, the assay training program was revised during the study. One clinical site entered the study later, and the site personnel were trained with the new program. This site had two invalid results of 82 patients tested (2.4%). Critical histologic data were missing for five of the 421 patients, thus the performance of the BLN Assay was validated based on comparison with OHR for 416 patients.

Difficult-to-Detect Metastases
BLN Assay performance was analyzed for metastases that were found on only one set of study slides or that were IHC-positive and HE-negative. In the validation study, there were 19 patients for whom the OHR was positive for metastasis, but either the final site slide result or the final central slide result was negative. The BLN Assay identified metastases in seven (87.5%) of eight of these patients with macrometastases and in six (54.5%) of 11 with micrometastasis.

The OHR of only one patient in the validation study was negative by HE but positive (micrometastasis) by IHC. This patient was found negative by the BLN Assay.

BLN Assay Performance and Size of Metastases
In the validation study, for OHR-positive patients with macrometastases (n = 94), the sensitivity of the BLN Assay was 97.9% (95% CI, 92.5 to 99.7%), and for patients with micrometastases (n = 23), sensitivity was 56.5% (95% CI, 34.5 to 76.8%). Micrometastases are less distributed throughout the node, and greater discrepancies between test results are expected when different parts of the node are being evaluated. Similar discrepancies in detection of micrometastases were seen when comparing results from different histologic sections from the same node pieces. Site pathologists found metastases on site slides only in 75.0% (95% CI, 47.6 to 92.7%) of the patients who had micrometastases identified by central pathologists on nearby central slides.

The majority (nine of 15) of BLN Assay false-negative results were observed in patients with only one OHR-positive node, and that node contained micrometastases. Eight of these same 15 patients had nodes that were confirmed positive on one set of histology slides (site or central), but not both. Similarly, the majority (15 of 17) of BLN Assay–positive/OHR-negative patients were BLN Assay–positive for only one node.

Histologic Intraoperative Test Performance
Performance of the BLN Assay was compared with the performance of the histologic intraoperative tests routinely used at some of the clinical sites (Table 3). In the validation study, the sensitivity of the BLN Assay was approximately 10 percentage points higher than that of frozen section evaluation (95.6% v 85.6%; P = .04), whereas specificity was not statistically different (P = .09).

View larger version (14K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig 1. High correlation between GeneSearch BLN Assay cycle threshold (Ct) values and level of metastasis determined by histology. The Ct values plotted for mammaglobin (MG) and cytokeratin-19 (CK-19) are the lowest Ct values (highest measure of positivity) found on any node for a given patient. Ct values falling in the upper right boxed section on each graph are interpreted as negative in the BLN Assay. There are 97 and 132 overlapping patient data points at the 40/40 points on the graphs (ie, no fluorescence detected for MG or CK-19) for the beta and validation studies, respectively. Data points represent the histology result for each patient as shown in the key within the figure.

Assay Time to Result
Timing data collected during the study indicted that when an operator is experienced with the assay (has tested at least 30 patients’ samples), assay results are typically available within 36 to 46 minutes for one to three nodes. Timing near the lower end of this range is more likely if the testing lab is near the surgery suite and if there are one or two sentinel nodes. Additional nodes take approximately 4 to 6 minutes more each. The final result is automated and available on the computer screen or by print out.

	DISCUSSION

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Our validation trial used an innovative Bayesian methodology to monitor the results, stopping accrual when the goal of the trial was achieved. In comparison with the maximum sample size of 700 patients, the trial's actual size of 416 patients represented a savings of 284 patients and approximately 9 months. The BLN Assay's true performance evaluation is limited by the intrinsic necessity of different tissue sections being tested by the BLN Assay and by histology with the practical limitations of histologic sampling. Despite these limitations, the results of the present study demonstrate high sensitivity and specificity of the BLN Assay for the molecular detection of SLN metastases greater than 0.2 mm. The Assay's agreement with moderately extensive permanent-section histology performed on adjacent node portions was similar to the agreement between central and site histology slides in the present study. The BLN Assay demonstrated higher sensitivity than intraoperative histologic techniques, despite the disadvantage of sampling different node sections than permanent-section histology.

Specificities for the histologic/cytologic intraoperative tests were slightly, but not statistically, higher than that of the BLN Assay. Because of limited sampling, intraoperative histologic tests rarely identify metastases that are later missed by adjacent and more thorough permanent-section histology.¹⁷ In terms of false positives, nevi found in SLNs rarely can be mistaken on frozen section for metastases from infiltrating lobular carcinoma. In current practice, when the permanent-section HE result is negative and the intraoperative frozen-section test result is positive, it is more likely that intraoperative sampling exhausted the metastasis, not that the intraoperative result was incorrect. In the present study, it is likely that at least some BLN Assay–positive/OHR-negative results were due to the metastases being restricted to the portion of the node tested by the assay.

Evidence for this supposition is provided by the independent molecular test analyses on the residual BLN Assay samples. For most (73%) BLN Assay–positive/OHR-negative samples, the independent test confirmed positivity, suggesting that the node portions tested by the BLN Assay did contain metastases. Thus the sensitivity and specificity of the BLN Assay reported here against histology on adjacent node pieces is likely an underestimation of the assay's true performance.

Although permanent-section HE histology is the current standard, the number of histologic disagreements reported in the literature^18-21 and observed in the present study between site slide and central slide results illustrate the limitations of histology owing to imperfect node analysis. These results point out the importance of more thorough evaluation of the node to detect metastases. Use of the BLN Assay would minimize the effect of sampling because a greater portion (50%) of the node is evaluated.

Other sources contribute to the generation of histologic false positives, which may or may not be sources of false positives with molecular tests. In a recent study, one source of false positives in IHC analysis was the presence of benign epithelial cells in the SLNs that test positive for cytokeratin.²² The authors suggested that these displaced cells originate in intraductal papillomas and are transported into the SLNs. Because the BLN Assay is designed not to recognize tumor cell deposits 0.2 mm, this is unlikely to be a source of error in the test. Another issue is the subjective nature of evaluating histologic slides.¹⁸ The subjective element is eliminated for a software-generated molecular result.

The BLN Assay and histologic results were in high agreement for the number of SLNs positive in a given patient, providing evidence that the Assay cutoff values are appropriately chosen to detect only clinically relevant metastases.

The present study's results indicate several potential clinical uses for the BLN Assay. It can be used as an accurate intraoperative test for metastatic spread to the SLNs and can also provide valuable postoperative information as an adjunct to permanent-section histology by testing more of the node than is practical by histology alone. A patient found negative by both histology and the BLN Assay is more likely to have an accurate diagnosis than one found negative only by histology. This is supported by the data shown in this article for those cases when histology was negative and the BLN Assay was positive. Additional molecular testing supported the positive BLN Assay result, suggesting that the negative histologic result was not representative of the entire node's status. When both histology and the assay results are negative, the surgeon could feel more confident that the entire node is likely negative. An added advantage of the BLN Assay is that it can be performed by trained technicians and thus may be more easily adopted by clinical sites with limited pathology support.

In summary, the combined data from two, large, prospective, multisite studies indicate that the BLN Assay is more sensitive than current intraoperative assays. The Assay's performance is similar to the current standard of care, permanent histologic analysis. It is an objective and reproducible assay and can easily be performed in most clinical laboratories, as shown by the high performance in this study where the assay was conducted soon after SLNB on fresh tissue at 12 different laboratories by a variety of personnel from histotechnologists to experienced pathologists. Using this new molecular pathology technique allows for a more thorough node evaluation and, when used intraoperatively, is likely to reduce the number of patients returning for a second operation for additional node removal because of delayed diagnosis.

	AUTHORS’ DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST

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Although all authors completed the disclosure declaration, the following author(s) indicated a financial or other interest that is relevant to the subject matter under consideration in this article. Certain relationships marked with a "U" are those for which no compensation was received; those relationships marked with a "C" were compensated. For a detailed description of the disclosure categories, or for more information about ASCO's conflict of interest policy, please refer to the Author Disclosure Declaration and the Disclosures of Potential Conflicts of Interest section in Information for Contributors.

Employment or Leadership Position: None Consultant or Advisory Role: Thomas B. Julian, Veridex (C); Peter Blumencranz, Veridex (C); Pat Whitworth, Veridex (C); Donald A. Berry, Veridex (C); Scott M. Berry, Veridex (C); Rache Simmons, Veridex (C) Stock Ownership: None Honoraria: Thomas B. Julian, Veridex; Peter Blumencranz, Veridex; Pat Whitworth, Veridex Research Funding: Thomas B. Julian, Veridex; Peter Blumencranz, Veridex; Kenneth Deck, Veridex; Pat Whitworth, Veridex; Anne Rosenberg, Veridex; Anees B. Chagpar, Veridex; Douglas Reintgen, Veridex; Peter Beitsch, Veridex; Rache Simmons, Veridex; Sukamal Saha, Veridex; Eleftherios P. Mamounas, Veridex; Armando Giuliano, Veridex Expert Testimony: None Other Remuneration: None

	AUTHOR CONTRIBUTIONS

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Provision of study materials or patients: Thomas B. Julian, Peter Blumencranz, Kenneth Deck, Pat Whitworth, Anne Rosenberg, Anees B. Chagpar, Douglas Reintgen, Peter Beitsch, Rache Simmons, Sukamal Saha, Eleftherios P. Mamounas, Armando Giuliano

Collection and assembly of data: Thomas B. Julian, Peter Blumencranz, Kenneth Deck, Pat Whitworth, Donald A. Berry, Scott M. Berry, Anne Rosenberg, Anees B. Chagpar, Douglas Reintgen, Peter Beitsch, Rache Simmons, Sukamal Saha, Eleftherios P. Mamounas, Armando Giuliano

Data analysis and interpretation: Donald A. Berry, Scott M. Berry

Manuscript writing: Thomas B. Julian, Peter Blumencranz, Kenneth Deck, Pat Whitworth, Donald A. Berry, Scott M. Berry, Anne Rosenberg, Anees B. Chagpar, Douglas Reintgen, Peter Beitsch, Rache Simmons, Sukamal Saha, Eleftherios P. Mamounas, Armando Giuliano

Final approval of manuscript: Thomas B. Julian, Peter Blumencranz, Kenneth Deck, Pat Whitworth, Donald A. Berry, Scott M. Berry, Anne Rosenberg, Anees B. Chagpar, Douglas Reintgen, Rache Simmons, Sukamal Saha, Eleftherios P. Mamounas, Armando Giuliano

	Glossary Terms

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Macrometastasis:

A cohesive cluster of metastatic tumor cells more than 2 mm in the largest dimension within a lymph node.

Micrometastasis:

A cohesive cluster of metastatic tumor cells measuring more than 0.2 mm and up to 2.0 mm in the largest dimension found within a lymph node.

CK-19 (cytokeratin-19):

CK-19 belongs to the intermediate filaments, which create a cytoskeleton in almost all cells. CK-19 is normally not expressed in the hematopoietic cells, although it is commonly expressed in epithelial cells such as mammary cells, either normal or neoplastic.

Mammaglobin:

Mammaglobin is a glycoprotein that is mostly, although not exclusively, confined to breast tissue. Expression of mammaglobin in nonbreast tissue is associated with the metastasis of breast cancer and may be upregulated in breast cancer.

RT-PCR (reverse-transcriptase polymerase chain reaction):

PCR is a method that allows logarithmic amplification of short DNA sequences within a longer, double-stranded DNA molecule. Gene expression can be measured after extraction of total RNA and preparation of cDNA by a reverse-transcription step.Thus, RT-PCR enables the detection of PCR products on a real-time basis, making it a sensitive technique for quantitating changes in gene expression.

Permanent-section histology:

Usually this refers to formalin-fixed, paraffin-embedded tissue sections stained by hematoxylin and eosin (HE) to reveal metastases. This technique takes an average of 2 to 3 days to complete.

Immunohistochemistry:

The application of antigen-antibody interactions to histochemical techniques. Typically, a tissue section is mounted on a slide and is incubated with antibodies (polyclonal or monoclonal) specific to the antigen (primary reaction). The antigen-antibody signal is then amplified using a second antibody conjugated to a complex of peroxidase-antiperoxidase (PAP), avidin-biotin-peroxidase (ABC) or avidin-biotin alkaline phosphatase. In the presence of substrate and chromogen, the enzyme forms a colored deposit at the sites of antibody-antigen binding. Immunofluorescence is an alternate approach to visualize antigens. In this technique, the primary antigen-antibody signal is amplified using a second antibody conjugated to a fluorochrome. On UV light absorption, the fluorochrome emits its own light at a longer wavelength (fluorescence), thus allowing localization of antibody-antigen complexes.

	NOTES

 
Supported by Veridex LLC.

Presented in part at the 12th Annual Meeting of the Association for Molecular Pathology, November 16-19, 2006, Orlando, FL; 8th Annual Meeting of the American Society of Breast Surgeons, May 2-6, 2007, Phoenix, AZ; 43rd Annual Meeting of the American Society of Clinical Oncology, June 1-5, 2007, Chicago, IL; American Society of Clinical Oncology Breast Cancer Symposium, September 7-8, 2007, San Francisco, CA; Annual Meeting of the College of American Pathology, September 9-13, 2006,San Diego, CA; 6th Biennial Meeting of International Sentinel Node Society, November 1-4, 2006, Rome, Italy; 29th Annual Meeting of the San Antonio Breast Cancer Symposium, December 14-17, 2006, San Antonio, TX; Annual Meeting of the Society of Surgical Oncology, March 15-18, 2007, Washington, DC; 30th Annual Meeting of the San Antonio Breast Cancer Symposium, December 13-16, 2007, San Antonio, TX.

Terms in blue are defined in the glossary, found at the end of this article and online at www.jco.org.

Authors’ disclosures of potential conflicts of interest and author contributions are found at the end of this article.

	REFERENCES

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION AUTHORS' DISCLOSURES OF... AUTHOR CONTRIBUTIONS Glossary Terms REFERENCES

 
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Submitted August 17, 2007; accepted March 28, 2008.

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