Intraoperative qRT-PCR for detection of Lymph Node Metastasis in Head and Neck Cancer.

This project is performed in collaboration with Dr. Robert Ferris at the University of Pittsburgh.

The project is funded through 2007 by an NIH R01 grant to Dr. Godfrey (R.L. Ferris Co-PI).

Project Summary:  Squamous cell carcinoma of the head and neck (SCCHN) frequently metastasizes to the regional lymphatics and this is the best predictor of disease prognosis and outcome. Accurate staging of lymph nodes in the neck is essential to optimal patient management, but current clinical methods are inadequate, and misdiagnose the presence or absence of cervical nodal metastasis in many patients. Consequently, the current standard of care, elective neck dissection, may represent overtreatment of over 50% of clinically tumor-negative patients. Sentinel lymph node (SLN) biopsy may obviate this problem, and is currently undergoing a multi-center validation trial. One problem with this approach is the current lack of a highly accurate, rapid (intraoperative) assay to assess the SLN. Frozen section analysis is only 60-80% sensitive, and false negatives due to frozen section analysis would frequently result in second procedures when final histology detects metastatic disease.  Re-operation on such SLN biopsied necks is likely to be poorly received by both patients and surgeons, due to increased technical difficulty, morbidity of a second surgery and anesthetic, anxiety due to diagnostic uncertainty and increased costs.  In addition, 7-10% of pathologically tumor negative patients manifest tumor recurrence, possibly indicating the presence of pathologically occult disease. We hypothesize that quantitative RT-PCR (qRT-PCR) can overcome these problems and can provide accurate, definitive staging of the neck in an intraoperative time-frame. In preliminary studies, we have identified outstanding markers for qRT-PCR in SCCHN. We have also developed methods to facilitate rapid, internally controlled and automated qRT-PCR in a clinical setting. In the current project, we hope to demonstrate that our fully-automated qRT-PCR assays can be optimized for use in an intraoperative time frame, and that they can be performed in a standardized and reprodicible manner.  These assays should prove to be useful tools for staging of lymph nodes from SCCHN patients.

Background:  The incidence of metastases to regional cervical lymphatics in SCCHN has been well established ^1-5 .  Even early cancers of the head and neck mucosa, including oral cavity, pharynx, and larynx, commonly metastasize to the cervical nodal basins ¹ and the presence of cervical metastasis in patients with SCCHN is the strongest prognostic factor available. Patients with cervical metastases have advanced stage of disease and are at increased risk for distant metastasis and locoregional failure ⁶ . Also, information obtained from staging the neck is used to direct the application of adjuvant therapies (radiation and chemotherapy) to reduce disease recurrence. Unfortunately, clinical staging, with physical examination and radiographic imaging, is inadequate for accurately assessing the tumor status of the neck.  Radiographic imaging studies such as CT and MRI scans are limited by their sensitivity and specificity: they are unable to detect metastases < 8-10 mm in size and their overall accuracy is only approximately 75% ⁷ .  Positron emission tomography (PET) scans have not been shown to significantly improve the radiographic staging of the neck in this disease. The inadequacy of clinical staging in accurately predicting tumor status of the neck is highlighted by the high rates of both downstaging and upstaging, when correlated with pathologic analysis of cervical lymph nodes. Indeed, 15% of clinically tumor-positive (cN+) necks are actually pathologically tumor-negative (pN0), and 25-30% of clinically tumor-negative (cN0) necks harbor occult disease on pathologic analysis, and are deemed pN+. Clearly, a more accurate staging technique would be desirable.

The inaccuracy of clinical staging results in a surgical treatment dilemma; to operate on the neck or not. Clearly, in a cN+ patient the answer is yes since it will be the correct choice 85% of the time. The decision in a cN0 patient however is not so easy, since neck dissection will be the correct choice only 25-30% of the time. As a result, for several decades the preferred approach to the cN0 patient was resection of the primary tumor followed by observation of the neck, otherwise referred to as “watchful waiting”. However, this was found to result in a large subset of patients who suffered regional recurrence in the neck and outcome for these patients was poor due to the frequent inability to perform successful surgical salvage. In addition, the extent of surgery following neck recurrence is more extensive than when performing neck dissection at the time of initial surgery and critical structures, such as jugular vein, sternomastoid muscle and/or accessory cranial nerve must often be sacrificed. The removal of one or more of these structures is directly responsible for the morbidity, functional deficits ^8-13 and decreased quality of life (QOL) after neck dissection ^8-13 . Finally, since this approach did not provide definitive staging of the neck, any decision to give adjuvant therapy relied only on pathologic features of the primary tumor.  As a result the current management of the cN0 neck commonly includes routine elective neck dissection (END) with pathologic examination of the removed lymph nodes. 

Large retrospective analyses from our and other centers have demonstrated that elective neck dissection done at the time of primary surgery for SCCHN for a clinically N0 neck, is associated with a decreased rate of regional failure (by three-fold compared with observation), increased regional recurrence-free survival, and perhaps, lower incidence of distant metastases ^3,4,14. This procedure also allows for a rational decision of when to give adjuvant therapies based on more accurate, pathologic evaluation of lymph nodes rather than on clinical staging alone ^15-17 .  However, because only 25-30% of clinically negative necks harbor pathologic evidence of disease, END in cN0 patients results in overtreatment of ³ 70% of  patients. In addition, END in cN+ patients results in unnecessary treatment 15% of the time, when final pathology is negative. Overall, the current practice of performing END results in overtreatment of 50% of SCCHN patients. The initial difficulty however is in predicting who needs treatment of the neck. To address this dilemma, there has recently been a great deal of interest in attempting to apply the technique of sentinel lymph node (SLN) mapping to SCCHN. This technique, when combined with the intraoperative analysis of the SLN(s), has the potential to define those cN0 patients in whom neck dissection is most appropriate (i.e. those likely to be pN+), and eliminate the need for END and its associated morbidities in node negative patients. Such intraoperative decision-making requires that SLN mapping accurately predicts the status of the neck, and presumes the existence of an accurate method to evaluate the presence of tumor in the SLN(s).

Use of SLN biopsy to stage SCCHN.  The concept of SLN biopsy as a minimally invasive method for evaluating the presence of regional nodal metastases was introduced by Morton et al. in 1992 for patients with cutaneous melanoma ¹⁸ . The technique is based on the theory that metastases progress from the primary tumor to the SLN before spreading further to involve other regional nodes. In melanoma, SLN biopsy has been shown to have accuracy greater than 95% and a false-negative rate of less than 2% ^19,20 . Similar results have been found for breast cancer ²¹ and the technique is now widely used in the staging and treatment of these diseases. Preliminary evidence from a number of single-institution studies, including our own, suggests that SLN biopsy may be clinically valid for SCCHN as well ^7,22-27 Shoaib et al. ⁷ evaluated the use of sentinel lymph node mapping in 40 patients with cN0 oral cavity cancers.  They found SLNs in 17 of 20 necks with pathologic disease, of which 16 SLN’s contained metastases.  Data from the University of Pittsburgh ^6,22,23 also suggest that SLN biopsy is useful for identification of subclinical, microscopic lymph node metastases, and enthusiasm is rapidly growing for the potential of this technique in SCCHN. In the United States, a trial evaluating SLN biopsy in SCCHN is currently being conducted by the American College of Surgeons Oncology Group, Head and Neck Organ Site, in which the University of Pittsburgh is participating. However, the eventual utility of SLN biopsy in SCCHN will be heavily dependent on the accuracy and timeliness of the technique employed for analysis of the SLN. Currently, the most accurate pathologic analysis consists of multiple level, serial sectioning of formalin-fixed, paraffin-embedded tissue, and staining with H&E plus immunohistochemistry to enhance detection of small tumor deposits. Unfortunately, this process takes several days to complete. Applying a more rapid (less than 30 minutes), but equally accurate, method for SLN analysis would allow the surgeon to perform immediate and definitive treatment of SLN positive patients in a single procedure.

Intra-operative frozen section analysis. The current method for intraoperative analysis of sentinel lymph nodes involves frozen sectioning, followed by staining with hematoxylin and eosin (H&E).  This method suffers from poor sampling (since only one or two sections are analyzed), lack of sensitivity (since immunohistochemistry is not used) and time stress on the pathologist. As a result, reports on intra-operative frozen section sensitivity in breast cancer range from 44% to 74% while in melanoma reports range from 38%-47%.  As a result, most institutions do not perform frozen section analysis for melanoma and all node positive patients have to undergo a second procedure for lymph node dissection. Most institutions perform some kind of intra-operative analysis on SLN from breast cancer but do so with the expectation that 30-40% of positive nodes will be missed and that these patients will then need a second surgery for axillary lymph node dissection (although we acknowledge that this is currently a highly debated issue). While there is very little data on the accuracy of intra-operative lymph node analysis in SCCHN, one small study found the technique to be only 60% sensitive (6/10) ²⁸ . This is clearly too small a sample size to make any conclusions (confidence interval is 26-88%) but it appears that the sensitivity of intra-operative SLN analysis in SCCHN may be similar to that in breast cancer.  In all diseases, the disadvantages of having to undergo a second operative procedure include the financial costs of a repeat operative procedure, the risks of a second anesthesia, and the considerable mental anguish to the patient. Furthermore, in SCCHN patients, acute inflammatory changes and wound-healing occurring in the neck postoperatively make the second procedure significantly more difficult technically and increase the risk of injury to major vessels and cranial nerves in the operative field. It is therefore evident that a more timely and accurate assay for detecting metastases in cervical lymph nodes would have great clinical utility, particularly in situations such as SLN mapping where intraoperative decision-making has obvious value.  Finally, staging of the neck by either SLN biopsy or END is only as predictive as permanent histologic analysis. Regional recurrence in 7-10% of pN0 patients suggests that any technical improvements that enhance detection of micrometastasis may also upstage an additional group of patients and preliminary studies suggest that these patients have the same poor outcome as those with gross metastatic disease ^29,30. In summary current methods of clinical (and pathologic) staging of cervical metastasis in SCCHN are inadequate. We hypothesize that molecular staging of the neck, including the SLN’s, can be performed intraoperatively, and with the same accuracy as final SLN pathology. Furthermore molecular staging may identify the pN0 patients with a higher risk of regional disease recurrence (i.e. those patients with occult LN disease).

Molecular detection of lymph node metastases.  While there have been a few studies on identification of tumor cells in lymph nodes using detection of tumor-related mutations in genomic DNA ^31,32 , the vast majority of studies have utilized reverse transcription-PCR (RT-PCR) to detect tumor-associated mRNA species.  Since it is unclear whether there are any truly tumor-specific mRNA’s, identification of metastases by RT-PCR typically relies on the detection of epithelial cell-specific mRNAs in a tissue (lymph node) that does not normally express them.  The presence of the epithelial mRNA is then presumed to indicate the presence of tumor cells.  RT-PCR has several theoretical advantages over current methods for analysis of lymph nodes. First, due to the power of PCR amplification, RT-PCR is extremely sensitive with some authors claiming the ability to detect one cancer cell in a background of 10⁷ normal cells ³³ .  Second, in most reports, up to one half of the lymph node has been used for RNA isolation and RT-PCR.  Since sampling error is one of the biggest limitations of pathologic examination (by IHC or H&E) ^34-39 , the ability to assess large amounts of tissue in a single assay may be the most significant advantage of RT-PCR.  Third, RT-PCR has the potential to provide an objective and standardizable result, as opposed to pathology, which is more subjective and can be variable from institution to institution or pathologist to pathologist ⁴⁰.

Despite these theoretical advantages however, there are significant hurdles to overcome before RT-PCR can be seriously considered as an alternative, or adjunct, to routine pathology.  With the exception of some recent reports ^41-44 , most RT-PCR studies to date have relied on gel-based assays and simple positive/negative detection of marker mRNAs as the criterion for the existence of occult metastatic disease. While results from these studies show that the sensitivity of RT-PCR is indeed high, the specificity has been unacceptably low ^45-48,48-50 and these assays would result in over-staging of many patients. One likely reason for this poor specificity, is the presence of background (often referred to as ectopic or illegitimate) expression of marker mRNAs in most lymph nodes.  Indeed, without quantitative studies, the extreme sensitivity of RT-PCR may actually interfere with its ability to discriminate between normal nodes and those with metastases.  With the widespread use of real-time, quantitative RT-PCR (qRT-PCR) however several groups, including ours, have shown that quantification can discriminate background expression from expression due to the presence of tumor cells ^41,42,44,51 .  Furthermore, quantification allows sensitivity and specificity to be optimized since the assay results are no longer binary (positive or negative), but a continuous variable for expression level ⁴⁴.

A second problem is that RT-PCR is very labor intensive, prone to contamination (resulting in false positive results), prone to RNA degradation (resulting in false negative results) and requires extensive laboratory expertise. In addition, reports in the literature have used a wide variety of experimental methods and very few have incorporated adequate quality controls. As a result, standardization and comparison of results between laboratories has proven extremely difficult ⁵² .  This issue was recently reviewed ⁵³ and the authors concluded that “The wide variety of RT-PCR assays makes it difficult to draw firm conclusions and impedes interlaboratory comparison. To translate RT-PCR approaches from the bench to the bedside, two requirements must be met: (1) standardization and quality control at the individual laboratory must be achieved; (2) multicenter trials must be conducted in which standardized assays are used and the RT-PCR results are correlated with the clinical outcome of the patients”. Ideally, this standardized assay should be simple to use, minimize technical steps, and be amenable to automated analysis. We believe that this standardized assay should use a fluorescence-based, multi-color, quantitative RT-PCR. In such an assay, internal controls can be used to verify not only that the assay worked, but that it worked within an acceptable sensitivity range ^54,55 . Furthermore, in a fluorescent assay, results can be analyzed immediately and objective results can be obtained automatically with no post-PCR handling. 

Finally, most lymph node RT-PCR studies report homogenizing whole portions of, or even complete lymph nodes for RNA isolation. In this situation, the ability to perform routine pathology analysis is eliminated and this is unacceptable in a clinical setting. We believe that instead of homogenizing a whole portion of the lymph node, lymph nodes should be processed in such a way that both RT-PCR and routine pathologic evaluation can be performed in parallel on immediately adjacent tissue sections. 

Over the past 3-4 years, research in our laboratory has focused on solving the problems associated with detection of lymph node metastases by RT-PCR, and developing assays and tools that will permit RT-PCR to be used in clinical settings. This research has been targeted towards two specific clinical scenarios. First, as with most RT-PCR studies, we are attempting to use RT-PCR to detect pathologically occult metastases (typically called micrometastases) with the goal of predicting outcome in early stage, pN0 patients. This work continues in the form of R01 funded projects on esophageal and lung cancer. The second scenario, and the one primarily addressed in this prject, is that of intra-operative detection of lymph node metastases using RT-PCR. For breast cancer and melanoma, this work is currently funded by an STTR grant in collaboration with a company called Cepheid in Sunnyvale CA.  Both scenarios require that we identify good molecular markers for the disease in question, that we incorporate adequate controls for the clinical setting and that the assays be as automated, standardized and reproducible as possible. The intra-operative scenario adds to this the requirement for a very rapid assay. In this project we intend to further develop and validate our preliminary work on SCCHN and demonstrate that molecular diagnostics can surpass current staging of the neck by establishing a fully automated, completely objective analysis that is at least as accurate as current techniques, but is superior because of the automation, objective nature, and timeliness of the assay.

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