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Five top stories in cytopathology.

THE CONTINUING VALUE OF CYTOMORPHOLOGY

Cytopathologists have been primarily morphologists, using the smallest possible biopsy specimen for diagnosis. It is unusual for new diagnostic morphologic features to be discovered. A rare example is the description of intranuclear cytoplasmic inclusions in at least a subset of pancreatic intraductal papillary mucinous neoplasms. (1) The new Bethesda system for reporting thyroid cytopathology is certainly important, but the actual morphologic criteria for diagnosing thyroid fine-needle aspirations (FNAs) have scarcely changed in the past several decades. It seems that classical cytomorphology has brought the field about as far as it can go.

Though new diagnostic cytomorphologies are not likely to help the productive evolution of cytology, the theme of this first story is that classical morphology will continue to have an essential value. In order to provide new useful clinical applications, cytopathology needs to be redefined as the field that uses the smallest possible biopsy specimen for diagnosis by using any technique. The full value of classical cytomorphology can be boosted by new technologies, including improvements in cell block techniques (2-4) and better microbiopsy needles. (5) The use of ultrasound guidance for cytopathologist-performed FNA and the development of new endoscopic techniques have provided new applications for classical cytomorphology. New immunofluorescent microscopy techniques are promising to synergize with classical cytomorphologic diagnosis by simultaneously allowing multiple antigens to be visualized. (6,7) There are emerging microscopy techniques for screening for abnormalities without even removing a sample from a patient. (8) Cytologic samples can be optically sectioned ex vivo within the specimen container, potentially at the bedside, to look like a hematoxylin-eosin-stained cell block without any tissue processing. (7) Development and application of these techniques would greatly benefit from cytopathologists' expertise. These new techniques all help fulfill the vision of enabling diagnosis based on the smallest possible biopsy specimen.

Molecular techniques are synergistic with cytomorphology, and cytology is a natural partner for the molecular diagnostic laboratory because relatively few cells are needed and cytology fixatives are naturally molecular-friendly. Importantly, molecular techniques cannot be applied in a morphologic vacuum. The value of mutation testing for predicting response to personalized therapies is dependent on assuring that the sample is morphologically representative. When molecular techniques have imperfect sensitivity and specificity (eg, thyroid molecular testing, as discussed in story 4), the value of molecular testing depends strongly on the pretest (ie, morphologic) probability of cancer. Human papillomavirus testing by itself has too little specificity to completely replace morphologic Papanicolaou testing.

Cytomorphology continues to have value because we still have almost no understanding of the molecular basis for diagnostic structural changes in cells. An understanding of the exact structural basis of diagnostic morphologic features of cancer should allow the development of an entirely new class of "structural diagnostic markers." For example, identification of the biochemical/structural basis of nuclear irregularity could improve the subjective appreciation of the features of papillary thyroid carcinoma. Lamin immunostaining shows promise for improving the detection of diagnostic nuclear irregularity. (9-11)

Understanding the precise molecular basis of diagnostic changes in cell and tissue morphology should also provide essential insights into cancer biology. It is instructive to review how classical cytomorphologic observations provided the insight required to reach our current understanding of cell biology. Examples of the fruitful discoveries based on cytomorphology include the inference by Walter Sutton at the beginning of the 1900s that chromosomes were the basis of Mendelian inheritance (12); Theodore Boveri's insight into the importance of chromosomal instability in cancer development (13); Barbara McClintock's (14) work showing that the nucleolus originated from discrete regions of the chromosomes, culminating in the demonstration that ribosomal RNA derives from the nucleolus (15); the nature and functional significance of the Barr body (16,17); and the importance of large-scale chromatin organization for gene regulation. (18,19)

Although cytomorphology drove these earlier discoveries, by the 1980s cytology was often dismissed as a "study of the artifacts of fixation and staining," devoid of any deeper biological meaning. Cytology appeared unattractive to cancer researchers and a major gap developed, separating cytologists from the cell biologists who otherwise share a common interest in understanding cancer.

With the advent of green fluorescent protein imaging in the mid-1990s and its application in live cell imaging (20,21) (culminating in the Nobel Prize in Chemistry in 2008), it became clear that the fixatives and stains of cytologists do in fact provide a very valid snapshot image of the organization and physiology of living cells, including cancer cells.

Altered cytomorphology is a premier hallmark of cancer because cancer is still diagnosed based on morphology. Yet morphology has been completely absent from the "hallmarks of cancer" (22,23) model that dominates the field of contemporary cancer research. The hallmarks of cancer model cannot explain how or why cancer cells are structured differently from normal cells, or why different forms of cancer would have different morphologic appearances. (24) It is a serious problem that the defining features of cancer--for now and for the foreseeable future--are not being more aggressively funded by the National Institutes of Health. Cytologists are in a better position than surgical pathologists to help promote research into morphology, because cellular-level diagnostic features are probably easier to characterize at a precise biochemical level compared with diagnostic tissue-level features. Engagement of cytopathologists in basic cell biology research, or active lobbying for more funding, will be important for improving cancer diagnosis, prevention and treatment.

A direct relation between cytomorphology and the molecular biology of cancer is becoming apparent in several examples. (24) The thyroid provides perhaps the best example of a direct relation between the morphologic criteria for diagnosing cancer and the underlying genetic abnormalities in cancer, validating the instincts of generations of cytopathologists that morphology provides essential insights into the underlying pathophysiology of cancer. (24,25) Activations of RET, TRK, and B-RAF have been shown to be sufficient to induce the nuclear morphologic features of papillary thyroid carcinoma when expressed in normal thyroid cells in culture (24,26,27) or in transgenic mice models. (28) Uncovering the histone code"29 that likely defines the diagnostic chromatin differences between follicular neoplasms (FNs) and papillary thyroid carcinoma, or discovering the alteration in the nuclear lamina that defines the diagnostic differences, will likely bring important insights into the underlying molecular mechanisms of cancer while helping improve diagnosis.

Currently, our entire armamentarium of diagnostic cytomorphology consists of snapshot images of dead cells, yet experience in every other level of biology predicts that dynamic properties of cancer cells will have powerful diagnostic value. An exciting synergy between cancer biologists and cytopathologists can be envisioned in which classical cytomorphology will be applied to living cancer cells in their native environment. It is entirely feasible with current technologies to capture the 3-dimensional, time-lapse, high-resolution appearance of tumor cells growing in situ. (7) Beyond improving diagnosis, new types of cancer cell physiologies will likely become apparent by visualizing "dynamic criteria of malignancy." Cytology is poised to lead these endeavors: The physics of live cell imaging, and the dyes that are needed to perform live cell imaging, mandate use of relatively small fragments of tissue, about 100 to 200 im in diameter, (7) and cytopathologists are the experts at using fragments of this size for diagnosis. In an era in which hypothesis-generating research is receiving funding support, it would seem important to engage a modern-day Charles Darwin in a Beagle-like voyage to catalogue dynamic live cell images of all of the major types of human cancer, within the tumor cells' native microenvironment.

NEW CLINICAL APPLICATIONS OF CYTOLOGY: ENDOSCOPIC BRONCHIAL ULTRASOUND, ENDOSCOPIC ULTRASOUND-GUIDED FNA, AND SENTINEL NODE SAMPLING

We read the "obituary" to breast FNA, (30) but FNA in combination with endoscopic ultrasound (EUS) and endobronchial ultrasound (EBUS) is placing the nail in the coffin for mediastinoscopy. (31) In this story we review new areas where cytology is replacing histologic biopsy, and examine the factors that caused the field of cytology to expand.

Staging Non Small Cell Lung Cancer

Management of non-small cell lung cancer depends on the stage. Surgical resection offers the chance of cure if patients do not have more than N1 nodal disease (involvement of ipsilateral lobar, interlobar, or hilar nodes--stations 10, 11 and 12, respectively). For patients with involvement of mediastinal nodes, patients may still be candidates for a curative resection, but only if neoadjuvant chemoradiotherapy can be shown to downstage the patients. Thus, evaluation of the mediastinal nodes is essential for the initial decision to perform surgical resection, and for evaluating patients for potential resection following neoadjuvant therapy. (32)

Positron emission tomography scanning is commonly used as a first step in the evaluation of newly diagnosed non-small cell lung cancers. Positron emission tomography and conventional computed tomography (CT) scanning are sensitive for detection of metastases in the mediastinum. Patients can undergo resection if both positron emission tomography and CT are negative. Positron emission tomography and CT scanning have low specificity, however, and positron emission tomography-positive or enlarged nodes need biopsy confirmation. (31,33) Mediastinoscopy has been the gold standard for mediastinal node staging in this setting. (34) It takes about 2.5 hours to perform, requires 1.5 days of hospitalization, and has a 2% morbidity and 0.08% mortality. (34) There is a much higher complication rate for repeat mediastinoscopy following neoadjuvant therapy, or for mediastinoscopy in patients with previous sternotomy.

Endobronchial ultrasound and EUS offer a safer, nonsurgical approach to evaluation of the mediastinum in this setting. Endoscopic ultrasound (via the esophagus) can sample the paraesophageal (station 8) and pulmonary ligament (station 9) lymph nodes. Both EUS and EBUS can sample station 2 (upper paratracheal), 4 (lower paratracheal), and 7 (subcarinal) lymph nodes, whereas only EBUS can sample station 10 (hilar), 11 (interlobar), and 12 (lobar) lymph nodes. (32) In a recent prospective study, there were no complications of EBUS/EUS in staging 153 patients, whereas minor morbidity from mediastinoscopy occurred in 3%.35 Importantly, sensitivity and specificity of EUS and EBUS were slightly superior to those of mediastinoscopy in this study. (35) Endobronchial ultrasound offers the most cost-effective initial approach to patients with suspected lung cancers because it can combine an initial diagnosis with staging. (36)

There are increasing requests for rapid on-site evaluation of adequacy (ROSE) by cytopathologists because of the increase in the number of EBUS or EUS procedures. Low reimbursement for ROSE is a problem for cytology. (37) Objective adequacy criteria for on-site evaluation have been developed for this scenario, potentially simplifying the work of ROSE. (38) Surprisingly, a recent, large multicenter study of EBUS/EUS for staging non-small cell lung cancer failed to show a benefit of ROSE. (33) The reasons for lack of value of ROSE in this study are not apparent, but could reflect heterogeneity in the handling of ROSE by the different institutions. Other reports have emphasized the importance of ROSE in this precise setting. (31,32,35) In settings in which the differential diagnosis is broader than merely positive or negative for metastatic non-small cell lung cancer, ROSE has clear value. (39)

Other Scenarios Seeing an Increase in FNA

Endobronchial ultrasound and EUS are not limited in their value to staging non small cell lung cancer; they have emerged as cost-effective and accurate diagnostic procedures for sarcoidosis (40,41) and a wide variety of other neoplastic and infectious diseases involving the lung and mediastinum. (32,39) Cell blocks are generally synergistic with monolayer preparations for diagnosis in these settings. (39,42) Bronchoscopic approaches are safer than transthoracic approaches for evaluation of pulmonary nodules. Transthoracic approaches have a risk of pneumothorax of about 20% with a need for chest-tube placement in 3% to 15%,43 whereas bronchoscopic approaches have morbidity of only about 0.15% to 0.3%.41 Bronchoscopic approaches are therefore particularly useful in patients at high risk for pneumothorax, or for whom pneumothorax would be life threatening.

Two new techniques, electromagnetic navigation bronchoscopy (44,45) and virtual bronchoscopic navigation, (43) allow the bronchoscopist to reach virtually any lung nodule greater than about 2 cm. (45) Electromagnetic navigation bronchoscopy is like maneuvering a car with a global positioning system. A patient first undergoes a conventional CT scan to set up a map of the anatomy. Bronchoscopy is then performed with the patient positioned on a platform that creates a 3-dimensional electromagnetic field. A special probe incorporated in the bronchoscope detects the electromagnetic field. Several reference marks (eg, the carina) are touched by the probe to register the location of the probe onto the CT scan. The bronchoscope is then wedged into the nearest bronchus and a 1.9-mm sleeve can then be inserted through lung tissue and guided to its target. Electromagnetic navigation bronchoscopy can also use transbronchial pinch biopsy devices as well as cytologic brushes and FNA needles. There are not yet studies comparing the accuracy and morbidity of histologic approaches compared with cytologic approaches with electromagnetic navigation bronchoscopy. (45)

Fine-needle aspiration via EUS has also become the primary means of diagnosis of pancreatic lesions and staging of upper gastrointestinal cancers, and it has increasing utility as a safe and accurate procedure for diagnosing a wide variety of lesions within a few centimeters of the gastrointestinal tract lumen. (39)

Ultrasound-guided axillary node sampling in patients with newly diagnosed breast cancer is another area with a dramatic increase in volume in recent years. The sensitivity for detecting metastases is as high as about 90%.46 The sensitivity of sentinel node biopsy for detecting lymph node metastases is estimated to be about 93%.47 Core biopsy is technically more challenging, with higher rates of hematoma and higher costs, and does not improve sensitivity beyond that of FNA for detection of positive nodes in women with newly diagnosed breast cancer. (48) Ultrasound-guided axillary node FNA has been modeled to be cost effective when performed on morphologically abnormal lymph nodes, even when the sensitivity of FNA is only 65%.47 Fine-needle aspiration is cost effective by reducing the surgical, anesthesia, and frozen section costs associated with sentinel node biopsy. (47)

What Factors Account for the Expansions of Cytology, and Will These New Applications Remain Within Cytology?

The difficulty in determining the presence of invasion was a major factor that led to the decline in breast FNA, (30) but invasion is a moot point for the body sites in which FNA has seen an expanded role. Improvement in breast core biopsy devices facilitated the transition away from FNA for breast, but so far EUS and EBUS scopes have limited or no ability to acquire cores of tissue. The EBUS scopes accommodate only a 22-gauge front-end collection needle that cannot acquire intact cores of tissue. (32) Endoscopic ultrasound scopes can accommodate a 19-gauge tru-cut, side-capture, core biopsy device. However, published experience with EUS tru-cut biopsies have not shown an advantage over FNA-type needles, (39,49,50) in part because angulation of the scope reportedly impairs the function of the biopsy device. (49,50) The new 22- and 25-gauge EchoTip ProCore front-capture device (Cook Medical, Inc, Bloomington, Indiana) shows promise, (51) and other core biopsy needles are said to be in development. (52)

Cytology should be able to retain its role in EBUS and EUS, even if better endoscopic biopsy devices emerge. Small core biopsies tend to become fragmented. Though there are few data, one would expect that fragmented biopsies should be more efficiently processed by the cytopathology laboratory as a combined monolayer preparation and a cell block than as a purely histologic sample. Histologic processing cannot easily retain fragments under 150 [micro]m (the approximate pore size of filter bags and lens paper). Cytologists can make diagnoses on material smaller than 150 [micro]m, and many key diagnostic features usually span less than the diameter of even a 23-gauge needle (320 [micro]m, or about two-thirds of a X400 field of view). For example, efficient cell blocks of breast FNAs permit histologic diagnosis of invasion in about 40% of invasive breast cancers, and allow an accurate histologic assessment of the degree of ductal hyperplasia. (2) With high-quality cell blocks, the limiting factor for breast diagnosis is not the size of the needle; it is the relative inefficiency of the existing FNA needle designs. (2) It seems likely that improvements in microbiopsy needle design will widely boost the value of cytology and could allow cytology to reemerge as the primary diagnostic tool for breast masses.

The expansion of cytology was heavily dependent on clinical trials that validated the use of cytology in EBUS, EUS, and sentinel node biopsy. Clinical validation in prospective studies is a key determinant of future practice patterns. Clinical validation means that the cytologic diagnosis allowed a clinical intervention with an equivalent or superior outcome compared with the use of other diagnostic techniques. This is a much higher bar than merely showing comparability between cytology and surgical pathology diagnoses, and it is essential to surpass this bar to change practice patterns. There is a need for active involvement of cytopathologists in prospective clinical validations, and professional organizations representing cytologists should actively advocate for the inclusion of cytology in future clinical trials.

TARGETED THERAPIES BASED ON CYTOLOGY

Molecular advances have led to new pharmacologic targeted therapies that benefit a subset of patients. For example, patients with advanced lung adenocarcinomas bearing mutations in the epidermal growth factor receptor have a high chance of responding to treatment with the epidermal growth factor receptor tyrosine kinase inhibitor erlotinib (Tarceva, Roche Genentech, South San Francisco, California). (53) Likewise, patients with advanced non-small cell lung cancer (NSCLC) bearing a rearrangement of the anaplastic lymphoma kinase (ALK) gene respond to the new tyrosine kinase inhibitor crizotinib (Xalkori, Pfizer, New York City, New York). (54,55) Vemurafenib (Zelboraf, Roche Genentech), an inhibitor of the serine-threonine kinase B-RAF, produces a 50% response against melanomas that harbor an activating mutation in B-RAF, whereas traditional nonselective chemotherapy produce a 5% response. (56) To avoid side effects and unnecessary costs associated with new targeted therapies, molecular diagnostic tests play an essential role in patient selection. This story discusses the opportunities and challenges for cytology to help guide targeted therapies.

The patients who benefit from the new therapies have incurable metastatic disease. For such patients, there is an obvious advantage in using minimally invasive cytologic approaches to obtain the cells and tissue needed for molecular characterization. Because about 60% of patients with NSCLC present with surgically unresectable disease, (57) and optimal techniques for diagnosis and staging such patients use EBUS or EUS that produce only cytology samples (see previous story), it certainly makes sense to adapt the molecular testing to cytology samples.

Many emerging studies are showing that cytology can provide excellent material for the molecular testing required for personalized therapies. (57-62) A variety of cytology platforms have been validated for mutation testing. (63) Ninety-eight percent of lung cytology samples were suitable for epidermal growth factor receptor and KRAS mutation testing in a recent study. (57) Evaluation of HER2 gene expression by fluorescence in situ hybridization (FISH) on cytologic samples from primary or distant metastases of breast cancer has been shown to be a cost-effective method with fewer procedure-related risks for the patients. (64) Compared with tissue sections that can truncate the nucleus, cytology smears improve FISH assays by displaying the entire nucleus of the cells. (65) Air-dried smears are particularly useful for FISH assays in our experience by flattening and spreading the chromatin and increasing the adhesion of the cells to the slide. Compared with bone marrow tissue sections, bone marrow touch imprints markedly improve the identification of chromosomal abnormalities. (66,67) Cytologists routinely use FISH studies to identify malignancies in urine, biliary tract brushings, and hematological preparations. Some alcohol fixatives have been shown to provide better FISH signals than formalin-fixed material. (68)

New Challenges for Cytology

A first challenge for cytology is to be able to provide the more detailed diagnoses needed to direct the appropriate molecular tests. Previously, most malignant pulmonary cytology specimens only needed to be reported as either NSCLC or small cell carcinoma. The overall specificity and overall positive predictive value of FNAB for diagnosis of pulmonarymalignancyareclose to 100% and 99%, respectively, (69,70) but in the era of targeted therapies, NSCLC needs to be subdivided into squamous cell carcinoma or adenocarcinoma to determine the appropriate testing and therapies. Several recent studies are showing that morphologic subclassification of NSCLS is accurate on cytological specimens. (57,71,72) In difficult cases, the use of immunohistochemical stains such as P63 and TTF-1, and rarely napsin and P40, can differentiate adenocarcinoma from squamous cell carcinoma. These immunostains work well in cytology material, allowing subclassification with up to 100% accuracy. (73,74)

A more difficult challenge for cytology emerged recently: The Food and Drug Administration (FDA) approved the Vysis (Abbott, Abbott Park, Illinois) break-apart FISH probe test to identify ALK-positive NSCLC patients who benefit from crizotinib therapy. The Vysis FISH Probe kit was designed to identify ALK gene rearrangements in sections of formalin-fixed, paraffin-embedded (FFPE) human NSCLC tissue specimens. According to the manufacturer's instructions, "the FDA-approved Abbott assay should be performed only on 10% neutral buffered FFPE human lung tumor tissue. Other types of specimens or fixatives should not be used." (75) There are similar requirements for using FFPE for the FDA-approved Cobas (Roche Molecular, Pleasanton, California) epidermal growth factor receptor and B-RAF mutation assays. Of note, Roche makes both the drug and the assay for the mutation that defines who should receive the therapy. The restriction for using FFPE in FDA-approved assays can make it difficult for cytology to provide the platform for personalized medicine, as explored below.

A Fixation on Formalin

The reason that FFPE has emerged as the platform for these FDA-approved molecular tests is that clinical trials used readily available FFPE tissue and did not include cytology arms. This is unarguably a great first step in discovering new molecular markers and diagnostic tests, but from a patient's perspective, less invasive cytology samples offer advantages. It is also cost effective to avoid having to obtain a second biopsy sample from those patients diagnosed primarily by EBUS and EUS who have provided only a cytology sample. Formalin can be used by cytologists, but it can pose unnecessary problems. Labs can collect FNA needle rinses in a saline solution and then split the sample between formalin and cytology fixatives for use in monolayer preparations. The time in saline before transfer of the rest of the sample into formalin is a potential variable in molecular or immunohistochemical studies that has not received much attention. The alternative of collecting cytology samples directly in formalin typically prohibits monolayer preparations and Papanicolaou staining unless trained individuals triage part of the sample. The Papanicolaou stain has advantages over hematoxylin-eosin staining because it specifically marks keratinized cells diagnostic of squamous differentiation. (72) Direct collection of cytology samples in formalin also increases the amount of red blood cells and precipitated blood proteins in cytology samples. The dilution of diagnostic fragments with red cells and precipitated debris, or with cell block binding agents such as fibrin or agarose, can lead to the erroneous impression that the cellular yield of FNA is low.

The use of FFPE for molecular testing raises more fundamental questions. It is not obvious that assays should perform as well on FFPE tissue as they can on alcohol-fixed cytology samples. Several studies have shown advantages of alcohol fixation for nucleic acid as well as proteomic work. (68,76-81) Formalin fixation introduces sequence alterations into DNA. (82) Formalin fixation is temperature dependent, and is a relatively slow process, whereas denaturation fixation by alcoholic fixatives appears to go to completion quickly with less apparent temperature dependence. Unlike formalin fixation, the time in alcohol fixatives does not seem to be a factor that affects protein preservation. (83,84) Alcohol-based fixatives therefore appear easier to standardize than formalin.

The discovery of inaccurate estrogen and progesterone receptor immunohistochemical assays (85) in 2005 ultimately led to rigorous guidelines for use of formalin fixation. It is important to note that the discovery of inaccurate results was made by restaining the same blocks of formalin-fixed tissue that had given erroneous results. Thus, the conclusion of the investigation does not seem logical: "Stated in general terms, the primary causes of the changes in testing results were poor fixation and tissue processing..." (86)

An overlooked observation is that formalin fixation impairs the yield of recovered nucleic acid from paraffin-embedded tissues, with estimates between 30% (82) and 99%. (87) If the goal of cytopathology is to make a diagnosis on the smallest possible biopsy specimen, these estimates argue against using formalin, because it would require biopsies to be 3 to 100 times bigger that they would need to be. It is unusual for a polymerase chain reaction-based assay to require more than about 200 ng of DNA. The DNA content of normal diploid cells is about 7 pg, so a sample of only about 30 000 cells is theoretically sufficient, and such a sample would weigh only about 150 [micro]g and be virtually invisible to the naked eye, with a total volume of only about 0.15 [micro]L (assuming a cell is spherical with a diameter of 20 im with a density of about 1 g/ml). In fact, an average of 7400 ng of DNA was recovered from air-dried or alcohol-fixed and stained cytology smears of FNAs in one study. (81) The poor yield of nucleic acids from formalin-fixed samples can explain the relatively high rates of insufficient samples in some studies of formalin-fixed cell blocks. (61,88) Cell blocks can be made without formalin, without risk of cross-contamination, and with efficient cell recovery. (2-4)

There is also a concern about the safety of formalin, and European countries are considering banning use of formalin because of its categorization by the World Health Organization as a carcinogen. (84,89) A ban on formalin use in Europe could force a global collaborative effort to develop safer and better fixatives that could also have advantages for cytology.

Validating or Verifying Molecular Tests on Cytology Samples

The FDA approval of FFPE as the platform for the emerging molecular tests does not prohibit labs from performing tests on non-formalin-fixed cytology samples, but it does necessitate a validation, a more extensive process compared with the required "verification" of FDA-approved tests. (90) Currently, each individual laboratory must validate modifications of FDA-approved tests, without reimbursement. The economic burden of validation hits smaller laboratories (or the relatively smaller cytology divisions of laboratories) disproportionately. The current validation process tends to inhibit innovation, because even an obvious improvement in a validated procedure poses a significant expense and time commitment for a laboratory. Although our system is designed to protect patients, it benefits larger organizational structures that can obtain or follow FDA approval.

Labs can use published data to justify only verification rather than a full validation. (90) However, in this early period of personalized cytology, it is likely that many individual labs will be independently scrambling to each prove that cytology platforms are valid for testing. Alternatively, clinical systems will settle on practices without cytology. Professional societies could help to establish a consortium approach to reduce the redundancy of validations and improve the efficiency of the process: Groups of laboratories could use their validation budget to collectively validate nonoverlapping aspects of assays and disseminate their validated protocols to the broader pathology community, who would then apparently only need to verify the assays for their laboratory.

Validation using clinical samples is intrinsically problematic, because by definition, clinical samples are generally unknown except as assessed by the last validated set of clinical samples. It would be useful to establish a national program to provide anatomic pathology controls, bearing known amounts of protein or known mutations. The best and simplest anatomic pathology controls are likely to be cell lines--that is, cytology samples. (91) Cell line standards could be characterized in several independent manners (eg, by Western blotting, immunohistochemistry, and various molecular assays). True anatomic controls would streamline validations and should be able to assure interlaboratory uniformity beyond what can be achieved with ad hoc clinical samples.

The field of targeted therapies is developing rapidly, expanding into new drugs targeting MEK, P13K, and mTOR. (54,55) Cytopathology has proven itself to be a capable partner for this era of personalized medicine, offering benefits to patients with inoperable cancers. It

will be crucial to include cytology platforms in emerging clinical trials that validate new targeted therapeutics, because the FDA approval process, and the cumbersome current means of test validation, can otherwise exclude cytology.

THYROID MOLECULAR TESTING

Fine-needle aspiration still provides the most effective means of distinguishing benign from malignant nodules. (92-96) Fine-needle aspiration biopsy, rather than surgical pathology, has such a stronghold in the initial evaluation of thyroid lesions because the criteria for diagnosis are primarily at the cellular level for papillary thyroid carcinoma, (24) and even follicular neoplasms (FNs) can be recognized on the basis of relatively small tissue fragments measuring well under the diameter of even a 27-gauge needle (190 [micro]m). Unlike the evolution of molecular testing in other areas of anatomic pathology, molecular testing is being developed specifically for application to cytology samples.

About 5% of adult women and 1% of adult men (approximately 5 000 000) have palpable thyroid nodules; the incidence of ultrasonographically detectable nodules is much higher. (97) From this large pool of lesions, there are about 275 00098 to 450 000 thyroid nodules (99) currently examined by FNA each year in the United States, from which an estimated 56 000 new thyroid cancers (approximately 10% of FNAs) are expected in 2012. (100)

The Bethesda system for reporting thyroid cytopathology (101,102) was designed to standardize the nomenclature within and across labs, and convey maximal clinically relevant information. Six categories of diagnoses are defined, with distinctly different risks of cancer, distinctive management implications, and different potential benefits for ancillary studies. (103) The 6 categories are benign, nondiagnostic, follicular lesion of undetermined significance (FLUS), FN, suspicious for malignancy (mostly papillary thyroid carcinoma), and malignant (mostly papillary thyroid carcinoma).

Cost Saving is a Key Goal

A key cost-saving measure is to reduce the chance of surgical resection of benign nodules, currently estimated to be 57%. (99) An ancillary test for thyroid FNAs with a high negative predictive value could help. Another important cost-saving measure is to reduce the need for a completion thyroidectomy (second surgery) in patients with cancer diagnosed only at lobectomy. (104) An ancillary test with a high positive predictive value would be able to triage patients for an appropriate 1-step total thyroidectomy. An additional significant cost in the evaluation of patients with thyroid nodules is the need for repeat FNAs, particularly for nondiagnostic FNAs. (105) It seems likely that cytopathologist-performed FNAs are the most efficient means of reducing this expense.

Ancillary Molecular Tests

There are 3 different ancillary molecular tests for thyroid FNAs: direct detection of mutations, messenger RNA expression profiling, and microRNA expression profiling. Although preliminary study of microRNA expression profiling appears promising, (106) it is troubling that results from different groups have identified completely non-overlapping sets of discriminatory microRNAs. (107) Numerous studies have demonstrated a value for direct mutation testing. (108-111)

Direct detection of the mutations of thyroid cancer is feasible because only about 7 genes are mutated in the vast majority of thyroid cancers. (112) The mutations in these genes are almost always mutually exclusive. (113) The various mutations correlate closely with tumor type: Mutations in B-RAF, RET and TRK are restricted to papillary thyroid carcinomas, N-RAS is strongly associated with the follicular variant of papillary thyroid carcinoma, (114,115) PAAX8/PPAARgamma is largely restricted to FNs, and H-RAS and K-RAS (less common) are more likely in FNs than papillary thyroid carcinomas. (113)

Though only 7 genes appear involved in initiation of the vast majority of thyroid follicular cancers, testing is complicated by the presence of multiple potential point mutations in RAS and B-RAF genes, and many different translocations of RET and TRK (at least 11 and 5, respectively). Testing for point mutations (eg, B-RAF, NRAS, H-RAS, and K-RAS) requires only a small amplicon size, an assay that works on the relatively poor substrates of FFPE material. (116) Detecting translocations is much more challenging. The translocations of RET and TRK are mostly intrachromosomal, (117) making them relatively difficult to detect by FISH. Studies using FISH to detect RET translocations have found alternating positive and negative areas within morphologically malignant cells of papillary thyroid carcinoma (118)--a finding that may reflect insensitivity of the FISH technique rather than the implausible alternative of multiple independent RET translocations. Polymerase chain reaction-based detection of the translocations is also difficult because the translocations can take place over a large distance. Use of messenger RNA as the basic substrate makes the translocations easier to detect, because relatively long introns are spliced out of the messenger RNAs, though working with RNA requires special attention to the method of preservation. (113)

Nikiforov et al (108,109,111) have pioneered thyroid mutation testing, using carefully analytically validated methods, and chose the 9 most common mutations that together are found in about 80% of thyroid cancers of follicular origin (but not medullary thyroid carcinoma or lymphomas). For their studies involving 1526 patients, they use only material collected in RNA preservative solutions (which cannot simultaneously preserve morphology), either from a dedicated pass or from residual needle rinses. In prospective tests, the mutation testing had an estimated sensitivity and specificity of about 63% and 98% respectively. (108,109,111) It is important to note that the few false-positives in these cohorts were all neoplasms (follicular adenomas), potentially precursors to invasive cancers. (113) Based on testing of the tumors after resection, there were no analytical false-positives and only 5% false-negatives, apparently due to inadequate sampling by FNA. (111) The assay has an analytical sensitivity of about 10% tumor cells for the point mutation assays, and 1% tumor cells for the translocation assays; higher sensitivity is achievable, but not desirable because of the chance for false-positive result. (116) Nikiforov et al (103) developed a molecular estimate of the proportion of epithelial cells in the sample to help avoid analyzing samples with less than about 10% epithelial cells, and a surprisingly high proportion of FNA samples (92%) have been sufficient for testing.

The Effects of Disease Prevalence on the Value of Mutation Testing

For the purposes of understanding the potential value of ancillary testing, data were abstracted (Table) from 2 large studies from 3 academic institutions, (93,94) representing 7290 FNAs classified according to the Bethesda System for reporting thyroid cytopathology. As summarized below, the value of mutation testing is largely restricted to thyroid FNAs in the FLUS and FN categories.

Cytologically benign nodules would not benefit from mutation testing because testing 65% of all patients would detect only about 1% of cancers in the whole cohort (about half of the cancers in cytologically benign nodules), with potentially 89% false-positive results.

Many of the approximately 10% of patients with insufficient cells for cytomorphology would be likely to have insufficient material for ancillary molecular testing. Even if material were available for successful molecular testing, only about 1% of cancers in the whole original cohort would be picked up by testing 9% of samples. Because only about 35% of mutation positive cases would be anticipated to have cancer, mutation-positive patients may be better served with lobectomy (or repeat FNA) rather than total thyroidectomy; a risk of cancer of greater than about 50% is a rough guideline for consideration of 1-step total thyroidectomy. (97)

In this academic cohort, only about 4% of patients have a Bethesda System for reporting thyroid cytopathology diagnosis of FLUS; the rate is much higher in other cohorts in the literature. (119) Ancillary mutation testing could allow most of the cancers in this cohort (about 3% of the total cancers) to forego repeat FNA and instead immediately undergo a 1-step total thyroidectomy with 78% chance of cancer. Importantly, mutation-negative FLUS cases would still need follow-up studies, probably repeat FNA, because their posttest risk of cancer is still about 4%.

The FN category contains about 20% of all cancers in the cohort. In the absence of ancillary testing, a diagnosis of FN generally leads to lobectomy only, because the prevalence of cancer is well below 50%. A positive mutation test would direct more than half of this cohort to total thyroidectomy (saving about 12% of the total cancer patients from having to undergo a 2-step thyroidectomy). Mutation-negative FN cases would still probably need lobectomy, however.

It is possible that mutation testing would have value in helping with the difficult decision about total thyroidectomy versus lobectomy in patients whose FNA is suspicious for papillary thyroid carcinoma (97,111) (comprising about 27% of all cancers).

There is no apparent advantage of mutation testing for the approximately 46% of cancers diagnosed as malignant by FNA. Although B-RAF-positive cases of papillary thyroid carcinoma may be more aggressive than other forms of papillary thyroid carcinoma, (112) there is no consensus about the significance of B-RAF positivity in terms of the extent of surgery or need for extended lymphadenectomy. (120)

Costs and Available Platforms for Mutation Testing

The mutation assay (111) was estimated to be cost effective when applied to FNAs with FLUS and FN diagnoses when the cost is less than about $870. (104) The estimated average reimbursement for the 9-mutation panel was estimated to be $650 in 2007. (104)

Asuragen, Inc (Austin, Texas) offers the 9-mutation panel (Inform Thyroid) using its own proprietary RNA preservative. One dedicated pass into the preservative is acceptable if it contains a minimum of 50 ng of tissue. Asuragen does third-party billing and does not bill for samples that have insufficient quality or quantity of nucleic acids. The Inform Thyroid test has been analytically validated, but there is not yet a published clinical validation.

Quest Diagnostics also offers the 9-mutation panel with third-party billing. Acceptable samples for Quest include "Needle washings in alcohol based fixative (eg, CytoLyt, Hologic Corporation, Bedford, Massachusetts), 4 slides, or formalin-fixed, paraffin-embedded tissue block, FNA, or formalin-fixed paraffin-embedded specimens." (121) Performance characteristics of the Quest test are not apparently published. The use of formalin-fixed materials suggests that many small amplicons must be simultaneously assayed for this test to work, and contradicts recommendations by Nikiforov (113) and Nikiforova and Nikiforov. (116)

In spite of using RNAlater for preserving RNA, some groups have reported "variable RNA quality and, at times, substantial RNA degradation." (122) The suggestion of adopting reverse-transcriptase-polymerase chain reaction to scraped cells from smears (123) is considered not feasible by experts in the field. (116)

The precise method for testing is an important variable. In a study that used RNA (preserved in RNA preservative) to make complementary DNA for all testing, 11 of the 50 mutations detected were in histologically benign (non-neoplastic and "adenomatoid" nodules), and the spectrum of mutations differed from previous cohorts by having about 80% NRAS mutations. (124) Careful validation is needed if the exact techniques or primers differ from established protocols.

Expression Profiling for Molecular Diagnosis of Thyroid FNAS

Direct mutation testing has a high positive predictive value, with particular value for sending patients with indeterminate findings for 1-step total thyroidectomy. A messenger RNA expression profiling assay has been developed by Veracyte, Inc (South San Francisco, California) to have high negative predictive value, with the goal of allowing patients with indeterminate findings to be safely followed without surgery. On January 9, 2012, Veracyte, Inc, announced that Medicare will pay a new Current Procedural Terminology code for the Afirma Gene Expression Classifier (Veracyte Website Press Releases. http://www.veracyte.com/ media/press-releases/?id=26 [accessed August 4, 2012]). Of major significance for practicing pathologists, clinicians can completely bypass their cytopathologists by sending in samples collected in collection tubes supplied by Veracyte. Veracyte processes a CytoLyt aliquot for morphologic diagnosis by its own cytopathologists and attempts a definitive diagnosis. If the diagnosis is FLUS or FN, and if the nodule is larger than 1 cm in a patient 21 years of older, Afirma is applied to a separate aliquot of sample sent on ice in RNA transport medium.

Veracyte's test was originally developed on surgical pathology specimens. Using 142 genes, the test was developed to have high negative predictive value. (122) Of some concern in this study funded by Veracyte, discrepancies were identified in applying the algorithm derived from surgical samples to cytology samples. The discrepancy was thought likely to be due to dilution of the cytology sample (about 5-fold dilution on average) with cells from outside the nodule, though this seems unlikely based on clinical experience. (122) With adjustments of the algorithm, they achieved a remarkable 96% negative predictive value for FNAs. (122) Four percent of samples have insufficient RNA for Afirma testing. In modeling a hypothetical cohort, the Afirma Gene Expression Classifier was estimated to be cost effective if it cost less than $3200, saving $1453 per patient and contributing 0.07 quality-adjusted life years. Cost savings is achieved primarily because of decreased numbers of thyroid surgeries.

An independent clinical trial funded by Veracyte, Inc, of the Afirma test has just been published. (125) Of some concern, the study used a new updated classifier algorithm from the previous study, (122) and there is only about one year of follow-up. Thirty percent of benign nodules had a positive Afirma test, indicating that care needs to be taken to avoid testing cytologically benign samples. There were 3 malignancies of 55 cases (5%) with a negative Afirma test and a referring cytologic diagnosis of FLUS. Longer-term follow-up may be needed to know how effective the assay is for reducing the need for surgery.

The Continuing Importance of the Primary Cytologic Diagnosis

High rates of FLUS diagnoses (for which the value of molecular testing is being marketed) can represent problems at many levels, including diagnostic expertise, the quality of the aspiration, and cytopreparation. (126) A recent nationwide educational survey by the College of American Pathologists found a troubling approximately 40% of circulated FNA slides felt to represent FNs were miscategorized as either benign or malignant. (127) An overall 18% diagnostic discordance was found on review of thyroid FNAs referred to a tertiary care center. (128) High FLUS rates erode the utility of mutation testing and especially gene expression profiling. The quality of the primary morphological cytology diagnosis dictates the relative clinical value of mutation testing, and efforts are still needed at a national level to raise expertise in thyroid cytopathology.

INTERVENTIONAL CYTOPATHOLOGISTS USING ULTRASOUND-GUIDED FNA

Fine-needle aspiration is the logical first step in evaluating most mass lesions because of its safety, simplicity, rapidity, and ability to provide definitive diagnoses. Fine-needle aspiration performance is not trivial, however, and the advantages of FNA disappear if the sample is sparse, diluted with blood, poorly preserved, or not appropriately triaged. Inefficient triaging of material for ancillary studies can raise costs, with some samples triaged for studies that are not needed (eg, flow cytometry for a metastatic carcinoma), or samples not triaged for studies that are needed to establish a firm diagnosis (eg, microbiology studies if mycobacterial infection is suspected). Any health care provider can perform FNAs with little or no specialized training, but getting a good sample, well triaged and well prepared, requires expertise. Poor quality and poor triaging of FNA samples by clinicians have contributed to the erroneous impression that FNA has limited usefulness, with a resultant shift toward use of larger biopsy samples than are actually needed, or the use of diagnostic alternatives (eg, radiology tests) that add cost to the workup but have less definitive results than a properly performed FNA.

Value of Cytopathologist-Performed FNA

Clinicians can use on-site assessment by a cytopathologist to help overcome these limitations, allowing procurement of an adequate sample, often with provision of an instant diagnosis, and with efficient triage of material for necessary ancillary studies. On-site assessment decreases the non-diagnostic rate to about 1% and is cost effective by preventing patients from having to return for a repeat procedure. (129) However, to provide an on-site assessment for another clinician involves simultaneously scheduling 2 professionals for the procedure, and the relatively low reimbursement for onsite evaluation, or even the FNA performance itself, makes this strategy difficult for health care organizations to fund. (130) The economic support for this model has become further eroded with recent changes in the coding and decreased reimbursement for on-site evaluation. (37) For patients to benefit from having the smallest possible biopsy specimen, it is most economical and diagnostically useful for the FNA to be performed whenever possible by the cytopathologist who will eventually make the diagnosis. It is not surprising that the results of cytopathologist-performed FNA are superior to the results of FNAs performed by clinicians. (131)

From the perspective of a patient presenting to their primary care physician with a concerning mass, a common practice pattern is referral to a specialist such as an endocrinologist, otolaryngologist, or surgeon. Often these specialists then refer the patient to radiology for initial scans, with follow-up biopsy by radiology in yet another visit. The patient then schedules a follow-up appointment to learn the biopsy results. This scenario can involve at least 4 different physicians and multiple appointments at different medical facilities. In the appropriate clinical setting it can be more efficient for patients to be referred by primary care directly to a cytopathologist to perform the FNA.

Value of Addition of Ultrasound Guidance for Cytopathologist-Performed FNA

Within the past 10 years, cytopathologists have begun to use ultrasound guidance as a tool for their performance of FNA. The incorporation of ultrasound allows an expansion of cytopathologist-performed FNA to nonpalpable lesions, and it allows greater assurance of safe sampling when masses are near vital structures (eg, a supraclavicular node near the pleura). Addition of ultrasound guidance decreases the nondiagnostic rate for thyroid FNAs (105) and allows specific targeting toward solid or suspicious areas in heterogeneous nodules. (132) Nondiagnostic FNAs are a major source of increased cost to the health care system. (105,129)

Perhaps the most important benefit of including ultrasound is that the ultrasound appearance itself can provide essential clues to the differential diagnosis. Most cytopathologists who use ultrasound for guidance still welcome a detailed ultrasound report from radiology. The radiology report is billed separately from the ultrasound guidance of the FNA. The radiology report provides a clear image of the lesion, documentation of its size and characteristics, and often a differential diagnosis. The radiology report is especially useful for cytopathologists less experienced in ultrasound anatomy and differential diagnosis. When a patient is seen by a specialist and referred to radiology for FNA, it can be difficult to integrate the complete clinical and radiologic picture and make it consistent with the FNA diagnosis. In the appropriate clinical setting, cytopathologist-performed FNA with ultrasound guidance can restore continuity of care and often allow this essential clinical integration to take place seamlessly. Based on the on-site evaluation of the sample, additional history can immediately be obtained, and useful confirmatory exams can be immediately initiated (eg, a skin examination when a metastatic melanoma is identified).

A new niche is developing for "interventional cytopathologists" using ultrasound and FNA as cost-effective tools. In comparing FNA performed with and without ultrasound guidance by the same cytopathologist, ultrasound guidance increased overall accuracy, especially the specificity and negative predictive value of FNA, (133) and this approach appears cost effective. When on-site evaluation is nondiagnostic, patients can be referred for core biopsies. Importantly, this strategy offers the patients the smallest, safest possible biopsy in as few clinic visits as possible. Rare cytopathologists are further extending their practice to the performance of core biopsies (132) and placement of radiologic clips when clinically indicated. (134) In one office visit, 87% of patients with breast masses were able to be diagnosed with only ultrasound-guided FNA; core biopsies were performed to enable diagnosis of invasion for patients with positive FNAs and to resolve nondiagnostic FNAs. (134) Interventional cytopathologists are appropriately resurrecting breast FNA.

What Are the Steps to Adopting Ultrasound-Guided FNA?

Ultrasound machines have become more compact and affordable, in the range of $20 000 to $60 000 for portable units with good resolution and high-quality imaging. Pathologists who already routinely perform FNAs based on palpation have proficiency in FNA technique, slide preparation, and adequacy assessment, and they are in the best position to acquire the skill to adopt ultrasound guidance. There is a need for more formal training in residency and fellowship programs because it can be more difficult to learn this technique later in practice. Some cytopathology fellowships have arranged for rotations through otorhinolaryngology, endocrinology, or radiology clinics to learn ultrasound-guided FNA. Several continuing medical education training opportunities in ultrasound interpretation and ultrasound-guided FNA are particularly appropriate for cytopathologists already in practice. The College of American Pathologists offers the Ultrasound-Guided Fine-Needle Aspiration Advanced Practical Pathology Program. There is also the American Association of Clinical Endocrinologists' Diagnostic Endocrine Neck Ultrasound and Ultrasound-Guided FNA Course. Newly trained cytopathologists in ultrasound often find it easiest to start by adding ultrasound guidance to the lesions that they are already proficient at biopsying by palpation. Commercial ultrasound phantom models can be purchased for training, or a homemade phantom using a boneless turkey breast stuffed with an olive is an inexpensive alternative. It is remarkable that in one study, the advantages of adding ultrasound to cytopathologist-performed FNA became apparent in a consecutive cohort that started only within 6 months after ultrasound training. (133)

Ultrasound imaging and anatomy are not trivial, but several basic textbooks are available. Appropriate judgment in nodule selection can be learned by working in parallel with an experienced ultrasonographer (technologist) or with another trained radiologist or pathologist. In general, the sonographic features suspicious for malignancy include heterogeneous hypoechogenicity, solid components in cystic masses, irregular margins, microcalcifications, nodules that are taller toward the skin than wide, and moderately increased intranodular vascularity. Lymph nodes that lack a fatty hilum, are relatively spherical in shape, or show nodular configurations of the cortex are generally more suspicious. A scoring system for the clinical and ultrasound features that predict malignancy in lymph nodes has been recently published. (135)

Ideally, when the patient is referred from the primary care provider or specialist for a suspected mass, the FNA cytopathologist reviews prior imaging reports and/or history/physical examination notes, obtains a medical history, and performs a focused physical examination. Real-time ultrasound scanning is then performed to evaluate the lesion in 3-dimensional views, using Doppler to assess the vascularity. Ultrasound-guided FNA is then performed, with direct smears for adequacy or preliminary diagnosis. Based on the assessment, material is triaged as needed to cell block preparations for immunohistochemistry and/or molecular studies, flow cytometry, microbiology cultures, cytogenetics, and/or air-dried smears for fluorescent in situ hybridization. The patient may be given a preliminary diagnosis, and is then referred back to the clinician for communication of the final diagnosis with discussion of the treatment or follow-up.

In building an ultrasound-guided FNA service, it is important to approach radiologists, endocrinologists, and surgeons as team members for patient care. In our experience, the actual billing for the FNA itself is hardly a cause for friction with radiologists or clinicians, yet the combination of the performance and adequacy assessment is economically viable when only the cytopathologist is involved. Some insurers may not reimburse for the small incremental increase of ultrasound guidance by nonradiologists. (133)

Patients with high-deductible health insurance policies (who pay out of pocket) appear to be particularly attracted to cytopathologist-performed ultrasound-guided FNA clinics, presumably because they are cost conscious and educated about the advantages of FNA over core biopsy. High-deductible health insurance policies--if they can remain an option--would seem to benefit cytology by making patients seek the value that our profession has promised for so many years. Alternatively, in the era of accountable care organizations, with evidence-based medicine and value-based outcomes, (136) the stars appear aligned for cytology to expand its turf: There should be enthusiasm for a nationally funded outcome study to compare the ideal practice pattern of cytopathologist-performed FNA with alternatives, such as referral to specialists or radiologists. Outcome data will help assure that cytopathologists can be reimbursed for their valuable service.

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Andrew H. Fischer, MD; Cynthia C. Benedict, MD; Mojgan Amrikachi, MD

Accepted for publication October 4, 2012.

From the Department of Pathology, University of Massachusetts Medical Center, Worcester, Massachusetts (Dr Fischer); the Department of Cytopathology, DCL Medical Laboratories, Inc, Indianapolis, Indiana (Dr Benedict); and the Department of Pathology and Genomic Medicine, The Methodist Hospital, Houston, Texas (Dr Amrikachi).

Dr Fischer is an inventor of the Cellient technology for making cell blocks and he receives royalties on sales of this technology. The other authors have no relevant financial interest in the products or companies described in this article.

Reprints:AndrewH.Fischer,MD, Department of Pathology, University of Massachusetts Medical Center, Biotech Three, 1 Innovation Dr, Worcester, MA 01605 (e-mail: Andrew.Fischer@ umassmemorial.org).
Representative Data Based on 2 Studies (93,94) to Illustrate
Value of Mutation Testing (111) of Thyroid Fine-Needle Aspirations
(FNAs) Classified According to the Bethesda System for Reporting
Thyroid Cytopathology (TBSRTC) (a)

                       Treatment
                    Implications of                 Approximate
                     First Cytology                  % of All
                      Diagnosis in      % of All    Follicular-
                       Absence of       FNAs in       Derived
     TBSRTC            Molecular         TBSRTC     Cancers in
    Category          Testing (97)      Category   Each Category

Benign             Clinical follow-        65           2.4
                    up

Non-               Repeat FNA              9            1.5
 diagnostic

FLUS               Repeat FNA              4            3.4

FN                 Lobectomy               10           20
                    versus total
                    thyroidectomy
                    based on
                    clinical factors

Suspicious for     Total                   6            27
 malignancy         thyroidectomy

Malignant          Total                   6            46
                    thyroidectomy

                    Approximate    Prevalence of
                   Prevalence of     Cancer if      Positive
                     Cancer in     Mutation Test   Predictive
                   Each Category    Is Negative     Value of
                    Based Only     (1--Negative     Mutation-
     TBSRTC         on Cytology     Predictive      Positive
    Category         Result, %     Value), % (b)   Result, %(b)

Benign                  0.4            0.15            11

Non-                    1.7            0.65            35
 diagnostic

FLUS                    10               4             78

FN                      30              14             93

Suspicious for          61              37             98
 malignancy

Malignant               98              95             .99

                     Potential Value for
                   Mutational Testing per
     TBSRTC         Protocol of Nikiforov
    Category             et al(111)

Benign             No value apparent at
                   this time

Non-               No value apparent at
 diagnostic        this time

FLUS               Mutation testing may
                   be useful: Negative
                   results may allow
                   clinical follow-up,
                   repeat FNA, or
                   lobectomy; positive
                   results may allow
                   total
                   thyroidectomy (111)

FN                 Mutation testing may
                   be useful: Negative
                   results should
                   probably have
                   lobectomy whereas
                   positive results
                   should have total
                   thyroidectomy (111)

Suspicious for     Mutation testing may
 malignancy        be useful: Negative
                   results should
                   probably have
                   lobectomy whereas
                   positive results
                   should have total
                   thyroidectomy

Malignant          No clear value at this
                   time

Abbreviations: FLUS, follicular lesion of undetermined significance;
FN, follicular neoplasm.

(a) This approximation is derived from the sum of 2 large studies
from 3 academic institutions (93,94) representing more than 7000
FNAs classified according to TBSRTC. Several simplifications
and assumptions were necessary: The data from Yassa et al (93)
data gave the "final" cytology FNA that preceded resections,
whereas the data from Yang et al (94) counted some cases with
multiple FNAs as separate nodules. Non-follicular-derived
malignancies were ignored from both studies. Only surgically
resected nodules with cytologic diagnoses of FN, suspicious
for papillary thyroid carcinoma, or malignant were used for
the total nodule count (which can overestimate specificity of
the cytology diagnosis), but all FNAs were counted as total
nodules for cases in the benign, nondiagnostic, and FLUS
categories in order to avoid overestimating prevalence in
these categories.

(b) Using Bayesian analysis modeling based on 63% sensitivity
and 98% specificity of molecular testing.
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Author:Fischer, Andrew H.; Benedict, Cynthia C.; Amrikachi, Mojgan
Publication:Archives of Pathology & Laboratory Medicine
Geographic Code:1USA
Date:Jul 1, 2013
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