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Vascular and Interventional Radiology |
1 From the Departments of Radiology (N.J.S., L.H.B.) and Histopathology (J.W.G.), Addenbrookes Hospital, Hills Rd, Cambridge CB2 2QQ, England. Received March 13, 2001; revision requested April 17; revision received October 9; accepted November 13. Address correspondence to N.J.S. (e-mail: nicholas.screaton@papworth-tr .anglox.nhs.uk).
| ABSTRACT |
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MATERIALS AND METHODS: Two hundred sixty US-guided core biopsies were performed in 247 patients with cervicofacial lymphadenopathy. The age of the patients ranged from 1 to 91 years (mean, 50 years). Seventy-four (30%) had a history of malignancy. Biopsies were performed as outpatient procedures with direct US guidance and nonadvancing 1618-gauge core needles. Hospital records were reviewed 6 months to 5 years after biopsy. Final diagnoses were rendered based on results of histologic examination of excised specimens, clinical course, or results of other laboratory studies.
RESULTS: Two hundred thirty-eight (92%) core biopsies yielded adequate material. In 28 (11%) patients, the histologic diagnosis was considered highly probable. In the 210 patients in whom adequate material was obtained and an unequivocal histologic diagnosis was given, the sensitivity, specificity, and accuracy of US-guided core needle biopsy in differentiating benign from malignant lymphadenopathy were 98.1%, 100%, and 98.7%, respectively. Seventy biopsies were performed in 66 patients with lymphoma. Sensitivity, specificity, and accuracy in differentiating lymphoma from reactive lymphadenopathy were 98.5%, 100%, and 98.7%, respectively. In 53 patients (80%) with lymphoma as a final diagnosis, histologic subclassification was sufficient to guide treatment without the need for surgical biopsy. There were no major complications and only three minor postbiopsy hematomas.
CONCLUSION: US-guided core biopsy in patients with head and neck lymphadenopathy is a safe outpatient procedure that has a high diagnostic yield and accuracy and frequently obviates surgery.
© RSNA, 2002
Index terms: Head and neck neoplasms, 20.342, 20.343, 20.375 Head and neck neoplasms, diagnosis Lymphatic system, biopsy, 997.12985 Lymphoma, diagnosis, 997.12985 Ultrasound (US), guidance, 20.12985
| INTRODUCTION |
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At our institution, patients with head and neck masses are referred for immediate ultrasonographic (US) assessment, which proceeds directly to US-guided cutting-needle core biopsy when appropriate. This process expedites referral of patients to the appropriate clinical team and eliminates the need for open lymph node biopsy performed solely for diagnosis.
Patients with cervicofacial lymphadenopathy comprise two broad groups: those with known malignancy in whom metastatic disease is a concern, and those with lymphadenopathy arising de novo. Imaging plays an important role in depicting the presence and distribution of cervical lymphadenopathy. Computed tomography, magnetic resonance imaging, and US have a 20%28% higher sensitivity for the detection of lymphadenopathy than does clinical assessment (1). Imaging techniques, however, have low specificity for differentiating benign lymphadenopathy from malignant lymphadenopathy and lymphoma from metastatic disease (2,3). A tissue diagnosis remains a standard requirement.
Fine-needle aspiration cytology (FNAC) is widely used in the assessment of patients with head and neck masses. It is a safe and inexpensive outpatient procedure with a reported diagnostic accuracy in malignant lymphadenopathy that exceeds 90% (4). Disadvantages to FNAC include a high rate of nondiagnostic samples and incomplete classification of lymphoma. The larger tissue sample obtained with core biopsy permits the use of a range of histochemical and immunohistochemical stains. This factor, combined with the preservation of tissue architecture in the core biopsy sample, enables a more precise histologic assessment.
Results of lymph node excision biopsy remain the diagnostic standard of reference, but this procedure is invasive and may entail general anesthesia and hospital admission. Excision biopsy is usually undertaken solely for diagnosis and may not be integral to the patients treatment. In addition, adverse outcomes resulting from diagnostic surgical biopsies have been described in the setting of squamous cell carcinoma (5).
Image-guided core tissue biopsy with automated cutting needles is a well-established technique. However, there are only a few small series in which the routine use of core biopsy in the assessment of head and neck masses was described (6,7). The purpose of our study was to evaluate our experience in 247 patients with cervicofacial lymphadenopathy who underwent US-guided core biopsy.
| MATERIALS AND METHODS |
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At the time of each biopsy, the following information was noted and entered into a database: the site, size, number, and echo pattern of lesions; the gauge of the biopsy needle used; the number of tissue cores obtained; and the presence of any immediate complications.
Hospital records were reviewed 6 months to 5 years after each biopsy to determine the final diagnosis; any delayed complications following the core biopsy were recorded. If a surgical excision biopsy proved necessary, either for diagnosis or as part of definitive therapy, this was documented. Final diagnoses were based on the results of histologic examination of excised specimens, the patients clinical course, or the results of other laboratory studies.
Patients ranged in age from 1 to 92 years (mean, 50 years). One hundred twenty-one patients were male and 126 were female. Seventy-four (30%) patients had a history of malignant disease. One hundred seventy-three (70%) had no previous malignant disease. Ten patients had previously undergone a nondiagnostic FNAC procedure that was performed by the referring physician without imaging guidance.
Biopsy Technique
A preliminary US evaluation, which included color Doppler studies, was performed with a 7.510.0-MHz linear array transducer (Toshiba SSD 270 and Powervision; Toshiba Medical Systems, Crawley, England). Patients suspected of having lymphoma also underwent abdominal US. Informed consent for the procedure was obtained. A coagulation screen was not routinely performed.
All patients underwent biopsy with local anesthesia except for one child who received intravenous anesthesia for a simultaneous bone biopsy. In two other pediatric patients, local anesthesia was augmented with light oral sedation.
Biopsies were performed by staff radiologists (including L.H.B.) or by radiology fellows (including N.J.S.) during their US attachment. A modified, nonadvancing, disposable, spring-loaded cutting biopsy needle (Temno; Allegiance Healthcare, McGaw Park, Ill) was used. All biopsies were performed with a freehand technique and direct US guidance. Further details of the technique have been previously described (7,8). An image was obtained with the biopsy needle in the lesion to document accurate sampling. A 16-gauge biopsy needle was used in larger lesions and in all patients suspected of having lymphoma. The use of 18-gauge needles was usually reserved for small and relatively inaccessible lesions. The number of tissue cores obtained ranged from one to four (mean, two), depending on the suspected cause of the lymphadenopathy, the quality of the core specimen as assessed by inspection with the naked eye, and whether additional specimens were required for microbiologic assessment.
Patients were examined 1560 minutes after the procedure, and if hemorrhage was suspected, a repeat US examination was performed.
Tissue cores were conventionally processed and were stained with hematoxylin and eosin and histochemical and immunohistochemical stains as appropriate. A single histopathologist (J.W.G.) reviewed all specimens, and, if necessary, the expertise of subspecialist histopathologists was sought both locally and at other institutions. Lymphoma was subclassified as Hodgkin or non-Hodgkin lymphoma. Non-Hodgkin lymphoma was further classified as high or low grade. When possible, full subclassification according to the Kiel or the Revised European-American Lymphoma (REAL) classification system was recorded.
Definition of Terminology
There is no consensus in the literature about the optimal presentation and analysis of data in this area. This has resulted in difficulty when comparing results and techniques from different centers. For the purpose of the current study, we have defined several potentially confusing terms as follows.
Adequate sample.Sample adequacy was a subjective judgement made by the histopathologist. A specimen was deemed adequate if the lesion was correctly sampled and if sufficient material was present for a diagnosis to be rendered.
Inadequate sample.An inadequate sample was one in which insufficient material was obtained for histologic diagnosis. This includes cases of sampling error, as well as cases in which the lesion was correctly sampled but the histopathologist was unable to render a diagnosis on the basis of the material supplied.
False-negative biopsy result.A biopsy result was considered to be false-negative when the tissue sample was thought to be adequate but histologic diagnosis indicated benign disease when the final diagnosis was of malignancy.
False-positive biopsy result.A biopsy result was considered to be false-positive when the tissue sample was thought to be adequate but histologic diagnosis indicated malignant disease when the final diagnosis was of benign disease.
Statistical Analysis
Sensitivity, specificity, and accuracy were calculated with standard methods. Previous studies of the sensitivity, specificity, and accuracy of biopsy in the evaluation of patients with head and neck masses have used different inclusion and exclusion criteria. This relates particularly to how inadequate samples and "probable" diagnoses were considered. We have presented our data by using three different analyses found in other studies: (a) an analysis that includes all biopsy results and considers inadequate and equivocal biopsy results as incorrect, (b) an analysis that includes results from only those biopsies in which adequate material was obtained, and (c) an analysis that includes results from only those biopsies in which adequate material was obtained and after which an unequivocal histologic report was issued. The
2 test was used for the comparison of two proportions.
| RESULTS |
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In 137 cases (53%), the node sampled during biopsy was one of several enlarged lymph nodes. In 105 cases (40%), a solitary enlarged node was present. In 18 (7%) cases, this information was not available. The size of the nodes sampled during biopsy ranged from 0.5 cm to 8.0 cm (mean, 2.2 cm).
On the basis of their appearance at US, 230 (88%) of the nodes were solid, three (1%) were mixed solid and cystic, and six (2%) were cystic. This information was not recorded for 21 (8%) of the nodes sampled during biopsy.
Histologic Findings
The final diagnosis in all cases was defined as the result of surgical resection, clinical follow-up, and/or serologic testing.
Adequate samples.Two hundred thirty-eight samples (92%) were considered adequate by the histopathologist. Of these 238 samples, 210 resulted in a confident histologic diagnosis, while 28 resulted in a probable diagnosis.
Inadequate samples.Twenty-two core biopsy samples (8%) were reported as inadequate. These included samples in which there was necrotic debris (n = 4), no nodal tissue (n = 5), or insufficient tissue from an accurately sampled node (n = 13).
In three of the 22 patients in whom inadequate biopsy samples were obtained, a repeat US-guided core biopsy was performed, which in each case provided adequate material for definitive diagnosis. Of the remaining 19 patients, nine underwent diagnostic surgical biopsy. In one patient in whom histologic results were nondiagnostic, Mycobacterium tuberculosis was cultured from a specimen that had been divided between the histology and microbiology departments. In nine patients, the enlarged node resolved spontaneously, obviating further biopsy.
Table 1 provides a detailed analysis of the 238 biopsy samples judged to be adequate by the histopathologist. Table 2 lists the final diagnosis in the inadequate samples.
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Patients who underwent surgical resection.Fifty-nine patients subsequently underwent open lymph node resection. The results of this procedure are detailed in Table 3. In 26 (44%) of the patients who underwent lymph node resection, it was performed for therapeutic purposes after a definitive diagnosis was rendered at core biopsy.
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False-negative diagnoses.Results of three core biopsies (1%) were reported as indicating inflammatory changes, but subsequent open biopsies performed because of continuing clinical suspicion revealed malignancy (Table 1).
False-positive diagnoses.There were no false-positive diagnoses at core biopsy.
Accuracy of core biopsy.Because of the confusion caused by the various inclusion and exclusion criteria that previous authors have used, we have presented our data by using three different analyses found in other studies.
1. If all 260 biopsies are included in our analyses and we define all inadequate samples or equivocal diagnoses as incorrect diagnoses (false-negative or false-positive), the overall accuracy of the procedure for differentiating benign from malignant lesions was 90% (235 of 260 samples). Sensitivity and specificity cannot be calculated from these data.
2. If we exclude from our analyses the 22 biopsies in which inadequate material was provided for histologic analysis, the sensitivity, specificity, and accuracy of core biopsy in differentiating benign from malignant disease were 98.1%, 100%, and 98.7%, respectively. There were 28 biopsies at which the histopathologist made a "highly probable" diagnosis; these biopsies have been considered as having provided adequate samples for the purpose of this analysis. If our group of patients is subdivided into those with and those without a history of malignancy, the following results are obtained: In patients with a previous diagnosis of malignant disease, the sensitivity, specificity, and accuracy of core biopsy were 96.8%, 100%, and 97.4%, respectively. These values did not differ significantly from the sensitivity, specificity, and accuracy of 98.9%, 100%, and 99.4% in patients with no relevant history who presented with lymphadenopathy of unknown cause.
3. If we analyze only the 210 biopsies in which an adequate sample was provided and an unequivocal histologic report was issued, the sensitivity, specificity, and accuracy of core biopsy in differentiating benign from malignant disease were 99.3%, 100%, and 99.5%, respectively.
Diagnosis of Lymphoma
A final clinical diagnosis of lymphoma was rendered in 66 patients who underwent a total of 70 core biopsies. There were 48 patients with non-Hodgkin lymphoma, and 18 with Hodgkin disease.
Seventy core biopsies were performed in patients with lymphoma; lymphoma was correctly identified in 66 of these biopsies. One patient who had non-Hodgkin lymphoma had been given a false-negative diagnosis of reactive lymphadenopathy at core biopsy; non-Hodgkin lymphoma was diagnosed after excision biopsy. Three biopsy samples were considered inadequate by the histopathologist. Repeat core biopsy in two of these three patients yielded a diagnosis of lymphoma (non-Hodgkin lymphoma in one, Hodgkin disease in one); in one case, non-Hodgkin lymphoma was diagnosed at excision biopsy. There were no false-positive diagnoses of lymphoma.
In 66 patients in whom lymphoma was correctly identified at core biopsy, an unequivocal histologic diagnosis was given in 54, while histologic features that were strongly suggestive of lymphoma were observed in 12. Ten of these 12 patients subsequently underwent surgical excision biopsy for histologic confirmation and subtyping, which confirmed lymphoma in all cases. In two patients, a diagnosis of recurrent lymphoma was made and treatment was initiated on the basis of the core biopsy results, which showed histologic features concordant with those observed at the patients initial presentation.
If the three inadequate samples are treated as false-negative diagnoses, the sensitivity, specificity, and accuracy of core biopsy in differentiating malignant lymphoma from reactive lymphadenopathy were 94.3%, 100%, and 95.2%, respectively. If only the 67 biopsies in which the samples were considered adequate for diagnosis by the reviewing histopathologist are included in our analysis, the sensitivity, specificity, and accuracy of core biopsy in differentiating malignant lymphoma from reactive lymphadenopathy were 98.5%, 100%, and 98.7%, respectively. In the 54 patients in whom an unequivocal diagnosis of lymphoma was rendered at core biopsy, the accuracy was 100%.
Classification of Lymphoma
Complete subclassification of lymphoma according to the Kiel or REAL system was available in 29 (60%) of the 48 patients with non-Hodgkin lymphoma. Full subclassification according to the Rye system was possible at five (28%) of the 18 biopsies that revealed Hodgkin disease.
In 53 (80%) of the 66 patients with lymphoma, histologic subclassification was sufficient for therapeutic purposes and treatment was instituted on the basis of the results of the core biopsy alone. In two patients with lymphoma, therapy was begun despite limited subclassification because high-grade transformation had been excluded at core biopsy.
In addition to the 11 open surgical biopsies performed for diagnosis or confirmation of lymphoma, another two excision biopsies were performed for further classification of disease in patients with an unequivocal diagnosis at core biopsy. In one of these patients, excision biopsy revealed nodular sclerosing Hodgkin disease, although the diagnosis at core biopsy had been non-Hodgkin lymphoma.
Complications
Three patients had minor postbiopsy hematomas that resolved spontaneously without intervention or the need for hospital admission. There were no other complications.
| DISCUSSION |
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In squamous cell carcinoma of the head and neck, cervical metastases have prognostic and therapeutic importance. A solitary ipsilateral cervical metastasis reduces 5-year survival by 50%; a contralateral metastasis reduces 5-year survival by nearly 75% (13). When the risk of metastasis exceeds 15%20%, neck dissection or radiation therapy is indicated (14,15).
Cross-sectional imaging is more accurate than clinical assessment in revealing neck lymphadenopathy (1618). Although imaging studies are sensitive, their specificity is inadequate for enabling the confident diagnosis or subclassification of malignancy (2,3,1822). A tissue diagnosis is usually required for planning the management of head and neck malignancy. Surgical lymph node resection has been the diagnostic standard, but it is invasive and often requires general anesthesia and hospital admission. Some authors also report that in squamous cell carcinoma of the head and neck, surgical biopsy prior to definitive surgery may adversely affect prognosis (23).
Nonsurgical approaches to tissue diagnosis, particularly FNAC, have been widely adopted. Core biopsy is an established technique, but there are few series that report its routine use in the head and neck. This may be due to a reluctance to use an automated biopsy device in an anatomic site that contains numerous major vessels and nerves.
Percutaneous biopsy procedures (ie, FNAC or tissue-core biopsy) are limited by the quality of the specimens they provide. A false-negative diagnosis may result from sampling error, particularly when neoplastic cells are scanty (eg, in Hodgkin disease). In a series of nonimage-guided FNAC procedures in head and neck lesions, inadequate samples were obtained in 21.5% and inconclusive samples in 12% of procedures, resulting in a frequency of nondiagnostic sampling of 33.3% (24). When a cytopathologist performs FNAC, the frequency of nondiagnostic samples is reduced (10) because sample adequacy can be assessed immediately and resampling can be performed if necessary.
The use of US guidance to enable the biopsy of nonpalpable lesions and the targeting of a favorable site (eg, the wall of a cystic lesion) increases sensitivity. With US guidance, the frequency of nondiagnostic FNAC procedures has been reduced to 6%11% (2528). However, these series have included highly selected patients with known malignancy.
Robinson and Cozens (24) combined US guidance with immediate cytologic assessment in a less-selected patient group. The frequency of nondiagnostic samples in their series was 15%. This dual-operator approach has implications for manpower, time, and cost. In one study, the mean duration of US-guided FNAC of the thyroid increased by almost 30 minutes when a cytologist was present for the procedure, but diagnostic yield was not increased (29).
Despite accurate sampling of a lesion, nonuniform distribution of disease within an affected node may result in sampling error. This possibility can be reduced when multiple samples are obtained or when larger specimens such as tissue cores are acquired.
Core biopsy provides an intermediate step between FNAC and open surgical biopsy. The histologic specimen obtained at core biopsy is also of sufficient size to permit extensive immunohistochemical staining. The use of image guidance and the larger size of the core biopsy sample also reduce sampling error, increasing sensitivity and negative predictive value. All of these advantages of core biopsy are particularly relevant to the diagnosis of lymphoma (3032). The possibility of sampling error does persist, however, and, if lymphoma remains a concern after a negative biopsy result, we recommend a repeat core or open biopsy.
In our study, a single operator acquired a biopsy core in approximately 15 minutes. Previous studies have demonstrated the cost-effectiveness of FNAC compared with surgical resection (33,34). To our knowledge, no studies have assessed the cost-effectiveness of core biopsy versus that of surgical resection.
In the current study of unselected patients with unexplained lymphadenopathy, the frequency of obtaining an inadequate sample at US-guided core biopsy was 8% (22 of 260 biopsies). At another 28 (11%) of 260 biopsies, histologic features were highly suggestive of the correct diagnosis but the histopathologist offered a differential diagnosis. The overall adequacy of 81% for the biopsies in our series is slightly lower than the 89%94% reported in studies of US-guided FNAC in selected patients with known malignant disease (2528). Several factors adversely affected the diagnostic yield in our series, including the relatively unselected nature of our patient groupover two-thirds of our patients had no history of malignancy. Twenty-seven biopsies (10%) were performed by radiology fellows who were learning the technique. Furthermore, we used an extremely rigorous classification system for pathologic findings. Only unequivocal histologic findings were reported as such.
In addition, our series included many patients in whom lymphoma was a consideration. At cytologic and histologic examination, it is considerably more challenging to differentiate reactive lymphoid hyperplasia from lymphoma than it is to establish the diagnosis of metastatic carcinoma. Of the 28 equivocal biopsy results, 12 were considered indeterminate because the histopathologist was unable to differentiate lymphoma from reactive lymphadenopathy. In each of these 12 cases, the probable diagnosis of lymphoma was later confirmed.
A comparison of the accuracy of core biopsy in our study with the accuracy of FNAC as described in previous studies was hindered by the use of different statistical methods, particularly different inclusion and exclusion criteria. In the current study, if inadequate samples and equivocal histologic reports are excluded, as is the case in many studies of FNAC (4,28,35,36), we achieve a sensitivity, specificity, and accuracy of 99.3%, 100%, and 99.5%, respectively. Our results compare with reported accuracies of FNAC, which are in the range of 85%96% (4,28,35,36), for diagnosing metastatic head and neck carcinoma.
Differences in patient selection criteria make comparison between series difficult. In the current study, the sensitivity, specificity, and accuracy of core biopsy in patients with known malignancy (when inadequate samples were excluded) were 96.8%, 100%, and 97.4%, respectively. Moreover, in the difficult-to-assess group of patients with lymphadenopathy and no known malignancy, sensitivity, specificity, and accuracy of core biopsy were 98.9%, 100%, and 99.4%, respectively. These results exceed even those reported in FNAC studies with selected patients suspected of having metastatic carcinoma of the head and neck, where accuracies of 85%96% were reported (4,28,35,36).
In patients with lymphoma, tissue diagnosis is essential for treatment both at initial presentation (37) and at possible relapse. The value of cytologic examination in lymphoma is controversial. In specialized laboratories, the diagnostic accuracy of FNAC in lymphoma approaches 79%90% (38,39). However, the required ancillary examination techniques, including immunocytochemistry, flow cytometry, cytogenetics, and molecular genetics, are not universally available. In practice, the accuracy of FNAC in the assessment of lymphoma is approximately 20% less than its accuracy in carcinoma (40). Although a high positive predictive value (86%) for lymphoma has been reported for FNAC (41), due to frequent false-negative results the negative predictive value and sensitivity in the same series were only 42% and 66%.
In the current study, the accuracy of core biopsy in differentiating lymphoma from reactive lymphadenopathy, given an adequate sample, was 98.5%. This approximates our overall accuracy for all malignancies (98.7%).
A further advantage of tissue core sampling is that it enables the subclassification of lymphoma. A panel of 10 to 15 immunohistochemical stains is frequently required to fully characterize lymphoma. A good core biopsy sample provides sufficient material to enable full classification and permits additional unfixed material to be preserved unfrozen for molecular diagnostic procedures (eg, gene rearrangement studies), should they be required.
In the study of Erwin et al (42) of patients with lymphoma who underwent core biopsy and FNAC, subclassification of lymphoma was possible in 38% of patients on the basis of the results of the core biopsy. In our series, full subclassification of lymphoma according to the International Working Formulation or REAL systems of classification of non-Hodgkin lymphoma and the Rye system of classification of Hodgkin disease was achieved in 51% of cases. Although in many institutions, surgical excision biopsy is seen as essential in lymphoma, in 80% of our patients classification of lymphoma on the basis of core biopsy results was sufficient to commence therapy. These results agree with those of other series, which demonstrate that core biopsy results are sufficient for guiding lymphoma treatment in 72%96% of patients (30,31,43,44).
In our opinion, needle-track metastasis has been overstated as a theoretical complication of percutaneous biopsy. To our knowledge, there are no reported cases following core biopsy of superficial lymph nodes (45), and there are only 12 reported cases in the literature following FNAC. In a large series of cutting-needle biopsies in the head and neck, Southam et al (46) found no cases of needle-track metastases after up to 7 years of follow-up.
A potential criticism of this study is the use of both results of excision biopsy and findings at clinical follow-up of between 6 months and 5 years as confirmation of correct findings at core biopsy. Although regression of lymphadenopathy over a follow-up period is not definitive proof of benignity, it is highly suggestive.
In summary, US-guided core biopsy in the head and neck is a safe, minimally invasive outpatient procedure with a high diagnostic yield. In the 81% of cases where US-guided core biopsy provided a diagnostic sample, the sensitivity, specificity, and accuracy of the procedure in differentiating benign from malignant disease were 98.1%, 100%, and 98.7%, respectively. Due to its high yield in lymphoma and its accuracy in differentiating lymphoma from reactive lymphadenopathy, there was no significant difference between the accuracy of US-guided core biopsy in patients with de novo lymphadenopathy and its accuracy in patients with a history of malignancy. In our series, 80% of patients with lymphoma were treated on the basis of US-guided core biopsy results, without the need for surgical biopsy.
Due to its high sensitivity and accuracy in the setting of metastatic carcinoma, we recommend FNAC as the initial investigation in patients with epithelial malignancies who are suspected of having lymph node metastases. In patients without a history of malignancy and in centers where cytologic expertise is not available, core biopsy should be adopted.
| FOOTNOTES |
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Author contributions: Guarantors of integrity of entire study, L.H.B., N.J.S.; study concepts and design, L.H.B., N.J.S.; literature research, N.J.S.; clinical studies, J.W.G., L.H.B., N.J.S.; data acquisition, L.H.B., N.J.S., J.W.G.; data analysis/interpretation, N.J.S.; statistical analysis, N.J.S.; manuscript preparation and definition of intellectual content, N.J.S., L.H.B.; manuscript editing, J.W.G., L.H.B.; manuscript revision/review, L.H.B., J.W.G.; manuscript final version approval, L.H.B., J.W.G., N.J.S.
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