Fine needle biopsy (FNB) is frequently used in the diagnostic workup of clinically or radiologically detected mass lesions that are suspicious for metastatic melanoma. By determining whether they represent metastatic melanoma, the use of FNB in melanoma patients can expedite detection of metastases, leading to earlier removal and treatment; facilitate the staging of patients enrolled in clinical trials of adjuvant therapies (particularly in deep-seated lesions); reduce the frequency of unnecessary surgery; and assist in the planning of the most appropriate surgery. 1 2 3 6 7 10 11 12 The objective of this retrospective cohort study was to evaluate the diagnostic accuracy of the FNB procedure in the detection of metastatic melanoma. To accomplish this, a very large consecutive sample was collected of FNBs performed on melanoma patients who attended the Sydney Melanoma Unit (SMU), Sydney, Australia. This large sample also allowed evaluation of the effect of several clinicopathologic features and factors related to the procedure on the diagnostic accuracy of FNB. MATERIALS AND METHODS Patients 1 TABLE 1. Patient characteristics Characteristic n % Sex   Male 888 62.7   Female 528 37.3 Age at melanoma diagnosis (y)   10–30 110 7.8   31–40 135 9.5   41–50 256 18.1   51–60 303 21.4   61–70 302 21.3   70–80 219 15.5   81+ 62 4.4   Unknown 29 2.0 Cancer diagnosis   Other cancer diagnoses 159 11.2   More than one other cancer diagnoses 15 1.1 No. of primary melanoma lesions   1 1236 87.3   2 148 10.4   ≥3 32 2.3 No. of FNBs   1 953 67.3   2 287 20.3   3 98 6.9   >3 78 5.5 Total no. of patients 1416 100.0 FNB, fine needle aspiration biopsy. FNB Procedure 2 2 Review of Clinical Material and Follow-up The accuracy of the FNB procedure in diagnosing metastatic melanoma was evaluated by two reference standards: a) histopathologic evaluation of the excised lesion (1120 cases) or b) follow-up in those cases for which histologic material was not available (462 cases). The duration of follow-up was 6 months or greater (mean 50.2 months, median 45.7 months, range 6.1–144.4 months) in 456 cases. In six cases, the length of follow-up was less than 6 months (mean 4.2 months, median 4.0 months, range 3.0–5.7 months). The mass was considered benign if it was stable in size or resolved after clinical follow-up. Cytodiagnosis 2 TABLE 2. Categories of cytodiagnoses Characteristic Total n % Confirmed Not confirmed Positive for Metastatic Melanoma   Malignant cells—melanoma 1089 49.4 805 284 Suspicious for Metastatic Melanoma   Malignant cells—suspicious for melanoma 44 2.0 39 5   Malignant cells—unknown cancer 33 1.5 22 11   Suspicious for malignancy 40 1.8 35 5 Negative for Metastatic Melanoma   Malignant cells—other cancer 92 4.2 47 45   No malignant cells—other cells present 416 18.9 308 108   No malignant cells—scant other cells 296 13.4 204 92   No malignant cells—no other cells 175 7.9 122 53   No malignant cells—procedure not performed 19 0.9 0 19 Total 2204 100 1582 622 Samples that contained cells from unclassified/unspecified malignancies or cases categorized as suspicious for melanoma (those with small numbers of atypical cells, poorly preserved cells, and cells that lacked specific features of melanoma, such as cytoplasmic pigment, and where insufficient material was present for immunochemistry) were classified as suspicious for metastatic melanoma. These were determined to be true suspicious (n = 78, 4.9%) or falsely suspicious (n = 18, 1.1%) results after verification. FNBs classified as negative for metastatic melanoma contained no material that could be diagnosed as metastatic melanoma. They included cases containing malignant cells diagnosed as another neoplasm or b) various amounts of cellular material from the tissue of the site that was sampled. After verification, these procedures were found to be either true negative (n = 612, 38.7%) or false negative (n = 69, 4.4%). Statistical Analysis All analyses were performed using the S-PLUS software package (Insightful Corporation, Seattle, WA) and Microsoft Excel, version 2000 (Microsoft, Redmond, WA). Diagnostic accuracy of FNB for metastatic melanoma was measured by the sensitivity and specificity of the test. 13 14 15 RESULTS 3 TABLE 3. Fine needle biopsy procedures that could not be performed Location Explanation for failure Confirmation Histology Left neck Unable to locate lesion Negative—follow-up – Left face (subcutis) Pain Not confirmed – Right face Unable to locate lesion Not confirmed – Left neck (LN) Unable to locate lesion Negative—surgery No evidence of malignancy Right breast Adjacent to prothesis Negative—follow-up – Right neck Pain Not confirmed – Right axilla (LN) Unable to locate lesion Negative—follow-up – Right neck Pain Negative—surgery No evidence of malignancy Left neck (LN) Pain Negative—follow-up – Left neck (LN) Unable to locate lesion Negative—follow-up – Right groin Pain Negative—surgery No evidence of malignancy Left axilla (LN) Unable to locate lesion Positive—surgery Melanoma Right axilla (LN) Unable to locate lesion Negative—follow-up – Right neck (LN) Pain Not confirmed – Left axilla (LN) Unable to locate lesion Not confirmed – Left neck Unable to locate lesion Negative—follow-up – Thyroid Unable to locate lesion Negative—follow-up – Right axilla (LN) Unable to locate lesion Negative—follow-up – Right sternum (LN) Unable to locate lesion Not confirmed – LN, lymph node. 1 FIG. 1. Flow diagram showing fine needle biopsy (FNB) result distribution.  A total of 603 FNBs (27.4%) could not be verified; this was due to loss to follow-up, particularly by death in patients with advanced metastatic melanoma, uncertainty regarding sites (where FNBs from more than one site were obtained at the same visit to the clinic), and where there were multiple metastases, which led to uncertainty in lesion location and the correlation of FNB and histopathologic results. Unconfirmed FNBs differed from confirmed procedures in several ways. They were more likely to be from lesions located in visceral organs or to be in patients with AJCC/UICC stage IV disease. FNBs with inconclusive cytodiagnoses were more likely to be followed up with clinical observation or further biopsy. Confirmation of FNBs was unaffected by the year of procedure, the number of needle passes during sampling, the use of immunostains, the reporting cytopathologist, or the age or sex of the patient. Approximately 12% of the patients were diagnosed with additional cancers (most commonly breast cancer, colorectal cancer, and chronic lymphocytic leukaemia), with some suffering from multiple types of other cancers. Multiple primary melanomas occurred in an eighth of this patient population. Almost a third of the patients underwent multiple FNBs. These procedures were performed both concurrently and sequentially. 4 2 TABLE 4. False-positive fine needle biopsy findings for metastatic melanoma Cytodiagnosis Histology Location Comment Melanoma Metastatic adenocarcinoma Right axillary (LN) – Melanoma Metastatic papillary carcinoma Left supraclavicular fossa (LN) – Melanoma Hematoma Left axilla – Melanoma Chronic osteomyelitis Left skull (bone) a Melanoma Metastatic adenocarcinoma Right axilla – LN, lymph node. a FIG. 2. Distribution of false-negative fine needle biopsy (FNB) cytodiagnoses.  5 TABLE 5. Diagnostic accuracy of FNB: Effect of tissue type and anatomic location Location n Confirmed % TP FN TS FS FP TN Sn (95% CI) Sp (95% CI) All FNB 2204 1582 71.8 800 69 78 18 5 612 0.92 (0.90–0.94) 0.99 (0.98–1.00) Lymph nodes 926 753 81.3 413 43 41 7 3 246 0.91 (0.88–0.93) 0.99 (0.97–1.00)   Neck 235 185 78.7 104 3 15 4 1 58 0.97 (0.92–0.99) 0.98 (0.91–1.00)   Axilla 383 313 81.7 155 29 12 2 2 113 a (0.78–0.89) 0.98 (0.94–1.00)   Groin 274 233 85.0 144 11 11 0 0 67 0.93 (0.88–0.96) 1.00 –   Other 34 22 64.7 10 0 3 1 0 8 1.00 – 1.00 – Skin and subcutis 711 504 70.9 270 17 26 4 0 187 b (0.91–0.96) 1.00 –   Head and neck 131 98 74.8 54 5 7 0 0 32 0.92 (0.82–0.96) 1.00 –   Trunk 288 196 68.1 101 4 6 2 0 83 0.96 (0.91–0.99) 1.00 –   Limbs 292 210 71.9 115 8 13 2 0 72 0.93 (0.88–0.97) 1.00 – Visceral organs 176 79 44.9 30 3 5 2 0 39 0.91 (0.76–0.97) 1.00 –   Liver 56 22 39.3 7 1 1 0 0 13 0.88 (0.53–0.98) 1.00 –   Lung 94 43 45.7 18 2 4 1 0 18 0.90 (0.70–0.97) 1.00 – Other 391 246 62.9 87 6 6 5 2 140 0.94 (0.87–0.97) 0.99 (0.95–1.00)  TP, true positive; FN, false negative; TS, true suspicious; FN, false suspicious; FP, false positive; TN, true negative; SN, sensitivity; SP, specificity; 95% CI, 95% confidence interval. a b 6 TABLE 6. Diagnostic accuracy of fine needle biopsy effect of clinicopathologic factors Factor N Confirmed % TP FN TS FS FP TN SN (95% CI) SP (95% CI) All FNB 2204 1582 71.8 800 69 78 18 5 612 0.92 (0.90–0.94) 0.99 (0.98–1.00) AJCC/UICC stage at FNB   Stage I 400 323 80.8 128 9 22 6 3 155 0.93 (0.88–0.97) 0.98 (0.95–0.99)   Stage II 705 569 80.7 283 31 24 6 1 224 0.90 (0.86–0.93) 1.00 (0.98–1.00)   Stage III 836 561 67.1 310 24 27 5 1 194 0.93 (0.90–0.95) 0.99 (0.97–1.00)   Stage IV 263 129 49.0 79 5 5 1 0 39 0.94 (0.87–0.97) 1.00 (0.91–1.00) Location   Regional 1340 1077 80.4 617 58 58 6 4 334 0.91 (0.89–0.93) 0.99 (0.97–1.00)   Distant 864 505 58.4 183 11 20 12 1 278 0.94 (0.90–0.97) 1.00 (0.98–1.00) Use of immunochemistry   Yes 583 406 69.6 316 6 26 8 1 49 a (0.96–0.99) 0.98 (0.90–1.00)   No 1621 1176 72.5 484 63 52 10 4 563 0.88 (0.86–0.91) 0.99 (0.98–1.00) Year   1992–1994 392 285 72.7 144 15 15 5 1 105 0.91 (0.85–0.94) 0.99 (0.95–1.00)   1995–1997 555 414 74.6 216 15 13 6 1 163 0.94 (0.90–0.96) 0.99 (0.97–1.00)   1998–2000 693 500 72.2 241 21 26 3 2 207 0.92 (0.88–0.95) 0.99 (0.97–1.00)   2001–2002 564 383 67.9 199 18 24 4 1 137 0.92 (0.87–0.95) 0.99 (0.96–1.00) Sex   Male 1370 994 72.6 500 48 45 10 1 390 0.91 (0.89–0.93) 1.00 (0.99–1.00)   Female 834 588 70.5 300 21 33 8 4 222 0.93 (0.90–0.96) 0.98 (0.96–0.99) Age at FNB   ≤50 y 572 414 72.4 198 12 22 1 2 179 0.94 (0.90–0.97) 0.99 (0.96–1.00)   >50 y 1632 1168 71.6 602 57 56 17 3 433 0.91 (0.89–0.93) 0.99 (0.98–1.00) No. of FNB attempts   1 426 309 72.5 232 4 3 2 0 68 b (0.96–0.99) 1.00 (0.95–1.00)   2 518 371 71.6 185 13 21 3 0 149 0.93 (0.89–0.96) 1.00 (0.97–1.00)   3 315 219 69.5 79 13 12 6 0 109 0.86 (0.77–0.92) 1.00 (0.97–1.00)   ≥4 192 136 70.8 33 17 21 1 1 63 0.66 (0.52–0.78) 0.98 (0.92–1.00)   Unknown 753 547 72.6 271 22 21 6 4 223 0.92 (0.89–0.95) 0.98 (0.96–0.99) c   22G 133 60 45.1 22 2 4 1 0 31 0.92 (0.74–0.98) 1.00 (0.89–1.00)   23G 173 138 79.8 66 7 8 2 0 55 0.90 (0.81–0.95) 1.00 (0.93–1.00)   25G 910 664 73.0 351 24 39 7 1 242 0.94 (0.91–0.96) 1.00 (0.98–1.00) Necrosis present   Yes 67 41 61.2 24 4 9 0 0 4 0.86 (0.69–0.94) 1.00 (0.51–1.00)   No 2137 1541 72.1 776 65 69 18 5 608 0.92 (0.90–0.94) 0.99 (0.98–1.00) Pathologist caseload   <100 cases 164 104 63.4 51 7 5 4 0 37 0.88 (0.77–0.94) 1.00 (0.91–1.00)   100–500 cases 651 474 72.8 227 29 27 3 2 186 0.89 (0.84–0.92) 0.99 (0.96–1.00)   >500 cases 1389 1004 72.3 522 33 46 11 3 389 d (0.92–0.96) 0.99 (0.08–1.00) First primary Breslow thickness    ≤2 mm 950 705 74.2 332 23 34 8 4 304 0.94 (0.90–0.96) 0.99 (0.97–0.99)   >2 mm 919 662 72.0 367 38 33 6 0 218 0.91 (0.87–0.93) 1.00 (0.98–1.00)   Unknown 335 215 64.2 101 8 11 4 1 90 0.93 (0.86–0.96) 0.99 (0.94–1.00) First primary ulceration   Yes 571 407 71.3 247 16 18 1 0 125 e (0.90–0.96) 1.00 (0.97–1.00)   No 1009 757 75.0 360 40 35 10 4 308 0.90 (0.87–0.93) 0.99 (0.97–1.00)   Unknown 624 418 67.0 193 13 25 7 1 179 0.94 (0.90–0.96) 0.99 (0.97–1.00) 2   ≤1 414 300 72.5 136 10 12 4 2 136 0.93 (0.88–0.96) 0.99 (0.95–1.00)   1 to <4 481 352 73.2 170 19 20 2 1 140 0.90 (0.85–0.93) 0.99 (0.96–1.00)   4 to <8 332 237 71.4 141 7 8 1 0 80 0.95 (0.91–0.98) 1.00 (0.95–1.00)   ≥8 392 296 75.5 175 22 14 4 0 81 0.89 (0.84–0.93) 1.00 (0.95–1.00)   Unknown 585 397 67.9 178 11 24 7 2 175 0.94 (0.90–0.97) 0.99 (0.96–1.00) First primary lesion histologic subtype   Desmoplastic 130 97 74.6 42 6 6 3 0 40 0.88 (0.75–0.94) 1.00 (0.91–1.00)   SSM 512 370 72.3 168 10 22 4 1 165 0.94 (0.90–0.97) 0.99 (0.97–1.00)   NM 461 343 74.4 198 20 14 4 1 106 0.91 (0.86–0.94) 0.99 (0.95–1.00)   Other 754 549 72.8 277 26 22 4 1 219 0.91 (0.88–0.94) 1.00 (0.97–1.00)   Unknown 347 223 64.3 115 7 14 3 2 82 0.94 (0.89–0.97) 0.98 (0.92–0.99) First primary lesion predominant cell type   Epithelioid 443 329 74.3 185 17 13 4 0 110 0.92 (0.87–0.95) 1.00 (0.97–1.00)   Spindle 137 89 65.0 47 6 9 0 0 27 0.89 (0.77–0.95) 1.00 (0.88–1.00)   Mixed 173 111 64.2 58 6 8 3 0 36 0.91 (0.81–0.96) 1.00 (0.90–1.00)   Unknown 1451 1053 72.6 510 40 48 11 5 439 0.93 (0.90–0.95) 0.99 (0.97–1.00) TP, true positive; FN, false negative; TS, true suspicious; FN, false negative; FP, false positive; TN, true negative; SN, sensitivity; SP, specificity; 95% CI, 95% confidence interval; SSM, superficial spreading melanoma; NM, nodular melanoma. a b c d e 6 3 6 FIG. 3. Sensitivity of fine needle biopsy (FNB) in the diagnosis of metastatic melanoma. Sensitivity is reduced in a linear manner as the number of FNB attempts needed to obtain the sample increases. Bars, 95% confidence intervals.  DISCUSSION Metastatic melanoma is an aggressive tumor with a high mortality rate. Patients with primary melanomas that are at high risk of metastasizing who attend the SMU are followed closely for evidence of metastatic disease with radiological investigations and regular clinical follow-up. Early surgery for metastatic disease may lead to longer disease-free periods and may ultimately improve survival in some of these patients. This underscores the importance of early detection of metastatic disease. 2 16 7 12 11 7 10 12 17 , 18 19 20 21 In this study, there were differences in those FNBs that were verified by the reference tests compared with those that were not. Unconfirmed FNBs were more often performed on nonpalpable lesions in visceral sites, and in patients with advanced disease. Verification rates of negative, positive, and suspicious FNBs were 68.2%, 73.9%, and 82.1%, respectively. 4 22 2 There were several sources of difficulty in performing the FNB procedure for metastatic melanoma. Often it occurred when there was a failure to locate the suspicious lesion because of its small size or its location was not communicated with adequate precision. Failure to identify metastatic melanoma occurred when cellular material showed too few typical morphologic characteristics of metastatic melanoma, or these characteristics were destroyed or masked by necrosis. The large number of confirmed FNB in this study permitted the analysis of subgroups. Most FNB for metastatic melanoma were performed in lymph nodes as well as skin and subcutaneous tissues. No difference in sensitivity was noted among these tissue groups. However, FNBs of lymph nodes of the axilla were significantly less sensitive compared with those performed at other sites and with FNBs performed in lymph nodes of the groin and neck. A number of factors are likely to contribute to the low sensitivity of FNBs performed in the axilla, including the greater difficulty in gaining access to and locating lymph nodes, particularly those high in the axilla, and the presence of large amounts of fatty tissue in axillary lymph nodes (“horseshoe” nodes). 23 24 25 26 Our study shows that FNB for metastatic melanoma is a procedure with very high specificity and good sensitivity. Several clinicopathologic factors were found to influence the diagnostic accuracy of the test for metastatic melanoma. These included factors relating to the original primary melanoma lesion, location of the sampled lesion, and factors relating to the performance of the test. The SMU employs an on-demand FNB service with assessment of the cytologic material and delivery of a provisional result to the clinician at the time of the patient’s visit. This helps guide subsequent diagnostic and therapeutic measures, and it reduces costs (e.g., by decreasing the need for additional patient visits to the clinic). A multidisciplinary approach involving clinicians, pathologists and radiologists enables an efficient and cost-effective management strategy in melanoma patients.