Case 4

Isolated tumor cells identified by cytokeratin stain only (AJCC pN0 [i +])

History: This sentinel lymph node is from a 49 year old woman with 2.2 cm infiltrating lobular carcinoma. The sentinel lymph node was reported as "negative" at the time of intra-operative examination. The H&E stained slide and the cytokeratin immunohistochemical stained slide are submitted for your review. How would you report the findings? What is the clinical significance? Case 4 - Figure 1 - H&E Case 4 - Figure 2 - CYTOKERATIN Diagnosis: Isolated tumor cells identified by cytokeratin stain only (AJCC pN0 [i +]) Discussion: Axillary dissection has been an integral part of breast cancer management, with axillary nodal status being the single most important predictor of disease-free survival and overall survival in breast cancer and the absolute number of nodes involved by tumor of prognostic importance. Furthermore, excellent regional control is achieved with axillary lymph node dissection, with subsequent axillary recurrence a rare event. The improvement in long-term survival remains debatable. However, axillary lymph node dissection may have substantial associated morbidity. Occasionally, injury to major vessels or motor nerves may occur. Reports of significant lymphedema after axillary lymph node dissection has been reported to occur early or years later, with the incidence as high as 30% in some series. More importantly, approximately 70-75% of breast cancer patients will be node negative. We have entered a new era where we are re-evaluating the management of patients with breast cancer in regard to the axillary lymph node dissection. The sentinel node biopsy is founded on two basic principles: (1) there is an orderly and predictable pattern of drainage to a regional lymph node basin, and (2) the first lymph node (sentinel node) is an effective filter for tumor cells. As more surgeons rely on the sentinel node biopsy, completion axillary lymph node dissections are not routinely performed if the sentinel lymph node is negative. However, if the sentinel lymph node is positive, the current recommendation is to continue with the completion axillary lymph node dissection. Sentinel node biopsy is not unique to breast cancer and was tested in other cancers before its application to breast cancer. Sentinel node biopsy was originally explored by Cabanas in 1977 for the treatment of penile carcinoma. Morton and colleagues independently introduced the sentinel lymph node concept for melanoma. It was not until 1993, when Dr. Krag and colleagues of the University of Vermont used a radioactive isotope and gamma probe to localize the sentinel node in breast cancer. Dr. Giuliano and colleagues at John Wayne Cancer Institute in 1994 applied the technique of using blue dye to help localize the sentinel node in breast cancer. Finally in 1996, Dr. Albertini and colleagues from the H. Lee Moffitt Cancer Center and Research Institute used a combination of radioisotope and blue dye for the intraoperative lymphatic mapping in breast cancer. Since then, sentinel node biopsy has emerged as the most significant advance in the surgical treatment of breast cancer since the advent of breast conservation and many institutions have reported their results. The success rate of the sentinel node procedure is operator and technique dependent. Reported success rates have ranged from 66 to 98%. This supports that investigators have been able to identify the sentinel node in almost all patients despite the technique used; however, this also informs us that sentinel lymph nodes are not detected in all patients. The majority of patients will have one sentinel node identified, however, between 8-35% of patients are considered to have more than one sentinel lymph node, with most studies reporting somewhere between one and three sentinel lymph nodes per patient. Pathologic Assessment The assessment of the sentinel node biopsy is a collaborative exercise between the surgeon and the pathologist. The pathologic methods used to assess the sentinel node is of paramount importance since lymph node status is the most powerful prognostic factor in breast cancer patients. Unfortunately, there is presently no consensus for the optimal handling of the sentinel node or to what extent the sentinel node should be sectioned. Most institutions have developed their own "in-house" protocols, and many of these protocols are based on the institution's research endeavors. Therefore, due to this non-uniformity, it is difficult to compare studies across institutions. Further studies are needed to reach a consensus on the most sensitive and cost-effective technique for pathologic examination of the sentinel lymph node for the staging of breast cancer. Many different techniques in assessing the sentinel node have been described in the literature and include: imprint cytology, frozen section, serial and step sections of the paraffin embedded sentinel node tissue, cytokeratin immunohistochemistry, and even sophisticated molecular biological techniques such as reverse transcription polymerase chain reaction. Radiation Safety The Surgical Pathology Committee of the College of American Pathologists and the Association of Directors of Anatomic and Surgical Pathology have written an excellent article that was recently published in The American Journal of Surgical Pathology. The half-life of technetium-99m is 6 hours with radiation levels dropping to background levels after 10 half-lives (60 hours). Title 10 of the Code of Federal Regulations states that the total effective dose limit for radiation workers is 5000 mrem per year or 50,000 mrem per year for skin or extremities. The mean radiation dose to the skin of the surgeon's hand during sentinel lymphadenectomy for breast cancer has been reported to be approxi-mately 10 mrem per case, whereas the total effective dose is estimated to be less than 0.1 mrem. Based on these figures, a surgeon could theoretically perform thousands of such operations each year and not exceed the statutory exposure limits. In terms of the pathology staff, the mean radiation dose has rarely been measured, but is most likely lower than that to the surgeon. Each institution should develop their own written protocols for handling radioactive materials in the pathology laboratory. These protocols should be developed in conjunction with the institution's radiation safety officer. The protocol should clearly state how specimen containers are labeled, how specimens are transported from the operating room to the laboratory, and how the specimen is handled once in the laboratory. If frozen sections are performed, one may consider using a dedicated cryostat or clean the cryostat between uses. However, due to the limited amount of radioactivity present, special precautions may not be warranted. Disposable gloves should be worn when handling radioactive tissue specimens. The protocol should also clearly specify storage and disposal of radioactive tissue specimens. Gross Examination In the gross examination of the sentinel node, the lymph node is either sectioned or bivalved depending on its size. Many institutions section the sentinel node at 2-3 mm intervals when the sentinel node is larger than 1 cm and bivalve the lymph node when the sentinel node is less than 1 cm. Still, other institutions have adopted one of these two methods as standard protocol regardless of the lymph node size. Intraoperative Examination The intraoperative techniques used in the histopathologic evaluation of the sentinel node include: imprint cytology, frozen section and rapid cytokeratin immunohistochemistry. However, unless an immediate decision is required for patient care, many believe that the uses of these techniques are discouraged. The exception to this might be if the sentinel node is macroscopically abnormal and imprint cytology or frozen section confirms the node to be positive for metastatic carcinoma, allowing further axillary surgery to be performed immediately. Cytologic Preparation Many studies have been undertaken to evaluate the reliability of imprint cytology or "touch prep" for the intraoperative evaluation of the sentinel node for breast cancer. Even with this technique, there is diversity in the work-up of the sentinel node. The cut surface of the sentinel node is either dabbed several times along a glass slide or scraped with a scalpel blade and the cellular material smeared onto a glass slide. The number of cut surfaces examined, the number of slides prepared, whether or not the slides are air-dried or fixed in alcohol and what stain is employed also varies among studies. Imprint cytology has the advantage of being a simple and rapid technique and less expensive than frozen section. In addition to good cytologic detail obtained by this method, no artifacts are left in the tissue sample. Many studies have claimed this technique to be very reliable without loss of valuable tissue. In contrast, the imprint cytology may be of limited value due to the high percentage of micrometastases missed in evaluating the sentinel node, thus requiring patients to undergo a second operation. Although rare, false positives have been reported. Many studies have stressed that the interpretation of imprint cytology requires experience in cytology by the pathologist evaluating the specimen. Therefore, this technique may not be appropriate in a hospital without an active cytology service. Since most reported studies have not calculated the false negative rate using the accepted definition (number of false negatives/number of false negatives plus true positives), the false negative rate has been recalculated. This table demonstrates the intraoperative false negative rate per sentinel lymph node. The definition of a false negative is a negative test result in a patient that has the disease. In some instances the number of patients with a positive sentinel node is not given, but information regarding the total number of sentinel nodes positive or negative is given, and I have stated when I have used per sentinel node rather than per patient with disease. These studies have compared the result of touch imprint cytology of the sentinel node with paraffin section of the same sentinel node using standard H&E and/or cytokeratin immunohistochemistry. As you can see, the false negative rate per sentinel node varies from 0-71%. Intraoperative Cytology Imprint False Negative Rate per SLN Intraoperative FN rate Reference Pt (no.) SLN (no.) No. % Ku (1997) 86 118 10/25 40 Usman (1999) 55 157 0/37 0 van Diest (1999) 54 59 10/26 38 Litz (2000) 28 47 5/7 71 Krishnamurthy (2001) 34 77 5/12 42 Massoll (2001) 206 206 6/33 18 Cserni (2001) 60 72 16/36 44 Lee (2002) 64 155 14/32 43 The next table demonstrates the intraoperative false negative rate per patient and is a more accurate approach to assessing false negative rate. The intraoperative false negative rate ranges from 6-62%. Micrometastases were the main contributors to undetected metastases and were often not seen. Other factors that have contributed are fatty replaced lymph nodes or patients with a known diagnosis of lobular carcinoma. False positive results are rare. Several authors described reactive epithelioid histiocytes, endothelial cells, and germinal center lymphocytes as pitfalls. But overall, the specificity of imprint cytology is high (>95%). Intraoperative Cytology Imprint False Negative Rate per Patient Intraoperative FN rate Reference Pt (no.) SLN (no.) No. % Rubio (1998) 55 93 1/17 6 Keshtgar (1999) 25 35 2/6 33 Ratanawichitrasin (1999) 55 138 1/15 7 Moes (2000) 66 175 6/17 35 Mathur (2000) 83 Not available 8/13 62 Motomura (2000) 101 158 3/33 9 Creager (2002) 646 1255 95/204 46 Frozen Section Some investigators have advocated intraoperative frozen section of sentinel nodes. The major benefit of intraoperative frozen section is that a positive result allows an immediate axillary dissection. The extent to which the sentinel node is evaluated intraoperatively varies among different institutions. In the majority of hospitals that perform a frozen section on the sentinel node, a single level from each cut surface is usually evaluated. However, more intensive intraoperative assessment with serial sections and rapid cytokeratin immunohistochemistry has been described. In comparison to imprint cytology, the nodal architecture of the sentinel node is easily visualized as well as the location of the metastasis by frozen section. Detecting metastatic disease on frozen section is less challenging and fewer indeterminate diagnoses are encountered in comparison to imprint cytology. The time to perform a frozen section can vary from several minutes to 15-20 minutes depending on the institution. A disadvantage includes frozen tissue artifact at the time of frozen as well as on subsequent paraffin-embedded sections. The cost of a frozen section is considerably more expensive than that of a touch prep. Many have argued against performing frozen sections on sentinel nodes stating that significant amounts of tissue may be consumed while facing the frozen sentinel node block and "small" metastases may end up undetected at the bottom of the cryostat. While others believe the advantages of finding a positive result at the time of surgery and the ability to complete the axillary dissection outweighs the small amount of tissue lost to this process. This table demonstrates the intraoperative false negative rate of frozen section in comparison to the final permanent H&E standard section and/or cytokeratin immunohistochemistry. The false negative rate is the number of patients with a negative frozen section divided by the total number of patients with a positive sentinel node by standard H&E or cytokeratin immunohistochemistry. Intraoperative Frozen Section False Negative Rate   Pt (no.) Intraoperative FN rate H&E CK-IHC H&E/CK-IHC No. % No. % No. % Veronesi (1997) 107 18/50 36 Flett (1998) 56 3/21 14 Turner (1999)* 278 19/72 26 58/111 52 van Diest (1999) 54 4/31 13 Veronesi (1999) 192 26/81 32 Chiu (2000) 129 7/49 14 16/58 28 Zavagno (2000) 81 12/25 48 Weiser (2000) 890 96/231 42 Brown (2001) 37 2/6 33 2/6 33 Mayo (unpub) 1729 117/404 28 163/450 36 *Frozen section/imprint cytology The intraoperative false negative rate varies from 13-52%. Cytokeratin immunohistochemistry on paraffin-embedded sections substantially increases the false negative rate of frozen section by converting approximately 10–25% of sentinel node negative patients to sentinel node positive patients. Based on our experience and the review of the literature, frozen section for the most part detects macrometastases (> 2.0 mm) and commonly misses the micrometastases (< 2.0 mm). In my experience, greater than 98% of our frozen section false negative rate is due to micrometastatic disease. This is likely to remain an inherent limitation to this staging procedure. However, patients with a small primary breast cancer and micrometastasis in the sentinel node are less likely to have positive non-sentinel nodes. Serial Frozen Sections and CK-IHC There are two groups that have looked at extensive intraoperative examination of frozen sentinel nodes as an effective technique in predicting axillary lymph node status and identifying patients in whom an axillary dissection may be avoided. Both groups report an intraoperative false negative rate of 0%. The technique used is serial sectioning of the sentinel node. Viale et al. examined 15 pairs of adjacent frozen sections (4 ųm thick @ 50 ųm intervals) for each lymph node moiety in 155 patients. If residual tissue was left, additional pairs were cut (100 um intervals) until the entire sentinel node was examined. One section from each pair was stained with H&E and the other section was stained for cytokeratin using a rapid staining method. A positive sentinel node was identified in 70 patients. The majority (64%) of patients with metastases was detected within the first paired section, and no metastases were found beyond the 15th cutting level. Similarly, Veronesi and his colleagues also examined 15 pairs of frozen sections (4 ųm thick @ 50 ųm levels) for each lymph node moiety in 119 patients. A positive sentinel node was found in 52 patients. Both groups believe that this new method is definitive in evaluating the sentinel node. However, this extensive intraoperative examination is labor intensive and time consuming, requiring approximately 30-40 minutes per sentinel node. In this era of managed care, more detailed analyses are required to determine if the cost of this type of exhaustive intraoperative frozen section is more beneficial than the patient undergoing a second operation due to a false negative result. Intraoperative False Negative Rate Using Serial Frozen Sections Reference Pt (no.) Intraoperative FN rate No. % Viale (1999) 155 0/70 0 Veronesi (1999) 119 0/52 0 Immunohistochemistry is a sensitive method for the detection of minimal metastatic disease and a rapid assay has been applied intraoperatively to the sentinel node. The time to perform this assay has been reported to be between 10-20 minutes per sentinel node. Several studies have reported this technique to be very sensitive; while Beach and colleagues recently reported rapid immunohistochemistry to be less sensitive (57%) in comparison to other methods used to detect metastases intraoperatively. They stated that single cell detection and very small tumor deposits were difficult to identify due to frozen section folding, background staining and weak membranous staining of metastatic carcinoma cells despite good controls. Viale and colleagues reported that rapid immunocytochemical staining for cytokeratin did not increase the detection rate of sentinel node metastases. Thus, it remains to be agreed upon whether this rapid immunohistochemistry assay adds to the detection of tumor foci and is sufficiently quick and reliable for general use. Histopathologic Examination We are spending much of our time asking the question how do we best optimize the histopathologic examination of the sentinel node. Numerous studies have looked at this question. Sentinel nodes grossly involved by tumor require no special treatment. However, there should be a systematic approach to sentinel nodes that are grossly negative. The approach taken should be based on the minimum size of metastasis one wishes to detect with a high degree of probability. Unfortunately, no current data exists to specify what size this should be. Therefore, the choice may be driven by considerations of labor and economics. Also of note, appropriate reimbursement codes for this type of extensive examination of the sentinel node have not yet been devised. The 1999 College of American Pathologist (CAP) Consensus Statement on prognostic factors in breast cancer was recently published. They recommended that sentinel nodes be sectioned at 2 mm intervals and entirely submitted for permanent histologic examination. Intraoperative assessment should include a careful gross examination of sentinel nodes, and if necessary, touch preps are preferable to frozen section. In term of postoperative examination, a single microscopic section from each sentinel node is considered sufficient. They believe that there is insufficient data to support routine serial step sectioning and strongly believe that routine cytokeratin staining of negative sentinel nodes should not be considered standard until the clinical significance is established. The Association of Directors of Anatomic and Surgical Pathology (ADASP) recommended that intraoperative examination is only appropriate in those clinical situations in which the results will influence immediate therapeutic management. In contrast to CAP consensus statement, ADASP recommended more than one section be performed on each negative node(s), but no direction as to the number of sections to be performed was given. They also stated that it was unclear if immunohistochemical studies added clinically relevant information, but stated that if metastases were identified only by immunohistochemistry, this should be stated in the pathology report. With that said, this table demonstrates several of the studies that have used extensive sectioning, ranging from multiple-step to serial sectioning, as well as cytokeratin immunohistochemistry to identify metastases. SLN Positivity at Postoperative Examination Reference Pt (no.) SLN Positive H&E IHC Giuliano (1997) 100 33 9 Czerniecki (1999) 41 12 3 Ku (1999) 478 93 41 Kelley (1999) 28 7 2 Liu (2000) 38 12 5 Reitsamer (2000) 110 41 6 Mayo (unpublished) 1729 404 46 Different cytokeratin antibodies have been used. The most widely cytokeratin antibodies used are cytokeratin AE1/AE3, CAM5.2 or a cytokeratin cocktail. Epithelial membrane antigen (EMA) has proven to have too low specificity and sensitivity. In these various studies, the routine use of cytokeratin immunohistochemistry on paraffin sections of H&E negative sentinel nodes enabled the detection of micrometastases and upstaging anywhere from 8-50% of patients from node negative to node positive patients. The majority of those metastases identified by cytokeratin immunohistochemistry were micrometastases. In a preliminary study by Dowlatshahi and colleagues, they performed exhaustive sectioning of sentinel nodes. A single paraffin section (5 ųm thick ) was stained with H&E and the remainder of the block was completely sectioned at 0.25 mm intervals and examined by standard H&E and cytokeratin immunohistochemistry. They reported that metastatic disease was found on the initial H&E section in only 6 patients. The additional 24 patients were found to have metastatic disease with the step sections and immunohistochemistry, for a conversion rate of approximately 50%. Of these 24 patients, 12 had isolated tumor cells and 12 had colonies of malignant cells. There is no question that enhanced pathological examination of the sentinel lymph node will upstage the patient Unfortunately, the impact of such enhanced detection of metastatic disease in sentinel nodes on breast cancer patient outcome, if any, remains to be characterized. Postoperative Findings with Multiple Sectioning and Cytokeratin IHC Reference Pt (no.) SLN Positive H&E IHC Jannink (1998) 19 6 3 Dowlatshahi (1999) 52 6 24 Turner (1999) 52 10 9 Bloom (2001) 222 35 57 The protocol that we have adopted at Mayo Clinic is as follows: The sentinel lymph node is initially bivalved and a frozen section is performed on each half. If the sentinel node is negative by intra-operative examination, then two levels are examined post-operatively by standard H&E and one level is examined by cytokeratin immunohistochemistry. Histologic Pitfalls Identifying metastatic carcinoma in lymph nodes usually does not present a challenge. However, there are elements other than tumor that can be found in the lymph node. Most small tumor deposits are found within the subcapsular sinus or adjacent nodal tissue. A possible source of confusion with metastatic tumor is the presence of nevus cell aggregates. These aggregates are usually found along the outer border of the lymph node capsule. Cytologically, the nevus cells are oval with finely granular chromatin. The cytoplasm is pale or clear, sometimes lightly pigmented, with indistinct borders. These nevus cell aggregates are immunoreactive for S100 protein. Recently, Carter and colleagues described the finding of epithelial cells in the subcapsular sinus of axillary lymph node dissections after biopsy and attributed this finding to benign lymphatic transport of these cells secondary to surgical manipulation. It is important to be aware of this histologic finding, since not everything that is keratin positive represent metastatic disease. The diagnosis of metastatic disease should be made with caution whenever keratin positive cells are associated with hemosiderin-laden macrophages and an inflammatory reaction. Vacuolated histiocytes can also present a difficult diagnostic problem when they resemble signet ring adenocarcinoma cells. Histochemical and immunohistochemical stains can be helpful in such instances. There are also pitfalls predominantly with over-interpretation of cytokeratin stained sections. These include desquamated keratinocytes from laboratory personnel, nonspecific staining with dendritic cells, and cytokeratin stain precipitate. To consider a sentinel node positive, the foci in question should show cohesive cluster of malignant appearing cells or cytologically atypical cells similar to the primary tumor. Histologic Pitfalls Pitfall Helpful Histologic Clues Nevus cells Capsular location, oval nuclei with granular chromatin, sometimes pigmented, pale cytoplasm, S100 positive Benign transport of breast epithelium Prior core biopsy or surgical manipulation, associated hemosiderin-laden macrophages, inflammatory reaction Benign glandular inclusion Bland nuclei without pleomorphism and normal N:C, some may have apocrine or squamous lining with keratin debris, two cell layer, location Vacuolated histiocytes Multinucleated cells, bland nuclei, abundant cytoplasm, cytokeratin negative Sentinel Lymph Node Micrometastasis Micrometastasis was first introduced by Huvos et al. as a metastatic foci less than or equal to 2.0 mm in greatest dimension and any tumor foci greater than 2.0 mm in diameter is defined as a macrometastasis. However, the term "micrometastasis" now encompasses tumor deposits down to a single tumor cell and the extent of metastasis varies from isolated tumor cells to large colonies of cells. In January 2003, the American Joint Commission on Cancer (AJCC) revised the staging system to distinguish between micrometastasis and isolated tumor cells based on the metastatic tumor characteristics and size. Micrometastasis is defined as implantation of tumor cells in the lymph node with extravasation, proliferation and stromal reaction with a defined tumor size of greater than 0.2 mm but not greater than 2.0 mm in diameter, while isolated tumor cell groups are defined as 0.2 mm in diameter or less, and do not typically show evidence of metastatic activity. Unfortunately, there is no guidance on how to classify isolated tumor cells or small foci of tumor cells at multiple locations within a single lymph node. The recent edition of AJCC Cancer Staging Manual adds the descriptor "i" for immunohistochemistry. For example, a sentinel lymph node that is negative for metastasis by H&E and positive for isolated tumor cells by immunohistochemistry would be designated as pN0(i+). However, this differs from the UICC's 6th ediction of the TNM Classification of Malignant Tumors in which they state that "i" stands for isolated tumor cells, which may or may not be identified by H&E, but by immunohistochemistry. Therefore, a sentinel node that shows isolated tumor cells whether by H&E or by both H&E and immunohistochemistry would be designated as pN0(i+), and if no isolated tumor cells are identified, then the sentinel node would be classified as pN0(i-). The significance of micrometastasis in the sentinel lymph node remains controversial. Some authors have shown that lymph node micrometastases have a small but significant adverse effect on distant recurrences and survival in some studies, but not in all, and clinicians are still uncertain how micrometastatic evidence will inform treatment decisions. The distinction between micrometastasis and macrometastasis is a quantitative difference rather than a qualitative difference, and it appears that the survival of patients depends more on the quantity of tumor metastasis in a lymph node. It is imperative that the prognostic significance of nodal micrometastasis be determined as there are important implications for systemic adjuvant chemotherapy and hormonal therapy. Presently, the data of sentinel lymph node metastasis do not posses sufficient follow up to observe any survival effects as retrospective studies have shown. Therefore, the prognostic significance of such occult metastases at this time is unknown. Prospective Randomized Clinical Trials Five randomized clinical trials are currently in progress to evaluate certain aspects of the sentinel node biopsy. The purpose/hypotheses of these trials are multifactorial. Some will test local and distant recurrence with and without axillary lymph node dissection, morbidity of sentinel lymph node versus axillary lymph node dissection, and evaluate whether occult micrometastasis affects survival and local recurrence. It is also hypothesized that completion axillary dissection will result in the same degree of regional control and survival as axillary radiotherapy. If the outcome of this study shows no difference in treatment modalities, patients with positive sentinel lymph node will most likely be treated more frequently with axillary radiotherapy. Clinical Trial Method NSABP B-32 Group 1 SLN and ALND Group 2 SLN (-) by H&E, randomize to ALND vs SLN biopsy alone ACOSOG Z0010 Group 1 SLN (-) by H&E, no ALND Group 2 SLN (+), randomize to Z0011 ACOSOG Z0011 SLN (+) by H&E, randomize to ALND vs no ALND ALMANAC Group 1 SLN (-) by H&E, no ALND Group 2 SLN (+) by H&E, randomize to ALND vs axillary radiotherapy EORTC SLN (+) by H&E, randomize to ALND vs axillary radiotherapy Predictors of Non-SLN Metastasis Multiple studies have shown that the sentinel node is the only positive node in 35-65% of the cases. Because of this, an intriguing question is whether sentinel node positive patients require a complete axillary dissection. The current practice is to perform a complete axillary dissection when the sentinel node is positive to minimize the chance of metastatic disease in the remainder of the axilla. Recently, clinicians have begun to question this practice. Many investigators are exploring different parameters of the primary tumor or characteristics of the sentinel node metastasis to examine if there is a single or combination of features that are associated with the finding of additional positive lymph nodes in the axilla. A recent meta-analysis was performed to examine clinicopathologic features of metastasis in nonsentinel lymph nodes of breast carcinoma patients with a positive sentinel lymph node. Eleven studies met the criteria for analysis. Despite differences in methodologies, the correlation between positive non-sentinel lymph nodes and certain pathologic features was found to be similar among studies. The 5 individual characteristics found to be associated with the highest likelihood of non-sentinel metastasis are sentinel node metastasis > 2mm in size, extranodal extension in the sentinel node, tumor size > 2 cm, > than 1 positive sentinel lymph node and angiolymphatic invasion in the primary tumor. Non-SLN Metastasis According to Tumor Size and SLN Micrometastasis Reference Tumor Size Incidence of Non-SLN Metastasis Czerniecki (1999) T1 T2 0 of 5 3 of 4 Chu (1999) T1 T2 2 of 46 2 of 20 Reynolds (1999) T1 T2 0 of 18 6 of 9 Viale (2001) T1 T2 20 of 93 4 of 17 Additional studies are needed to examine whether the number of sentinel nodes involved by tumor or the histologic tumor type is predictive of additional lymph node metastasis. More importantly, the question still remains whether the additional information obtained by complete axillary dissection can improve the treatment and survival of patients or whether a positive sentinel lymph node in itself is sufficient information for patient management and prognosis. Future Questions Sentinel node biopsy has raised more questions than answers. One of the most important questions to be answered is the clinical significance of isolated tumor cells and micrometastases. 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