Hypomethylation of LINE-1 in primary tumor has poor prognosis in young breast cancer patients: a retrospective cohort study.

Long interspersed element 1 (LINE-1), a non-coding genomic repeat sequence, methylation status can influence tumor progression. In this study, the clinical significance of LINE-1 methylation status was assessed in primary breast cancer in young versus old breast cancer patients. LINE-1 methylation index (MI) was assessed by absolute quantitative assessment of methylated alleles (AQAMA) PCR assay. Initially, LINE-1 MI was assessed in a preliminary study of 235 tissues representing different stages of ductal breast cancer development. Next, an independent cohort of 379 primary ductal breast cancer patients (median follow-up 18.9 years) was studied. LINE-1 hypomethylation was shown to occur in DCIS and invasive breast cancer. In primary breast cancer it was associated with pathological tumor stage (p = 0.026), lymph node metastasis (p = 0.022), and higher age at diagnosis (>55, p < 0.001). In multivariate analysis, LINE-1 hypomethylation was associated with decreased OS (HR 2.19, 95 % CI 1.17-4.09, log-rank p = 0.014), DFS (HR 2.05, 95 % CI 1.14-3.67, log-rank p = 0.016) and increased DR (HR 2.83, 95 % CI 1.53-5.21, log-rank p = 0.001) in younger (≤55 years), but not older patients (>55 years). LINE-1 analysis of primary breast cancer demonstrated cancer-related age-dependent hypomethylation. In patients ≤55 years, LINE-1 hypomethylation portends a high-risk of DR.

Development of sporadic microsatellite instability in colorectal tumors involves hypermethylation at methylated-in-tumor loci in adenoma.

Microsatellite instability (MSI) and genomic hypermethylation of methylated-in-tumor (MINT) loci are both strong prognostic indicators in a subgroup of patients with sporadic colorectal cancer (CRC). The present study was designed to determine whether the methylation of MINT loci during the progression of adenoma to CRC is related to MSI in CRC cases. Methylation index (MI) was measured by absolute quantitative assessment of methylated alleles at seven MINT loci in primary CRC with contiguous adenomatous and normal tissues of 79 patients. Results were then validated in primary CRC tissues from an independent group of 54 patients. Increased MI of both MINT loci 1 and 31 was significantly associated with MSI in CRC and was specific for adenoma. Total MI and the number of methylated loci were threefold (P=0.02) and fivefold (P=0.004) higher, respectively, in adenomas associated with microsatellite-stable CRC versus microsatellite-unstable CRC. MINT MI was found to be correlated with mismatch repair protein expression, MSI, BRAF (V600E) mutation status, mut-L homologue 1 methylation status, and disease-specific survival in the second independent validation group of patients. MI of specific MINT loci may be prognostic indicators of colorectal adenomas that will develop into sporadic microsatellite-unstable CRCs. Increased MINT locus methylation appears to precede MSI and may have utility in defining clinical pathology in the absence of features of malignant invasive tumors.

Sentinel lymph node biopsy for papillary thyroid cancer: 12 years of experience at a single institution.

BACKGROUND: The purpose of this study was to report our experience with sentinel lymph node dissection (SLND) for papillary thyroid carcinoma, to evaluate the feasibility and safety of the procedure, and to examine its potential utility as a guide for central neck dissection. MATERIALS AND METHODS: A retrospective chart review of patients undergoing total thyroidectomy from January 1998 thru January 2010 was conducted. Intratumoral injection of blue dye was used to identify the SLN. Central neck dissection (CND) was performed if the SLN was positive on frozen section. Locally advanced disease, previous thyroid surgery, or lymphadenopathy on preoperative imaging or intraoperative palpation were exclusion criteria. RESULTS: A total of 211 patients underwent SLN mapping. Of these, 165 patients (78%) were female and 46 (22%) were male. Also, 75 (36%) were ≤45 years of age, and 136 (64%) were older than 45. Tumors were ≤2.0 cm (T1) in 142 patients (67%), 2-4 cm (T2) in 35 patients (17%), >4 cm with minimal invasion (T3) in 32 patients (15%), and locally invasive (T4) in 2 patients (1%). At least 1 blue node was found in 192 patients (91%). Also, 47 patients had a positive SLN on frozen section, with an additional 24 node-positive patients on permanent section, for a total of 71 (37%). There were 43 patients (91%) who underwent central neck dissection; 26 (60%) had additional metastases. CONCLUSIONS: Sentinel lymphadenectomy for papillary thyroid carcinoma is feasible, safe, and can identify patients who may benefit from central neck dissection.

Massive cotyledenoid leiomyoma treated with uterine-conserving surgery.

OBJECTIVE: To describe and illustrate a massive cotyledenoid leiomyoma treated with uterine-conserving surgery. DESIGN: Case report. SETTING: Medical center. PATIENTS: A 39-year-old woman with a large abdominal mass and a magnetic resonance imaging scan showing a 28-cm multi-lobulated mass. INTERVENTIONS: Laparotomy and myomectomy. MAIN OUTCOME MEASURES: Recurrence and need for repeat surgery. RESULTS: No recurrence at 8 years of follow-up. CONCLUSIONS: Cotyledonoid leiomyomas are rare. These benign tumors may be suspected preoperatively based on MRI appearance. Frozen section suggests a benign process and uterine-conserving surgery may be successfully accomplished.

Human high molecular weight-melanoma-associated antigen: utility for detection of metastatic melanoma in sentinel lymph nodes.

PURPOSE: Detection of micrometastasis in melanoma-draining lymph nodes is important for staging and prognosis. Immunohistochemical staining (IHC) using S-100p-HMB-45-, and MART-1-specific antibodies is used for detecting metastases in sentinel lymph nodes (SLN). However, improvement in IHC is needed for melanoma micrometastasis detection. EXPERIMENTAL DESIGN: Paraffin-embedded archival tissue (PEAT) specimens were obtained from 42 non-SLN macrometastases, 42 SLN metastases, and 16 tumor-negative SLNs of 100 melanoma patients who underwent SLN biopsy. PEAT specimens were assessed by IHC with high molecular weight-melanoma-associated antigen (HMW-MAA)-specific monoclonal antibodies (mAb) and with S-100p-, HMB-45-, and MART-1-specific antibodies. Quantitative real-time reverse-transcriptase PCR assay was used for HMW-MAA and MART-1 mRNA detection. RESULTS: Expression frequency and immunostaining intensity were higher for HMW-MAA than MART-1 in nodal macrometastases (P < 0.0001 and P < 0.0001, respectively) and micrometastases (P < 0.0001 and P = 0.004, respectively). All 52 (100%) macrometastases were positive with HMW-MAA-specific mAbs, whereas 43 (83%) were positive with MART-1-specific mAbs. In a comparison analysis, 23 of 23 (100%) micrometastases were HMW-MAA-positive, whereas 21 (91%) and 18 (78%) specimens were S-100p- and HMB-45-positive, respectively. Quantitative real-time reverse-transcriptase PCR analysis of 48 nodal metastases showed HMW-MAA mRNA detection in SLNs with metastases. CONCLUSIONS: HMW-MAA is more sensitive and specific than MART-1, S-100p, and HMB-45 for IHC-based detection of SLN micrometastases. SLN PEAT-based detection specificity of melanoma micrometastases can be improved by IHC with HMW-MAA-specific mAbs.

Prognostic relevance of occult nodal micrometastases and circulating tumor cells in colorectal cancer in a prospective multicenter trial.

PURPOSE: Nodal micrometastasis and circulating tumor cells detected by multimarker quantitative real-time reverse transcription-PCR (qRT-PCR) may have prognostic importance in patients with colorectal cancer. EXPERIMENTAL DESIGN: Paraffin-embedded sentinel lymph nodes from 67 patients and blood from 34 of these patients were evaluated in a prospective multicenter trial of sentinel lymph node mapping in colorectal cancer. Sentinel lymph nodes were examined by H&E staining and cytokeratin immunohistochemistry. Sentinel lymph nodes and blood were examined by a four-marker qRT-PCR assay (c-MET, melanoma antigen gene-A3 family, beta1-->4-N-acetylgalactosaminyltransferase, and cytokeratin-20); qRT-PCR results were correlated with disease stage and outcome. RESULTS: In H&E-negative sentinel lymph node patients that recurred, cytokeratin immunohistochemistry and qRT-PCR detected metastasis in 30% and 60% of patients, respectively. Disease-free survival differed significantly by multimarker qRT-PCR upstaged sentinel lymph node (P = 0.014). qRT-PCR analysis of blood for circulating tumor cells correlated with overall survival (P = 0.040). CONCLUSION: Molecular assessment for micrometastasis in sentinel lymph node and blood specimens may help identify patients at high risk for recurrent colorectal cancer, who could benefit from adjuvant therapy.

Assessment of methylation events during colorectal tumor progression by absolute quantitative analysis of methylated alleles.

To date, the epigenetic events involved in the progression of colorectal cancer are not well described. To study, in detail, methylation during colorectal cancer development in high-risk adenomas, we developed an assay combining in situ (on-slide) sodium bisulfite modification (SBM) of paraffin-embedded archival tissue sections with absolute quantitative assessment of methylated alleles (AQAMA). We tested the performance of the assay to detect methylation level differences between paired pre-malignant and malignant colorectal cancer stages. AQAMA assays were used to measure methylation levels at MINT (methylated in tumor) loci MINT1, MINT2, MINT12, and MINT31. Assay performance was verified on cell line DNA and standard cDNA. On-slide SBM, allowing DNA methylation assessment of 1 to 2 mm(2) of paraffin-embedded archival tissue, was employed. Methylation levels of adenomatous and cancerous components within a single tissue section in 72 colorectal cancer patients were analyzed. AQAMA was verified as accurately assessing CpG island methylation status in cell lines. The correlation between expected and measured cDNA methylation levels was high for all four MINT AQAMA assays (R >or= 0.966, P<0.001). Methylation levels at the four loci increased in 11% and decreased in 36% of specimens comparing paired adenoma and cancer tissues (P<0.0001 by Kolmogorov-Smirnov test). Single-PCR AQAMA provided accurate methylation level measurement. Variable MINT locus methylation level changes occur during malignant progression of colorectal adenoma. Combining AQAMA with on-slide SBM provides a sensitive assay that allows detailed histology-oriented analysis of DNA methylation levels and may give new, accurate insights into understanding development of epigenetic aberrancies in colorectal cancer progression.

Newer pathologic assessment techniques for colorectal carcinoma.

The pathogenesis of colorectal carcinoma is characterized by progressive genetic abnormalities, which lead to proteomic and cellular changes that determine the cancer malignant phenotype. Phenotypic characteristics seen on histopathologic examination (e.g., tumor stage, histologic grade, and vasoinvasiveness) are essential to planning patient management and should continue to be the major focus of pathologists' efforts. Nonetheless, additional markers that improve the prognostic and predictive power of the pathologic analysis of the primary tumor have been the focus of intense research in recent years. Improved prognostic power may derive from advancements in histopathologic evaluation, more sensitive lymph node staging techniques, and specific molecular analysis methods, such as genetic tests or immunophenotypic profiles. Histopathologic improvements are needed to better standardize histologic grade determination and recognize tumor budding at the invasive front as a marker of aggressive biological behavior and an adverse parameter. Ultrastaging of mesenteric lymph nodes remains a controversial area. Genotypic studies are well developed in the areas of microsatellite instability and chromosome 18q deletion/loss of heterozygosity. Immunophenotypic studies are available in a range of areas including tumor suppressor gene/oncogene expression, proliferation/apoptosis, angiogenesis, and cell adhesion and signaling. Gene expression profiles identified by microarray techniques may help to subtype the large category of microsatellite-stable colorectal carcinoma and define immunophenotypic panels to subclassify tumors into prognostic and therapeutic groups. This brief review discusses the most promising of these approaches and evidence supporting their potential clinical utility.

New approaches to assessing and treating early-stage colon and rectal cancer: summary statement from 2007 Santa Monica Conference.

The 2007 Santa Monica Conference on Assessing and Treating Early-Stage Colon and Rectal Cancer, a multidisciplinary meeting of leaders in surgery, medical and radiation oncology, and pathology, was convened on January 12 to 13, 2007. The purpose of the meeting was to assess current data and issues in the field and to develop recommendations for advancing patient care and clinical research. Topics included pathologic assessment and staging, transanal versus laparoscopic versus open resection, adjuvant therapy, genetic testing and counseling, cooperative group strategies, and the use of biological therapies and novel agents. A review of the key issues discussed, as well as conclusions and recommendations considered significant to the field, is summarized below and presented at greater length in the individual manuscripts and accompanying discussion that comprise the full conference proceedings. Although the management of early-stage colon and rectal cancers remains a challenge for all of us, the development and use of new technologies and methods of assessment and treatment over the past several decades is yielding encouraging results. A variety of opportunities to further improve outcomes were addressed in this forum, including recommendations that specific protocols be adopted regarding surgical and pathologic dissection and reporting, particularly for stage II disease; the corollary need to increase active multidisciplinary collaboration; and the development of comprehensive consensus guidelines and recommendations to standardize care in early-stage colorectal cancer.

Proteomic profiling of primary breast cancer predicts axillary lymph node metastasis.

To determine if protein expression in primary breast cancers can predict axillary lymph node (ALN) metastasis, we assessed differences in protein expression between primary breast cancers with and without ALN metastasis using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS). Laser capture microdissection was performed on invasive breast cancer frozen sections from 65 patients undergoing resection with sentinel lymph node (SLN) or level I and II ALN dissection. Isolated proteins from these tumors were applied to immobilized metal affinity capture (IMAC-3) ProteinChip arrays and analyzed by SELDI-TOF-MS to generate unique protein profiles. Correlations between unique protein peaks and histologically confirmed ALN status and other known clinicopathologic factors were examined using ANOVA and multivariate logistic regression. Two metal-binding polypeptides at 4,871 and 8,596 Da were identified as significant risk factors for nodal metastasis (P = 0.034 and 0.015, respectively) in a multivariate analysis. Lymphovascular invasion (LVI) was the only clinicopathologic factor predictive of ALN metastasis (P = 0.0038). In a logistic regression model combining the 4,871 and 8,596 Da peaks with LVI, the area under the receiver operating characteristic curve was 0.87. Compared with patients with negative ALN, those with > or =2 positive ALN or non-SLN metastases were significantly more likely to have an increased peak at 4,871 Da (P = 0.016 and 0.0083, respectively). ProteinChip array analysis identified differential protein peaks in primary breast cancers that predict the presence and number of ALN metastases and non-SLN status.

Chemokine receptor CXCR4 expression in colorectal cancer patients increases the risk for recurrence and for poor survival.

PURPOSE: Liver metastasis is the predominant cause of colorectal cancer (CRC) related mortality. Chemokines, soluble factors that orchestrate hematopoetic cell movement, have been implicated in directing cancer metastasis, although their clinical relevance in CRC has not been defined. Our hypothesis was that the chemokine receptor CXCR4 expressed by CRC is a prognostic factor for poor disease outcome. METHODS: CRC cell lines (n = 6) and tumor specimens (n = 139) from patients with different American Joint Committee on Cancer (AJCC) stages of CRC were assessed. Microarray screening of select specimens and cell lines identified CXCR4 as a prominent chemokine receptor. CXCR4 expression in tumor and benign specimens was assessed by quantitative real-time reverse transcription polymerase chain reaction and correlated with disease recurrence and overall survival. RESULTS: High CXCR4 expression in tumor specimens (n = 57) from AJCC stage I/II patients was associated with increased risk for local recurrence and/or distant metastasis (risk ratio, 1.35; 95% CI, 1.09 to 1.68; P = .0065). High CXCR4 expression in primary tumor specimens (n = 35) from AJCC stage IV patients correlated with worse overall median survival (9 months v 23 months; RR, 2.53; 95% CI, 1.19 to 5.40; P = .016). CXCR4 expression was significantly higher in liver metastases (n = 39) compared with primary CRC tumors (n = 100; P < .0001). CONCLUSION: CXCR4, a well-characterized chemokine receptor for T-cells, is differentially expressed in CRC. CXCR4 gene expression in primary CRC demonstrated significant associations with recurrence, survival, and liver metastasis. The CXCR4-CXCL12 signaling mechanism may be clinically relevant for patients with CRC and represents a potential novel target for disease-directed therapy.

American Society of Clinical Oncology guideline recommendations for sentinel lymph node biopsy in early-stage breast cancer.

PURPOSE: To develop a guideline for the use of sentinel node biopsy (SNB) in early stage breast cancer. METHODS: An American Society of Clinical Oncology (ASCO) Expert Panel conducted a systematic review of the literature available through February 2004 on the use of SNB in early-stage breast cancer. The panel developed a guideline for clinicians and patients regarding the appropriate use of a sentinel lymph node identification and sampling procedure from hereon referred to as SNB. The guideline was reviewed by selected experts in the field and the ASCO Health Services Committee and was approved by the ASCO Board of Directors. RESULTS: The literature review identified one published prospective randomized controlled trial in which SNB was compared with axillary lymph node dissection (ALND), four limited meta-analyses, and 69 published single-institution and multicenter trials in which the test performance of SNB was evaluated with respect to the results of ALND (completion axillary dissection). There are currently no data on the effect of SLN biopsy on long-term survival of patients with breast cancer. However, a review of the available evidence demonstrates that, when performed by experienced clinicians, SNB appears to be a safe and acceptably accurate method for identifying early-stage breast cancer without involvement of the axillary lymph nodes. CONCLUSION: SNB is an appropriate initial alternative to routine staging ALND for patients with early-stage breast cancer with clinically negative axillary nodes. Completion ALND remains standard treatment for patients with axillary metastases identified on SNB. Appropriately identified patients with negative results of SNB, when done under the direction of an experienced surgeon, need not have completion ALND. Isolated cancer cells detected by pathologic examination of the SLN with use of specialized techniques are currently of unknown clinical significance. Although such specialized techniques are often used, they are not a required part of SLN evaluation for breast cancer at this time. Data suggest that SNB is associated with less morbidity than ALND, but the comparative effects of these two approaches on tumor recurrence or patient survival are unknown.

Prospective multicenter trial of staging adequacy in colon cancer: preliminary results.

HYPOTHESIS: Lymph node evaluation is an important prognostic factor in colorectal cancer (CRC). A 25% recurrence rate in patients with node-negative CRC suggests that current staging practices are inadequate. Focused analysis of the sentinel node (SN) by multiple sectioning and immunohistochemistry improves staging accuracy. DESIGN: Prospective phase 2 multicenter trial. SETTING: Tertiary referral cancer centers. PATIENTS: Between March 2001 and June 2005, 132 patients were enrolled with clinical stage I and II CRC in a prospective multicenter trial (R01-CA90484). INTERVENTION: During a standard oncologic resection, lymphatic mapping was performed and the SN identified either by the surgeon or the pathologist. Hematoxylin-eosin staining was performed on all lymph nodes and immunohistochemistry, on lymph nodes negative by hematoxylin-eosin staining. MAIN OUTCOME MEASURES: Micrometastases greater than 0.2 mm but less than 2 mm and isolated tumor cells less than 0.2 mm were defined according to the sixth edition of the American Joint Committee on Cancer Cancer Staging Manual. RESULTS: The 63 men and 69 women had a median age of 74 years. Sixty-eight patients (52%) underwent a right hemicolectomy; 3 (2.3%), a transverse colectomy; 9 (7%), a left colectomy; 15 (11%), a sigmoid colectomy; 34 (26%), a low anterior resection; 1 (1%), an abdominal perineal resection; and 2 (2%), a total colectomy. Of the 111 evaluable primary tumors, 19 (17%) were T1 lesions; 17 (15%), T2; 72 (65%), T3; and 3 (2.7%), T4 tumors. Thirty-three patients (30%) were classified as stage I; 46 (41%), stage II, and 32 (29%), stage III. The SN was identified by the surgeon in 127 patients (96%) and by the pathologist in 5 patients (4%). The median number of SNs and total lymph nodes examined were 3 and 14.5, respectively. The sensitivity of lymphatic mapping and SN analysis was 88.2% and the false-negative rate, 7.4% (6/81). Of the 6 false-negative results, 4 were attributed to lymphatic channels obliterated by tumor. Upstaging occurred in 28 patients (23.6%). CONCLUSIONS: In a multicenter trial, ultrastaging of colon cancer is feasible and accurate. In stage II CRC, 24% of patients had nodal carcinoma cells not detected by conventional staging methods. Surgical technique (adequate lymph node retrieval) and focused pathological analysis may improve staging accuracy and the selection of patients for chemotherapy. The unnecessary toxicity and expense of chemotherapy may be avoided in those patients who are truly node negative.

Distinct hypermethylation profile of primary breast cancer is associated with sentinel lymph node metastasis.

PURPOSE: Gene promoter region hypermethylation is a significant event in primary breast cancer. However, its impact on tumor progression and potential predictive implications remain relatively unknown. EXPERIMENTAL DESIGN: We conducted hypermethylation profiling of 151 primary breast tumors with association to known prognostic factors in breast cancer using methylation-specific PCR for six known tumor suppressor and related genes: RASSF1A, APC, TWIST, CDH1, GSTP1, and RAR-beta2. Furthermore, correlation with sentinel lymph node (SLN) tumor status was assessed as it represents the earliest stage of metastasis that is readily detected. Hypermethylation for any one gene was identified in 147 (97%) of 151 primary breast tumors. The most frequently hypermethylated gene was RASSF1A (81%). RESULTS: Hypermethylation of the CDH1 was significantly associated with primary breast tumors demonstrating lymphovascular invasion (P = 0.008), infiltrating ductal histology (P = 0.03), and negative for the estrogen receptor (P = 0.005), whereas RASSF1A and RAR-beta2 gene hypermethylation were significantly more common in estrogen receptor-positive (P < 0.001) and human epidermal growth factor receptor 2-positive (P < 0.001) tumors, respectively. In multivariate analysis, hypermethylation of GSTP1 and/or RAR-beta2 was significantly associated with patients having macroscopic SLN metastasis compared with those with microscopic or no sentinel node metastasis (odds ratio, 4.59; 95% confidence interval, 2.02-10.4; P < 0.001). In paired SLN metastasis, CDH1 was the most frequently methylated gene (90%) and provides evidence in patients corroborating its role in the clinical development of metastasis. CONCLUSION: Hypermethylation profiling of primary breast tumors is significantly associated with known pathologic prognostic factors and may have additional clinical and pathologic utility for assessing patient prognosis and predicting early regional metastasis.

Interpretive Diagnostic Error Reduction in Surgical Pathology and Cytology: Guideline From the College of American Pathologists Pathology and Laboratory Quality Center and the Association of Directors of Anatomic and Surgical Pathology.

CONTEXT: Additional reviews of diagnostic surgical and cytology cases have been shown to detect diagnostic discrepancies. OBJECTIVE: To develop, through a systematic review of the literature, recommendations for the review of pathology cases to detect or prevent interpretive diagnostic errors. DESIGN: The College of American Pathologists Pathology and Laboratory Quality Center in association with the Association of Directors of Anatomic and Surgical Pathology convened an expert panel to develop an evidence-based guideline to help define the role of case reviews in surgical pathology and cytology. A literature search was conducted to gather data on the review of cases in surgical pathology and cytology. RESULTS: The panel drafted 5 recommendations, with strong agreement from open comment period participants ranging from 87% to 93%. The recommendations are: (1) anatomic pathologists should develop procedures for the review of selected pathology cases to detect disagreements and potential interpretive errors; (2) anatomic pathologists should perform case reviews in a timely manner to avoid having a negative impact on patient care; (3) anatomic pathologists should have documented case review procedures that are relevant to their practice setting; (4) anatomic pathologists should continuously monitor and document the results of case reviews; and (5) if pathology case reviews show poor agreement within a defined case type, anatomic pathologists should take steps to improve agreement. CONCLUSIONS: Evidence exists that case reviews detect errors; therefore, the expert panel recommends that anatomic pathologists develop procedures for the review of pathology cases to detect disagreements and potential interpretive errors, in order to improve the quality of patient care.

Prognostic significance of molecular upstaging of paraffin-embedded sentinel lymph nodes in melanoma patients.

PURPOSE: Detection of micrometastases in sentinel lymph nodes (SLNs) is important for accurate staging and prognosis in melanoma patients. However, a significant number of patients with histopathology-negative SLNs subsequently develop recurrent disease. We hypothesized that a quantitative realtime reverse transcriptase polymerase chain reaction (qRT) assay using multiple specific mRNA markers could detect occult metastasis in paraffin-embedded (PE) SLNs to upstage and predict disease outcome. PATIENTS AND METHODS: qRT was performed on retrospectively collected PE SLNs from 215 clinically node-negative patients who underwent lymphatic mapping and sentinel lymphadenectomy for melanoma and were followed up for at least 8 years. PE SLNs (n = 308) from these patients were sectioned and assessed by qRT for mRNA of four melanoma-associated genes: MART-1 (antigen recognized by T cells-1), MAGE-A3 (melanoma antigen gene-A3 family), GalNAc-T (beta1-->4-N-acetylgalactosaminyl-transferase), and Pax3 (paired-box homeotic gene transcription factor 3). RESULTS: Fifty-three (25%) patients had histopathology-positive SLNs by hemotoxylin and eosin and/or immunohistochemistry. Of the 162 patients with histopathology-negative SLNs, 48 (30%) had nodes that expressed at least one of the four qRT markers, and these 48 patients also had a significantly increased risk of disease recurrence by a Cox proportional hazards model analysis (P <.0001; risk ratio, 7.48; 95% CI, 3.70 to 15.15). The presence of > or = one marker in histopathology-negative SLNs was also a significant independent prognostic factor by multivariate analysis for overall survival (P =.0002; risk ratio, 11.42; 95% CI, 3.17 to 41.1). CONCLUSION: Molecular upstaging of PE histopathology-negative SLNs by multiple-marker qRT assay is a significant independent prognostic factor for long-term disease recurrence and overall survival of patients with early-stage melanoma.

Effect of lymphatic mapping on the new tumor-node-metastasis classification for colorectal cancer.

PURPOSE: Sensitive detection methods and accurate reporting are necessary to determine the prognostic significance of micrometastases (MM) and isolated tumor cells (ITCs) in lymph nodes that drain colorectal cancers (CRCs). This study examined the role of lymphatic mapping (LM) in the application of the new tumor-node-metastasis (TNM) classification for MM and ITC. PATIENTS AND METHODS: All patients at the John Wayne Cancer Institute underwent LM immediately before standard resection of primary CRC between 1996 and 2001. Sentinel nodes (SNs) were identified using blue dye and/or radiotracer and were examined by hematoxylin-eosin (H&E) staining, cytokeratin immunohistochemistry, and multilevel sectioning. The comparison group comprised 370 patients whose primary CRCs were resected without LM during the same period at the same institution. RESULTS: LM was successfully performed in 115 of 120 (96%) patients and correctly predicted the tumor status of the nodal basin in 110 of 115 (96%) patients. Thirty-seven patients (32%) were lymph node-positive by H&E, ITC and MM were found in 23 patients (29.4%) whose lymph nodes were negative by H&E. Tumor deposits were found in the SN only in 29 patients (50%). Nodal involvement was identified for 14.3%, 30%, 74.6%, and 83.3% of T1, T2, T3, and T4 tumors, respectively, in the study group, and for 6.8%, 8.5%, 49.3%, and 41.8% of T1, T2, T3, and T4 tumors, respectively, in the comparison group. The study group had a higher percentage of nodal metastases (53% v 36%; P <.01) and a higher incidence of MM and ITC (29.4% v 1.9%; P <.0001). The mean number of lymph nodes found in the study group (14) was also significantly more than the number found in the comparison group (10; P <.00001). CONCLUSION: Conventional examination of lymph nodes for CRC is inadequate for the detection of MM and ITC as described in the new TNM classification. Thus, LM and focused SN analysis should be considered to fully stage CRC.

Prediction of disease outcome in melanoma patients by molecular analysis of paraffin-embedded sentinel lymph nodes.

PURPOSE: A significant number of patients who develop recurrence after a histopathologically negative sentinel lymph node (SLN) biopsy will demonstrate occult metastases on re-evaluation of the SLNs with serial sectioning and immunohistochemistry. Reverse transcriptase polymerase chain reaction (RT-PCR) has been evaluated to improve disease staging and avoid false-negative findings in fresh or frozen-section SLNs. The purpose of this study was to develop a multimarker RT-PCR assay for assessing melanoma patients' archived paraffin-embedded (PE) SLNs. PATIENTS AND METHODS: Archived PE histopathologically positive (n = 37) and negative (n = 40) SLNs from patients with primary melanoma were analyzed using a semiquantitative multimarker RT-PCR assay. RESULTS: Marker expression in histopathologically positive and negative SLNs were as follows: 89%, 92%, 35%, and 43% (positive) and 40%, 33%, 5%, and 13% (negative) for tyrosinase, melanoma antigen recognized by T cells-1, tyrosinase-related protein-1, and tyrosinase-related protein-2, respectively. Twenty-five percent of histopathologically negative SLN patients were upstaged using at least two markers. Of these, 80% developed a recurrence. Furthermore, at a median follow-up of 55 months, patients with histopathologically negative SLNs who expressed zero or one marker had a significantly improved disease-free (P <.002) and overall (P <.03) survival versus those expressing two or more markers. CONCLUSION: These findings demonstrate the feasibility of a multimarker RT-PCR assay for evaluating archived PE SLNs. More significantly, identification of molecular risk factors can be detected in histopathologically negative SLNs for distinguishing early-stage melanoma patients with a worse prognosis.

Melanoma recurrence patterns after negative sentinel lymphadenectomy.

HYPOTHESIS: A tumor-negative sentinel node (SN) does not eliminate the chance of melanoma recurrence. Patterns of metastasis can be identified and characterized in patients with tumor-negative SNs. DESIGN: Retrospective review. SETTING: Melanoma referral center. PATIENTS: Patients who underwent lymphatic mapping and sentinel lymphadenectomy between 1995 and 2002 and whose SNs were negative for metastasis by hematoxylin-eosin and immunohistochemistry staining were included in the study. The SN specimens from patients with recurrent disease were reexamined for missed metastasis. MAIN OUTCOME MEASURES: Differences in survival related to sites of recurrence and the rate of false-negative histopathologic SN diagnosis were determined. RESULTS: At a median follow-up of 36.7 months, 69 (8.9%) of 773 patients with tumor-negative SNs had recurrent disease. Three-year survival after first recurrence was 17.1% in the 37 patients with distant recurrence, 48.7% in the 19 patients with local or in-transit recurrence, and 63.5% in the 13 patients with regional basin recurrence; the difference in survival between patients with local or regional and distant recurrences was statistically significant (P<.001). Histopathologic reexamination of SNs from the 69 patients identified 9 patients with false-negative SNs; 2 of these had same-basin recurrences. CONCLUSIONS: The SN is a valuable prognostic indicator because only 8.9% of patients with tumor-negative SNs will develop recurrence. The low incidence (1.7%) of regional basin recurrence in patients with negative SNs supports the accuracy of our current method of lymphatic mapping and sentinel lymphadenectomy to identify occult regional nodal basin metastasis.

Nonsentinel node metastases in breast cancer patients with isolated tumor cells in the sentinel node: implications for completion axillary node dissection.

BACKGROUND: Controversy exists regarding axillary dissection (ALND) for sentinel node (SLN) metastases detected as isolated tumor cells (ITC). We hypothesized that the number of positive non-SLNs is low and ALND is unnecessary for most patients with ITC. METHODS: From 1995 to 1999, 634 breast cancer patients underwent SLND. SLNs were examined using immunohistochemistry if hematoxylin and eosin was negative. ALND was recommended for ITC-positive SLNs. RESULTS: Seventy-eight patients (12.3%) with ITC-positive SLNs were offered ALND. Sixty-one consented, whereas 17 refused. Fifty-eight (95.1%) had negative non-SLNs. Three (4.9%) had non-SLN metastases. One patient (1.6%) had macrometastatic disease, whereas 2 (3.3%) had micrometastases. No ITC-only-positive SLN patient experienced axillary recurrence. CONCLUSIONS: When ALND was performed for ITC, 1.6% of non-SLNs harbored macrometastases and 3.3% had micrometastases. When ALND was not performed, axillary recurrence was not seen. The low risk of non-SLN disease in this study fails to support the routine use of ALND for ITC-positive SLNs.