Detection of occult metastatic breast cancer cells in blood by a multimolecular marker assay: correlation with clinical stage of disease.

Currently, molecular markers offer the unique opportunity to identify occult metastasis in early stage cancer patients not otherwise detected with conventional staging techniques. To date, well-characterized molecular tumor markers to detect occult breast cancer cells in blood are limited. Because breast tumors are heterogeneous in tumor marker expression, we developed a "multimarker" reverse transcription-PCR assay combined with the highly sensitive electrochemiluminescence automated detection system. Breast cancer cell lines (n = 7), primary breast tumors (n = 25), and blood from normal donors (n = 40) and breast cancer patients [n = 65; American Joint Committee on Cancer (AJCC) stages I-IV] were assessed for four mRNA tumor markers: beta-human chorionic gonadotropin (beta-hCG), oncogene receptor (c-Met), beta 1-->4-N-acetylgalactosaminyl-transferase, and a tumor-associated antigen (MAGE-A3). None of the tumor markers were expressed in any normal donor bloods. Breast cancer cell lines and primary breast tumors expressed beta-hCG, c-Met, beta 1-->4-N-acetylgalactosaminyl-transferase, and MAGE-A3 mRNA. Of the 65 breast cancer patient blood samples assessed, 2, 3, 15, 49, and 31% expressed 4, 3, 2, 1, and 0 of the mRNA tumor markers, respectively. At least two markers were expressed in 20% of the blood specimens. The addition of a combination of markers enhanced detection of systemic metastasis by 32%. In patient blood samples, the MAGE-A3 marker correlated significantly with tumor size (P = 0.0004) and AJCC stage (P = 0.007). The combination of beta-hCG and MAGE-A3 mRNA markers correlated significantly with tumor size (P = 0.04), and the marker combination c-Met and MAGE-A3 showed a significant correlation with tumor size (P = 0.005) as well as AJCC stage (P = 0.018). A multimarker reverse transcription-PCR assay that correlates with known clinicopathological prognostic parameters may have potential clinical utility by monitoring tumor progression with a blood test.

Prognostic significance of occult metastases detected by sentinel lymphadenectomy and reverse transcriptase-polymerase chain reaction in early-stage melanoma patients.

PURPOSE: Detection of micrometastases in the regional tumor-draining lymph nodes is critical for accurate staging and prognosis in melanoma patients. We hypothesized that a multiple-mRNA marker (MM) reverse transcriptase-polymerase chain reaction (RT-PCR) assay would improve the detection of occult metastases in the sentinel node (SN), compared with hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC), and that MM expression is predictive of disease relapse. PATIENTS AND METHODS: Seventy-two consecutive patients with clinical early-stage melanoma underwent sentinel lymphadenectomy (SLND). Their SNs were serially sectioned and assessed for MAGE-3, MART-1, and tyrosinase mRNA expression by RT-PCR, in parallel with H&E staining and IHC, for melanoma metastases. MM expression in the SNs was correlated with H&E and IHC assay results, standard prognostic factors, and disease-free survival. RESULTS: In 17 patients with H&E- and/or IHC-positive SNs, 16 (94%) expressed two or more mRNA markers. Twenty (36%) of 55 patients with histopathologically negative SNs expressed two or more mRNA markers. By multivariate analysis, patients at increased risk of metastases to the SN had thicker lesions (P =.03), were 60 years of age or younger (P <.05), and/or were MM-positive (P <.001). Patients with histopathologically melanoma-free SNs who were MM-positive, compared with those who were positive for one or fewer mRNA markers, were at increased risk of recurrence (P =.02). Patients who were MM-positive with histopathologically proven metastases in the SN were at greatest risk of disease relapse (P =. 01). CONCLUSION: H&E staining and IHC underestimate the true incidence of melanoma metastases. MM expression in the SN more accurately reflects melanoma micrometastases and is also a more powerful predictor of disease relapse than are H&E staining and IHC alone.

Limitations of specific reverse-transcriptase polymerase chain reaction markers in the detection of metastases in the lymph nodes and blood of breast cancer patients.

PURPOSE: This study was performed to evaluate the potential of specific mRNA markers to detect micrometastases by reverse-transcriptase polymerase chain reaction (RT-PCR) and Southern blot analysis of sentinel lymph nodes (SNs) and blood from patients with breast cancer. PATIENTS AND METHODS: We assessed the specificity of carcinoembryonic antigen (CEA), cytokeratin-19 (CK-19), CK-20, gastrointestinal tumor-associated antigen-733.2 (GA733.2), and mucin-1 (MUC-1) in the blood of healthy donors (n = 13) and lymph nodes from patients without cancer (n = 3) by RT-PCR assay. The sensitivity of the RT-PCR assay for the target mRNA markers was assessed in breast cancer cell lines (n = 4), primary breast tumors (n = 8), and the frozen sections of SNs (n = 22) from 22 patients with American Joint Committee on Cancer (AJCC) stages I to IIIA breast cancer. RESULTS: CK-20 was the only mRNA marker not detected in lymph nodes or blood from patients without cancer. Both the blood and lymph nodes from patients without cancer expressed CEA, CK-19, GA733.2, and MUC-1 mRNA. All four breast cancer cell lines and six of eight primary breast tumors expressed all five mRNA markers. Expression of mRNA by the RT-PCR assay in the frozen-section SNs (n = 12) without metastases by conventional histopathology ranged from 8% (CK-20) to 92% (GA733.2). Detection of RT-PCR cDNA products in frozen-section SNs was increased with Southern blot analysis compared with ethidium bromide gel electrophoresis (EtBr) for all mRNA markers except CK-19. CONCLUSION: CEA, CK-19, GA733.2, and MUC-1 show no diagnostic value as mRNA markers for the detection of micrometastases by the RT-PCR assay because they are expressed in the blood and lymph nodes of patients without cancer. Further studies are needed to assess the sensitivity of CK-20 to detect micrometastases by the RT-PCR assay in the blood and frozen-section SNs of patients with breast cancer.

Assessment of messenger RNA of beta 1-->4-N-acetylgalactosaminyl-transferase as a molecular marker for metastatic melanoma.

Gangliosides GM2 [GalNAc beta 1-4(NeuAc alpha 2-3)Gal beta 1-4Glc beta 1-1Cer] and GD2 [GalNAc beta 1-4(NeuAc alpha 2-8NeuAc alpha 2-3)Gal beta 1-4Glc beta 1-1Cer] are cell surface tumor-associated antigens and have been demonstrated to be important markers of human malignant melanoma progression. Expression of these glycolipid antigens on melanoma tissues can be assessed by immunohistochemistry or biochemical analysis. These methodologies, however, are not logistically practical or sensitive for testing metastatic melanoma cells in blood or in tissue biopsies. In the present study, we hypothesized that the enzyme involved in GM2 and GD2 synthesis, beta 1-->4-N-acetylgalactosaminyltransferase (beta 1-->4GalNac-T), can be a useful marker for detection of occult metastatic melanoma. A reverse transcription PCR and Southern blot assay to detect beta 1-->4GalNac-T mRNA expression was developed. Beta 1-->4GalNac-T mRNA was detected in all 13 melanoma cell lines tested. Metastatic melanoma of lymph nodes and different organ sites expressed beta 1-->4GalNac-T mRNA at various levels. Detection sensitivity of the reverse transcription PCR assay was 1 ng of total RNA extracted from tumor specimens and approximately 5 melanoma cells in 20 million normal donor peripheral blood lymphocytes. In assessment of blood from 126 melanoma patients, beta 1-->4GalNac-T mRNA was more frequently found in advanced-stage melanomas and in patients showing more aggressive tumor progression. Normal donor blood samples (n = 37) were all negative for beta 1-->4GalNac-T mRNA expression. These results suggest that beta 1-->4GalNac-T mRNA is a promising molecular marker for detecting melanoma cells, characterizing antigen expression, and monitoring tumor progression.

Vital dyes in sentinel node localization.

Vital blue dyes were used to show the feasibility and accuracy of intraoperative lymphatic mapping of the sentinel node (SN) in patients with melanoma, breast cancer, and other solid tumors. Surgeons who have successfully completed an adequate number of cases of intraoperative mapping and sentinel lymph node dissection (SLND) can use blue dye alone to localize the SN. However, radiopharmaceutical agents can facilitate intraoperative mapping; preoperative lymphoscintigraphy can identify the location of the SN, and intraoperative mapping with the gamma probe can provide an auditory signal that complements the visual guide provided by the blue dye. Studies are required to establish more clearly the intralymphatic kinetics of the various radiopharmaceutical agents. An ongoing international Phase III trial in melanoma, the 2 upcoming trials in breast cancer, and similar trials for other solid tumors will further clarify the future role of SLND.

Standardized probe-directed sentinel node dissection in melanoma.

BACKGROUND: Radiopharmaceutical agents appear to improve the accuracy of sentinel node (SN) identification in patients with early-stage melanoma, but the optimal radiopharmaceutical agent and its timing from injection to surgery remain controversial. We undertook this investigation to examine the utility of 3 methods of intraoperative lymphatic mapping with radiopharmaceutical-directed sentinel lymphadenectomy (LM/SL). We suspected that concurrent injection of radiopharmaceutical and blue-dye would lead to the greatest success of SN identification. METHODS: The study was composed of 247 consecutive patients who had American Joint Committee on Cancer stage I or II melanoma. Before LM/SL, all patients underwent cutaneous lymphoscintigraphy by 1 of 3 techniques: technetium 99m (Tc 99m) human serum albumin (HSA) injected at least 24 hours before LM/SL (124 patients), Tc 99m filtered sulfur colloid (SC) injected no more than 4 hours before LM/SL (same-day SC, 95 patients), or Tc 99m SC injected at least 18 hours before LM/SL (prior-day SC, 28 patients). At the time of LM/SL, isosulfan blue dye was injected alone (SC groups) or with a second dose of HSA (HSA group). A hand-held gamma probe was used to determine the radioactive (hot) counts of blue-stained and nonstained nodes, and the in vivo and ex vivo node-to-background count ratios of the nodes were compared. RESULTS: Preoperative LS identified 299 drainage basins; LM/SL identified at least 1 SN in 119 (98%) of 121 basins using same-day SC, 142 (97%) of 146 basins using HSA, and 32 (100%) of 32 basins using prior-day SC. There was no difference (P = .62) in the accuracy rate between the 3 techniques. The total number of SNs was 463. Same-day SC yielded higher intraoperative node-to-background count ratios than did either of the other techniques (P < .0001). Same-day SC also had the greatest relative change in radioactivity between the blue sentinel node and the post-excision basin (P < .0001), and the highest rate of SNs that were both blue and hot (in vivo or ex vivo ratio > or = 2, P = .05). CONCLUSIONS: LS and LM/SL performed on the same day with a single injection of filtered Tc 99m SC serves as the most useful method for probe-directed LM/SL. This technique demonstrated the highest in vivo and ex vivo count ratios, fall-off of radioactivity between the excised nodes and post-excision basin, and concordance between blue dye and hot nodes. It should be recommended as the method of choice for probe-directed LM/SL.

Radiofrequency ablation: a novel primary and adjunctive ablative technique for hepatic malignancies.

The majority of primary and metastatic tumors of the liver are not amenable to surgical resection at presentation. Radiofrequency ablation (RFA) is a new modality for local tumor destruction with minimal local and systemic complications. We prospectively reviewed the experience with RFA at a single institute as a primary or adjunctive ablative technique in the treatment of hepatic malignancies. Between November 1997 and December 1998, 30 patients with primary or metastatic hepatic lesions were treated with RFA at the John Wayne Cancer Institute and the Cancer Center at Century City Hospital. Pathology of the treated lesions included colorectal metastases (29 in 14 patients), neuroendocrine metastases (29 in 4 patients), noncolorectal metastases (29 in 9 patients), and hepatocellular carcinoma (6 in 3 patients). Twelve patients underwent RFA laparoscopically, 12 at celiotomy, and the remaining 6 patients had percutaneous ablation. RFA was the only procedure in 17 patients, whereas the remainder underwent a combination of RFA and other procedures including resection, cryosurgical ablation, and hepatic artery infusion pump placement. Median length of stay for all patients was 6 days (2 days for laparoscopic patients). A single complication of a delayed intrahepatic abscess was noted in this series (3%). There have been no deaths associated with RFA. At a median follow-up of 5 months, 16 patients remain disease free, and 10 are alive with disease. RFA is a safe and effective method of tumor ablation for hepatic malignancies. This technique can be performed laparoscopically, at celiotomy, or percutaneously and can be used as a primary technique or in conjunction with other interventional procedures.

Management of penetrating colon injuries.

The management of colon injuries remains an area of major controversy. Selecting the patients who can undergo primary repair safely remains undefined. To address this issue, 231 consecutive patients with penetrating colon injuries were reviewed to determine those factors that affected outcome. Overall, there were 54 (25.2%) septic complications, with 36 (16.8%) wound infections and 18 (8.4%) intra-abdominal abscesses. There were seven (3.3%) deaths in the entire series. The surgical management method of the colon injury was not significant in wound infections (P > .39), intra-abdominal abscesses (P > .24), or mortality (P > .39). A more aggressive approach of primary repair should be performed for civilian colon injuries.

Management of extraperitoneal rectal injuries.

Twenty-eight consecutive extraperitoneal rectal injuries for a period of 34 months ending in May 1990 were reviewed retrospectively. All injuries were due to penetrating gunshot wounds. The rectal exam was positive in 75% of patients versus 80.8% with proctosigmoidoscopy. All 28 patients had diversion of the fecal stream. Diverting colostomies were performed in 17 patients, Hartmann's colostomies in 7 patients, and proximal loop colostomies in 4 patients. Presacral drainage was used in 25 patients (89.3%). Distal irrigation was performed in 13 patients (46.4%) and primary repair in 9 patients (32.1%). There was one infectious complication (3.6%) and no deaths (0%). Fecal diversion and presacral drainage are the mainstay of therapy for civilian rectal injuries. The importance of distal irrigation of the rectum has not been established. Primary repair of the rectum has no effect on morbidity and mortality.

Radiofrequency ablation: a minimally invasive technique with multiple applications.

PURPOSE: Radiofrequency ablation (RFA) of soft tissue, which has recently been approved by the United States Food and Drug Administration, destroys tumor cells by delivering an electrical current through a 15-gauge needle. This study evaluated RFA for patients with hepatic malignancies considered unresectable because of their distribution, their number, and/or the presence of liver dysfunction. PATIENTS AND METHODS: Between November 1997 and February 1999, 50 patients with 132 unresectable hepatic metastases underwent RFA of tumors from 0.5 to 9 cm in diameter. There were 41 colorectal metastases in 22 patients, 13 hepatomas in seven patients, 37 neuroendocrine metastases in six patients, and 41 noncolorectal metastases in 15 patients. Real-time ultrasonography was used to guide RFA, and lesions were ablated by applying temperatures of approximately 100 degrees C for 8 minutes. Overlapping ablations were used for larger lesions. In patients with multiple lesions, RFA was performed simultaneously with cryosurgery, resection, and/or hepatic arterial infusion. RESULTS: RFA was undertaken percutaneously on an outpatient basis in 13 patients (25 lesions). The remaining patients underwent RFA via laparoscopy (21 patients; 58 lesions) or celiotomy (16 patients; 49 lesions); mean hospital stay was 1 and 5 days, respectively. RFA was the sole therapy in 28 patients and was additional therapy in 22 patients. At a median follow-up of 6 months, 27 patients were free of disease, 17 were alive with disease, and six had died of their disease (three colon, three melanoma). Three patients whose disease recurred at a prior RFA site underwent successful percutaneous RFA. Overall, there was a significant postoperative reduction in levels of carcinoembryonic antigen, alpha-fetoprotein, serotonin, and 5-hydroxyindoleacetic acid. Intraoperative ultrasonography identified unrecognized hepatic lesions in 12 of 37 patients (32%); these lesions were successfully ablated. When performed with cryosurgery, RFA reduced the morbidity of multiple freezes. DISCUSSION: RFA is a safe and effective alternative for the ablation of unresectable hepatic malignancies and when used adjunctively can reduce the morbidity of cryosurgery. Percutaneous and laparoscopic RFA can be performed effectively with less than 24 hours of hospitalization. Intraoperative ultrasonography is essential for accurate staging.

Detection of metastases in sentinel lymph nodes of breast cancer patients by multiple-marker RT-PCR.

This study was undertaken to assess a multiple-marker RT-PCR and Southern blot assay for detection of metastases in frozen sections of sentinel lymph nodes from breast cancer patients. Sentinel lymphadenectomy was performed in 41 AJCC (American Joint Committee on Cancer) stage I-IIIA breast cancer patients and 57 sentinel nodes (SNs) were excised. The SN, which is the first node in the lymphatic basin to receive metastases from the primary tumor, was identified using isosulfan blue dye. Hematoxylin and eosin (H&E), immuno-histochemistry (IHC) and RT-PCR were performed on adjacent sections of the SN. Six consecutive 12-microm frozen sections of each SN were obtained for the RT-PCR assay to determine expression of mRNA tumor markers C-Met, beta1 --> 4GalNAc-T and P97. Metastatic breast cancer was detected by H&E in 10 of 57 (18%) SNs and by IHC in an additional 7 (12%). Only 1 of 17 (6%) SNs with metastases did not express any of the 3 tumor mRNA markers. C-Met, beta1 --> 4GalNAc-T and P97 tumor mRNA markers were expressed in 31 (78%), 21 (53%) and 25 (63%) of 40 SNs without metastases, respectively. At least 2 mRNA tumor markers were expressed in 25/40 (63%) histo-pathologically tumor-free SNs, whereas all 3 mRNA tumor markers were expressed in 17/40 (43%) SNs. Expression of all 3 mRNA tumor markers in a SN was significantly higher in patients with a family history of breast cancer (p = 0.05), prior history of breast cancer (p < 0.05), infiltrating lobular carcinoma (p = 0.06), estrogen receptor-negative (p = 0.04) tumor or a higher Bloom Richardson score (p = 0.04). The multiple-marker RT-PCR and Southern blot assay improves the detection of occult metastases in the SN when compared to conventional H&E and IHC analysis. Expression of all 3 tumor mRNA markers in the SN correlated with poor prognostic clinico-pathologic factors compared to expression of 0 to 2 markers.

Carbon dye histologically confirms the identity of sentinel lymph nodes in cutaneous melanoma.

BACKGROUND: False-negative results from lymphatic mapping and sentinel lymphadenectomy (LM/SL) are associated with technical failures in nuclear medicine and surgery or with erroneous histologic evaluation. Any method that can confirm sentinel lymph node (SN) identity might decrease the false-negative rate. Carbon dye has been used as an adjunct to assist lymphadenectomy for some tumors, and the authors hypothesized that it could be used for the histologic verification of SNs removed during LM/SL. The current study assessed the clinical utility of carbon dye as a histopathologic adjunct for the identification of SNs in patients with melanoma and correlated the presence of carbon particles with the histopathologic status of the SNs. METHODS: LM/SL was performed using carbon dye (India ink) combined with isosulfan blue dye and sulfur colloid. Blue-stained and/or radioactive lymph nodes (two times background) were defined as SNs. Lymph nodes were evaluated for the presence of carbon particles and melanoma cells. If an SN lacked carbon dye in the initial histologic sections, four additional levels were obtained with S-100 protein and HMB-45 immunohistochemistry. Completion lymph node dissection (CLND) was performed if any SN contained melanoma cells. RESULTS: One hundred patients underwent successful LM/SL in 120 lymph node regions. Carbon particles were identified in 199 SNs from 111 lymph node regions of 96 patients. Sixteen patients had tumor-positive SNs, all of which contained carbon particles. The anatomic location of the carbon particles within these tumor-positive SNs was found to be correlated with the location of tumor cells in the SNs. The presence of carbon particles appeared to be correlated with blue-black staining (P = 0.0001) and with tumor foci (P = 0.028). All 35 non-SNs that were removed during LM/SL were tumor-negative, and only 2 contained carbon particles. Of the 272 non-SNs removed during CLND, 5 contained metastases; 3 of these 5 were the only non-SNs that had carbon particles. The use of carbon particles during LM/SL was found to be safe and nontoxic. CONCLUSIONS: Carbon dye used in LM/SL for melanoma permits the histologic confirmation of SNs. Carbon particles facilitate histologic evaluation by directing the pathologist to the SNs most likely to contain tumor. The location of carbon particles within SNs may assist the pathologist in the detection of metastases, thereby decreasing the histopathologic false-negative rate of LM/SL and subsequently reducing the same-basin recurrence rate.

Detection of MAGE-A3 in breast cancer patients' sentinel lymph nodes.

The detection of occult metastatic breast cancer cells by RT-PCR is limited by the poor specificity of most tumour mRNA markers. MAGE-A3 is a highly specific tumour mRNA marker that is not expressed in non-cancer cells. This study assesses MAGE-A3 mRNA as a molecular marker for the detection of tumour cells in the sentinel lymph nodes (SLN) of breast cancer patients. Serial frozen sections of SLN (n = 121) were obtained from 77 AJCC (American Joint Committee on Cancer) Stage I-IIIA breast cancer patients. MAGE-A3 mRNA analysis of SLN was performed by RT-PCR and Southern blot analysis. Tumour cells were detected in 48 of 121 (40%) SLN from 77 patients by H&E or IHC staining, and 35 of 77 (45%) patients, overall, had histopathologically (H&E and/or IHC) positive SLN. Among histopathologically negative SLN, 28 of 73 (38%) SLN were MAGE-A3 mRNA positive by RT-PCR. Overall, 41 of 77 (53%) patients and 50 of 121 (41%) SLN were positive for MAGE-A3. MAGE-A3 mRNA expression in the SLN occurred more frequently with infiltrating lobular carcinoma (P < 0.001) than with infiltrating ductal carcinoma, adding further evidence of possible phenotypic differences between these 2 subtypes of breast cancer. Due to its high specificity, MAGE-A3 mRNA is a potentially useful marker for detecting breast cancer cells in the SLN. One half of breast tumours expressed MAGE-A3 mRNA, which has important potential implications for antigen-specific targeted immunotherapy.