Multimarker reverse transcriptase-polymerase chain reaction assay in lymphatic drainage and sentinel node tumor burden.

PURPOSE: We assessed molecular (presence of melanoma cells markers in lymph fluid [LY]) and pathological features (sentinel lymph node [SN] tumor burden according to Rotterdam criteria, metastases microanatomic location) and correlated them with survival and melanoma prognostic factors in a group of patients with positive SN biopsy. METHODS: We analyzed 368 consecutive SN-positive patients after completion lymph node dissection (CLND). In 321 patients we obtained data on SLN microanatomic location/tumor burden (only 7 cases had metastases <0.1 mm); in 137 we additionally analyzed 24-hour collected LY after CLND (multimarker reverse transcriptase-polymerase chain reaction [MM-RT-PCR] with primers for tyrosinase, MART1 (MelanA), and uMAGE mRNA (27.7% positive samples)]. Median follow-up time was 41 months. RESULTS: According to univariate analysis, the following factors had a negative impact on overall survival (OS): higher Breslow thickness (P = .0001), ulceration (P < .0001), higher Clark level (P = .008), male gender (P = .0001), metastatic lymph nodes >1 (P < .0001), nodal metastases extracapsular extension (P < .0001), metastases to additional non-SNs (P = .0004), micrometastases size ≥ 0.1 mm (P = .0006), and positive LY MM-RT-PCR (P = .0007). SN tumor burden showed linear correlation with increasing Breslow thickness (P = .01). The 5-year OS rates for SLN tumor burden <0.1 mm, 1-1.0 mm, and >1.0 mm were 84%/66%/44%, respectively, and for positive and negative LY MM-RT-PCR 47%/0%, respectively. The independent factors for shorter OS (multivariate analysis): male gender, primary tumor ulceration, number of involved nodes ≥ 4, micrometastases size >1.0 mm, and, in additional model including molecular analysis-positive MM-RT-PCR results (hazard ratio [HR] 3.2), micrometastases size >1.0 mm (HR 1.13), and primary tumor ulceration (HR 2.17). Similar results were demonstrated for disease-free survival (DFS) data. CONCLUSIONS: SN tumor burden categories according to Rotterdam criteria and the positive result of LY MM-RT-PCR assay demonstrated additional, independent prognostic value in SN-positive melanoma patients, showing significant correlation with shorter DFS and OS.

Molecular staging by multimarker reverse transcriptase-polymerase chain reaction assay of lymphatic drainage and blood from melanoma patients after lymph node dissection.

Reverse transcriptase-polymerase chain reaction (RT-PCR)-mediated detection of melanoma cells may be a prognostic factor for disease outcome. We investigated the presence of melanoma cells in lymphatic drainage and blood in melanoma patients after lymph node dissection (LND) via the highly sensitive multimarker (MM) RT-PCR assay. We collected 24-h lymph fluid (LY) and peripheral blood (BL) from 107 stage III melanoma patients after radical LND (59 axillary and 48 ilioinguinal LND). Tyrosinase, MART1 and uMAGE mRNA levels were determined by RT-PCR to detect melanoma cells, and the presence of at least one marker signified a positive result. All patients underwent follow-up (median for survivors, 21 months, range: 4-37 months). Forty patients (37.4%) were positive for LY MM RT-PCR and 28 (26.2%) were positive based on BL MM RT-PCR. No differences for disease-free survival (DFS) curves according to BL MM RT-PCR were observed, but we found significant differences in the estimated 24-month DFS rate for patients with at least one marker and those without any marker in lymph fluid [18.9% (95% confidence interval: 1.4-37.5%) and 42.1% (95% confidence interval: 29.7-54.5%), median: 9.9 and 15.3 months, respectively] (P=0.04). Detection of multiple markers in lymph fluid correlated with shorter DFS. Approximately 37% of lymph fluid after radical LND were positive by MM RT-PCR, which correlated significantly with early melanoma recurrences and shorter survival. The LY MM RT-PCR seems to be an effective prognostic tool for stage III melanoma patients. The MM RT-PCR analysis of single peripheral blood sample in these patients did not have additional prognostic value.

Usefulness of real-time PCR in long-term follow-up of follicular lymphoma patients.

The aim of this study was to evaluate the usefulness of quantitative real-time PCR (RQ-PCR) for the monitoring of molecular remission in follicular lymphoma (FL) patients during long-term follow-up. RQ-PCR by the use of TaqMan detection system is a sensitive tool to monitor minimal residual disease (MRD) in FL through amplification of the t(14;18) fusion gene during and post-therapy. In most cases the breakpoint region occurs within the major breakpoint region (MBR). Among 75 patients diagnosed with FL, cells harboring the fusion gene BCL2/JH were found in peripheral blood of 31 patients (41%). We further monitored 30 of these patients in a period varying from 6 months to 5 years by RQ-PCR. In our study the level indicating the possibility of the presence of MRD was established at more than five t(14;18)-positive cells in the background of 83,000 normal cells. The results of this work also confirmed that the presence of MRD detected by RQ-PCR is an indication for careful observation of patients because of a higher risk of disease recurrence.

Unusual IgD+/CD38-follicular lymphoma with leukemic presentation.

Follicular lymphoma (FL) is a low-grade lymphoma, with rare presentation of leukemic phase in peripheral blood at the diagnosis. We describe a 49-yr-old woman who developed leukemic phase of FL in a 3 mo period after histological diagnosis of peripheral lymph node. To confirm the final diagnosis, flow cytometry (FCM) of peripheral blood, nested PCR with bcl-2 rearrangement, and cytogenetic analysis of peripheral blood and bone marrow cells were done. Lymphoma cells were negative for CD38 and expressed monoclonal surface immunoglobulins with relatively strong and "bright" IgD and "dim" kappa-chain by FCM analysis. Although the patient presented with generalized lymphadenopathy, massive peripheral blood and bone marrow involvement, she achieved complete clinical response after first-line chemotherapy COP and rituximab. She is still in a good condition with follow up over 2 yr.

Detection of carbonic anhydrase 9-expressing tumor cells in the lymph nodes of vulvar carcinoma patients by RT-PCR.

Regional lymph node status is an important prognostic factor for vulvar cancer. The goal of our study was to elaborate a reliable test for detecting micrometastases, undetectable by traditional methods, in the lymph nodes of vulvar squamous carcinoma patients. For this purpose, carbonic anhydrase-9 (CA9) was investigated as a cancer-related marker by RT-PCR. Firstly, primary carcinoma specimens were examined for CA9 expression by immunohistochemistry with M75 monoclonal antibody. All 19 tissues exhibited a variable degree of staining, which was mostly confined to the plasma membranes of tumor cells. Correspondingly, all primary tumor specimens and the control A-431 vulvar cancer cell line gave a positive signal in the nested RT-PCR assay designed to detect CA9-expressing cells with a high sensitivity. Analysis of 77 lymph node specimens from 20 patients revealed a full correlation between RT-PCR results and standard hematoxylin-eosin staining in 75% of samples, whereas 25% of specimens were negative by the standard method and positive for CA9 mRNA, accounting for 28% of all histologically negative lymph nodes. There were no false-negatives with RT-PCR. A positive inguinal lymph node with a negative sentinel node was observed in the same groin only once in 38 specimens. Our findings clearly indicate potential value of CA9 as a molecular marker for the assessment of regional lymph node status in vulvar cancer patients and support a possible utility of our RT-PCR assay in the detection of micrometastases.

Detection of melanoma cells in the lymphatic drainage after lymph node dissection in melanoma patients by using two-marker reverse transcriptase-polymerase chain reaction assay.

BACKGROUND: The aim of this study was to evaluate the role of melanoma gene expression as a marker of the presence of melanoma cells in lymphatic drainage routinely collected after lymphadenectomy and to correlate reverse transcriptase-polymerase chain reaction (RT-PCR) assay results with recurrence, survival, and prognostic factors. METHODS: We collected 24-hour postoperative lymphatic drainage samples (between days 2 and 4) from 93 patients with stage III melanoma who underwent radical lymphadenectomy between May 2002 and November 2003. We used RT-PCR assays with primers specific for the tyrosinase and MART-1 (Melan-A) genes. The samples were considered positive if at least one marker was expressed. Median follow-up time was 12.8 months. RESULTS: In 18 (19.4%) of 93 patients, the RT-PCR assay results were positive: in 8 of 18 for tyrosinase only, in 7 of 18 for MART-1 only, and in 3 of 18 for both markers. We observed a significantly higher recurrence rate in patients with positive RT-PCR results (15 of 18; 83%) than negative results (26 of 75; 35%; P = .0001). Positive results of RT-PCR correlated with the number of involved lymph nodes (P = .0001) and extracapsular extension of nodal metastases (P = .03). We observed significant differences in overall and disease-free survival for RT-PCR-positive and -negative patients in univariate and multivariate analyses. CONCLUSIONS: We observed positive RT-PCR assay results for melanoma cells in the lymphatic drainages of approximately 20% of patients after lymphadenectomy. This correlated significantly with early recurrence and shorter survival. These results may suggest that the RT-PCR assay could be useful for routinely analyzing postoperatively collected lymphatic drainage in stage III melanoma patients and for predicting disease progression.

Detection of circulating breast cancer cells in peripheral blood by a two-marker reverse transcriptase-polymerase chain reaction assay.

The aim of this study was to use a two-marker assay for the detection of breast cancer cells circulating in patients' blood. We have applied a PCR-based methodology to follow up the possibility of the development of metastatic disease in stage I and II patients who had undergone curative surgery. Since the number of circulating cancer cells in peripheral blood is very low, the technique for their detection needs to be not only highly sensitive, but also very specific. The reverse transcriptase-polymerase chain reaction (RT-PCR) technique may improve the sensitivity of breast cancer cell detection up to only a few cells per one million. The principle of the RT-PCR assay is to amplify a messenger RNA characteristic for breast epithelial cells in a blood sample. Since we do not expect such cells to be circulating in peripheral blood of healthy subjects, detection of the characteristic mRNA should indicate the presence of circulating breast cancer cells. We analyzed the usefulness of three mRNA markers: cytokeratin 19 (CK19), mammaglobin (hMAM) and beta subunit of human chorionic gonadotropin (beta-hCG) for this test. Blood samples (112) were obtained from 55 patients, in stages I and II, with or without metastasis to regional lymph nodes (N0 or N1). We found that a two-marker assay increases the sensitivity of detection of breast cancer cells in comparison with a single-marker one. Combination of two tumor-specific mRNA markers, hMAM/CK19 or beta-hCG/CK19, allowed the detection of circulating breast cancer cells in 65% of N1 patients and 38% of N0 patients. By comparison, the combination hMAM/beta-hCG allowed the detection of circulating breast cancer cells in the blood of 68% of N1 patients and 46% of N0 patients. Addition of the third marker did not significantly increase the detection sensitivity.

[Preliminary results of the use of MRD test among children with acute lymphoblastic leukaemia]. / Wstepne wyniki wykorzystania testu MRD u dzieci z ostra bialaczka limfoblastyczna.

UNLABELLED: The purpose of the study was to monitor minimal residue disease (MRD) among children with ALL and to evaluate the possibility of using this test to detect and monitor the minimal residual disease (MRD test) in the stratification of risk groups on a parallel basis with other recognised prognostic factors. MATERIALS AND METHODS: 56 children, with de novo diagnosed ALL, comprised test group. Control group consisted of 10 healthy persons. DNA was isolated from peripheral blood and bone marrow. During research applied was the PCR method with the use of starters specific for conservative IgH and TCR delta gene fragments. ALL therapy was monitored by evaluation of MRD in the 15th and 33rd day of treatment and prior to supportive treatment. Stratification into risk groups, based on the MRD test results was compared with the stratification conducted in accordance with the recognised prognostic factors, such as: primary leucocytosis, steroid therapy response, drug resistance and karyotype. RESULTS: Among 52 children (92.8%) of the 56, which were tested prior to the start of treatment, obtained the sought for rearrangement, 90-120bp size stripe being an amplified regrouping of the VDJ fragment. Among all the tested healthy persons, the test result was negative. Testing the IgH and TCR delta gene regrouping allowed to demonstrate clonality of the neoplastic process during the diagnosis in 92.8% of the cases. In some of them the regrouping had a bi- or oligoclonal character. The number of patients with a positive MRD test result was dependent on the phase of treatment. Positive results were recorded among 10% of the patients (4/39) during clinical remission, among 86% (6/7) deceased and among 80% (4/5) suffering from relapse. Among 16.7% (2/13) of the patients with determined relapse or who died due to the course of illness process were also observed negative MRD test results. Steroid resistance was stated among 18/56 children, out of which 8/56 obtained a positive MRD test result in the 33rd day of treatment. Among 3 of them the result was maintained prior to supportive treatment. Leucocytosis during diagnosis above 50 thousand was recorded among 10/56 patients. Among 5 of them determined was rearrangement on the 33rd day of treatment, and for 1 prior to supportive treatment. Cytogenetic tests showed a correct karyotype for 19 out of 56 tested children. In this group steroid resistance was determined among 8 children, high preliminary leucocytosis among 5 and in 17 cases of the tested rearrangement. Stratification into therapeutic groups based on classical prognostic factors in most cases was the same with the stratification based on the MRD test results. The accuracy of the latter method was greater. CONCLUSIONS: The use of MRD test has its application in risk group qualification. MRD test is helpful in determining relapses prior to the appearance of clinical symptoms. Among the tested group of children with ALL a positive MRD test result was a bad prognostic factor. It seams that the use of a combined analysis of the risk factors and the MRD test results in a significant manner contribute to a more complex evaluation of the patient's condition.

Prognostic value of multiple reverse transcription-PCR tyrosinase testing for circulating neoplastic cells in malignant melanoma.

BACKGROUND: The reverse transcription-PCR tyrosinase assay (TYR test) cannot reliably detect malignant melanoma (MM) cells in blood as the cells often circulate at low concentrations. We evaluated the prognostic value of multiple TYR testing, the prognostic significance of individual positive TYR test results (TYR+) in asymptomatic melanoma patients, and whether statistical analysis could help in the interpretation of results of a test that measures phenomena that typically occur below its detection threshold. METHODS: MM patients in stages I-IV (n = 150) underwent multiple testing with the TYR test during the course of their disease. TYR testing was performed as described by Smith et al. (Lancet 1991;38:1227-9). Statistical analyses were performed with the logistic function and t-test procedures. RESULTS: The relationship between MM stage and the frequency of TYR+ was statistically significant (P = 0.011). Higher frequency of TYR+ in clinically asymptomatic patients after complete resection of the primary tumor was associated with an increased risk of recurrence of MM (prognostic sensitivity, 62%; specificity, 78%). CONCLUSIONS: A single positive TYR test provides a warning for disease relapse, suggesting that multiple TYR testing might provide more reliable predictions of disease progression. Multiple testing and statistical analysis using a logistic function might allow for the interpretation of apparently inconsistent results of tests for very rare cells.