Clinical significance of molecular detection of carcinoma cells in lymph nodes and peripheral blood by reverse transcription-polymerase chain reaction in patients with gastrointestinal or breast carcinomas.

1998 ◽  
Vol 16 (1) ◽  
pp. 128-132 ◽  
Author(s):  
M Mori ◽  
K Mimori ◽  
H Ueo ◽  
K Tsuji ◽  
T Shiraishi ◽  
...  
Keyword(s):  
Lymph Nodes ◽  
Peripheral Blood ◽  
Detection Rate ◽  
Breast Carcinomas ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Blood Samples ◽  
Polymerase Chain ◽  
Cea Mrna ◽  
Positive Results

PURPOSE This study evaluates the clinical significance of detection of carcinoembryonic antigen (CEA) mRNA in the dissected lymph nodes and peripheral blood samples of patients with gastrointestinal or breast carcinomas. PATIENTS AND METHODS A total of 406 lymph nodes obtained from 65 patients were analyzed by both histologic and molecular examination of CEA-specific reverse transcriptase-polymerase chain reaction (RT-PCR). Peripheral blood samples from another 102 patients were also analyzed by CEA-specific RT-PCR. Patients were followed up prospectively for 24 +/- 12 months. RESULTS Of 406 lymph nodes, the positive detection rate increased from 20% by histologic examination to 60% by RT-PCR examination. The recurrence rate was 40% in 15 cases showing positive results in both examinations, 14% in 29 cases showing histologically negative but RT-PCR positive results, and none in 21 cases showing negative results in both examinations. The positive detection rate for CEA mRNA in peripheral blood samples increased with advancing stage of disease. With respect to 62 curatively operated cases, CEA mRNA was detected in 12 cases. Four of these 12 cases developed metastatic disease after surgery whereas none of 50 cases negative by RT-PCR developed metastasis. CONCLUSION It has been shown that RT-PCR is a powerful tool to detect CEA mRNA in the lymph nodes or the peripheral blood. This is potentially very useful to determine high-risk patients for metastasis. Serial analysis is warranted to assess the long-term significance of this method and its therapeutic and prognostic implications.

Prognostic Value of Circulating Melanoma Cells Detected by Reverse Transcriptase–Polymerase Chain Reaction

2003 ◽  
Vol 21 (5) ◽  
pp. 767-773 ◽  
Author(s):  
Giuseppe Palmieri ◽  
Paolo A. Ascierto ◽  
Francesco Perrone ◽  
Sabrina M.R. Satriano ◽  
Alessandro Ottaiano ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Subgroup Analysis ◽  
Peripheral Blood ◽  
Predictive Value ◽  
Prognostic Value ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Blood Samples ◽  
Stage Of Disease ◽  
Polymerase Chain

Purpose: Factors that are predictive of prognosis in patients who are diagnosed with malignant melanoma (MM) are widely awaited. Detection of circulating melanoma cells (CMCs) by reverse transcriptase-polymerase chain reaction (RT-PCR) has recently been postulated as a possible negative prognostic factor. Two main questions were addressed: first, whether the presence of CMCs, defined as the patient being positive for any of the three markers, had a prognostic role; and second, what the predictive value of each individual marker was. Patients and Methods: A consecutive series of 200 melanoma patients observed between January 1997 and December 1997, with stage of disease ranging from I to IV, was analyzed by semiquantitative RT-PCR. Tyrosinase, p97, and MelanA/MART1 were used as markers to CMCs on baseline peripheral blood samples. Progression-free survival (PFS) was used as a unique end point and was described by the product limit method. Multivariable analysis was applied to verify whether the auspicated prognostic value of these markers was independent of the stage of disease, and a subgroup analysis was performed that excluded patients with stage IV disease. Results: Overall, 32% (64 of 200) of patients progressed, and a median PFS of 52 months in the whole series was observed. The presence of CMCs and the markers individually or combined was predictive of prognosis in the univariate analysis but did not provide additional prognostic information to the stage of disease in multivariable models. In the subgroup analysis of stage (ie, I–III subgroup), similar results were observed. Conclusion: Detection of CMCs in peripheral blood samples at the time of MM diagnosis by semiquantitative RT-PCR does not add any significant predictive value to the stage of disease. Thus, this approach should not be used in clinical practice, and further studies are required to determine its usefulness.

Impact of Molecular Staging Methods in Primary Melanoma: Reverse-Transcriptase Polymerase Chain Reaction (RT-PCR) of Ultrasound-Guided Aspirate of the Sentinel Node Does Not Improve Diagnostic Accuracy, But RT-PCR of Peripheral Blood Does Predict Survival

2008 ◽  
Vol 26 (35) ◽  
pp. 5742-5747 ◽  
Author(s):  
Christiane A. Voit ◽  
Gregor Schäfer-Hesterberg ◽  
Martina Kron ◽  
Alexander C.J. van Akkooi ◽  
Juergen Rademaker ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Reverse Transcriptase ◽  
Peripheral Blood ◽  
Primary Melanoma ◽  
Disease Free Survival ◽  
Rt Pcr ◽  
Ultrasound Guided ◽  
Chain Reaction ◽  
Blood Samples ◽  
Polymerase Chain

Purpose This study analyzes (1) the value of tyrosinase reverse-transcriptase polymerase chain reaction (RT-PCR) of aspirates obtained by ultrasound-guided fine-needle aspiration cytology (US-FNAC) of sentinel nodes (SNs) in patients with melanoma before sentinel lymph node biopsy (SLNB) and (2) the value of RT-PCR of blood samples of all SLNB patients. Patients and Methods Between 2001 and 2003, 127 patients with melanoma (median Breslow depth, 2.1 mm) underwent SLNB. FNAC was performed in all SNs of all patients pre- and post-SLNB. The aspirates were partly shock-frozen for RT-PCR and were partly used for standard cytology. Peripheral blood was collected at the time of SLNB and at every outpatient visit thereafter. Results Thirty-four (23%) of 120 SNs were positive for melanoma. SN involvement was predicted by US-FNAC with a sensitivity of 82% and a specificity of 72%. Additional tyrosinase RT-PCR revealed the same sensitivity of 82% and a specificity of 72%. At a median follow-up time of 40 months from first blood sample, peripheral-blood RT-PCR was a significant independent predictor of disease-free survival (DFS) and overall survival (OS; P < .001). Conclusion US-FNAC is highly accurate and eliminates the need for SLNB in 16% of all SLNB patients. RT-PCR of the aspirate or excised SN does not improve sensitivity or specificity. RT-PCR of blood samples predicts DFS and OS.

Investigations on the prevalence of tortoise picorna-virus in captive tortoises in Germany

2018 ◽  
Vol 46 (05) ◽  
pp. 304-308 ◽  
Author(s):  
Svenja Funcke ◽  
Michael Lierz ◽  
Susanne Paries
Keyword(s):  
Rt Pcr ◽  
Chain Reaction ◽  
Blood Samples ◽  
Specific Antibodies ◽  
Testudo Graeca ◽  
The Family ◽  
High Prevalence ◽  
Disease Pattern ◽  
Polymerase Chain ◽  
Positive Results

Summary Objective: Tortoise picornavirus (ToPV) has been speculated to play an important role in the frequently seen disease pattern of juvenile shell softening. This study aimed to determine ToPV prevalence among German tortoise collections. Material and methods: A total of 334 animals selected from 27 different collections were included. Seven species of four genera of the family Testudinidae (Testudo graeca, T. hermanni, T. marginata, T. horsfieldii, Centrochelys sulcata, Stigmochelys pardalis, Chelonoidis carbonarius) were sampled. The tortoises were clinically investigated and none of the adults showed any signs of shell softening. Seven hatchlings of a ToPV-positive T. graeca breeding pair showed retarded growth and a progressive shell weakness that resulted in death. Each animal was sampled by conjunctival, pharyngeal and cloacal swabs (990 swabs in total) and blood sampling (293 in total). All three swabs of one animal were pooled and tested by reverse transcriptase polymerase chain reaction (RT-PCR) for tortoise picornavirus RNA. Blood samples were investigated by virus neutralisation test (VNT) for specific anti ToPV antibodies. All titres equal to or higher than log2 = 2 were considered positive. Results: In total, 35 adult and 11 juvenile animals were tested positive for ToPV RNA. The serological investigation did detect specific antibodies against ToPV in 44 adult tortoises and one juvenile. In total, 76 animals were tested positive in either one of the investigations, 16 animals in both. The highest number of ToPV-positive animals was found for T. graeca, with a prevalence of 32 %. No specimens of C. carbonarius, C. sulcata, or S. pardalis were tested positive. Conclusion and clinical relevance: The results propose a predisposition in T. graeca, as well as a high prevalence of ToPV in T. graeca, whereas other species showed only single or no positive animals, but may function as virus carriers.

Quantitative Polymerase Chain Reaction for the Detection of Micrometastases in Patients With Breast Cancer

1999 ◽  
Vol 17 (3) ◽  
pp. 870-870 ◽  
Author(s):  
Martin J. Slade ◽  
Brendan M. Smith ◽  
H. Dudley Sinnett ◽  
Nicholas C.P. Cross ◽  
R. Charles Coombes
Keyword(s):  
Breast Cancer ◽  
Bone Marrow ◽  
Metastatic Breast Cancer ◽  
Peripheral Blood ◽  
Primary Breast Cancer ◽  
Metastatic Breast ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Blood Samples ◽  
Polymerase Chain

PURPOSE: Previous reports have indicated that reverse transcriptase polymerase chain reaction (RT-PCR) for cytokeratin 19 (CK-19) may be useful in the management of patients with breast cancer. However, the specificity of this technique is low, principally because of a high rate of false-positive results. To improve the specificity of this assay, we developed a quantitative RT-PCR methodology that enables an estimate to be made of the number of CK-19 transcripts in blood and bone marrow samples. PATIENTS AND METHODS: We examined 45 peripheral-blood samples and 30 bone marrow samples from patients with a variety of nonneoplastic conditions using nested RT-PCR for CK-19. We also examined bone marrow and peripheral-blood samples from 23 patients with primary breast cancer and peripheral-blood samples from 37 patients with metastatic breast cancer. The number of CK-19 transcripts was estimated in positive specimens by competitive PCR and normalized to the number of ABL transcripts as an internal control for the quality and quantity of cDNA. RT-PCR results were compared with the numbers of CK-19–positive cells detected by immunocytochemistry. RESULTS: Analysis of samples from patients without cancer enabled us to define an upper limit for the background ratio of CK-19 to ABL transcripts (1:1,000 for blood samples and 1:1,600 for bone marrow samples). Using these figures as cut-off points, elevated CK-19: ABL ratios were detected in peripheral-blood samples of 20 of 37 (54%) patients with metastatic breast cancer and in bone marrow samples of 14 of 23 (61%) patients with primary breast cancer. Only three of 23 (13%) primary breast cancer peripheral-blood samples and none of the control samples were positive by these criteria. Only two of 23 patients (9%) with primary breast cancer showed immunocytochemically detectable cells in the blood; 10 of 23 (43%) showed immunocytochemically detectable cells in the bone marrow. Of 36 patients with metastatic breast cancer, eight (22%) showed positive events. CONCLUSION: Quantitative RT-PCR for CK-19 detects a percentage of patients with breast cancer and may enable the progression or regression of the disease to be monitored.

Detection of circulating neoplastic cells by reverse-transcriptase polymerase chain reaction in malignant melanoma: association with clinical stage and prognosis.

1996 ◽  
Vol 14 (7) ◽  
pp. 2091-2097 ◽  
Author(s):  
B Mellado ◽  
D Colomer ◽  
T Castel ◽  
M Muñoz ◽  
E Carballo ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Malignant Melanoma ◽  
Prognostic Factor ◽  
Peripheral Blood ◽  
Clinical Stage ◽  
Melanoma Cells ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Circulating Neoplastic Cells

PURPOSE Circulating melanoma cells can be detected in peripheral blood by means of tyrosinase mRNA amplification by reverse-transcriptase polymerase chain reaction (RT-PCR). We conducted a prospective study to evaluate the clinical significance of the presence of circulating neoplastic cells in the blood of patients with malignant melanoma (MM). METHODS A sensitive RT-PCR assay was used to detect tyrosinase mRNA in the peripheral blood of patients with stages I to IV melanoma. Healthy subjects or patients with other malignancies were used as negative controls. RESULTS Ninety-one assessable patients were included in the study. There was a statistically significant association between RT-PCR positivity and clinical stage. Circulating melanoma cells were detected in 36% of patients with localized disease (stages I and II), in 45% of patients with regional nodal involvement (stage III), and in 94% of patients with metastatic disease (stage IV) (P < .001). In stage II-III patients who were RT-PCR-positive for mRNA tyrosinase in blood, the recurrence rate and disease-free survival were significantly worse than patients who were RT-PCR-negative. In multivariate analysis, RT-PCR was an independent prognostic factor for recurrence in patients with nonmetastatic disease (P = .002). CONCLUSION The detection of circulating melanoma cells in peripheral blood by RT-PCR correlated with the clinical stage of patients with melanoma and was an independent prognostic factor for recurrence. Further studies are warranted to better assess the significance of this test in the evaluation of prognosis, early detection of relapse, and in monitoring the effectiveness of systemic therapy.

Polymerase Chain Reaction-Based Detection of Circulating Melanoma Cells as an Effective Marker of Tumor Progression

1999 ◽  
Vol 17 (1) ◽  
pp. 304-304 ◽  
Author(s):  
Giuseppe Palmieri ◽  
Maria Strazzullo ◽  
Paolo A. Ascierto ◽  
Sabrina M.R. Satriano ◽  
Antonio Daponte ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Tumor Progression ◽  
Peripheral Blood ◽  
Melanoma Cells ◽  
Disease Stage ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Gene Markers ◽  
Risk Of Recurrence ◽  
Polymerase Chain

PURPOSE: Reverse transcriptase (RT) polymerase chain reaction (PCR) with multiple markers has been demonstrated to be highly sensitive in detecting circulating cells from patients with malignant melanoma (MM). We evaluated the clinical significance of the presence in peripheral blood of specific PCR-positive mRNA markers as an expression of circulating melanoma cells. PATIENTS AND METHODS: Total cellular RNA was obtained from the peripheral blood of 235 patients with either localized (n = 154) or metastatic (n = 81) melanoma. We performed RT-PCR using tyrosinase, p97, MUC18, and MelanA/MART1 as gene markers. The PCR products were analyzed by gel electrophoresis and Southern blot hybridization. In addition, 20 healthy subjects and 21 patients with nonmelanoma cancer were used as negative controls. RESULTS: Although detected at various levels among assessable patients, each mRNA marker was significantly correlated with disease stage. A significant correlation with disease stage was demonstrated for patients who were positive to all four markers (P < .0001) or to at least three markers (P < .001). Univariate analysis showed a significant correlation between risk of recurrence (evaluated in stage I, II, and III patients) and increasing number of PCR-positive markers (P = .0002). Logistic regression multivariate analysis indicated that each single marker (except tyrosinase) and, more especially, the presence of four PCR-positive markers remained statistically independent prognostic factors for tumor progression. CONCLUSION: Our data establish the existence of a significant correlation among clinical stages, tumor progression, and presence of circulating melanoma-associated antigens in peripheral blood of MM patients. Preliminary assessment of a subset of patients with a higher risk of recurrence needs longer follow-up and further studies to define the role of RT-PCR in monitoring MM patients.

Prospective Multi-Institutional Study of Reverse Transcriptase Polymerase Chain Reaction for Molecular Staging of Melanoma

2006 ◽  
Vol 24 (18) ◽  
pp. 2849-2857 ◽  
Author(s):  
Charles R. Scoggins ◽  
Merrick I. Ross ◽  
Douglas S. Reintgen ◽  
R. Dirk Noyes ◽  
James S. Goydos ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Reverse Transcriptase ◽  
Peripheral Blood ◽  
Prognostic Significance ◽  
Disease Free Survival ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Molecular Staging ◽  
Polymerase Chain

Purpose To evaluate the prognostic significance of molecular staging using reverse transcriptase polymerase chain reaction (RT-PCR) in detecting occult melanoma cells in sentinel lymph nodes (SLNs) and circulating bloodstream. Patients and Methods In this multicenter study, eligibility criteria included patient age 18 to 71 years, invasive melanoma ≥ 1.0 mm Breslow thickness, and no clinical evidence of metastasis. SLN biopsy and wide excision of the primary tumor were performed. SLNs were examined by serial-section histopathology and S-100 immunohistochemistry. A portion of each SLN was frozen for RT-PCR. In addition, RT-PCR was performed on peripheral-blood mononuclear cells (PBMCs). RT-PCR analysis was performed using four markers: tyrosinase, MART1, MAGE3, and GP-100. Disease-free survival (DFS), distant–DFS (DDFS), and overall survival (OS) were analyzed. Results A total of 1,446 patients with histologically negative SLNs underwent RT-PCR analysis. At a median follow-up of 30 months, there was no difference in DFS, DDFS, or OS between the RT-PCR–positive (n = 620) and RT-PCR–negative (n = 826) patients. Analysis of PBMC from 820 patients revealed significant differences in DFS and DDFS, but not OS, for patients with detection of more than one RT-PCR marker in peripheral blood. Conclusion In this large, prospective, multi-institutional study, RT-PCR analysis on SLNs and PBMCs provides no additional prognostic information beyond standard histopathologic analysis of SLNs. Detection of more than one marker in PBMC is associated with a worse prognosis. RT-PCR remains investigational and should not be used to direct adjuvant therapy at this time.

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