Sentinel Lymph Node Molecular Pathology in Breast Carcinoma (original) (raw)
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Experimental and Molecular Pathology, 2010
Background: Sentinel lymph node (SLN) processing remains variable in terms of performing multiple tissue levels and immunohistochemical (IHC) or PCR-based assays. A rapid and reliable molecular pathology assay, as an adjunct to routine SLN processing, could minimize and standardize the histologic evaluation needed for an accurate and clinically significant diagnosis. We compared the recently FDA-approved Veridex GeneSearch™ Breast Lymph Node (BLN) Assay (Veridex, LLC; Warren, NJ), a real time reverse transcriptase-polymerase chain reaction assay that is designed to detect metastases N 0.2 mm, with our standard lymph node processing. Materials and methods: The GeneSearch™ BLN assay evaluates RNA expression data for three target genes (mammaglobin, cytokeratin 19, and internal control porphobilinogen deaminase), and provides a qualitative (positive/negative) result. In 59 patients, the assay was performed on SLN tissue that would normally be deep within the tissue block and not routinely evaluated histologically. Two 1-mm slices from the outer node portions were submitted fresh for RNA extraction; the remaining tissue was submitted for routine histology. Results: Of the 59 patients, the assay determined 43 as true negative, eight as true positive, one as falsenegative, three as false-positive, and four as invalid. Assay sensitivity was 88.9%, specificity 93.5%. Discussion: The sensitivity of the assay sampling from the outer node tissue was high (88.9%) and identical to that validated in the large registration study in which half of the node was assessed as alternate slices (87.6%). Our protocol uses this assay as an adjunct to traditional histologic evaluation, to reduce and standardize the number of tissue sections needed for thorough SLN evaluation, and to enhance our ability to bank RNA.
Breast Cancer Research and Treatment, 2010
2 Abstract Purpose: A RT-PCR assay (GeneSearch TM , Veridex, LLC), FDA approved and CE marked to detect metastases >0.2-mm in sentinel lymph nodes (SLNs) is used intra-operatively for breast cancer patient management. The assay provides qualitative results by applying cut-off values to cycle times (Ct) for mammaglobin (MG) and cytokeratin-19 (CK19) genes. Aims of this study were to evaluate the performance of the quantitative Ct values to estimate the size of nodal metastases and the risk of additional disease in non-SLNs. Methods: SLNs from 367 patients were clinically processed using both BLN assay and post-operative histology. Complementary axillary lymph node dissection (ALND) was performed concurrently in case of BLN assay positivity or tumour size >2-cm. Results: BLN positivity was reported in 19.6% of the patients for a sensitivity of 89%. BLN specificity (94.5%) and negative predictive value (97.5%) clearly demonstrated its reliability to guide ALND decision. All, except one, residual axillary metastases were found in BLN-positive patients. Considering the 78 patients with SLN positivity or discordant status according to both criteria, the metastases histological size was significantly correlated to the expression level of MG (ρ=0.62) and CK19 (ρ=0.64) genes (p<10E-6). Moreover, ALND status positivity was significantly associated to Ct value of MG (z=2.4; p=0.018) and CK19 (z=3.2; p=0.001).
Journal of Cellular and Molecular Medicine, 2009
The optimal pathological assessment of sentinel nodes (SLNs) in breast cancer is a matter of debate. Currently, multilevel histological evaluation and immunohistochemistry (IHC) are recommended, but alternative RT-PCR procedures have been developed. To assess the reliability of these different procedures, we devised a step-sectioning protocol at 100 micron-intervals of 74 SLNs using methacarn fixation. mRNA was extracted from sections collected from levels 4 to 5. Mammaglobin, CEA and CK19 were used for RT-PCR. mRNA extraction was successful in 69 SLNs. Of these, 7 showed macrometastases (Ͼ2mm), 2 showed micrometastases (Ͻ2 mm) and 7 showed isolated tumour cells (ITC) by IHC. RT-PCR was positive for the three markers in 6 of 7 macrometastases and in 1 of 2 micrometastases. In the 2 RT-PCR negative cases, metastases were detected only on sections distant from those analysed by RT-PCR. CEA and/or CK19 were positive by RT-PCR in 3 of 7 ITC and in 23 morphologically negative SLNs. In conclusion, the main goal of our study was to show that the use of alternate sections of the same sample for different procedures is the key reason for the discrepancies between molecular and morphological analyses of SLN. We believe that only prospective studies with quantitative mRNA analysis of specific metastatic markers on the whole lymph node can elucidate the utility of molecular assessments of SLN.
Breast Cancer Research and Treatment, 2008
Introduction Human mammaglobin (hMAM) mRNA is a sensitive and specific marker of breast cancer cells. We evaluated if hMAM mRNA detection in serial peripheral blood samples from non-metastatic breast cancer patients predicts for disease recurrence. Methods Patients scheduled for adjuvant or neoadjuvant chemotherapy were eligible. Serial blood samples were collected up to 5 years, the first before (neo)adjuvant chemotherapy. hMAM gene expression was analysed by RT-PCR. Specificity was evaluated in blood samples from healthy volunteers. A total of 321 patients were included. Results The incidence of pre-chemotherapy hMAM-positive samples was similar in patients who latter experienced cancer recurrence (22.4%) and those who remained disease-free (17.9%; P = 0.46). Similarly, the mean number of positive follow-up samples was similar in both groups (0.15 ± 0.22 and 0.13 ± 013; P = 0.29). Furthermore, there was no difference in disease-free (P = 0.63) or overall survival (P = 0.57) in patients with and without positive baseline samples or between patients whose follow-up samples were always hMAM negative and those with at least one positive sample. Multivariate survival analysis confirmed that hMAM mRNA detection before or after (neo)adjuvant chemotherapy was not predictive of recurrence. Discussion There is no evidence that hMAM mRNA detection at diagnosis or during follow-up predicts for breast cancer recurrence.
Cancer, 2005
BACKGROUNDThe ideal pathologic assessment of sentinel lymph nodes (SLNs) in patients with breast carcinoma remains controversial. The authors evaluated how detailed assessment of SLNs using immunohistochemistry (IHC) and serial sectioning would affect treatment decisions and outcomes in patients with breast carcinoma who had negative SLNs on standard hematoxylin and eosin staining.The ideal pathologic assessment of sentinel lymph nodes (SLNs) in patients with breast carcinoma remains controversial. The authors evaluated how detailed assessment of SLNs using immunohistochemistry (IHC) and serial sectioning would affect treatment decisions and outcomes in patients with breast carcinoma who had negative SLNs on standard hematoxylin and eosin staining.METHODSThe SLNs from patients who were treated between June 1998 and June, 1999 and who had negative lymph node status determined by hematoxylin and eosin staining (n = 84 patients) were evaluated further with serial sectioning and cytokeratin IHC. Patients were offered adjuvant therapy based on primary tumor factors.The SLNs from patients who were treated between June 1998 and June, 1999 and who had negative lymph node status determined by hematoxylin and eosin staining (n = 84 patients) were evaluated further with serial sectioning and cytokeratin IHC. Patients were offered adjuvant therapy based on primary tumor factors.RESULTSThe median patient age was 57 years, and the median tumor size was 1.2 cm. At a median follow-up of 40.2 months, 81 patients (96%) were alive with no evidence of disease, 1 patient was alive with disease, 1 patient had died of disease, and 1 patient had died of other causes. Fifteen patients (18%) had micrometastases identified on IHC. Of the total 84 patients, information regarding adjuvant therapy was not available for 5 patients. Of the remaining 79 patients, 10 patients (13%) were not offered adjuvant chemotherapy but had positive SLN status determined by IHC. SLN status based on IHC evaluation did not correlate with age (P = 0.077), tumor size (P = 0.717), grade (P = 0.148), estrogen receptor status (P = 1.000), or lymphovascular invasion (P = 0.274). Furthermore, IHC-detected positive SLN status did not correlate with distant metastasis (P = 0.372) or overall or distant metastasis-free survival (P = 0.543 and P = 0.540, respectively).The median patient age was 57 years, and the median tumor size was 1.2 cm. At a median follow-up of 40.2 months, 81 patients (96%) were alive with no evidence of disease, 1 patient was alive with disease, 1 patient had died of disease, and 1 patient had died of other causes. Fifteen patients (18%) had micrometastases identified on IHC. Of the total 84 patients, information regarding adjuvant therapy was not available for 5 patients. Of the remaining 79 patients, 10 patients (13%) were not offered adjuvant chemotherapy but had positive SLN status determined by IHC. SLN status based on IHC evaluation did not correlate with age (P = 0.077), tumor size (P = 0.717), grade (P = 0.148), estrogen receptor status (P = 1.000), or lymphovascular invasion (P = 0.274). Furthermore, IHC-detected positive SLN status did not correlate with distant metastasis (P = 0.372) or overall or distant metastasis-free survival (P = 0.543 and P = 0.540, respectively).CONCLUSIONSAlthough the finding of SLN micrometastases by IHC may change management in > 12% of patients, preliminary results suggested that such micrometastases do not affect outcomes significantly. Cancer 2005;103:1581–6. © 2005 American Cancer Society.Although the finding of SLN micrometastases by IHC may change management in > 12% of patients, preliminary results suggested that such micrometastases do not affect outcomes significantly. Cancer 2005;103:1581–6. © 2005 American Cancer Society.
European Journal of Surgical Oncology (EJSO), 2007
Aims: To evaluate the clinical significance of tumour metastases detected using real-time reverse transcription-PCR (RTePCR) in sentinel lymph nodes (SLN) of breast cancer patients. Methods: Sixty-seven patients with T1eT2 primary breast cancer were included in a prospective study. SLN were analysed for the presence of metastatic tumour cells using standard histopathology staining, immunochemistry (IHC) and multimarker real-time RTePCR assay for mammaglobin (MMG), carcinoembryonic antigen (CEA) and cytokeratin-19 (CK19) mRNA expression. Correlations between molecular metastases and traditional clinicopathological prognostic factors, including St Gallen risk categories were studied. Results: Of the 67 patients, 15 (22.3%) had one or more pathology-positive SLN. Five (9.6%) pathology-negative SLN were positive by IHC and 19 (36.5%) by RTePCR. Of note, RTePCR analysis was also positive in all cases with pathology-or IHC-positive SLN. MMG was the most informative tumour marker in the panel. Molecularly detected metastases were significantly associated with intermediate St Gallen risk category (p ¼ 0.023). Conclusion: Molecular staging of SLN using real-time RTePCR for early breast cancer could serve as a useful complement to standard clinicopathological risk factors. Studies with long-term follow-up are necessary to define the impact of molecular metastases on disease free survival and overall survival.
Annals of Surgical Oncology, 2010
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 transcriptasepolymerase 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 C0.1 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 C4, 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.
Annals of the New York Academy of Sciences, 2006
The detection of circulating tumor cells in the peripheral blood (pB) of breast cancer (BC) patients might become an important factor for the prognosis of BC patients. Sensitive molecular techniques, primarily based upon the reverse-transcriptase polymerase chain reaction (RT-PCR), have been developed using the expression of tissue-and/or tumor-specific genes as a marker for the presence of tumor cells.