Minoru Kitago

Predictive Utility of Circulating Methylated DNA in Serum of Melanoma Patients Receiving Biochemotherapy

PURPOSEnCurrently, no validated blood-based assays accurately predict treatment response or outcome in melanoma patients. We hypothesized that methylation of tumor-related genes detected in serum DNA could predict disease outcome and therapeutic response in patients receiving concurrent biochemotherapy (BC) for metastatic melanoma.nnnPATIENTS AND METHODSnAmerican Joint Committee on Cancer stage IV melanoma patients (N = 50) had blood drawn before administration of BC. Patients (n = 47) were classified as BC responders or nonresponders. Responders (n = 23) demonstrated a complete or partial response following BC; nonresponders (n = 24) demonstrated progressive disease. Hypermethylation of Ras association domain family 1 (RASSF1A), retinoic acid receptor-beta2 (RAR-beta2), and O6-methylguanine DNA methyltransferase (MGMT) genes were assessed by methylation-specific polymerase chain reaction.nnnRESULTSnCirculating methylated RASSF1A was significantly less frequent for responders (three of 23 patients; 13%) than nonresponders (10 of 24 patients; 42%; P = .028). Patients with RASSF1A, RAR-beta2, or at least one serum methylated gene had significantly worse overall survival than patients with no methylated genes (log-rank, P = .013, .021, and .01, respectively). Methylated RASSF1A was the only factor that significantly correlated with overall survival and BC response (risk ratio, 2.38; 95% CI, 1.16 to 4.86; P = .018; odds ratio = 0.21; 95% CI, 0.05 to 0.90; P = .036).nnnCONCLUSIONnDetection of circulating methylated DNA in serum can predict response to BC and disease outcome.

Utility of Circulating B-RAF DNA Mutation in Serum for Monitoring Melanoma Patients Receiving Biochemotherapy

Purpose: Somatic B-RAF gene mutation has been identified in many malignancies and detected at a high frequency in cutaneous malignant melanoma. However, the significance of the B-RAF mutation (B-RAFmt) in terms of its prognostic and predictive capabilities for treatment response or disease outcome is not known. We hypothesized that circulating serum B-RAFmt (B-RAFsmt) at V600E, detected in serum, predicts response in melanoma patients receiving concurrent biochemotherapy. Experimental Design: A real-time clamp quantitative reverse transcription-PCR assay was designed to assess B-RAFsmt by peptide nucleic acid clamping and a locked nucleic acid hybrid probe. Normal (n = 18) and American Joint Committee on Cancer stage I to IV melanoma patients (n = 103) were evaluated. These included stage IV patients (n = 48) with blood drawn before and after biochemotherapy. Patients were classified as biochemotherapy responders or nonresponders. Responders (n = 24) had a complete or partial response following biochemotherapy; nonresponders (n = 24) developed progressive disease. Results: Of the 103 melanoma patients, 38 (37%) had B-RAFsmt DNA, of which 11 of 34 (32%) were stage I or II, and 27 of 69 (39%) were stage III or IV. Of the 48 biochemotherapy patients, 10 of 24 (42%) patients were positive for the B-RAFsmt in the respective responder and nonresponder groups before treatment. After biochemotherapy, B-RAFsmt was detected in only 1 of 10 patients (10%) in the responder group and 7 of 10 patients (70%) in the nonresponder group. B-RAFsmt is associated with significantly worse (P = 0.039) overall survival in patients receiving biochemotherapy. Conclusion: These studies show the presence and utility of circulating B-RAFsmt DNA in melanoma patients.

Detection of circulating tumor cells in early-stage breast cancer metastasis to axillary lymph nodes.

Purpose: Clinical and pathologic prognostic factors do not always accurately predict disease outcome. Patients with early-stage breast cancer may harbor clinically significant but undetected systemic disease. We hypothesized that a multimarker quantitative real-time reverse transcription-PCR (qRT) assay could detect circulating tumor cells (CTC) in patients with early-stage breast cancer and correlate with sentinel lymph node (SLN) and non-SLN metastasis status. Experimental Design: Blood samples from 90 women with the American Joint Committee on Cancer stages I to III breast cancer and 39 age-matched normal healthy volunteers were assessed by qRT for mRNA expression of three markers: stanniocalcin-1 (STC-1), N-acetylgalactosaminyltransferase (GalNacT), and melanoma antigen gene family-A3 (MAGE-A3). CTC biomarker detection was correlated with overall axillary LN (ALN), SLN, and non-SLN histopathology status. Results: CTCs were detected in 39 of 90 (43%) patients, but not in normal volunteers. At least one CTC biomarker was detected in 10 of 35 (29%) stage I patients, 19 of 42 (45%) stage II patients, and 10 of 13 (77%) stage III patients. In multivariate analysis, only lymphovascular invasion and ≥2 CTC biomarkers detected significantly correlated with ALN metastasis [odds ratio (OR), 12.42; 95% confidence interval (95% CI), 3.52-43.77, P < 0.0001; and OR, 3.88; 95% CI, 1.69-8.89, P = 0.001, respectively]. The number of CTC biomarkers detected similarly correlated with SLN and non-SLN metastasis status (P = 0.0004). At least one CTC biomarker was detected in 10 of 11 (91%) patients with non-SLN metastases. Conclusion: The detection of CTCs offers a novel means to assess the presence of systemic disease spreading relative to SLN and ALN histopathology status.

Lymphatic Mapping Establishes the Role of BRAF Gene Mutation in Papillary Thyroid Carcinoma

Objective:To define the role of BRAF gene mutation in the progression of papillary thyroid carcinoma. Summary Background Data:BRAF gene mutation is frequently detected in papillary thyroid carcinoma. Its role in pathogenesis or progression is under investigation. Methods:Patients who underwent thyroidectomy and sentinel lymph node biopsy for papillary thyroid cancer were accrued. BRAF mutation was assessed in primary tumors and matched sentinel lymph nodes by a quantitative real-time PCR assay. Results:Tissue specimens from 103 consecutive patients were evaluated. BRAF mutation of the primary tumor was detected in 34 (33%) patients. In 26 of 34 (76%) patients with BRAF mutation, concomitant lymph node metastasis was detected. On the contrary, in 69 patients with BRAF mutation-negative primary tumors, only 12 (17%) patients had lymph node metastasis (&khgr;2, P < 0.0001). BRAF mutation was detected in 20 of 26 (77%) lymph node metastases matched to BRAF mutation-positive primary tumors; it was not detected in lymph node metastases matched to BRAF mutation-negative primary tumors. Univariate analysis identified age, stage, tumor size, and BRAF mutation as prognostic factors for lymph node metastasis. In multivariate analysis, only BRAF mutation remained a significant prognostic factor for lymph node metastasis (odds ratio = 10.8, 95% confidence interval, 3.5–34.0, P < 0.0001). Conclusions:BRAF mutation may be a key genetic factor for the metastatic progression of papillary thyroid carcinoma. The study demonstrates that this gene mutation is a significant risk factor for locoregional lymph node metastasis and has potential utility as a surrogate marker.

Activation of toll-like receptors 2, 3, and 4 on human melanoma cells induces inflammatory factors

Toll-like receptors (TLR) have been shown to be expressed on various types of cancers; however, their functional activity is not known. We examined TLR profiles of human melanoma cells and showed that TLR2, TLR3, and TLR4 were found to be highly expressed. By PCR array analysis, specific stimulation of TLR2, TLR3, and TLR4 on melanoma cells showed significant activation of the adaptor protein MyD88, as well as downstream signal transduction factors nuclear factor-κB and inflammatory response–related factors. Specific ligand activation of TLR2, TLR3, and TLR4 was shown to induce cell migration. Peripheral blood lymphocytes and melanoma purified RNA was shown to activate TLR3 on melanoma cells. These studies show expression and functional activity of specific TLRs on melanoma cells and as potential therapeutic targets to control tumor progression. [Mol Cancer Ther 2008;7(11):3642–53]

Unfavourable prognosis associated with K-ras gene mutation in pancreatic cancer surgical margins

Background: Despite intent to cure surgery with negative resection margins, locoregional recurrence is common in pancreatic cancer. Aims: To determine whether detection of K-ras gene mutation in the histologically negative surgical margins of pancreatic cancer reflects unrecognised disease. Patients: Seventy patients who underwent curative resection for pancreatic ductal adenocarcinoma were evaluated. Methods: All patients had surgical resection margins (pancreatic transection and retroperitoneal) that were histologically free of invasive cancer. DNA was extracted from these paraffin embedded surgical margins and assessed by quantitative real time polymerase chain reaction to detect the K-ras gene mutation at codon 12. Detection of K-ras mutation was correlated with standard clinicopathological factors. Results: K-ras mutation was detected in histologically negative surgical margins of 37 of 70 (53%) patients. A significant difference in overall survival was demonstrated between patients with margins that were K-ras mutation positive compared with negative (median 15 v 55 months, respectively; pu200a=u200a0.0008). By univariate and multivariate analyses, detection of K-ras mutation in the margins was a significant prognostic factor for poor survival (hazard ratio (HR) 2.8 (95% confidence interval (CI) 1.5–5.3), pu200a=u200a0.0009; and HR 2.8 (95% CI 1.4–5.5), pu200a=u200a0.004, respectively). Conclusions: Detection of cells harbouring K-ras mutation in histologically negative surgical margins of pancreatic cancer may represent unrecognised disease and correlates with poor disease outcome. The study demonstrates that molecular-genetic evaluation of surgical resection margins can improve pathological staging and prognostic evaluation of patients with pancreatic ductal adenocarcinoma.

mRNA Expression and BRAF Mutation in Circulating Melanoma Cells Isolated from Peripheral Blood with High Molecular Weight Melanoma-Associated Antigen–Specific Monoclonal Antibody Beads

BACKGROUNDnThe detection of circulating tumor cells (CTCs) in the peripheral blood of melanoma patients by quantitative real-time reverse-transcription PCR (qRT-PCR) analysis correlates with a poor prognosis. The assessment of CTCs from blood has been difficult because of lack of a good monoclonal antibody (mAb) directed against surface cell antigens to capture melanoma cells.nnnMETHODSnBlood was collected prospectively from 57 melanoma patients (43 test and 14 test-development cases) and 5 healthy donors. High molecular weight melanoma-associated antigen (HMW-MAA)-specific mAbs bound to immunomagnetic beads were used to isolate CTCs. mRNA and/or DNA were extracted from CTCs. Testing for the expression of a melanoma-associated gene panel (MLANA, MAGEA3, and MITF) with qRT-PCR and for the presence of BRAFmt (a BRAF gene variant encoding the V600E mutant protein) verified the beads-isolated CTCs to be melanoma cells. A peptide nucleic acid-clamping PCR assay was used for BRAFmt analysis.nnnRESULTSnSpiking of peripheral blood cells (PBCs) with melanoma cells showed that the beads-based detection assay can detect approximately 1 melanoma cell in 5 x 10(6) PBCs. qRT-PCR analysis detected MLANA, MAGEA3, and MITF expression in 19 (44%), 29 (67%), and 19 (44%) of the patients, respectively. At least one biomarker of the panel was positive in 40 (93%) of the 43 melanoma patients. BRAFmt was detected in 17 (81%) of the 21 assessed stage IV melanoma patients.nnnCONCLUSIONnThe assay of bead capture coupled with the PCR has utility for assessing CTCs in melanoma patients, which can then be characterized for both genomic and transcriptome expression.

Molecular mechanisms of metastasis.

A major topic covered at the First International Symposium on Cancer Metastasis and the Lymphovascular System was the molecular mechanisms of metastasis. This has become of major interest in recent years as we have discovered new metastasis-related genes and gained understanding of the molecular events of lymphatic metastasis. The symposium covered new aspects and important questions related to the events of metastasis in both humans and animals. The basic and clinical related research covered in this topic represented many disciplines. The presentations showed novel findings and at the same time, raised many new unanswered questions, indicating the limited knowledge we still have regarding the molecular events of metastasis. The hope is that further unraveling of the direct and indirect molecular events of lymphatic metastasis will lead to new approaches in developing effective therapeutics.

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–specfic 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.