RESUMO
The restriction enzyme-based digital methylation-specific polymerase chain reaction (RE-dMSP) assay is useful for diagnosing sentinel lymph node (SN) metastasis in patients with breast cancer, by detecting tumor-derived methylated Ras association domain-containing protein 1 (RASSF1A). In addition, this assay has high concordance (95.0%) with one-step nucleic acid amplification (OSNA). The present study aimed to perform RE-dMSP using OSNA lysate from more patients and to re-evaluate its clinical usage. Overall, 418 SNs from 347 patients were evaluated using both OSNA and RE-dMSP. The concordance rate was 83.3% (348/418). RASSF1A methylation of the primary tumors was negative in 36 patients. When these patients were excluded, the concordance rate improved to 88.2% (330/374). Of the 79 OSNA-negative cases, 19 were RE-dMSP-positive, although all were positive for cytokeratin 19 expression in the primary tumor, suggesting that RE-dMSP can detect tumor-derived DNA with a higher sensitivity. The percent of methylated reference of the breast tumors showed a wide variety in the 16 OSNA-positive/RE-dMSP-negative cases, and such variability of methylation could have affected the results in these patients. In conclusion, although RE-dMSP can diagnose SN metastasis with high sensitivity and accuracy, and can be a supplementary tool to OSNA in breast cancer, RE-dMSP showed certain discordance with OSNA and critically depended on the absence or heterogeneity of DNA methylation in breast tumors. Further research is expected to develop an assay targeting other DNA alterations, such as mutations.
RESUMO
ESR1 mutations in breast cancer are one of the mechanisms of resistance to aromatase inhibitors. These mutations are common in metastatic breast cancer; however, these are rare in primary breast cancer. However, these data have been analyzed mainly in formalin-fixed, paraffin-embedded tissue; thus, rare mutations that may be present in primary breast cancer may be overlooked. In this study, we developed a highly sensitive mutation detection method called locked nucleic acid (LNA)-clamp droplet digital PCR (ddPCR) and validated it. The mutation detection sensitivity was substantiated to 0.003%. Then, we used this method to analyze ESR1 mutations in fresh-frozen (FF) tissues of primary breast cancer. cDNA extracted from the FF tissues of 212 patients with primary breast cancers were measured. Twenty-eight ESR1 mutations were found in twenty-seven (12.7%) patients. Sixteen (7.5%) patients had Y537S mutations and twelve (5.7%) had D538G mutations. Two mutations with a variant allele frequency (VAF) of ≥0.1% and twenty-six mutations with a VAF of <0.1% were found. By using this LNA-clamp ddPCR, this study demonstrated the presence of minor clones with a VAF of <0.1% in primary breast cancer.
RESUMO
PURPOSE: We have shown that "Click-to-sense" (CTS) assay based on the visualization of cancer cells by fluorescence probe targeted for acrolein is useful for differentiating between the malignant and benign lesions of the breast. In the present study, we aimed to apply CTS assay to the examination of the simulated surgical margins, being compared with frozen section (FS) analysis. EXPERIMENTAL DESIGN: The simulated surgical margin samples (n = 300) were obtained from 1 to 2 cm distant sites from the tumor margin in the mastectomy specimens of breast cancer patients, and divided into the training (n = 150) and validation (n = 150) set. The samples were subjected to CTS assay, subsequently to FS analysis and finally to permanent section (PS) analysis. RESULTS: Diagnostic accuracy of the CTS assay and FS analysis was evaluated in the examination of the simulated surgical margin status finally determined by the PS analysis. In the training set, sensitivity, specificity, and accuracy was 89.3%, 98.4%, and 96.7% for the CTS assay and 89.3%, 98.4%, and 96.7% for the FS analysis. In the validation set, sensitivity, specificity, and accuracy was 93.3%, 98.3%, and 97.3% for the CTS assay, and 93.3%, 99.2%, and 98.0% for the FS analysis. CONCLUSIONS: The CTS assay is as accurate as the FS analysis in the examination of the simulated surgical margins in breast cancer patients, and it seems to have a potential to replace the FS analysis for the intra-operative examination of surgical margins in breast-conserving surgery since it is less labor-intensive and more time-saving than the FS analysis.
Assuntos
Neoplasias da Mama , Secções Congeladas , Neoplasias da Mama/patologia , Neoplasias da Mama/cirurgia , Feminino , Humanos , Margens de Excisão , Mastectomia , Mastectomia Segmentar , Estudos RetrospectivosRESUMO
BACKGROUND: Everolimus is a mechanistic-target-of-rapamycin (mTOR) inhibitor bearing a potent antitumor effect against hormone receptor-positive breast cancer. Here, we report the case of a patient with recurrent breast cancer who developed osteomyelitis during the treatment with everolimus plus exemestane. CASE PRESENTATION: A 56-year-old woman with early-stage breast cancer underwent right mastectomy and axillary lymph node dissection at the age of 45. Four years after the surgery, she experienced relapse at the chest wall. Radiotherapy was performed on the chest wall, including the sternum, and denosumab was administered. After several regimens of hormonal therapies, everolimus in combination with exemestane was administered. Three months later, the patient visited our clinic because of continuous fever. A computed tomography scan showed an osteolytic change in the sternal bone with pneumomediastinum, which indicated sternal osteomyelitis. Extensive debridement followed by secondary reconstruction of the chest wall was successfully performed. CONCLUSIONS: Everolimus may cause osteomyelitis of the affected bone as a result of tumor necrosis. Everolimus-induced osteomyelitis may be manageable by extensive debridement performed without delay.
RESUMO
The diagnostic accuracy of the multigene panel test (MPT) and OncoScan™ in the determination of HER2 amplification in breast tumors remains controversial. In the present study, HER2 copy number was analyzed using both MPT and OncoScan™ in 45 breast tumors and was compared with that in fluorescent in situ hybridization (FISH) analysis. Tumors with low cellularity were examined using tumor cell enrichment and fluorescenceactivated cell sorting. Both MPT and OncoScan™ exhibited significant correlations with FISH with respect to the determination of HER2 amplification in breast tumors. However, the correlation coefficient was significantly higher for the comparison of MPT and FISH (r=0.770) compared with that between OncoScan™ and FISH (r=0.564). The accuracy of MPT (93.3%) was slightly higher compared with that in OncoScan™ (84.4%) in determining the HER2 status, which was mostly explained by the higher sensitivity of MPT in tumors with low cellularity (83.3 vs. 33.3%), but not in those with high cellularity (81.8 vs. 72.7%). The specificity was 100% for both tests. The MPT exhibited higher sensitivity in the determination of the amplification of other genes, including MYC, fibroblast growth factor receptor 1 and GATA binding protein 3 in tumors with low cellularity compared with that in tumors with high cellularity. OncoScan™ exhibited low sensitivity without tumor cell enrichment. The results suggested that MPT could be a promising method to determine HER2 status in breast tumors and that it could exhibit improved accuracy compared with that in OncoScan™ in tumors with low cellularity.
Assuntos
Neoplasias da Mama/genética , Análise Mutacional de DNA/métodos , Amplificação de Genes/genética , Genes erbB-2/genética , Hibridização in Situ Fluorescente/métodos , Receptor ErbB-2/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Neoplasias da Mama/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Pessoa de Meia-Idade , Receptor ErbB-2/metabolismoRESUMO
We aimed to validate the cosmetic utility of addition of nipple-areola recentralization (NAR) to rotation flap according to nipple tumor distance (NTD) as a volume displacement technique after breast conserving surgery (BCS) for lower-outer and upper-inner breast cancers. Twenty breast cancer patients who had been treated with rotation flap with (Group 1; nâ¯=â¯6) or without (Group 2; nâ¯=â¯14) NAR after BCS for lower-outer or upper-inner located tumors, and those who had undergone BCS without oncoplastic surgical technique for tumors in the same area (Control group; nâ¯=â¯43), were retrospectively investigated. Cosmetic outcome was evaluated using Harvard scale and/or BCCT.core. As a result, the ratio of patients categorized as excellent/good was 83% in Group 1 and 93% in Group 2, respectively, and there was no significant difference between them (Pâ¯=â¯0.521). In addition, Group 1â¯+â¯2 showed a significantly higher ratio of patients classified as excellent/good than the control group (90% vs. 56%; Pâ¯=â¯0.009). After adjustment of clinical background parameters using propensity score matching analysis between Group 1â¯+â¯2 and the control group, 12 pairs with similar background factors were matched. Among them, Group 1â¯+â¯2 showed a higher ratio of patients categorized as excellent/good than the control group (92% vs. 42%; Pâ¯=â¯0.034). In conclusion, addition of NAR to rotation technique according to NTD may enable us to perform a volume displacement after BCS for lower-outer or upper-inner located tumors irrespective of NTD without sacrificing postoperative breast appearance.
Assuntos
Neoplasias da Mama/cirurgia , Mamoplastia/métodos , Mastectomia Segmentar , Mamilos/cirurgia , Retalhos Cirúrgicos/cirurgia , Adulto , Idoso , Idoso de 80 Anos ou mais , Estética , Feminino , Humanos , Pessoa de Meia-Idade , Avaliação de Resultados em Cuidados de Saúde , Satisfação do Paciente/estatística & dados numéricos , Pontuação de Propensão , Estudos RetrospectivosRESUMO
HER2 amplification is seen in 20-25% of primary breast cancer cases, and HER2 detection is performed routinely in primary operable, as well as metastatic breast cancer patients. Currently, HER2 is the only gene of which amplification is routinely assayed by fluorescent in situ hybridization (FISH) and/or immunohistochemistry (IHC). However, histochemical assay (FISH/IHC) of multiple target genes is laborious and time-consuming, and simultaneous amplification by microarray is preferred. OncoScan™ is a microarray-based assay capable of whole-genome copy number analysis using DNA extracted from formalin-fixed paraffin-embedded (FFPE) tissues. In the current study, we aimed to investigate the impact of tumor cellularity on the accuracy of OncoScan™ in the determination of HER2 amplification. Our results demonstrated that HER2 amplification by OncoScan™ is accurate, and has a high concordance rate of 93.3% with FISH. However, the concordance rate is poor (66.7%) in cases with a tumor cellularity < 20%. Nevertheless, the addition of FISH to breast tumors with a tumor cellularity < 20% and a HER2 copy number of 4 appears to be useful to minimize false-negative results by OncoScan™.
Assuntos
Neoplasias da Mama/genética , Amplificação de Genes , Biomarcadores Tumorais/genética , Neoplasias da Mama/patologia , Feminino , Humanos , Receptor ErbB-2 , Estudos Retrospectivos , Análise Serial de Tecidos/instrumentação , Análise Serial de Tecidos/normasRESUMO
Ten stage I/II breast cancer patients with one or two suspicious lymph nodes (LNs) detected on ultrasonography underwent sentinel LN biopsy (SLNB) after the introduction of Twirl® breast markers into each suspicious LN revealed that each Twirl®-marked LN was SLN and was likely the first SLN. Three patients had less than three SLN metastases and were candidates for sparing completion axillary lymph node dissection (cALND). Indeed, of them, one underwent breast-conserving surgery without cALND and the other two underwent total mastectomy with cALND. These results suggest that, as all suspicious LNs are SLNs, patients can be treated with SLNB alone if they fulfill the ACOSOG Z-0011 criteria, despite suspicious LNs yielding positive results on preoperative fine-needle aspiration cytology.
Assuntos
Neoplasias da Mama/patologia , Metástase Linfática/diagnóstico , Biópsia de Linfonodo Sentinela/métodos , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/cirurgia , Feminino , HumanosRESUMO
Humoral immunity plays a substantial role in the suppression of breast cancer. We have revealed that a high serum concentration of anti-HER2 autoantibody (HER2-AAb) is associated with favorable outcomes in patients with invasive breast cancer. Thus, we aimed to clarify the association between high serum concentration of HER2-AAb and humoral immune response in the tumor microenvironment. Out of 500 consecutive patients with invasive breast cancer, we selected those whose HER2-AAb values were high (n = 33) or low (n = 20) based on the distribution of HER2-AAb values of 100 healthy individuals. Tumor and regional lymph node formalin-fixed paraffin-embedded samples prepared from the surgical specimens were subjected to immunohistochemistry. We confirmed that the recurrence-free survival of the high HER2-AAb group was significantly longer than that of the low HER2-AAb group (p = 0.015). The numbers of tumor-infiltrating CD20+ immune cells (ICs) (p < 0.001), IGKC+ICs (p = 0.023), and CXCL13+ ICs (p = 0.044) were significantly higher in the high HER2-AAb group than in the low HER2-AAb group. The number of CD4+ ICs in the B-cell follicles of the regional lymph nodes was also significantly greater in the high HER2-AAb group than in the low HER2-AAb group (p = 0.026). Our findings indicate that a high level of HER2-AAb is associated with enhanced humoral immunity against breast cancer and thus may provide a rationale for the association of HER2-AAb with favorable prognosis.
Assuntos
Autoanticorpos/sangue , Biomarcadores Tumorais/sangue , Neoplasias da Mama/imunologia , Carcinoma Ductal de Mama/imunologia , Carcinoma Lobular/imunologia , Receptor ErbB-2/imunologia , Adulto , Idoso , Autoanticorpos/imunologia , Biomarcadores Tumorais/imunologia , Neoplasias da Mama/sangue , Neoplasias da Mama/patologia , Neoplasias da Mama/cirurgia , Carcinoma Ductal de Mama/sangue , Carcinoma Ductal de Mama/patologia , Carcinoma Ductal de Mama/cirurgia , Carcinoma Lobular/sangue , Carcinoma Lobular/patologia , Carcinoma Lobular/cirurgia , Estudos de Casos e Controles , Feminino , Seguimentos , Humanos , Imunidade Humoral/imunologia , Linfócitos do Interstício Tumoral/imunologia , Pessoa de Meia-Idade , Prognóstico , Taxa de Sobrevida , Microambiente Tumoral/imunologiaRESUMO
BACKGROUND: The aim of our study is to find microRNAs (miRNAs) associated with sentinel lymph node metastasis (SLNM) and to develop a prediction model for SLNM in ER-positive and HER2-negative (ER+/HER2-) breast cancer. PATIENTS AND METHODS: In the present study, only ER+/HER2- primary breast cancer was considered. The discovery set for SLNM-associated miRNAs included 10 tumors with and 10 tumors without SLNM. The training and validation sets both included 100 tumors. miRNA expression in tumors was examined comprehensively by miRNA microarray in the discovery set and by droplet digital PCR in the training and validation sets. RESULTS: In the discovery set, miR-98, miR-22, and miR-223 were found to be significantly (P < 0.001, fold-change > 2.5) associated with SLNM. In the training set, we constructed the prediction model for SLNM using miR-98, tumor size, and lymphovascular invasion (LVI) with high accuracy (AUC, 0.877). The accuracy of this prediction model was confirmed in the validation set (AUC, 0.883), and it outperformed the conventional Memorial Sloan Kettering Cancer Center nomogram. In situ hybridization revealed the localization of miR-98 expression in tumor cells. CONCLUSIONS: We developed a prediction model consisting of miR-98, tumor size, and LVI for SLNM with high accuracy in ER+/HER2- breast cancer. This model might help decide the indication for SLN biopsy in this subtype.
Assuntos
Neoplasias da Mama , MicroRNAs , Linfonodo Sentinela , Neoplasias da Mama/genética , Neoplasias da Mama/cirurgia , Humanos , Linfonodos , Metástase Linfática , MicroRNAs/genética , Nomogramas , Curva ROC , Linfonodo Sentinela/cirurgia , Biópsia de Linfonodo SentinelaRESUMO
Recent studies demonstrated that homologous repair deficiency (HRD) score is a useful marker for response to poly (ADP-ribose) polymerase inhibitors or platinum-based chemotherapy. We determined HRD scores and elucidated the clinicopathologic characteristics of HRD-high tumors and their response to non-platinum-based chemotherapy. Primary breast cancer patients (nâ¯=â¯120) were pre-operatively treated with paclitaxel followed by 5-fluorouracil/epirubicin/cyclophosphamide (P-FEC). Germline and somatic homologous recombination related gene mutations (gHRRm and sHRRm, respectively) and HRD scores were analyzed using whole exome sequencing (WES) in tumor tissues obtained before chemotherapy. Of 120 tumors, 30 were determined to be HRD-high tumors, significantly associated with high Ki-67 (Pâ¯=â¯0.014), ER negativity (Pâ¯=â¯0.007), and PR negativity (Pâ¯=â¯0.021). Triple-negative cancers showed significantly higher HRD scores than the luminal, luminal-HER2, and HER2 subtypes (Pâ¯=â¯0.023, 0.016, and 0.033, respectively). HRD scores were significantly higher in tumors with gHRRm than in those with sHRRm (Pâ¯=â¯0.002) or wild-type HRR genes (Pâ¯=â¯1.44e-4), but no significant difference was found in HRD scores between tumors with sHRRm and wild-type HRR genes (Pâ¯=â¯0.206). HRD-high tumors had significantly (Pâ¯=â¯0.003) higher pCR rates and higher near-pCR rates (Pâ¯=â¯0.049) compared with those of the HRD-low tumors in all tumors and the luminal subtype, respectively. HRD-high tumors were associated with aggressive phenotypes and gHRRm, but not sHRRm. Our findings suggested that HRD scores might be useful in predicting response to P-FEC in the luminal subtype.
RESUMO
BACKGROUND/AIM: Magnetic resonance imaging (MRI)-guided breast biopsy is a complex and time-consuming procedure. This study aimed to clarify the factors that affect the duration of the procedure. PATIENTS AND METHODS: Twenty-eight examinations performed at our institute for 27 lesions detected solely on MRI were analyzed. The correlations between the clinicopathological factors and duration of the procedure were estimated. RESULTS: The needle guidance method was the only factor that significantly affected the duration of the MRI-guided vacuum-assisted breast biopsy (VAB) (p=0.012). The use of a computer-aided detection (CAD) system with grid breast compression plates had significantly shorter durations (62±12 min) than the manual calculation of coordinates with pillar-type compression plates (76±13 min). CONCLUSION: This preliminary study showed that the use of a CAD system might shorten the duration of MRI-guided VAB.
Assuntos
Neoplasias da Mama/diagnóstico , Mama/diagnóstico por imagem , Biópsia Guiada por Imagem , Imagem por Ressonância Magnética Intervencionista , Adulto , Idoso , Biópsia por Agulha/métodos , Mama/patologia , Neoplasias da Mama/diagnóstico por imagem , Neoplasias da Mama/patologia , Feminino , Humanos , Pessoa de Meia-Idade , Fatores de Tempo , VácuoRESUMO
We attempted to detect circulating tumor DNA (ctDNA), taking advantage of molecular barcode next-generation sequencing (MB-NGS), which can be more easily customized to detect a variety of mutations with a high sensitivity than PCR-based methods. Sequencing with a gene panel consisting of the 13 most frequently mutated genes in breast tumors from stage I or II patients revealed 95 somatic mutations in the 12 genes in 62% (62/100) of tumors. Then, plasma DNA from each patient (n = 62) before surgery was analyzed via MB-NGS customized to each somatic mutation, resulting in the detection of ctDNA in 16.1% (10/62) of patients. ctDNA was significantly associated with biologically aggressive phenotypes, including large tumor size (P = .004), positive lymph node (P = .009), high histological grade (P < .001), negative ER (P = .018), negative PR (P = .017), and positive HER2 (P = .046). Furthermore, distant disease-free survival was significantly worse in patients with ctDNA (n = 10) than those without ctDNA (n = 52) (P < .001). Our results demonstrate that MB-NGS personalized to each mutation can detect ctDNA with a high sensitivity in early breast cancer patients at diagnosis, and it seems to have a potential to serve as a clinically useful tumor marker for predicting their prognosis.
RESUMO
ESR1 mutations in breast cancer are known as one of the mechanisms of resistance to aromatase inhibitors. These mutations often occur in the hotspot regions in the ligand binding domain (LBD), but comprehensive mutational analysis has shown that mutations are observed throughout the whole LBD. We previously developed a molecular barcode sequencing (MB-NGS) technique to detect ESR1 hotspot mutations in plasma with high sensitivity. In this study, we have developed a multiplex MB-NGS assay that covers the whole LBD of ESR1. The assay demonstrated that the background errors in the plasma DNA of 10 healthy controls were below 0.1%; thus, the limit of detection was set at 0.1%. We analyzed the plasma DNA of 54 patients with estrogen receptor-positive metastatic breast cancer. Seventeen mutations were detected in 13 patients (24%), with variant allele frequencies ranging from 0.13% to 10.67%, including six rare mutations with a variant allele frequency <1.0% and a novel nonhotspot mutation (A312V). Three patients had double mutations located in the same amplicons, and it was revealed that the double mutations were located in different alleles. ESR1 hotspot mutations were associated with a longer duration of aromatase inhibitor treatment under metastatic conditions and to liver metastasis. The multiplex MB-NGS assay is useful for the sensitive and comprehensive detection of mutations throughout the whole LBD of ESR1. Our assay can be applied to any specific target region of interest using tailor-made primers and can result in minimized sequencing volume and cost.
RESUMO
AIM: To investigate the feasibility of hookwire-guided sentinel lymph node biopsy (SLNB) using contrast-enhanced ultrasonography (CEUS) followed by a one-step nucleic acid amplification (OSNA) assay. PATIENTS AND METHODS: Clinical T1-2N0M0 breast cancer patients scheduled to undergo SLNB participated in this study. Both Sonazoid® and dye were used as tracers, and the most upstream sentinel lymph node (SLN) at each lymphatic flow detected by CEUS (First-SLN) was sampled under hookwire guidance, a procedure called "Sona-Hook". RESULTS: In each of the 50 cases, at least one First-SLN was extracted by "Sona-Hook". All contrast-enhanced SLNs (CE-SLNs) were dye-positive, and the mean number of CE-SLNs sampled per patient was lower than that of dye-positive SLNs (1.48 vs. 1.88, p<0.01). Through OSNA, qualitative assessment of tumor metastasis between First-SLNs and all SLNs completely matched together. CONCLUSION: "Sona-Hook" for First-SLN followed by an OSNA assay may be a feasible minimally invasive SLNB strategy.
Assuntos
Neoplasias da Mama/patologia , Meios de Contraste , Biópsia Guiada por Imagem/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Linfonodo Sentinela/patologia , Ultrassonografia Mamária/métodos , Adenocarcinoma Mucinoso/diagnóstico por imagem , Adenocarcinoma Mucinoso/genética , Adenocarcinoma Mucinoso/secundário , Adenocarcinoma Mucinoso/cirurgia , Biomarcadores Tumorais/genética , Neoplasias da Mama/diagnóstico por imagem , Neoplasias da Mama/genética , Neoplasias da Mama/cirurgia , Carcinoma Ductal de Mama/diagnóstico por imagem , Carcinoma Ductal de Mama/genética , Carcinoma Ductal de Mama/secundário , Carcinoma Ductal de Mama/cirurgia , Carcinoma Lobular/diagnóstico por imagem , Carcinoma Lobular/genética , Carcinoma Lobular/secundário , Carcinoma Lobular/cirurgia , Feminino , Seguimentos , Humanos , Metástase Linfática , Prognóstico , Linfonodo Sentinela/diagnóstico por imagem , Linfonodo Sentinela/cirurgiaRESUMO
Onestep nucleic acid amplification (OSNA) targeting cytokeratin 19 (CK19) mRNA expression and pathological examination are widely used for the intraoperative diagnosis of sentinel node (SN) metastasis. The aim of the present study was to develop a novel assay for detecting SN metastasis by targeting Ras association domaincontaining protein 1 (RASSF1A) methylation in tumor cells, and to compare its performance with OSNA. Using digital PCR with methylationspecific restriction enzymes (REdMSP), our assay was able to detect ≥3 copies of methylated DNA per well, and was ≥10 times more sensitive than realtime PCR with bisulfite modification. OSNA lysates were examined using REdMSP and digital PCR for PIK3CA mutation, in the event that primary tumors were PIK3CA mutationpositive. REdMSP revealed a high concordance of 95.0% (153/161) with OSNA, and 100% (59/59) with PIK3CA mutation for detecting SN metastasis. In 11 breast cancer cell lines, the variation in methylated RASSF1A copy number was significantly lower than that of CK19 mRNA (2.8 vs. 10.5fold; P<0.01). REdMSP has the potential to more accurately detect SN metastasis, and to more precisely estimate total tumor loads in SN, compared with OSNA.
Assuntos
Neoplasias da Mama/secundário , Metilação de DNA , Recidiva Local de Neoplasia/patologia , Reação em Cadeia da Polimerase/métodos , Linfonodo Sentinela/patologia , Proteínas Supressoras de Tumor/genética , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Feminino , Humanos , Queratina-19/análise , Queratina-19/genética , Metástase Linfática , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/genética , Prognóstico , Curva ROC , Linfonodo Sentinela/metabolismo , Proteínas Supressoras de Tumor/análiseRESUMO
We previously developed a 95gene classifier (95GC) to classify ERpositive/HER2negative/nodenegative (ER+/HER2/N0) breast cancer as high and lowrisk. The present study aimed to devise a 95GC recurrence score (95GCRS) to estimate recurrence risk more precisely and, although the 95GC was originally developed using freshfrozen (FF) tissues, this was applied to formalinfixed paraffinembedded (FFPE) tissues. 95GCRS was calculated using betweengroup analysis and denominated as a value from 0 to 100. Correlation of 95GCRS with distant recurrence rate and response to neoadjuvant chemotherapy (NAC) was evaluated in 257 patients with ER+/HER2-/N0 breast cancer treated with adjuvant hormonal therapy at Osaka University Hospital and in 425 patients with ER+ breast cancer treated with NAC at Osaka University Hospital and the University of Texas MD Anderson Cancer Center (GSE25066 dataset). Correlation of 95GCRS between FF and FFPE tissues was evaluated in paired tissues from 56 ER+/HER2/N0 breast cancer types obtained from patients without NAC treatment. Distant recurrence rates were remarkably low in patients with 95GCRS ≤50 and increased proportionally in patients with 95GCRS >50. Pathological complete response (pCR) rates to NAC were increased in proportion to 95GCRS, indicating a greater sensitivity of breast cancers with high 95GCRS to chemotherapy. 95GCRS was highly correlated (R=0.92) between FF and FFPE tissues, and the concordance rate (94.6%) of high and lowrisk groups was also considerably high. Overall, the present study developed a 95GCRS that correlated with distant recurrence rate and pCR rate to NAC. The 95GC was applicable to FFPE tissues with a high concordance rate in FF tissues.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Proteínas de Neoplasias/genética , Recidiva Local de Neoplasia/tratamento farmacológico , Transcriptoma/genética , Adulto , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Quimioterapia Adjuvante/efeitos adversos , Intervalo Livre de Doença , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Estimativa de Kaplan-Meier , Pessoa de Meia-Idade , Terapia Neoadjuvante/efeitos adversos , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/patologia , Inclusão em Parafina , Receptor ErbB-2/genética , Receptores de Estrogênio/genética , Resultado do TratamentoRESUMO
BACKGROUND: Trastuzumab is a drug that targets the receptor tyrosine kinase HER2 and is essential for the treatment of HER2-positive breast cancer. Resistance to the drug leads to severe consequences, including disease recurrence, tumor enlargement, and metastasis. We hypothesized that trastuzumab treatment might be associated with phenotypic switching in HER2-positive breast cancer cells (BCCs), enabling them to escape and survive the effect of trastuzumab. METHODS: We conducted comprehensive immunophenotyping to detect phenotypic changes in HER2-positive BCCs treated with trastuzumab, based on criteria determined a priori. Based on immunophenotyping results, we characterized the vascular phenotypes of HER2-positive BCCs by western blotting, real-time RT-PCR, and tube formation assay. The vascular phenotype of tumor cells from clinical samples was evaluated by staining with periodic acid-Schiff and an anti-CD31 antibody. We explored small molecule inhibitors that suppress tube formation and determined the inhibitory mechanism. RESULTS: Out of 242 cell surface antigens, 9 antigens were significantly upregulated and 3 were significantly downregulated by trastuzumab treatment. All upregulated antigens were related to endothelial and stem cell phenotypes, suggesting that trastuzumab treatment might be correlated to switching to a vascular phenotype, namely, vasculogenic mimicry (VM). Several VM markers were upregulated in trastuzumab-treated cells, but these cells did not form tubes on Matrigel, a functional hallmark of VM. Upon analysis of three trastuzumab-resistant HER2-positive cell lines, we found that all three cell lines showed tube formation on Matrigel in the presence of angiogenic growth factors including EGF, FGF2, IGF1, or VEGF. Clinically, VM channels significantly increased in surviving cancer cell clusters of surgically removed tumors pretreated with trastuzumab and chemotherapy compared to both surgically removed tumors without prior systemic treatment and tumors biopsied before presurgical treatment with trastuzumab. Finally, we found that salinomycin completely suppressed VM in all three trastuzumab-resistant cell lines through disruption of actin cytoskeletal integrity. CONCLUSIONS: VM promotes metastasis and worsens patient outcomes. The present study indicates that HER2-positive BCCs can exhibit VM in an angiogenic microenvironment after eventually acquiring trastuzumab resistance. The clinical finding supports this in vitro observation. Thus, targeting VM might provide a therapeutic benefit to patients with HER2-positive breast cancer.
Assuntos
Antineoplásicos Imunológicos/farmacologia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Neovascularização Patológica/genética , Receptor ErbB-2/genética , Trastuzumab/farmacologia , Biomarcadores Tumorais , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Humanos , Imunofenotipagem , Metástase Neoplásica , Estadiamento de Neoplasias , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/metabolismo , Imagem com Lapso de Tempo , Microambiente Tumoral/genéticaRESUMO
Contrast-enhanced ultrasonography with Sonazoid (SNZ) enables real-time visualization, resulting in more precise identification of lymphatic flow to sentinel lymph nodes (SLNs). This study aimed to classify lymphatic drainage patterns to SLNs. Patients (nâ¯=â¯75) with T1-2 N0 M0 breast cancer received a periareolar injection of SNZ to identify SNZ-enhanced SLNs (SNZ-SLNs), followed by SLN biopsy with blue dye. The lymphatic drainage patterns were classified into four types: type A, single lymphatic route/single SLN; type B, multiple lymphatic routes/single SLN; type C, single lymphatic route/multiple SLNs; and type D, multiple lymphatic routes/multiple SLNs. SLNs were successfully identified in all patients using both blue dye and SNZ. The drainage lymphatic pathways identified were as follows: type A in 53 cases (70.7%), type B in seven (9.3%), type C in eight cases (10.7%) and type D in seven (9.3%). SNZ-SLN biopsy is a technically simple method with a 100% identification rate, enabling the real-time visualization of lymphatic flow to SNZ-SLNs.
Assuntos
Neoplasias da Mama/fisiopatologia , Compostos Férricos , Aumento da Imagem/métodos , Ferro , Vasos Linfáticos/diagnóstico por imagem , Óxidos , Linfonodo Sentinela/diagnóstico por imagem , Ultrassonografia/métodos , Feminino , Humanos , Vasos Linfáticos/fisiopatologia , Pessoa de Meia-IdadeRESUMO
PURPOSE: The use of formalin-fixed paraffin-embedded (FFPE) tumor tissues in flow cytometry (FCM)-based determination of tumor cell DNA content is more complicated than the use of fresh-frozen tissues. This study aimed to accurately measure tumor cell DNA content from FFPE tissues by separating tumor cells from stromal cells through FCM and investigating its prognostic impact. METHODS: We separately measured the DNA contents of tumor cells and stromal cells by gating with pan-cytokeratin and vimentin (FCMC/V). We evaluated tumor cell DNA contents [DNA index (DI)] of 290 FFPE tumor tissues and classified them into low and high DI groups, using a cutoff DI value determined through an unbiased computational method. RESULTS: The distribution of DI was bimodal, and a cutoff value was determined at a DI of 1.26. The high-DI tumors were associated with aggressive phenotypes and had significantly worse distant recurrence-free intervals (DRFI) than low-DI tumors. Multivariate analysis revealed that lymph node metastasis, Ki67, and DI were independent factors affecting DRFI. Accordingly, patients with low-DI/low-Ki67 tumors had excellent outcomes compared with other tumor types. Multiploid tumors were associated with increased lymphocytic infiltration and aggressive phenotypes. CONCLUSIONS: The DI of FFPE tumors could be precisely determined through FCMC/V. A combination of DI and Ki67 analyses may be able to predict the prognoses of breast cancer patients with greater accuracy.