Completion Dissection or Observation for Sentinel-Node Metastasis in Melanoma.

Sentinel-lymph-node biopsy is associated with increased melanoma-specific survival (i.e., survival until death from melanoma) among patients with node-positive intermediate-thickness melanomas (1.2 to 3.5 mm). The value of completion lymph-node dissection for patients with sentinel-node metastases is not clear. In an international trial, we randomly assigned patients with sentinel-node metastases detected by means of standard pathological assessment or a multimarker molecular assay to immediate completion lymph-node dissection (dissection group) or nodal observation with ultrasonography (observation group). The primary end point was melanoma-specific survival. Secondary end points included disease-free survival and the cumulative rate of nonsentinel-node metastasis. Immediate completion lymph-node dissection was not associated with increased melanoma-specific survival among 1934 patients with data that could be evaluated in an intention-to-treat analysis or among 1755 patients in the per-protocol analysis. In the per-protocol analysis, the mean (±SE) 3-year rate of melanoma-specific survival was similar in the dissection group and the observation group (86±1.3% and 86±1.2%, respectively; P=0.42 by the log-rank test) at a median follow-up of 43 months. The rate of disease-free survival was slightly higher in the dissection group than in the observation group (68±1.7% and 63±1.7%, respectively; P=0.05 by the log-rank test) at 3 years, based on an increased rate of disease control in the regional nodes at 3 years (92±1.0% vs. 77±1.5%; P<0.001 by the log-rank test); these results must be interpreted with caution. Nonsentinel-node metastases, identified in 11.5% of the patients in the dissection group, were a strong, independent prognostic factor for recurrence (hazard ratio, 1.78; P=0.005). Lymphedema was observed in 24.1% of the patients in the dissection group and in 6.3% of those in the observation group. Immediate completion lymph-node dissection increased the rate of regional disease control and provided prognostic information but did not increase melanoma-specific survival among patients with melanoma and sentinel-node metastases. (Funded by the National Cancer Institute and others; MSLT-II ClinicalTrials.gov number, NCT00297895 .)

Cytotoxic T lymphocytes that recognize decameric peptide sequences of retinoblastoma binding protein 1 (RBP-1) associated with human breast cancer

Retinoblastoma binding protein 1 (RBP-1) is a 143-kDa nuclear phosphoprotein that promotes cell growth by inhibiting the product of retinoblastoma tumour suppressor gene (pRB). We recently found that RBP-1 contains KASIFLK, a heptameric peptide (250–256) recognized by human antibodies and overexpressed by breast cancer cells. In the present study, we demonstrate that human T-cells stimulated with RBP-1 decameric peptides containing KASIFLK can kill human breast cancer cells. These decamers, GLQKASIFLK (247–256) and KASIFLKTRV (250–259), have anchor motifs for both HLA-A2 and HLA-A3. Peripheral blood lymphocytes from 41 normal donors were stimulated by these peptides in culture media containing 15 IU ml−1 interleukin-2, 25 IU ml−1 interleukin-7 and 500 IU ml−1 granulocyte–macrophage colony-stimulating factor. Cytotoxic activity of the T-cells was assessed against autologous B lymphoblastoid cells pulsed with each peptide. Stimulation by GLQKASIFLK generated specific cytotoxic T lymphocyte (CTL) lines from HLA-A2, A3 donors, HLA-A2 donors and HLA-A3 donors. Stimulation with KASIFLKTRV generated specific CTL lines from HLA-A2 donors. No HLA-A2−, A3− CTL line showed specific cytotoxicity against these target cells. These CTL lines were also cytotoxic against HLA-A2 and HLA-A3 breast cancer cells but not against normal fibroblastoid cell lines, normal epidermal cell lines, or a melanoma cell line. RBP-1 peptide antigens may be of clinical significance as a potential peptide vaccine against human breast cancer. © 1999 Cancer Research Campaig

Abstract P1-05-02: CRISPR/Cas9-guided editing of spliceosome factors enhances major histocompatibility complex proteins in triple-negative breast cancer

Abstract Triple-negative breast cancer (TNBC) exhibits an extraordinary plasticity allowing adaptation to unfamiliar microenvironments and survival despite hindrances imposed by aggressive therapeutic approaches and the immune system. Alternative mRNA splicing (AS), catalyzed by spliceosome factors (SFs), is a major source of transcriptional diversity and phenotypic plasticity for normal and cancer cells. We ascertained that patients with TNBC present up-regulation of specific subsets of SFs mainly involving the epithelial splicing regulatory proteins 1 and 2 (ESRP1/2) and the polypyrimidine tract binding proteins (PTBP1/2). Methods and Results: Through the integration of in-house and publicly-available gene expression profiles (n=890), we evaluated the correlation between the levels of 290 SFs in patients with TNBC. This analysis identified nine subsets of TNBC based on SFs expression profiles with diverse clinical and pathological characteristics. These findings were further validated using data generated by the TCGA-BRCA (n=1,105) and METABRIC projects (n=2,509). Interestingly, up-regulation of PTBP1 was significantly associated with a shorter relapse-free survival interval for patients with TNBC (n=305; HR=1.58 (1.07 − 2.33); p-value=0.02). To systematically identify PTBP1-regulated AS events, we generated and clonally selected CRISPR/Cas9-guided PTBP1 knock-out (KO) TNBC cell lines. Analysis of our RNA-sequencing data at the gene level revealed a significant enrichment of inflammatory response and antigen presentation pathways. Evaluation of potential upstream transcriptional regulators for the enriched molecular pathways and predicted PTBP1 targets identified SMARCA4, a member of the SWI/SNF chromatin remodeling complex, to be significantly activated after PTBP1KO (q-value&amp;lt;0.01). Integration of ENCODE ChIP-sequencing and JASPAR transcription factor binding profile databases revealed clusters of SMARCA4 binding sites upstream of several members of the major histocompatibility (MHC) class I and MHC class II genes. Mechanistically, we identified that PTBP1 induces SMARCA4 exon 30 retention leading to the full length transcript variant 1, which has lower affinity for HLA gene promoter regions. Significant up-regulation of HLA-A, HLA-B, HLA-DPA1, and HLA-DRA genes in CRIPSR-guided PTBP1KO TNBC cells was further demonstrated by either western blot or indirect immunofluorescence. Finally, a negative correlation between PTBP1 and HLA genes expression was also identified in multiple breast cancer gene expression datasets. Conclusions: This study suggests that alterations in the PTBP1-associated splicing programming lead to a reduction of the antigen presentation capability of TNBC cells. Due to the limited therapeutic alternatives for patients with TNBC, beyond chemotherapy, further understanding and modulation of this novel alteration may expand the applications of immunotherapy for patients with TNBC. Citation Format: Bustos MA, Orozco JIJ, Salomon MP, Hoon DSB, Marzese DM. CRISPR/Cas9-guided editing of spliceosome factors enhances major histocompatibility complex proteins in triple-negative breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-05-02.</jats:p