Probes for INS

Abstract

PURPOSE:

The most significant prognostic factor in early breast cancer is lymph node involvement. This stage between localised and systemic disease is key to understanding breast cancer progression, however our knowledge of the evolution of lymph node malignant invasion remains limited, as most currently available data derive from primary tumours.

EXPERIMENTAL DESIGN:

In 11 treatment-naïve node positive early breast cancer patients without clinical evidence of distant metastasis, we investigated lymph node evolution using spatial multi-region sequencing (n=78 samples) of primary and lymph node deposits and genomic profiling of matched longitudinal circulating tumour DNA (ctDNA).

RESULTS:

Linear evolution from primary to lymph node was rare (1/11) whereas the majority of cases displayed either early divergence between primary and nodes (4/11), or no detectable divergence (6/11) where both primary and nodal cells belonged to a single recent expansion of a metastatic clone. Divergence of metastatic subclones was driven in part by APOBEC. Longitudinal ctDNA samples from 2 of 7 subjects with evaluable plasma taken peri-operatively reflected the two major evolutionary patterns and demonstrate that private mutations can be detected even from early metastatic nodal deposits. Moreover, node removal resulted in disappearance of private lymph node mutations in ctDNA.

CONCLUSIONS:

This study sheds new light on a crucial evolutionary step in the natural history of breast cancer, demonstrating early establishment of axillary lymph node metastasis in a substantial proportion of patients.

Intra-tumor heterogeneity (ITH) is a major underlying cause of therapy resistance and disease recurrence, and is a read-out of tumor growth. Current genetic ITH analysis methods do not preserve spatial context and may not detect rare subclones. Here, we address these shortfalls by developing and validating BaseScope-a novel mutation-specific RNA in situ hybridization assay. We target common point mutations in the BRAF, KRAS and PIK3CA oncogenes in archival colorectal cancer samples to precisely map the spatial and morphological context of mutant subclones. Computational modeling suggests that subclones must arise sufficiently early, or carry a considerable fitness advantage, to form large or spatially disparate subclones. Examples of putative treatment-resistant cells isolated in small topographical areas are observed. The BaseScope assay represents a significant technical advance for in situ mutation detection that provides new insight into tumor evolution, and could have ramifications for selecting patients for treatment.

We have previously demonstrated that transcriptionally active high-risk HPV (hr-HPV) is strongly incriminated in Barrett's dysplasia (BD) and oesophageal adenocarcinoma (OAC) using mainly fresh frozen tissue. This study aimed to identify biomarkers of active HPV infection in Barrett's metaplasia, (BM)/BD/OAC by immunohistochemical staining (IHC) of formalin-fixed paraffin embedded (FFPE) tissue for aberrations of p53 and the retinoblastoma (pRb) pathway which are targets for the viral oncoproteins, E6/E7 respectively. Prospectively, BM(n=81)/BD(n=72)/OAC(n=65) FFPE specimens were subjected to IHC staining for pRb, p16INK4A , cyclin D1 , p53 and RNA in-situ hybridization (ISH) for E6/E7 transcripts. HPV DNA was determined via PCR in fresh frozen specimens. Viral load measurement (real-time PCR) and Next Generation Sequencing of TP53 was also performed. Of 218 patients, 56 were HPV DNA positive [HPV16 (n=42), 18 (n=13), 6 (n=1)]. Viral load was low. Transcriptionally active HPV (DNA+ /RNA+ ) was only found in the dysplastic and adenocarcinoma group (n=21). The majority of HPV DNA+ /RNA+ BD/OAC were characterized by p16INK4Ahigh (14/21, 66.7%), pRblow (15/21, 71.4%) and p53low (20/21, 95%) and was significantly different to controls [combination of HPV DNA- /RNA- (n=94) and HPV DNA+ /RNA- cohorts (n=22)]. p53low had the strongest association with DNA+ /RNA+ oesophageal lesions (OR=23.5, 95% CI=2.94-187.8, p=0.0029). Seventeen HPV DNA+ /RNA+BD/OAC identified as p53low, were sequenced and all but one exhibited wild-type status. pRblow /p53low provided the best balance of strength of association (OR=8.0, 95% CI=2.6-25.0, p=0.0003) and sensitivity (71.4%)/specificity (71.6%) for DNA+ /RNA+ BD/OAC. Active HPV involvement in BD/OAC is characterized by wild-type p53 and aberrations of the retinoblastoma protein pathway.

Abstract

PURPOSE:

Clinical responses with programmed death (PD-1) receptor directed antibodies occur in about 20% of patients with advanced head and neck squamous cell cancer (HNSCCa). Viral neoantigens, such as the E6/E7 proteins of HPV16/18 are attractive targets for therapeutic immunization, and offer an immune activation strategy that may be complementary to PD-1 inhibition.

EXPERIMENTAL DESIGN:

We report Phase Ib/II safety, tolerability and immunogenicity results of immunotherapy with MEDI0457 (DNA immunotherapy targeting HPV16/18 E6/E7 with IL-12 encoding plasmids) delivered by electroporation with CELLECTRA^® constant current device. Twenty-two patients with locally advanced, p16+ HNSCCa received MEDI0457.

RESULTS:

MEDI0457 was associated with mild injection site reactions but no treatment related grade 3-5 adverse events (AEs). Eighteen of 21 evaluable patients showed elevated antigen specific T cell activity by IFNg ELISpot and persistent cellular responses surpassing 100 SFU/106 PBMC were noted out to one year. Induction of HPV-specific CD8+ T cells was observed. MEDI0457 shifted the CD8+/FoxP3+ ratio in 4/5 post-immunotherapy tumor samples and increased the number of perforin+ immune infiltrates in all five patients. One patient developed metastatic disease and was treated with anti-PD-1 therapy with a rapid and durable complete response. Flow cytometric analyses revealed induction of HPV16 specific PD-1+ CD8+ T cells that were not found prior to MEDI0547 (0% vs. 1.8%).

CONCLUSIONS:

These data demonstrate that MEDI0457 can generate durable HPV16/18 antigen-specific peripheral and tumor immune responses. This approach may be used as a complementary strategy to PD-1/PD-L1 inhibition in HPV-associated HNSCCa to improve therapeutic outcomes.

Genetic alterations that potentiate PI3K signalling are frequent in prostate cancer, yet how different genetic drivers of the PI3K cascade contribute to prostate cancer is unclear. Here, we report PIK3CA mutation/amplification correlates with poor prostate cancer patient survival. To interrogate the requirement of different PI3K genetic drivers in prostate cancer, we employed a genetic approach to mutate Pik3ca in mouse prostate epithelium. We show Pik3caH1047R mutation causes p110α-dependent invasive prostate carcinoma in-vivo. Furthermore, we report PIK3CA mutation and PTEN loss co-exist in prostate cancer patients, and can cooperate in-vivo to accelerate disease progressionvia AKT-mTORC1/2 hyperactivation. Contrasting single mutants that slowly acquire castration-resistant prostate cancer (CRPC), concomitant Pik3ca mutation and Pten loss caused de-novo CRPC. Thus, Pik3ca mutation and Pten deletion are not functionally redundant. Our findings indicate that PIK3CA mutation is an attractive prognostic indicator for prostate cancer that may cooperate with PTEN loss to facilitate CRPC in patients.