Lung cancer is the leading cause of cancer-related deaths in the world, and non-small cell lung cancer (NSCLC) is the most common subset. We previously found that infiltration of tumor inflammatory monocytes (TIMs) into lung squamous carcinoma (LUSC) tumors is associated with increased metastases and poor survival. To further understand how TIMs promote metastases, we compared RNA-Seq profiles of TIMs from several LUSC metastatic models with inflammatory monocytes (IMs) of non-tumor-bearing controls. We identified Spon1 as upregulated in TIMs and found that Spon1 expression in LUSC tumors corresponded with poor survival and enrichment of collagen extracellular matrix signatures. We observed SPON1+ TIMs mediate their effects directly through LRP8 on NSCLC cells, which resulted in TGF-β1 activation and robust production of fibrillar collagens. Using several orthogonal approaches, we demonstrated that SPON1+ TIMs were sufficient to promote NSCLC metastases. Additionally, we found that Spon1 loss in the host, or Lrp8 loss in cancer cells, resulted in a significant decrease of both high-density collagen matrices and metastases. Finally, we confirmed the relevance of the SPON1/LRP8/TGF-β1 axis with collagen production and survival in patients with NSCLC. Taken together, our study describes how SPON1+ TIMs promote collagen remodeling and NSCLC metastases through an LRP8/TGF-β1 signaling axis.

Table S4 includes immune signature and iCluster results.

Table S7 contains microbe screening results.

Table S1 contains cohort description, Master Patient Table and MutSigCV results.

Table S2 contains BAP1 analysis results, as well as detailed lists of YY1 and IRF8 target genes.

Table S6 contains results from the analysis of DNA methylation in SETD2 mutated and BAP1 inactivated samples.

Supplementary materials and methods, as well as supplementary figures S1-S7.

Labile ctHPV16DNA clearance kinetics in patients with low ({less than or equal to}200 copies/mL) baseline ctHPVDNA levels

Primers, probes, plasmids and gBlocks used in the HPV assay

Multiplexed dPCR assay for non-16 HPV strains

Lack of correlation between ctHPV16DNA clearance at week 6 and persistent/recurrent disease

Baseline ctHPV16DNA levels stratified by AJCC 8th edition N staging

Specificity, sensitivity and linearity of HPV16 dPCR assay

Table S3 contains the karyotypes of 16 genome-wide LOH MPM cases from the BWH cohort.

Table S5 contains detailed miRs and lncRNAs results.

Purpose/Objective(s) HPV-positive squamous cell carcinoma of the oropharynx (HPV+ OPSCC) is the most prevalent HPV-associated malignancy in the United States and is primarily caused by HPV16. Favorable treatment outcomes have led to increasing interest in treatment de-escalation to reduce treatment-related morbidity. Prognostic biomarkers are needed to identify appropriately low-risk patients for reduced treatment intensity. Large series of complete HPV16 genome sequencing from HPV+ OPSCC tumors are lacking in the literature. Therefore, we sought to test the hypothesis that HPV16 genotype is prognostic of recurrence-free survival (RFS) in HPV16+ OPSCC. Materials/Methods Targeted sequencing of 104 patients with HPV16+ OPSCC tumors was performed, providing complete coverage of all HPV16 open reading frames. Clinical features were retrospectively extracted from the medical record. A second cohort of OPSCC patients was sequenced using total RNA sequencing, which identified 89 patients with HPV16+ OPSCC for analysis. Results A high degree of coding diversity in the HPV16 was identified, with 93 distinct protein-coding HPV16 genotypes amongst the 104 patients subject to HPV (DNA) sequencing. As found in uterine cervical carcinoma, E7 was the most conserved amongst HPV16 viral genes. Sub-clonal variants were more likely to be non-synonymous and were enhanced for APOBEC-related mutagenesis. The HPV16-A1 sub-lineage was the most prevalent (approximately 70%). Genotypes closely related to HPV16-A1 were associated with increased numbers of copy-number variants in the human genome. Genotypes divergent from HPV16-A1 were strongly associated with favorable RFS as compared to HPV16-A1 (or similar genotypes); this finding was independent of tobacco smoke exposure. HPV16 genotypes divergent from HPV16-A1 were subsequently validated in an independent cohort (subject to RNA sequencing), to be associated with improved RFS in patients with moderate (less than 30 pack-years) and low (no more than 10 pack-years) of tobacco smoke exposure. Conclusion HPV16 viral genotype is highly diverse in HPV associated OPSCC. Sequence divergence from the HPV16-A1 reference sequence is strongly associated with improved RFS in patients with moderate to no tobacco smoke exposure. This finding was confirmed in two independent cohorts. HPV16 genotype is a promising potential biomarker that could be easily adopted to guide therapeutic decision-making related to de-escalation therapy. Prognostic genotypic information can be obtained from clinical samples stored in FFPE applying either DNA or RNA sequencing technology. HPV-positive squamous cell carcinoma of the oropharynx (HPV+ OPSCC) is the most prevalent HPV-associated malignancy in the United States and is primarily caused by HPV16. Favorable treatment outcomes have led to increasing interest in treatment de-escalation to reduce treatment-related morbidity. Prognostic biomarkers are needed to identify appropriately low-risk patients for reduced treatment intensity. Large series of complete HPV16 genome sequencing from HPV+ OPSCC tumors are lacking in the literature. Therefore, we sought to test the hypothesis that HPV16 genotype is prognostic of recurrence-free survival (RFS) in HPV16+ OPSCC. Targeted sequencing of 104 patients with HPV16+ OPSCC tumors was performed, providing complete coverage of all HPV16 open reading frames. Clinical features were retrospectively extracted from the medical record. A second cohort of OPSCC patients was sequenced using total RNA sequencing, which identified 89 patients with HPV16+ OPSCC for analysis. A high degree of coding diversity in the HPV16 was identified, with 93 distinct protein-coding HPV16 genotypes amongst the 104 patients subject to HPV (DNA) sequencing. As found in uterine cervical carcinoma, E7 was the most conserved amongst HPV16 viral genes. Sub-clonal variants were more likely to be non-synonymous and were enhanced for APOBEC-related mutagenesis. The HPV16-A1 sub-lineage was the most prevalent (approximately 70%). Genotypes closely related to HPV16-A1 were associated with increased numbers of copy-number variants in the human genome. Genotypes divergent from HPV16-A1 were strongly associated with favorable RFS as compared to HPV16-A1 (or similar genotypes); this finding was independent of tobacco smoke exposure. HPV16 genotypes divergent from HPV16-A1 were subsequently validated in an independent cohort (subject to RNA sequencing), to be associated with improved RFS in patients with moderate (less than 30 pack-years) and low (no more than 10 pack-years) of tobacco smoke exposure. HPV16 viral genotype is highly diverse in HPV associated OPSCC. Sequence divergence from the HPV16-A1 reference sequence is strongly associated with improved RFS in patients with moderate to no tobacco smoke exposure. This finding was confirmed in two independent cohorts. HPV16 genotype is a promising potential biomarker that could be easily adopted to guide therapeutic decision-making related to de-escalation therapy. Prognostic genotypic information can be obtained from clinical samples stored in FFPE applying either DNA or RNA sequencing technology.

Supplementary Figure from Comprehensive Viral Genotyping Reveals Prognostic Viral Phylogenetic Groups in HPV16-Associated Squamous Cell Carcinoma of the Oropharynx

Human papillomavirus-positive (HPV thorn ) squamous cell carci-noma of the oropharynx (OPSCC) is the most prevalent HPV-associated malignancy in the United States and is primarily caused by HPV subtype 16 (HPV16). Favorable treatment outcomes have led to increasing interest in treatment deescalation to reduce treatment-related morbidity. Prognostic biomarkers are needed to identify appropriately low-risk patients for reduced treatment intensity. Targeted DNA sequencing including all HPV16 open reading frames was performed on tumors from 104 patients with HPV16 thorn OPSCC treated at a single center. Genotypes closely related to the HPV16-A1 reference were associated with increased numbers of somatic copy-number variants in the human genome and poor recurrence-free survival (RFS). Genotypes divergent from HPV16-A1 were associated with favorable RFS. These findings wereindependent of tobacco smoke exposure. Total RNA sequencing was performed on a second independent cohort of 89 HPV16 thorn OPSCC cases. HPV16 genotypes divergent from HPV16-A1 were again validated in this independent cohort, to be prognostic of improved RFS in patients with moderate (less than 30 pack-years) or low (no more than 10 pack-years) of tobacco smoke exposure. In summary, we show in two independent cohorts that viral sequence divergence from the HPV16-A1 reference is correlated with improved RFS in patients with moderate or low tobacco smoke exposure. Implications: HPV16 genotype is a potential biomarker that could be easily adopted to guide therapeutic decision-making related to deescalation therapy.

Inhibition of the HER2/ERBB2 receptor is a keystone to treating HER2-positive malignancies, particularly breast cancer, but a significant fraction of HER2-positive (HER2+) breast cancers recur or fail to respond. Anti-HER2 monoclonal antibodies, like trastuzumab or pertuzumab, and ATP active site inhibitors like lapatinib, commonly lack durability because of adaptive changes in the tumor leading to resistance. HER2+ cell line responses to inhibition with lapatinib were analyzed by RNAseq and ChIPseq to characterize transcriptional and epigenetic changes. Motif analysis of lapatinib-responsive genomic regions implicated the pioneer transcription factor FOXA1 as a mediator of adaptive responses. Lapatinib in combination with FOXA1 depletion led to dysregulation of enhancers, impaired adaptive upregulation of HER3, and decreased proliferation. HER2-directed therapy using clinically relevant drugs (trastuzumab with or without lapatinib or pertuzumab) in a 7-day clinical trial designed to examine early pharmacodynamic response to antibody-based anti-HER2 therapy showed reduced FOXA1 expression was coincident with decreased HER2 and HER3 levels, decreased proliferation gene signatures, and increased immune gene signatures. This highlights the importance of the immune response to anti-HER2 antibodies and suggests that inhibiting FOXA1-mediated adaptive responses in combination with HER2 targeting is a potential therapeutic strategy.