Tumor progression and the efficacy of immunotherapy are strongly influenced by the composition and abundance of immune cells in the tumor microenvironment. Due to the limitations of direct measurement methods, computational algorithms are often used to infer immune cell composition from bulk tumor transcriptome profiles. These estimated tumor immune infiltrate populations have been associated with genomic and transcriptomic changes in the tumors, providing insight into tumor–immune interactions. However, such investigations on large-scale public data remain challenging. To lower the barriers for the analysis of complex tumor–immune interactions, we significantly improved our previous web platform TIMER. Instead of just using one algorithm, TIMER2.0 (http://timer.cistrome.org/) provides more robust estimation of immune infiltration levels for The Cancer Genome Atlas (TCGA) or user-provided tumor profiles using six state-of-the-art algorithms. TIMER2.0 provides four modules for investigating the associations between immune infiltrates and genetic or clinical features, and four modules for exploring cancer-related associations in the TCGA cohorts. Each module can generate a functional heatmap table, enabling the user to easily identify significant associations in multiple cancer types simultaneously. Overall, the TIMER2.0 web server provides comprehensive analysis and visualization functions of tumor infiltrating immune cells.

Epidermal growth factor receptor (EGFR) gene mutations (G719X, exon 19 deletions/insertions, L858R and L861Q) predict favorable responses to EGFR tyrosine kinase inhibitors (TKIs) in advanced non-small-cell lung cancer (NSCLC). However, EGFR exon 20 insertion mutations (∼10% of all EGFR mutations) are generally associated with insensitivity to available TKIs (gefitinib, erlotinib and afatinib). The basis of this primary resistance is poorly understood.  We study a broad subset of exon 20 insertion mutations, comparing in vitro TKI sensitivity with responses to gefitinib and erlotinib in NSCLC patients; and find that most are resistant to EGFR TKIs. The crystal structure of a representative TKI-insensitive mutant (D770_N771insNPG) reveals an unaltered ATP-binding pocket and the inserted residues form a wedge at the end of the C-helix that promotes the active kinase conformation. Unlike EGFR-L858R, D770_N771insNPG activates EGFR without increasing its affinity for EGFR TKIs. Unexpectedly, we find that EGFR-A763_Y764insFQEA is highly sensitive to EGFR TKIs in vitro; and patients whose NSCLCs harbor this mutation respond to erlotinib. Analysis of the A763_Y764insFQEA mutant indicates that the inserted residues shift the register of the C-helix in the N-terminal direction, altering the structure in the region that is also affected by the TKI-sensitive EGFR-L858R. Our studies reveal intricate differences between EGFR mutations, their biology and their response to EGFR TKIs.

Purpose: Approximately 5% of lung adenocarcinomas harbor an EML4-ALK gene fusion and define a unique tumor group that may be responsive to targeted therapy. However ALK-rearranged lung adenocarcinomas are difficult to detect by either standard fluorescence in situ hybridization or immunohistochemistry (IHC) assays. In the present study, we used novel antibodies to compare ALK protein expression in genetically defined lung cancers and anaplastic large cell lymphomas.Experimental Design: We analyzed 174 tumors with one standard and two novel monoclonal antibodies recognizing the ALK protein. Immunostained tissue sections were assessed for the level of tumorspecific ALK expression by objective quantitative image analysis and independently by three pathologists.Results: ALK protein is invariably and exclusively expressed in ALK-rearranged lung adenocarcinomas but at much lower levels than in the prototypic ALK-rearranged tumor, anaplastic large cell lymphoma, and as a result, is often not detected by conventional IHC. We further validate a novel IHC that shows excellent sensitivity and specificity (100% and 99%, respectively) for the detection of ALK-rearranged lung adenocarcinomas in biopsy specimens, with excellent interobserver agreement between pathologists (κ statistic, 0.94).Conclusions: Low levels of ALK protein expression is a characteristic feature of ALK-rearranged lung adenocarcinomas. However, a novel, highly sensitive IHC assay reliably detects lung adenocarcinomas with ALK rearrangements and obviates the need for fluorescence in situ hybridization analysis for the majority of cases, and therefore could be routinely applicable in clinical practice to detect lung cancers that may be responsive to ALK inhibitors. Clin Cancer Res; 16(5); 1561-71. ©2010 AACR.

We introduce the TRUST4 open-source algorithm for reconstruction of immune receptor repertoires in αβ/γδ T cells and B cells from RNA-sequencing (RNA-seq) data. Compared with competing methods, TRUST4 supports both FASTQ and BAM format and is faster and more sensitive in assembling longereven full-length-receptor repertoires. TRUST4 can also call repertoire sequences from single-cell RNA-seq (scRNA-seq) data without V(D)J enrichment, and is compatible with both SMART-seq and 5′ 10x Genomics platforms.Both T and B cells can generate diverse receptor (TCR and BCR, respectively) repertoires, through somatic V(D)J recombination, to recognize various external antigens or tumor neoantigens. Following antigen recognition, BCRs also undergo somatic hypermutations (SHMs) to further improve antigen-binding affinity. Repertoire sequencing has been increasingly adopted in infectious disease 1 , allergy 2 , autoimmune 3 , tumor immuology 4 and cancer immunotherapy 5 studies, but it is an expensive assay and consumes valuable tissue samples. Alternatively, RNA-seq data contain expressed TCR and BCR sequences in tissues or peripheral blood mononuclear cells (PBMC). However, because repertoire sequences from V(D)J recombination and SHM are different from the germline, they are often eliminated in the read-mapping step.Previously we developed the TRUST algorithm 6-8 , utilized to de novo assemble immune receptor repertories directly from tissue or blood RNA-seq data. When applied to The Cancer Genome Atlas (TCGA) tumor RNA-seq data, TRUST revealed profound biological insights into the repertoires of tumor-infiltrating T cells 6 and B cells 8 , as well as their associated tumor immunity. Although less sensitive than TCR-seq and BCR-seq, TRUST is able to identify the abundantly expressed and potentially more clonally expanded TCRs/BCRs in the RNA-seq data that are more likely to be involved in antigen binding 9 . Recent years have also seen other computational methods introduced for immune repertoire construction from RNA-seq data, including V'DJer 10 , MiXCR 11 , CATT 12 and ImRep 13 . These methods focus on reconstruction of complementary-determining region 3 (CDR3), with limited ability to assemble full-length V(D)J receptor sequences, although CDR1 and CDR2 on the V sequence still contribute considerably to antigen recognition and binding. For example, five out of six mutations predicted in a recent study to influence antibody affinity in the acidic tumor environment are located in CDR1 and CDR2 (ref. 14 ), and four out of nine positions contributing most to 4A8 antibody binding to the SARS-CoV-2 spike protein are in CDR1 and CDR2 (ref. 15 ). Therefore, algorithms that can infer full-length immune receptor repertoires can facilitate better receptor-antigen interaction modeling.

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Three-dimensional (3D) tissue imaging methods are expected to improve surgical management of cancer. In this study, we examined the feasibility of two 3D imaging technologies, optical coherence tomography (OCT) and optical coherence microscopy (OCM), to view human breast specimens based on intrinsic optical contrast. Specifically, we imaged 44 ex vivo breast specimens including 34 benign and 10 malignant lesions with an integrated OCT and OCM system developed in our laboratory. The system enabled 4-mm axial resolution (OCT and OCM) with 14-mm (OCT) and 2-mm (OCM) transverse resolutions, respectively. OCT and OCM images were compared with corresponding histologic sections to identify characteristic features from benign and malignant breast lesions at multiple resolution scales. OCT and OCM provide complimentary information about tissue microstructure, thus showing distinctive patterns for adipose tissue, fibrous stroma, breast lobules and ducts, cysts and microcysts, as well as in situ and invasive carcinomas. The 3D imaging capability of OCT and OCM provided complementary information to individual 2D images, thereby allowing tracking features from different levels to identify low-contrast structures that were difficult to appreciate from single images alone. Our results lay the foundation for future in vivo optical evaluation of breast tissues, using OCT and OCM, which has the potential to guide core needle biopsies, assess surgical margins, and evaluate nodal involvement in breast cancer.

We demonstrate photothermal optical coherence tomography (OCT) imaging in highly scattering human breast tissue ex vivo. A 120 kHz axial scan rate, swept-source phase-sensitive OCT system at 1300 nm was used to detect phase changes induced by 830 nm photothermal excitation of gold nanoshells. Localized phase modulation was observed 300-600 μm deep in scattering tissue using an excitation power of only 22 mW at modulation frequencies up to 20 kHz. This technique enables integrated structural and molecular-targeted imaging for cancer markers using nanoshells.Optical coherence tomography (OCT) is a powerful tool for assessing tissue architectural morphology [1]. It enables three-dimensional (3D) imaging with resolutions approaching that of histopathology but can be performed in vivo and in real time without the need to remove and process specimens. Conventional OCT imaging is based on contrast from spatial variations in tissue scattering. The use of exogenous contrast agents would allow targeted imaging of specific cells, receptors, or functional processes. OCT contrast enhancement has been demonstrated using scattering microspheres [2], iron oxide microparticles [3], and nanoparticles [4][5][6][7][8][9][10]. Gold nanoparticles are attractive for OCT owing to their customizable absorption and scattering properties, biocompatibility, and ease of conjugation to antibodies and peptides that bind selectively with proteins associated with specific diseases [11]. Gold nanoparticles have also been used as photothermal therapy agents [12,13]. These characteristics offer gold nanoparticles "three-in-one" [14] functionality as targeting probes, image contrast enhancers, and therapeutic agents.Our group demonstrated phase-sensitive OCT to detect gold nanoshells as contrast agents in phantoms with high signal-to-noise ratio (SNR) [9]. The technique uses nanoshells with absorption at 780 nm, where tissue absorption is inherently low. An excitation laser at 808 nm induces small temperature gradients in sample regions containing nanoparticles. These thermal variations modulate the sample's optical path length, which is detected using phase-sensitive OCT. Photothermal OCT selectively detects regions containing nanoshells, potentially enabling molecular contrast. Other groups working in parallel demonstrated photothermal OCT imaging in 3D cell constructs using immunotargeted gold nanospheres [10]. Photothermal OCT imaging is challenging in tissues where scattering plays an important role. In this Letter, we * Corresponding author: jgfuji@mit.edu. demonstrate the feasibility of photothermal OCT in human breast tissue. By modulating the thermal excitation beam at high frequencies, localized photothermal phase contrast can be observed 300-600 μm deep in a tissue using a photothermal modulation power of only 22 mW at 830 nm. NIH Public AccessA swept-source OCT system ( Fig. 1) with a buffered Fourier-domain mode-locked (FDML) laser running at 1310 nm and 120 kHz was used for these studies. The laser tuning range is 170 nm. Phot...

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