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Supplementary Figures S1-S6

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Supplementary Tables S1-S9

Lynch syndrome is the most common cause of hereditary colorectal cancer and is secondary to germline alterations in one of four DNA mismatch repair (MMR) genes. Here we aimed to provide novel insights into the initiation of MMR-deficient (MMRd) colorectal carcinogenesis by characterizing the expression profile of MMRd intestinal stem cells (ISC). A tissue-specific MMRd mouse model (Villin-Cre;Msh2LoxP/LoxP) was crossed with a reporter mouse (Lgr5-EGFP-IRES-creERT2) to trace and isolate ISCs (Lgr5+) using flow cytometry. Three different ISC genotypes (Msh2-KO, Msh2-HET, and Msh2-WT) were isolated and processed for mRNA-seq and mass spectrometry, followed by bioinformatic analyses to identify expression signatures of complete MMRd and haplo-insufficiency. These findings were validated using qRT-PCR, IHC, and whole transcriptomic sequencing in mouse tissues, organoids, and a cohort of human samples, including normal colorectal mucosa, premalignant lesions, and early-stage colorectal cancers from patients with Lynch syndrome and patients with familial adenomatous polyposis (FAP) as controls. Msh2-KO ISCs clustered together with differentiated intestinal epithelial cells from all genotypes. Gene-set enrichment analysis indicated inhibition of replication, cell-cycle progression, and the Wnt pathway and activation of epithelial signaling and immune reaction. An expression signature derived from MMRd ISCs successfully distinguished MMRd neoplastic lesions of patients with Lynch syndrome from FAP controls. SPP1 was specifically upregulated in MMRd ISCs and colocalized with LGR5 in Lynch syndrome colorectal premalignant lesions and tumors. These results show that expression signatures of MMRd ISC recapitulate the initial steps of Lynch syndrome carcinogenesis and have the potential to unveil novel biomarkers of early cancer initiation.Significance:The transcriptomic and proteomic profile of MMR-deficient intestinal stem cells displays a unique set of genes with potential roles as biomarkers of cancer initiation and early progression.

BACKGROUND In cardiac amyloidosis (CA), misfolded proteins deposit in the extracellular space of cardiac tissue. These deposits classically cause restrictive cardiomyopathy with diastolic dysfunction. Although there are at least 30 proteins known to cause amyloid aggregates, 2 main types make up most diagnosed cases: light chain amyloidosis (AL) and transthyretin amyloidosis (ATTR). Since CA is considered a rare condition, it is often underdiagnosed or recognized in the advanced stages. Once amyloid deposits involve the heart tissue, they are associated with a worse outcome and higher mortality rates, especially in patients presenting symptoms of heart failure. CASE REPORT We report a case of a 22-year-old man presenting with acute severe mitral regurgitation, secondary to posterior mitral leaflet chordae tendineae rupture (CTR). Surgical mitral valve replacement with a mechanical prosthesis was performed, and cardiac tissue biopsy samples were obtained. After surgery, the patient improved significantly but suddenly presented with hemodynamic deterioration, until he died due to severe hemodynamic compromise and multiorgan failure. Although the etiology of the CTR was not established before surgical intervention, the histopathological analysis suggested CA. CONCLUSIONS CA diagnosis can be complex, especially in a 22-year-old-man with atypical clinical and imaging manifestations. In this patient, other differential diagnoses were considered, since CA presenting in a young patient is a rare phenomenon and acute mitral regurgitation secondary to CTR presents more frequently in other heart conditions. Furthermore, rapid postoperative deterioration resulted in the patient's death before biopsy samples were available because suspicion of amyloidosis had not been raised until that point.

Lynch syndrome is the most common cause of hereditary colorectal cancer and is secondary to germline alterations in one of four DNA mismatch repair (MMR) genes. Here we aimed to provide novel insights into the initiation of MMR-deficient (MMRd) colorectal carcinogenesis by characterizing the expression profile of MMRd intestinal stem cells (ISC). A tissue-specific MMRd mouse model (Villin-Cre;Msh2LoxP/LoxP) was crossed with a reporter mouse (Lgr5-EGFP-IRES-creERT2) to trace and isolate ISCs (Lgr5+) using flow cytometry. Three different ISC genotypes (Msh2-KO, Msh2-HET, and Msh2-WT) were isolated and processed for mRNA-seq and mass spectrometry, followed by bioinformatic analyses to identify expression signatures of complete MMRd and haplo-insufficiency. These findings were validated using qRT-PCR, IHC, and whole transcriptomic sequencing in mouse tissues, organoids, and a cohort of human samples, including normal colorectal mucosa, premalignant lesions, and early-stage colorectal cancers from patients with Lynch syndrome and patients with familial adenomatous polyposis (FAP) as controls. Msh2-KO ISCs clustered together with differentiated intestinal epithelial cells from all genotypes. Gene-set enrichment analysis indicated inhibition of replication, cell-cycle progression, and the Wnt pathway and activation of epithelial signaling and immune reaction. An expression signature derived from MMRd ISCs successfully distinguished MMRd neoplastic lesions of patients with Lynch syndrome from FAP controls. SPP1 was specifically upregulated in MMRd ISCs and colocalized with LGR5 in Lynch syndrome colorectal premalignant lesions and tumors. These results show that expression signatures of MMRd ISC recapitulate the initial steps of Lynch syndrome carcinogenesis and have the potential to unveil novel biomarkers of early cancer initiation. Significance: The transcriptomic and proteomic profile of MMR-deficient intestinal stem cells displays a unique set of genes with potential roles as biomarkers of cancer initiation and early progression.

Ipilimumab improves clinical outcomes when combined with nivolumab in metastatic non-small cell lung cancer (NSCLC), but its efficacy and impact on the immune microenvironment in operable NSCLC remain unclear. We report the results of the phase 2 randomized NEOSTAR trial (NCT03158129) of neoadjuvant nivolumab or nivolumab + ipilimumab followed by surgery in 44 patients with operable NSCLC, using major pathologic response (MPR) as the primary endpoint. The MPR rate for each treatment arm was tested against historical controls of neoadjuvant chemotherapy. The nivolumab + ipilimumab arm met the prespecified primary endpoint threshold of 6 MPRs in 21 patients, achieving a 38% MPR rate (8/21). We observed a 22% MPR rate (5/23) in the nivolumab arm. In 37 patients resected on trial, nivolumab and nivolumab + ipilimumab produced MPR rates of 24% (5/21) and 50% (8/16), respectively. Compared with nivolumab, nivolumab + ipilimumab resulted in higher pathologic complete response rates (10% versus 38%), less viable tumor (median 50% versus 9%), and greater frequencies of effector, tissue-resident memory and effector memory T cells. Increased abundance of gut Ruminococcus and Akkermansia spp. was associated with MPR to dual therapy. Our data indicate that neoadjuvant nivolumab + ipilimumab-based therapy enhances pathologic responses, tumor immune infiltrates and immunologic memory, and merits further investigation in operable NSCLC. Neoadjuvant treatment with nivolumab plus ipilimumab is well tolerated and demonstrates clinical efficacy in patients with early stage lung cancer.

Objective Patients with Lynch syndrome (LS) are at markedly increased risk for colorectal cancer. It is being increasingly recognised that the immune system plays an essential role in LS tumour development, thus making an ideal target for cancer prevention. Our objective was to evaluate the safety, assess the activity and discover novel molecular pathways involved in the activity of naproxen as primary and secondary chemoprevention in patients with LS. Design We conducted a Phase Ib, placebo-controlled, randomised clinical trial of two dose levels of naproxen sodium (440 and 220 mg) administered daily for 6 months to 80 participants with LS, and a co-clinical trial using a genetically engineered mouse model of LS and patient-derived organoids (PDOs). Results Overall, the total number of adverse events was not different across treatment arms with excellent tolerance of the intervention. The level of prostaglandin E2 in the colorectal mucosa was significantly decreased after treatment with naproxen when compared with placebo. Naproxen activated different resident immune cell types without any increase in lymphoid cellularity, and changed the expression patterns of the intestinal crypt towards epithelial differentiation and stem cell regulation. Naproxen demonstrated robust chemopreventive activity in a mouse co-clinical trial and gene expression profiles induced by naproxen in humans showed perfect discrimination of mice specimens with LS and PDOs treated with naproxen and control. Conclusions Naproxen is a promising strategy for immune interception in LS. We have discovered naproxen-induced gene expression profiles for their potential use as predictive biomarkers of drug activity. Trial registration number gov Identifier: NCT02052908

Immune checkpoint blockade (ICB) for cancer is associated with high response rates but also high rates of adverse events. To elucidate the underlying immunobiology, we profiled gene expression in intestinal, colitis, and tumor tissue from ICB-treated patients, with parallel studies in preclinical models, and validated our findings in a review of clinical cohort treated with interleukin-6 blockade. Expression of interleukin-6, neutrophil and chemotactic markers was higher in colitis than in normal intestinal tissue. The genes upregulated in colitis were not upregulated in responding tumors from patients receiving ICB. In murine models, interleukin-6 blockade was associated with improved tumor control and a higher density of CD4/CD8 effector T-cells, with reduced Th17, macrophages, and myeloid cells. In an experimental autoimmune encephalomyelitis (EAE) model with tumors, combined interleukin-6 blockade and ICB enhanced tumor rejection while simultaneously mitigating EAE symptoms versus ICB alone. Interleukin-6 blockade with ICB could de-couple autoimmunity from antitumor immunity.

As components of the innate immune response, antimicrobial peptides (AMPs) efficiently contribute to infection control and maintenance of a latent state in pulmonary tuberculosis (TB). As a therapeutic strategy, the administration of recombinant AMPs could be limited by enzymatic degradation and high production costs. Likewise, strategies based on the induction of AMPs have generated controversial results. In this study, 2 recombinant type-5 adenoviruses (Ad) expressing the human β-defensin 3 (HβD3) or cathelicidin (LL37) were assessed in a murine pulmonary TB model. Mice infected with either a high dose of a drug-sensitive (H37Rv) or a multidrug-resistant (MDR) strain of Mycobacterium tuberculosis (Mtb) were treated with a single administration of AdHβD3, AdLL37, AdGFP (control vector expressing a green fluorescent protein), or saline solution (SS). Lungs were obtained to determine the bacterial burden, histologic damage, and cytokine expression at different time points. Mice treated with AdHβD3 or AdLL37 showed significantly lower bacterial load and pneumonia, and higher proinflammatory cytokine expression than the control groups AdGFP and SS. A synergistic therapeutic effect could be observed when first- or second-line antibiotics (ABs) were administered with adenoviral therapy in animals infected with H37Rv or MDR strains, respectively. Adenovirus-delivered AMP's administration constitutes a promising adjuvant therapy for current anti-TB drugs by enhancing a protective immune response and potentially reducing current AB regimes' duration.

We report the first case series of immune checkpoint inhibitors (ICI)-associated colitis successfully treated with fecal microbiota transplantation, with reconstitution of the gut microbiome and a relative increase in the proportion of regulatory T-cells within the colonic mucosa. These preliminary data provide evidence that modulation of the gut microbiome may abrogate ICI-associated colitis.

Cerebral malaria (CM) is a neurological complication derived from the Plasmodium falciparum infection in humans. The mechanisms involved in the disease progression are still not fully understood, but both the sequestration of infected red blood cells (iRBC) and leukocytes and an exacerbated host inflammatory immune response are significant factors. In this study, we investigated the effect of Monocyte Locomotion Inhibitory Factor (MLIF), an anti-inflammatory peptide, in a well-characterized murine model of CM. Our data showed that the administration of MLIF increased the survival and avoided the neurological signs of CM in Plasmodium berghei ANKA (PbA) infected C57BL/6 mice. MLIF administration down-regulated systemic inflammatory mediators such as IFN-γ, TNF-α, IL-6, CXCL2, and CCL2, as well as the in situ expression of TNF-α in the brain. In the same way, MLIF reduced the expression of CD31, CD36, CD54, and CD106 in the cerebral endothelium of infected animals and prevented the sequestration of iRBC and leucocytes in the brain microvasculature. Furthermore, MLIF inhibited the activation of astrocytes and microglia and preserved the integrity of the blood-brain barrier (BBB). In conclusion, our results demonstrated that the administration of MLIF increased survival and conferred neuroprotection by decreasing neuroinflammation in murine CM.

Introduction: Despite the recent approval of immune checkpoints inhibitors (ICI) as a treatment option in the extensive-stage small cell lung cancer (SCLC) setting, survival has not significantly changed in the last decades. Recent scientific efforts have led to the identification of 4 major subtypes defined by expression of three transcription factors: ASCL1 (SCLC-A), NEUROD1 (SCLC-N), POU2F3 (SCLC-P) with the fourth subtype characterized by increased expression of immune genes (Inflamed subtype - SCLC-I). Transcriptomic results, along with recent immunotherapy trials, suggest that modulation of tumor immune microenvironment (TIME) could potential be critical to achieving clinical responses in a subset of patients, hence a comprehensive study of the TIME in SCLC is imperative. Here we report the feasibility of a multiplex immunofluorescence (mIF) methodology to analyze the TIME in SCLC. Methods: FFPE sections from surgically resected SCLC (N=4, one representative case across all SCLC subtypes) were identified from the ICON Project at UT MD Anderson Cancer Center. We used mIF to identify and quantify immune markers grouped into two 6-antibody panels: Panel 1: cytokeratin (CK, AE1/AE3), CD3, CD8, PD-1, PD-L1 and CD68; Panel 2: CK, CD20, granzyme B, FOXP3, CD45RO, and CD57. Finally, genomic (WES), transcriptomic (RNA sequencing) and proteomic (RPPA) data from these cases were integrated with the mIF data. Results: SCLC molecular subtypes (SCLC-A, N, P, I) were classified using transcriptomic and proteomic data. Analysis of TIME unveils a higher immune cell infiltration within SCLC-I subtype compared with the other cases representing, immune “cold” SCLC subtypes. SCLC-I subtype showed a 2-13 folder higher (range) immune cell density than SCLC-A, N and P subtypes (measured as a median of cell density). Specifically, T cells (CD3+) (695 and 242 cells/mm2, for SCLC-I and the median for the other subtypes respectively), T cytotoxic cells (CD3+ CD8+) (206 and 105), activated T cells (CD3+ CD8+ granzyme+) (20 and 2), antigen experienced ‘like' T cells (CD3+ PD-1+) (17 and 0), memory T cells (CD3+ CD45RO+) (328 and 91) and macrophages (CD68+) (773 and 57). PD-L1 expression in malignant cells did not show significant differences within the 4 SCLC subtypes. However, PD-L1 expression in macrophages was significantly higher in the SCLC-I subtype, suggesting an increase of IFN-gamma in the TIME. Conclusions: TIME study show the use of mIF in SCLC tumors to be feasible, and could potentially provide key information towards the identification of SCLC patients that could benefit from ICI. For the first time we complemented transcriptomic data from SCLC tumors with mIF analysis unveiling the complex interplay of the host immune response and malignant cells. Our preliminary results warrant further studies to explore the role of TIME in immunotherapeutic response in SCLC. Citation Format: Pedro Rocha, C. Allison Stewart, Edwin Parra, Luisa M. Solis, Carl M. Gay, Robert J. Cardnell, Naohiro Uraoka, Alejandro Francisco-Cruz, Hitoshi Dejima, Yuanxin Xi, Lixia Diao, Jing Wang, Marcelo V. Negrao, Jianjun Zhang, Ignacio Wistuba, Don L. Gibbons, Lauren A. Byers. Multiplex immunofluorescence (mIF) reveals differences in tumor immune microenvironment between molecularly-defined subsets of small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2758.

Multiplexed imaging platforms to simultaneously detect multiple epitopes in the same tissue section emerged in the last years as very powerful tools to study tumor immune contexture. These revolutionary technologies are providing a deep methodology for tumor evaluation in formalin-fixed and paraffin-embedded (FFPE) to improve the understanding of tumor microenvironment, new targets for treatment, prognostic and predictive biomarkers, and translational studies. Multiplexed imaging platforms allow for the identification of several antigens simultaneously from a single tissue section, core needle biopsies, and tissue microarrays. In recent years, multiplexed imaging has improved the abilities to characterize the different types of cell populations in malignant and non-malignant tissues, and their spatial distribution in relationship to clinical outcomes. Multiplexed technologies associated with digital image analysis software offer a high-quality throughput assay to study cancer specimens at multiple time points before, during and after treatment. The aim of this chapter is to provide a review of multiplexed imaging covering its fundamentals, advantages, disadvantages, and material and methods for staining applied to FFPE tumor tissues and focusing on the use of multiplex immunofluorescence with tyramine signal amplification staining for immune profiling and translational research.

Tuberculosis (TB), although a curable disease, remains a major cause of morbidity and mortality worldwide. It is necessary to develop a short-term therapy with reduced drug toxicity in order to improve adherence rate and control disease burden. Granulocyte-macrophage colony-stimulating factor (GM-CSF) may be a key cytokine in the treatment of pulmonary TB since it primes the activation and differentiation of myeloid and non-myeloid precursor cells, inducing the release of protective Th1 cytokines. In this work, we administrated by intratracheal route recombinant adenoviruses encoding GM-CSF (AdGM-CSF). This treatment produced significant bacterial elimination when administered in a single dose at 60 days of infection with drug sensitive or drug resistant Mtb strains in a murine model of progressive disease. Moreover, AdGM-CSF combined with primary antibiotics produced more rapid elimination of pulmonary bacterial burdens than conventional chemotherapy suggesting that this form of treatment could shorten the conventional treatment.

Patients diagnosed with germline mutations in MMR genes (Lynch Syndrome, LS) have up to 70-80% lifetime risk of colorectal cancer. Therefore, this high-risk population has the potential to benefit from effective chemopreventive strategies. Naproxen is an NSAID widely used for pain treatment with an excellent safety profile that has demonstrated to be more efficacious preventing colorectal cancer compared to aspirin in vivo using an intestinal tissue-specific mouse model of LS (VC-Msh2-LoxP). The ‘Naproxen trial' was designed to evaluate the modulation of PGE2 levels in colorectal mucosa, evaluate safety and tolerability, and discover novel molecular pathways involved in the chemopreventive activity of naproxen in LS patients. Methods: Participants were randomized to naproxen 440 mg (HD), 220 mg (LD) and placebo by mouth daily for 6 months. Modulation of prostaglandin levels, number of adverse events (AEs) observed in each treatment arm and gene expression profiles by next-generation sequencing (mRNAseq) in normal colorectal mucosa of LS patients after 6 months of intervention were examined. Results: Eighty participants diagnosed with LS were randomized, 25 participants to HD, 27 to LD, and 28 to placebo. From these patients, 54 were considered evaluable per-protocol analysis: 16 in the HD group, 15 in the LD and 23 in placebo. The level of prostaglandin E2 in the colorectal mucosa decreased significantly after treatment with both LD and HD naproxen when compared to placebo (-91.2%±14.1, -93.6%±7.9, and 23.8%±108.4, P-value Citation Format: Laura Reyes Uribe, Wenhui Wu, Ozkan Gelincik, Prashant V. Bommi, Alejandro Francisco-Cruz, Luisa M. Solis, Patrick M. Lynch, Ramona Lim, Elena Stoffel, Priyanka Kanth, N. Jewel Samadder, Maureen E. Mork, Melissa W. Taggart, Ginger L. Milne, Lawrence J. Marnett, Lana Vornik, Diane D. Liu, Maria Revuelta, Kyle Chang, Y. Nancy You, Levy Kopelovich, Ignacio I. Wistuba, J. Jack Lee, Shizuko Sei, Robert H. Shoemaker, Eva Szabo, Ellen Richmond, Asad Umar, Marjorie Perloff, Powell H. Brown, Steven M. Lipkin, Eduardo Vilar. Naproxen chemoprevention promotes immune activation in Lynch syndrome colorectal mucosa [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT111.

Introduction: Tertiary lymphoid structures (TLS) are ectopic lymphoid structures organized in a nodular pattern secondary to chronic inflammation. The presence of TLS has been associated with efficacious response to immune checkpoint blockade in a wide array of tumor types, including Non-Small Cell Lung Carcinoma (NSCLC). At early stages of NSCLC, TLS are observed in up to 70% of primary tumors where they are also associated with effective anti-tumor immune-responses and response to anti-PD-1 therapy. Despite complete surgical resection, however, up to 50% of patients with early stage NSCLC will eventually relapse. A systematic histomorphologic analysis of TLS and their association with relapse has not been well characterized. Design: Serial sections from Formalin-Fixed Paraffin-Embedded (FFPE) tissue specimens from 33 patients with stage I NSCLC were obtained (23 adenocarcinomas and 10 squamous cell carcinomas). Slides were stained with hematoxylin and eosin (H&E), immunohistochemistry for Vimentin, and 2 multiplex immunofluorescence (mIF) panels (Panel 1: cytokeratin (CK), CD3, CD8, CD68, PD-1, PD-L1 and DAPI Panel 2: CK, CD3 CD8, CD45RO, FOXP3, Granzyme B and DAPI). TLS were classified based on the H&E and Vimentin analysis into lymphoid aggregates (LA), immature TLS (iTLS), and mature TLS (mTLS). Morphometric analysis (number, area, and distance to nearest malignant cell) of intratumoral (IT) and peritumoral (PT) TLS was performed. mIF slides were scanned and IT representative areas were selected for cell densities and percentage quantification of immune-phenotypes. Morphometric analysis of TLSs was correlated with clinical outcomes, and tumor infiltrating immune cells. Results: Patients who recurred had fewer IT mTLS (1.8 vs 6; p=0.02) and smaller area of mTLS (64541.7μm2 vs 149870.5 μm2; p=0.004) compared with patients who did not recurr.The cell density of IT antigen-experienced cytotoxic T-lymphocytes (CTLs), regulatory T-cells, memory CTLs, and memory CTLs expressing FOXP3 were inversely correlated with the mTLS area (r=-0.6, p≤0.02). Antigen-experienced CTLs (r=-0.61; p≤0.05) and non-CTLs (r=-0.63; p≤0.05) were inversely correlated with the number of IT mTLS. Conclusion: Detailed morphometric analysis of mTLS offers relevant prognostic information for recurrence at stage I of NSCLC. mTLSs are associated with reduced IT infiltration by PD-1+ TILs. Taken together, germinal center development in mTLSs might convey a protective immune response due to immuno-dominant neoantigen presentation. Supported in part by CPRIT RP160668 grant and UT Lung SPORE. Citation Format: Rossana Natalia Lazcano Segura, Andre Catao, Luisa M. Solis, Mei Jiang, Auriole Tamegnon, Junya Fujimoto, Jaime Rodriguez-Canales, Chi-Wan B. Chow, Carmen Behrens, Neda Kalhor, Annika Weissferdt, John Heymach, Stephen Swisher, Boris Sepesi, Jack Lee, Cesar Moran, Andrew Futreal, Jianjun Zhang, Edwin R. Parra, Ignacio I. Wistuba, Michael T. Tetzlaff, Alejandro Francisco-Cruz. Tertiary lymphoid structures features associate with outcome in non-small cell lung carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 399.

Introduction: Imaging mass cytometry (IMC) enables highly multiplexed assessment of more than 30 proteins at a time for the quantification of cell phenotypes within the tumor microenvironment of formaldehyde fixed-paraffin embedded (FFPE) tumor tissue. This methodology provides a deep understanding of the immune-contexture while preserving their spatial information in regards to malignant cells. We report a detailed workflow for optimization of a 27-plex immune-oncology (IO) panel, immune-phenotyping, and spatial analysis of tumor tissue. Methods: IO-panel includes 27 antibodies targeting lymphoid (CD3e, CD4, CD8a, CD19, CD45RO, LAG3, ICOS, Granzyme-B), myeloid (CD11b, CD14, CD33, CD68, CSF1R, IDO-1), immune-regulatory (HLA-DR, OX-40, VISTA, TIM-3, CD73, B7-H3), epithelial (Cytokeratin), proliferative (Ki-67), and constitutive (GAPDH, NaKATPase, Vimentin, aSMA, Histone H3) markers that were selected by singlet automated chromogenic immunohistochemistry (IHC) staining. BSA-free formulation from the same clones of antibodies were isotope-metal conjugated and manual indirect immunofluorescence (IF) staining was used to confirm the stability of metal-tagged antibodies. The optimization by singlet-IF and multiplexed-IMC staining was performed with normal and malignant tissues (4mm core TMA with tonsil, placenta, prostate, breast carcinoma, ovarian carcinoma, and endometrioid carcinoma tissues). All images from IHC (HALO software), indirect IF (InForm software), and IMC (MCD viewer software) slides were evaluated by two pathologists. Cell densities and spatial analysis of a breast carcinoma core was performed using HALO software. Results: All antibodies included in the panel were individually optimized by IHC under the same protocol conditions. A significant correlation of optimal antibody concentrations where observed across the different staining techniques (IHC vs IF, r=0.51; IHC vs IMC, r= 0.54; IF vs IMC, r=0.992; all p values Conclusions: IMC is a powerful platform for highly multiplexed detection of protein and cell phenotypes quantification in FFPE samples. Our developed IMC panel and digital image analysis of tissues allow the quantification of immune cells and spatial analysis of the tumor tissue microenvironment. Citation Format: Pedro Rocha, Luisa Solis, Edwin Parra, Ou Shi, Barbara Mino, Vidya C. Sinha, Amanda Rinkenbaugh, Auriole Tamegnon, Mak Duncan, Wei Lu, Mei Jiang, Lakshimi Kakarala, Jared K. Burks, Helen Piwnica-Worms, Cara Haymacker, Michael Teztlaff, Ignacio Wistuba, Alejandro Francisco-Cruz. Immuno-oncology panel optimization for imaging mass cytometry and digital image analysis of tumor tissues [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2679.

High dose of Mycobacterium tuberculosis (Mtb) strain H37Rv administered by intratracheal injection in BALB/c mice induce progressive tuberculosis (TB). In this model, during the first month there is a temporal control of bacillary growth, in coexistence with macrophage activation, granuloma formation and Th-1 response. Then, bacterial proliferation recommences, accompanied by progressive pneumonia and decreasing expression of protective cytokines (IFN-γ and TNF-α). In this model, we studied the IL-12 gene expression kinetics and cellular source. There is a rapid and progressive IL-12 expression peaking at day 14, when granulomas start their formation and numerous macrophages show strong IL-12 immunostaining, while during progressive TB there is a significant decrease of IL-12 expression and occasional macrophages showed IL-12 immunolabeling. In the second part of this study, we determined the immunotherapeutic effect of recombinant adenoviruses that codify IL-12 (AdIL-12). Intratracheal administration of only one dose of AdIL-12 one day before Mtb infection produced significant decrease of bacterial loads, lesser pneumonia and higher expression of TNF-α, IFN-γ and iNOS. When only one dose of AdIL-12 was given in healthy mice cohoused with infected mice with highly virulent and transmissible Mtb, total prevention of infection was conferred. Moreover, when AdIL-12 was administered by intranasal route in animals suffering late active TB after 2 months of infection, a very low pulmonary bacilli burdens was detected. These experimental data confirm that IL-12 is a significant cytokine in the immune protection against Mtb, and gene therapy based in adenoviruses coding this cytokine increased protective immunity and prevent Mtb transmission.

Mycobacterium habana was isolated in Cuba in 1971. Later, was demonstrated its protection capacity in mycobacterial infection. Here we determined the level of virulence, immunogenicity and the efficacy of three different M. habana strains as attenuated live vaccines. Intratracheal infection of BALB/c mice with high dose M. habana TMC 5135 or IPK-337 strains permitted 100% survival and limited tissue damage. Mice infected with M. habana IPK-220 showed lower attenuation, so it was discarded for the vaccination experiments. Strains IPK-337 and TMC 5135 were used as subcutaneous vaccine and compared with BCG. Nude mice vaccinated with strain 5135 showed longer but non-significant survival than BCG vaccinated animals. Cell suspensions from M. habana vaccinated mice produced higher IFNγ after stimulation with mycobacterial antigens than BCG recipients. After four months of challenge with Mycobacterium tuberculosis strain H37Rv, mice vaccinated with BCG substrain Phipps or strain TMC 5135 showed total survival, while 60% survival was exhibited by animals vaccinated with M. habana IPK-337. Both M. habana strains do not prevent the infection with M. tuberculosis but avoided the progression of the experimental disease; strain TMC 5135 showed similar level of protection than BCG.

Introduction. PanNETs are characterized by heterogeneous but largely indolent growth, leading to advanced stage at diagnosis, difficulty predicting outcomes, and insufficiently effective treatments. A better understanding of PanNETs immune context is needed for rational immunotherapy strategies. The aim of this study was to characterize immune cell infiltrates within primary tumors, understand the correlation of immune infiltration with genes associated with PanNET development, and clinical-pathological features. Material and methods. Formalin-fixed paraffin-embedded (FFPE) surgically resected primary tumor specimens from 53 patients with metastatic PanNETs were evaluated for DAXX, ATRX, and immune-cell markers (CD8, CD4, CD45RO, FOXP3, ICOS, OX40, PD-1, LAG3, TIM-3, B7-H3, B7-H4, PD-L1, VISTA, and CD68) by immunohistochemistry (IHC). Intratumoral lymphocyte-enriched areas (LEA), defined by CD8 hot-spots, and macrophage-enriched areas (MEA), defined by CD68 hot spots, were selected for image analysis and cell densities were quantitated. We considered higher densities more than the third quartile value and low density as less or equal than the third quartile value for all the markers. 47 cases from the same FFPE tumor tissue blocks were used for paired-end RNA sequencing (HiSeq 4000 Sequencing System), and exome sequencing (T200 Platform) to MEN1, SETD2, MUTYH, CHEK2, BRCA2, ALT, DAXX, ATRX, PTEN, TSC1, and TSC2 genes. Differences between variables were analyzed by non-parametric t-test and Kaplan-Meier curves for time-to-event using SPSS statistical software version 24. Results. Overall, higher densities of CD8, CD4, CD68, and B7-H3 were found compared with the other markers. We found a significant correlation between CD8 in LEA with CD4 (r=0.7), FOXP3 (r=0.5), CD45RO (r=0.6), ICOS (r=0.5) and PD-1 (r=0.5) cell densities. In addition, CD68 in MEA had significant and positive correlation with TIM-3 (r=0.6) cell densities. Higher TIM-3 cell densities correlated with higher levels of TIM-3 (P= Conclusions. TAMs were significantly correlated with inferior DSS in PanNETs. TAM depletion may, therefore, present an appealing and rational target for immunotherapeutic approaches in NETs. This work was funded by a Conquer Cancer Foundation of ASCO Career Development Award, and a grant from the Neuroendocrine Tumor Research Foundation. Citation Format: Alejandro Francisco-Cruz, Naohiro Uraoka, Suyu Liu, Edwin R. Parra, Luisa M. Solis, Barbara Mino, Arvind Dasari, Jaime Rodriguez-Canales, Michael J. Overman, Jonathan M. Loree, James C. Yao, Ignacio I. Wistuba, Daniel M. Halperin, Jeannelyn S. Estrella. Study of the immune contexture in advanced pancreatic neuroendocrine tumors reveals tumor-associated macrophages as promoters of poor survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4576.

Introduction. The interaction between malignant cells (MCs), stromal cells, tumor-associated lymphocytes (TILs), and tumor-associated macrophages (TAMs) is relevant for non-small cell lung carcinoma (NSCLC) progression. The spatial distribution of those cells may affect the prognosis and can be related to genetic intra-tumor heterogeneity (ITH). The aim of this study was to characterize the immunologic ITH and the spatial distribution of immune cells to MCs in primary NSCLC tumors at early stages using multiplex immunofluorescence (mIF) and image analysis approaches. Material and methods. We studied 33 surgically resected NSCLC cases (adenocarcinomas=23; squamous-cell carcinomas=10) with a history of recurrence in a follow-up of at least 60 months (recurrence, N=13; non-recurrence, N=15). Consecutive FFPE tissue sections were stained with two mIF panels (panel 1: cytokeratin (AE1/AE3), PD-L1, PD-1, CD3, CD8, and CD68; panel 2: AE1/AE3, CD3, CD8, granzyme-B, CD45RO, and FOXP3). Three intra-tumor regions (3mm2 each) per case were selected after gridding the whole tumor section. A total of 99 intratumor regions were scanned and analyzed using Vectra Multispectral-Microscope and InForm-software. From each intratumor region, TILs and TAMs densities, as well as the coefficient of variation, were evaluated. The median distance and the G-Cross area under the curve (AUC) for specific radial distances (10µm, 20µm, and 40µm) were obtained between TILs and TAMs phenotypes to MCs. Results. Recurrence was associated with higher MCs density and TAMs/TILs ratio, and lower TIL densities. A high ITH of cytotoxic T-cells (CTLs) PD-L1+ was associated with worse survival. The distance of TAMs PD-L1+ to MCs PD-L1 negative (60µm vs 25µm) or to MCs PD-L1 positive (25µm vs 13µm) was higher in the non-recurrence group than in recurrence group. Close TAMs PD-L1+ to MCs was associated with worst survival. In a radial distance of 10µm, 20µm, and 40µm, a higher infiltration of CTLs PD-1+, was observed in the group of recurrence than non-recurrence group, surrounding MCs PD-L1 negative (AUC 0.49, 3.80, and 20.03; vs AUC 0.01, 0.16, and 1.29, respectively), and MCs PD-L1 positive (AUC 0.60, 4.35, and 19.90; vs AUC 0.01, 0.20, and 2.20, respectively). A high infiltration of CTLs PD-1+ surrounding MCs, with or without expression of PD-L1, was associated with worse survival. All the differences were statistically significant (P Conclusion. Close spatial proximity of antigen-experienced CTLs and TAMs PD-L1+ to MCs are associated with recurrence and poor survival in early stages of NSCLC. We determined that ITH of immune cell densities is associated with recurrence of surgically resected NSCLC. Tumor-immune cell spatial modeling offers a deep understanding of tumor microenvironment that impacts on clinical outcomes. Supported by CPRITRP160668 and UT Lung SPORE grants Citation Format: Alejandro Francisco-Cruz, Edwin R. Parra, Santhoshi N. Krishnan, Souptik Barua, Mei Jiang, Junya Fujimoto, Christine B. Peterson, Priyam Das, Chi-Wan Chow, Jaime Rodriguez-Canales, Carmen Behrens, Neda Kalhor, Annikka Weissferdt, John Heymach, Stephen Swisher, Boris Sepesi, Arvind Rao, J. Jack Lee, Cesar Moran, Andrew Futreal, Jianjun Zhang, Ignacio I. Wistuba. Impact of the spatial analysis of tumor-associated lymphocytes and tumor-associated macrophages on recurrence at early stage of non-small cell lung carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1180.

Multiplexed platforms for multiple epitope detection have emerged in the last years as very powerful tools to study tumor tissues. These revolutionary technologies provide important visual techniques for tumor examination in formalin-fixed paraffin-embedded specimens to improve the understanding of the tumor microenvironment, promote new treatment discoveries, aid in cancer prevention, as well as allowing translational studies to be carried out. The aim of this review is to highlight the more recent methodologies that use multiplexed staining to study simultaneous protein identification in formalin-fixed paraffin-embedded tumor tissues for immune profiling, clinical research, and potential translational analysis. New multiplexed methodologies, which permit the identification of several proteins at the same time in one single tissue section, have been developed in recent years with the ability to study different cell populations, cells by cells, and their spatial distribution in different tumor specimens including whole sections, core needle biopsies, and tissue microarrays. Multiplexed technologies associated with image analysis software can be performed with a high-quality throughput assay to study cancer specimens and are important tools for new discoveries. The different multiplexed technologies described in this review have shown their utility in the study of cancer tissues and their advantages for translational research studies and application in cancer prevention and treatments.

Summary BALB/c mice with pulmonary tuberculosis (TB) develop a T helper cell type 1 that temporarily controls bacterial growth. Bacterial proliferation increases, accompanied by decreasing expression of interferon (IFN)-γ, tumour necrosis factor (TNF)-α and inducible nitric oxide synthase (iNOS). Activation of dendritic cells (DCs) is delayed. Intratracheal administration of only one dose of recombinant adenoviruses encoding granulocyte–macrophage colony-stimulating factor (AdGM-CSF) 1 day before Mycobacterium tuberculosis (Mtb) infection produced a significant decrease of pulmonary bacterial loads, higher activated DCs and increased expression of TNF-α, IFN-γ and iNOS. When AdGM-CSF was given in female mice B6D2F1 (C57BL/6J X DBA/2J) infected with a low Mtb dose to induce chronic infection similar to latent infection and corticosterone was used to induce reactivation, a very low bacilli burden in lungs was detected, and the same effect was observed in healthy mice co-housed with mice infected with mild and highly virulent bacteria in a model of transmissibility. Thus, GM-CSF is a significant cytokine in the immune protection against Mtb and gene therapy with AdGM-CSF increased protective immunity when administered in a single dose 1 day before Mtb infection in a model of progressive disease, and when used to prevent reactivation of latent infection or transmission.

Aim: Mycobacterium tuberculosis (Mtb) latent infection can lead to reactivation. The design of new strategies to prevent it is an important subject. Material & Methods: B6D2F1 mice were infected intratracheally with a low dose of Mtb H37 Rv to induce chronic infection. After 7 months, mice were treated with one dose of recombinant adenoviruses encoding TNFα, β defensin-3 and LL37. Immunosupression was induced one month after with corticosterone. Results: In comparison with the control group, mice treated with adenoviruses showed significantly lesser bacterial loads and pneumonia, being the adenoviruses that encode TNFα and LL37 the most efficient. Conclusion: Gene therapy based in a proinflammatory cytokine or antimicrobial peptides are a potential useful system to prevent reactivation of latent tuberculosis.

In the version of this article originally published, an author was missing from the author list. Alexander J. Lazar should have been included between Jorge M. Blando and James P. Allison. The author has been added to the list, and the author contributions section has been updated to include Alexander J. Lazar's contribution to the study. The error has been corrected in the print, PDF and HTML versions of the manuscript.

Introduction. Recurrence of non-small cell lung carcinoma (NSCLC) is associated with genetic and epigenetic intra-tumor heterogeneity (ITH). The interaction between malignant cells, stromal cells, and tumor-associated immune-cells (TAICs), such as T-cell lymphocytes (TCLs) and tumor-associated macrophages (TAMs), is important for progression of NSCLC and the characterization of the immunologic ITH might be relevant to predict recurrence in surgically treated patients at early stages of NSCLC. The aim of this study was to characterize the immunologic ITH of primary NSCLC tumors at early stages using image analysis and multiplex immunofluorescence (mIF) approaches. Material and methods. Eight cases of stage IA and 8 cases of stage IB surgically resected NSCLC (11 adenocarcinomas, ADCs; and 5 squamous-cell carcinomas, SCCs) with a history of early recurrence were selected for this preliminary analysis. Formalin-fixed, paraffin-embedded (FFPE) blocks were obtained and consecutive sections were stained with two panels of mIF for immune profiling, panel 1: pan-cytokeratin (AE1/AE3), PD-L1, PD-1, CD3, CD8, and CD68; panel 2: AE1/AE3, CD3, CD8, granzyme-B (GB), CD45RO, and FOXP3. Three not adjacent, intra-tumor regions (3mm2 each) per case were randomly selected after gridding the whole tumor section. A total of 41 intra-tumor regions were scanned by Vectra multispectral-microscope (PerkinElmer) and analyzed using InForm-software (PerkinElmer). TAICs were quantified in the epithelial and stromal compartments from each intra-tumor region. Results. The median density of TCLs and TAMs were 1527 cells/mm2 and 635 cells/mm2, respectively, without significant differences between histologic subtypes. TCLs were predominantly concentered in the stromal compartment (median, 2222 cells/mm2) when compared with epithelial compartment (median, 332 cells/mm2). The percentage and density of TCLs and TAMs varied 4 and 8 times, respectively, between cases and regions. Non-cytotoxic T-cells and inactive cytotoxic T-cells were the most prevalent phenotypes. Higher density of TAMs and antigen-experienced TCLs were observed in stage IB than stage IA in the primary tumor of patients with NSCLC. Conclusion. The characterization of the immunologic ITH of NSCLC is able by mIF and image analysis with FFPE tumor tissue. There is a variability of TAICs densities between regions from the same tumor and different subpopulations were observed. TAMs and exhausted T-cells were more prominent in stage IB (tumor >3cm) suggesting these cells may play an important role in recurrence. Ongoing studies with a larger cohort and comparison with non-recurrent surgically treated patients are warranted. Supported in part by CPRIT RP160668 grant Citation Format: Alejandro Francisco Cruz, Edwin R. Parra, Mei Jiang, Junya Fujimoto, Chi-Wan Chow, Jaime Rodriguez-Canales, Carmen Behrens, Neda Kalhor, Annikka Weissferdt, John Heymach, Stephen Swisher, Boris Sepesi, J. Jack Lee, Cesar Moran, P. Andrew Futreal, Jianjun Zhang, Ignacio I. Wistuba. Characterization of the immunologic intra-tumor heterogeneity in early stages of non-small cell lung carcinoma using multiplex immunofluorescence and image analysis approaches [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1174.

e16178Background: Neuroendocrine tumors (NETs) are a highly heterogeneous group of malignancies with variable clinical course. Checkpoint inhibitors have shown modest antitumor activity, but ration...