Intratumoral IL12 mRNA Therapy Promotes TH1 Transformation of the Tumor Microenvironment.

PURPOSE: While immune checkpoint inhibitors such as anti-PD-L1 are rapidly becoming the standard of care in the treatment of many cancers, only a subset of treated patients have long-term responses. IL12 promotes antitumor immunity in mouse models; however, systemic recombinant IL12 had significant toxicity and limited efficacy in early clinical trials. EXPERIMENTAL DESIGN: We therefore designed a novel intratumoral IL12 mRNA therapy to promote local IL12 tumor production while mitigating systemic effects. RESULTS: A single intratumoral dose of mouse (m)IL12 mRNA induced IFNγ and CD8+ T-cell-dependent tumor regression in multiple syngeneic mouse models, and animals with a complete response demonstrated immunity to rechallenge. Antitumor activity of mIL12 mRNA did not require NK and NKT cells. mIL12 mRNA antitumor activity correlated with TH1 tumor microenvironment (TME) transformation. In a PD-L1 blockade monotherapy-resistant model, antitumor immunity induced by mIL12 mRNA was enhanced by anti-PD-L1. mIL12 mRNA also drove regression of uninjected distal lesions, and anti-PD-L1 potentiated this response. Importantly, intratumoral delivery of mRNA encoding membrane-tethered mIL12 also drove rejection of uninjected lesions with very limited circulating IL12p70, supporting the hypothesis that local IL12 could induce a systemic antitumor immune response against distal lesions. Furthermore, in ex vivo patient tumor slice cultures, human IL12 mRNA (MEDI1191) induced dose-dependent IL12 production, downstream IFNγ expression and TH1 gene expression. CONCLUSIONS: These data demonstrate the potential for intratumorally delivered IL12 mRNA to promote TH1 TME transformation and robust antitumor immunity.See related commentary by Cirella et al., p. 6080.

The Combined Effect of FGFR Inhibition and PD-1 Blockade Promotes Tumor-Intrinsic Induction of Antitumor Immunity.

The success of targeted or immune therapies is often hampered by the emergence of resistance and/or clinical benefit in only a subset of patients. We hypothesized that combining targeted therapy with immune modulation would show enhanced antitumor responses. Here, we explored the combination potential of erdafitinib, a fibroblast growth factor receptor (FGFR) inhibitor under clinical development, with PD-1 blockade in an autochthonous FGFR2K660N/p53mut lung cancer mouse model. Erdafitinib monotherapy treatment resulted in substantial tumor control but no significant survival benefit. Although anti-PD-1 alone was ineffective, the erdafitinib and anti-PD-1 combination induced significant tumor regression and improved survival. For both erdafitinib monotherapy and combination treatments, tumor control was accompanied by tumor-intrinsic, FGFR pathway inhibition, increased T-cell infiltration, decreased regulatory T cells, and downregulation of PD-L1 expression on tumor cells. These effects were not observed in a KRASG12C-mutant genetically engineered mouse model, which is insensitive to FGFR inhibition, indicating that the immune changes mediated by erdafitinib may be initiated as a consequence of tumor cell killing. A decreased fraction of tumor-associated macrophages also occurred but only in combination-treated tumors. Treatment with erdafitinib decreased T-cell receptor (TCR) clonality, reflecting a broadening of the TCR repertoire induced by tumor cell death, whereas combination with anti-PD-1 led to increased TCR clonality, suggesting a more focused antitumor T-cell response. Our results showed that the combination of erdafitinib and anti-PD-1 drives expansion of T-cell clones and immunologic changes in the tumor microenvironment to support enhanced antitumor immunity and survival.

Durable anticancer immunity from intratumoral administration of IL-23, IL-36γ, and OX40L mRNAs.

Many solid cancers contain dysfunctional immune microenvironments. Immune system modulators that initiate responses to foreign pathogens could be promising candidates for reigniting productive responses toward tumors. Interleukin-1 (IL-1) and IL-12 cytokine family members cooperate at barrier tissues after microbial invasion, in human inflammatory diseases, and in antitumoral immunity. IL-36γ, in classic alarmin fashion, acts in damaged tissues, whereas IL-23 centrally coordinates immune responses to danger signals. In this study, direct intratumoral delivery of messenger RNAs (mRNAs) encoding these cytokines produced robust anticancer responses in a broad range of tumor microenvironments. The addition of mRNA encoding the T cell costimulator OX40L increased complete response rates in treated and untreated distal tumors compared to the cytokine mRNAs alone. Mice exhibiting complete responses were subsequently protected from tumor rechallenge. Treatments with these mRNA mixtures induced downstream cytokine and chemokine expression, and also activated multiple dendritic cell (DC) and T cell types. Consistent with this, efficacy was dependent on Batf3-dependent cross-presenting DCs and cytotoxic CD8+ T cells. IL-23/IL-36γ/OX40L triplet mRNA mixture triggered substantial immune cell recruitment into tumors, enabling effective tumor destruction irrespective of previous tumoral immune infiltrates. Last, combining triplet mRNA with checkpoint blockade led to efficacy in models otherwise resistant to systemic immune checkpoint inhibition. Human cell studies showed similar cytokine responses to the individual components of this mRNA mixture, suggesting translatability of immunomodulatory activity to human patients.

SPINK1 protein expression and prostate cancer progression.

PURPOSE: SPINK1 overexpression has been described in prostate cancer and is linked with poor prognosis in many cancers. The objective of this study was to characterize the association between SPINK1 overexpression and prostate cancer-specific survival. EXPERIMENTAL DESIGN: The study included 879 participants in the U.S. Physicians' Health Study and Health Professionals Follow-Up Study, diagnosed with prostate cancer (1983-2004) and treated by radical prostatectomy. Protein tumor expression of SPINK1 was evaluated by immunohistochemistry on tumor tissue microarrays. RESULTS: Seventy-four of 879 (8%) prostate cancer tumors were SPINK1 positive. Immunohistochemical data were available for PTEN, p-Akt, pS6, stathmin, androgen receptor (AR), and ERG (as a measure of the TMPRSS2:ERG translocation). Compared with SPINK1-negative tumors, SPINK1-positive tumors showed higher PTEN and stathmin expression, and lower expression of AR (P < 0.01). SPINK1 overexpression was seen in 47 of 427 (11%) ERG-negative samples and in 19 of 427 (4%) ERG-positive cases (P = 0.0003). We found no significant associations between SPINK1 status and Gleason grade or tumor stage. There was no association between SPINK1 expression and biochemical recurrence (P = 0.56). Moreover, there was no association between SPINK1 expression and prostate cancer mortality (there were 75 lethal cases of prostate cancer during a mean of 13.5 years follow-up; HR = 0.71; 95% confidence interval, 0.29-1.76). CONCLUSIONS: Our results suggest that SPINK1 protein expression may not be a predictor of recurrence or lethal prostate cancer amongst men treated by radical prostatectomy. SPINK1 and ERG protein expression do not seem to be entirely mutually exclusive, as some previous studies have suggested.

Using glow stick chemistry for biological imaging.

PURPOSE: This study describes an imaging strategy based on glow stick chemistry to non-invasively image oxidative stress and reactive oxygen species (ROS) production in living animals. PROCEDURES: Upon stimulation, phagocytes produce toxic levels of ROS to kill engulfed microorganisms. The mitochondrial respiratory chain continually generates low levels of superoxide (O2·(-)) that serve as a source for generation of downstream ROS, which function as regulatory signaling intermediaries. A ROS-reacting substrate, 2-methyl-6-[4-methoxyphenyl]-3,7-dihydroimidazo[1,2-a]pyrazin-3-one hydrochloride, was used as the chemical energy donor for generating energy transfer luminescence in phagosomes and mitochondria. RESULTS: Using targeted photoluminescent dyes with specific subcellular localization that serve as chemical energy recipients, our imaging data demonstrate proof-of-concept for using glow stick chemistry to visualize ROS production associated with phagocytosis and mitochondrial respiration in living mice. CONCLUSIONS: Glow stick imaging is a complementary hybrid of chemiluminescence and photoluminescence imaging, capable of generating red or far-red emission for deep tissue imaging.

Utility of multispectral imaging in automated quantitative scoring of immunohistochemistry.

BACKGROUND: Automated scanning devices and image analysis software provide a means to overcome the limitations of manual semiquantitative scoring of immunohistochemistry. Common drawbacks to automated imaging systems include an inability to classify tissue type and an inability to segregate cytoplasmic and nuclear staining. METHODS: Immunohistochemistry for the membranous marker α-catenin, the cytoplasmic marker stathmin and the nuclear marker Ki-67 was performed on tissue microarrays (TMA) of archival formalin-fixed paraffin-embedded tissue comprising 471 (α-catenin and stathmin) and 511 (Ki-67) cases of prostate adenocarcinoma. These TMA were quantitatively analysed using two commercially available automated image analysers, the Ariol SL-50 system and the Nuance system from CRi. Both systems use brightfield microscopy for automated, unbiased and standardised quantification of immunohistochemistry, while the Nuance system has spectral deconvolution capabilities. RESULTS: Overall concordance between scores from both systems was excellent (r=0.90; 0.83-0.95). The software associated with the multispectral imager allowed accurate automated classification of tissue type into epithelial glandular structures and stroma, and a single-step segmentation of staining into cytoplasmic or nuclear compartments allowing independent evaluation of these areas. The Nuance system, however, was not able to distinguish reliably between tumour and non-tumour tissue. In addition, variance in the labour and time required for analysis between the two systems was also noted. CONCLUSION: Despite limitations, this study suggests some beneficial role for the use of a multispectral imaging system in automated analysis of immunohistochemistry.

ß-Catenin is a useful adjunct immunohistochemical marker for the diagnosis of pulmonary lymphangioleiomyomatosis.

Lymphangioleiomyomatosis (LAM) is a rare multisystem disease leading to cystic destruction of the lung parenchyma and is associated with abnormal smooth muscle proliferation affecting airways, lymphatics, and blood vessels. LAM occurs sporadically or in association with the tuberous sclerosis complex (TSC). Recent evidence demonstrates the role of aberrant ß-catenin signaling in TSC. To further understand the pathogenesis of LAM and to examine the diagnostic usefulness of ß-catenin, we examined protein expression in 28 pulmonary LAM cases and 10 cases of renal angiomyolipoma resected from patients with sporadic LAM. Immunohistochemical analysis was performed for established markers of LAM cells (HMB45, estrogen receptor [ER]-α, and progesterone receptor [PR]) and ß-catenin. All LAM cases were positive for ß-catenin and demonstrated high specificity with overall immunoreactivity superior to HMB45, ER-α, and PR. Similar expression was demonstrated in renal angiomyolipoma. Our results indicate that ß-catenin is a useful marker of LAM and may be clinically useful in the diagnostic setting.

Met activation in non-small cell lung cancer is associated with de novo resistance to EGFR inhibitors and the development of brain metastasis.

Most non-small cell lung cancer (NSCLC) patients harboring activating epidermal growth factor receptor (EGFR) mutations respond to tyrosine kinase inhibitor (TKI) therapy. However, about 30% exhibit primary resistance to EGFR TKI therapy. Here we report that Met protein expression and phosphorylation were associated with primary resistance to EGFR TKI therapy in NSCLC patients harboring EGFR mutations, implicating Met as a de novo mechanism of resistance. In a separate patient cohort, Met expression and phosphorylation were also associated with development of NSCLC brain metastasis and were selectively enriched in brain metastases relative to paired primary lung tumors. A similar metastasis-specific activation of Met occurred in vitro in the isogenous cell lines H2073 and H1993, which are derived from the primary lung tumor and a metastasis, respectively, from the same patient. We conclude that Met activation is found in NSCLC before EGFR-targeted therapy and is associated with both primary resistance to EGFR inhibitor therapy and with the development of metastases. If confirmed in larger cohorts, our analysis suggests that patient tumors harboring both Met activation and EGFR mutation could potentially benefit from early intervention with a combination of EGFR and Met inhibitors.

Preclinical model of organotypic culture for pharmacodynamic profiling of human tumors.

Predicting drug response in cancer patients remains a major challenge in the clinic. We have perfected an ex vivo, reproducible, rapid and personalized culture method to investigate antitumoral pharmacological properties that preserves the original cancer microenvironment. Response to signal transduction inhibitors in cancer is determined not only by properties of the drug target but also by mutations in other signaling molecules and the tumor microenvironment. As a proof of concept, we, therefore, focused on the PI3K/Akt signaling pathway, because it plays a prominent role in cancer and its activity is affected by epithelial-stromal interactions. Our results show that this culture model preserves tissue 3D architecture, cell viability, pathway activity, and global gene-expression profiles up to 5 days ex vivo. In addition, we show pathway modulation in tumor cells resulting from pharmacologic intervention in ex vivo culture. This technology may have a significant impact on patient selection for clinical trials and in predicting response to small-molecule inhibitor therapy.

Identification of CD44v6(+)/CD24- breast carcinoma cells in primary human tumors by quantum dot-conjugated antibodies.

Breast carcinoma cells with the CD44+/CD24(low) phenotype have been reported to exhibit 'cancer stem cell' (CSC) characteristics on the basis of their enhanced tumorigenicity and self-renewal potential in immunodeficient mice. We used immunohistochemistry to study the expression of these proteins in whole tissue sections of human breast carcinoma. We found that the fraction of CD44v6+ cells is higher in estrogen receptor-positive carcinomas after neoadjuvant chemotherapy. We also performed double immunohistochemistry for CD44v6 and for the proliferation marker Ki67. We found that the relative number of quiescent carcinoma cells is higher in the CD44v6+ population than in the CD44v6- population in specific carcinoma subtypes. We then used quantum dots and spectral imaging to increase the number of antigens that could be visualized in a single tissue section. We found that anti-CD44v6 and CD24 antibodies that were directly conjugated to quantum dots retained their ability to recognize antigen in formalin-fixed, paraffin-embedded tissue sections. We then performed triple staining for CD44v6, CD24 and Ki67 to assess the proliferation of each sub-population of breast carcinoma cells. Our results identify differences between CD44v6-positive and CD44v6-negative breast carcinoma cells in vivo and provide a proof of principle that quantum dot-conjugated antibodies can be used to study specific sub-populations of cancer cells defined by multiple markers in a single tissue section.

c-Jun amplification and overexpression are oncogenic in liposarcoma but not always sufficient to inhibit the adipocytic differentiation programme.

Genomic amplification of c-Jun and its upstream kinases have been implicated as a mechanism of progression from well-differentiated to dedifferentiated liposarcoma. To further define the role of c-Jun in liposarcoma progression, we performed immunohistochemistry for c-Jun and its activating kinase ASK1 on a series of liposarcomas (n = 81). We correlated the results with fluorescence in situ hybridization to detect c-Jun amplification. We also derived new cell lines from dedifferentiated liposarcomas with c-Jun amplification. c-Jun protein is expressed in the majority of dedifferentiated liposarcomas (91%) and their well-differentiated components (59%), but only in the minority of pure well-differentiated liposarcomas (27%). When c-Jun is amplified in dedifferentiated liposarcoma, it is interspersed with amplified MDM2 on ring and giant marker chromosomes. MDM2 amplification is one of the earliest events in liposarcoma development, and these results suggest that c-Jun was amplified at a similar time in the evolution of the tumour. In addition, shRNA to c-Jun in c-Jun-amplified liposarcoma cells reduces cell number in vitro and inhibits tumour formation in vivo without an observable effect on the differentiation state of the liposarcoma cells. Thus, c-Jun amplification is oncogenic in liposarcomas but not always sufficient for inhibition of adipocytic differentiation.

Overexpression of fatty acid synthase is associated with palmitoylation of Wnt1 and cytoplasmic stabilization of beta-catenin in prostate cancer.

Fatty acid synthase (FASN), a key metabolic enzyme for liponeogenesis highly expressed in several human cancers, displays oncogenic properties such as resistance to apoptosis and induction of proliferation when overexpressed. To date, no mechanism has been identified to explain the oncogenicity of FASN in prostate cancer. We generated immortalized prostate epithelial cells (iPrECs) overexpressing FASN, and found that (14)C-acetate incorporation into palmitate synthesized de novo by FASN was significantly elevated in immunoprecipitated Wnt-1 when compared to isogenic cells not overexpressing FASN. Overexpression of FASN caused membranous and cytoplasmic beta-catenin protein accumulation and activation, whereas FASN knockdown by short-hairpin RNA resulted in a reduction in the extent of beta-catenin activation. Orthotopic transplantation of iPrECs overexpressing FASN in nude mice resulted in invasive tumors that overexpressed beta-catenin. A strong significant association between FASN and cytoplasmic (stabilized) beta-catenin immunostaining was found in 862 cases of human prostate cancer after computerized subtraction of the membranous beta-catenin signal (P<0.001, Spearman's rho=0.33). We propose that cytoplasmic stabilization of beta-catenin through palmitoylation of Wnt-1 and subsequent activation of the pathway is a potential mechanism of FASN oncogenicity in prostate cancer.