T Cells Engineered to Express Immunoreceptors Targeting the Frequently Expressed Medullary Thyroid Cancer Antigens Calcitonin, CEA, and RET M918T.

Background: Medullary thyroid cancer (MTC) is a rare malignancy originating from the calcitonin-producing C cells of the thyroid. Despite recent therapeutic advances, metastatic MTC remains incurable. Adoptive cell therapy (ACT) using genetically engineered T cells targeting either tissue-restricted tumor-associated antigens or mutated neoantigens has led to durable remissions in other metastatic solid tumors. The majority of MTC express the tumor-associated antigens calcitonin and carcinoembryonic antigen (CEA), and ∼40% of MTC harbor the RET M918T oncogenic driver mutation. Methods: We developed and characterized three immunoreceptors that recognize extracellular CEA, a calcitonin epitope presented by HLA-A*24:02, or an RET M918T neoepitope restricted by HLA-DPB1*04:01/02. The chimeric antigen receptor (CAR) targeting CEA was synthetically designed, while the T cell receptors (TCRs) targeting calcitonin and RET M918T were isolated from a transgenic mouse and patient with MTC, respectively. These immunoreceptors were genetically engineered into peripheral blood T cells and tested for antigen specificity and antitumor activity. Results: T cells expressing the anti-CEA CAR or the calcitonin-reactive TCR produced effector cytokines and displayed cytotoxicity against cell lines expressing their cognate antigen in vitro. In immunodeficient mice harboring a human MTC cell line, the adoptive transfer of T cells engineered to express the anti-CEA CAR or calcitonin-reactive TCR led to complete tumor regression. T cells expressing the HLA-DPB1*04:01/02-restricted TCR targeting RET M918T, which was cloned from peripheral blood CD4+ T cells of a patient with MTC, demonstrated specific reactivity against cells pulsed with the mutated peptide and MTC tumor cells that expressed HLA-DPB1*04:01 and RET M918T. Conclusion: The preclinical data presented herein demonstrate the potential of using genetically engineered T cells targeting CEA, calcitonin, and/or RET M918T to treat metastatic MTC.

Using pan-TRK and RET Immunohistochemistry for the Detection of Fusion Types of Salivary Gland Secretory Carcinoma.

Secretory carcinoma (SC) is a low-grade salivary gland carcinoma characterized by recurrent ETV6 rearrangements. Most cases have ETV6-NTRK3 fusions, while the minority of cases have non-NTRK3 fusions, including ETV6-RET and ETV6-MET. Detection of the fusion partner has become important, as there are TRK or RET inhibitors that may benefit patients with advanced SC. Currently, there are different methods to detect gene rearrangement in SCs, such as next-generation sequencing, reverse transcription-polymerase chain reaction, or fluorescence in situ hybridization. Immunohistochemistry (IHC) has greater accessibility, quick turnaround time, and can serve as a screening tool for confirmatory molecular tests. Pan-TRK and RET antibodies have been used to detect gene fusions in different tumors. Here, pan-TRK and RET IHC assays were performed on 28 salivary gland SCs, including 27 cases with ETV6-NTRK3 and one with ETV6-RET fusion confirmed by fluorescence in situ hybridization. Pan-TRK staining was positive in 26/27 (96.3%) of NTRK3 fusion-positive SCs with a nuclear staining pattern in more than 50% of tumor cells, and negative in the RET-rearranged case. RET IHC showed positive staining in most cases (26/28), but only three cases (including the RET-rearranged case) had diffuse and strong staining. RET IHC can be considered an effective screening test when diffuse/strong reactivity is present in pan-TRK IHC-negative cases. This study showed that pan-TRK staining has high sensitivity and specificity for SC with NTRK3 fusion. Whereas pan-TRK IHC is a useful screening method, further studies are needed to assess the value of RET IHC as a second sequential step.

Pan-tropomyosin receptor kinase immunoreactivity, ETV6-NTRK3 fusion subtypes, and RET rearrangement in salivary secretory carcinoma.

Salivary secretory carcinoma (SASC) is frequently associated with ETV6-neurotrophic tyrosine receptor kinase (NTRK) 3 fusion and more rarely with RET, MET, or ALK rearrangement. We aimed to elucidate the potential diagnostic utility of pan-tropomyosin receptor kinase (Trk) immunohistochemistry and its relationship with the fusion gene subtype in SASC. We examined 33 cases of SASC for immunoexpression of pan-Trk, ALK and ROS1, and gene rearrangement of the ETV6, NTRK3, and RET genes using fluorescence in situ hybridization (FISH) and reverse transcription-polymerase chain reaction (RT-PCR). Thirty (90.9%) of 33 SASCs harbored ETV6-NTRK3 fusion gene transcripts by RT-PCR and/or both ETV6 and NTRK3 gene rearrangements by FISH, and 3 cases (9.1%) had RET gene rearrangement. Most NTRK3-rearranged SASCs (27/33 cases; 81.8%) had conventional ETV6 exon 5-NTRK3 exon 15 fusion, whereas 2 cases (6.1%) had both the conventional fusion and a novel ETV6 exon 4-NTRK3 exon 15 fusion variant. In the remaining one case (3%), only FISH revealed both ETV6 and NTRK3 rearrangements, suggesting an ETV6-NTRK3 fusion with an as yet undetermined break point. All 30 SASCs with ETV6-NTRK3 fusion and/or NTRK3 rearrangement showed nuclear and cytoplasmic immunoreactivity for pan-Trk. In contrast, 3 SASCs with RET rearrangement showed negative or only weak cytoplasmic staining for pan-Trk. There was no case harboring ALK and ROS1 rearrangements. All 17 non-SASC tumors were negative for pan-Trk. The results suggest that nuclear and cytoplasmic immunoreactivity for pan-TRK may be helpful to identify ETV6-NTRK3-fused SASCs and to distinguish them from RET-rearranged SASCs and morphological mimics.

Immuno-PET Detects Changes in Multi-RTK Tumor Cell Expression Levels in Response to Targeted Kinase Inhibition.

Receptor tyrosine kinase (RTK) coexpression facilitates tumor resistance due to redundancies in the phosphatidylinositol-3'-kinase/protein kinase B and KRAS/extracellular-signal-regulated kinase signaling pathways, among others. Crosstalk between the oncogenic RTK hepatocyte growth factor receptor (MET), epidermal growth factor receptor (EGFR), and human epidermal growth factor receptor 2 (HER2) are involved in tumor resistance to RTK-targeted therapies. Methods: In a relevant renal cell carcinoma patient-derived xenograft model, we use the 89Zr-labeled anti-RTK antibodies (immuno-PET) onartuzumab, panitumumab, and trastuzumab to monitor MET, EGFR, and HER2 protein levels, respectively, during treatment with agents to which the model was resistant (cetuximab) or sensitive (INC280 and trametinib). Results: Cetuximab treatment resulted in continued tumor growth, as well as an increase in all RTK protein levels at the tumor in vivo on immuno-PET and ex vivo at the cellular level. Conversely, after dual MET/mitogen-activated protein kinase inhibition, tumor growth was significantly blunted and corresponded to a decrease in RTK levels. Conclusion: These data show the utility of RTK-targeted immuno-PET to annotate RTK changes in protein expression and inform tumor response to targeted therapies.

Advances in Targeting RET-Dependent Cancers.

RET alterations have been characterized as oncogenic drivers in multiple cancers. The clinical validation of highly selective RET inhibitors demonstrates the utility of specific targeting of aberrantly activated RET in patients with cancers such as medullary thyroid cancer or non-small cell lung cancer. The remarkable responses observed have opened the field of RET-targeted inhibitors. In this review, we seek to focus on the impact of therapeutic RET targeting in cancers. SIGNIFICANCE: Successful clinical translation of selective RET inhibitors is poised to alter the therapeutic landscape of altered cancers. Questions that clearly need to be addressed relate to the ability to maintain long-term inhibition of tumor cell growth, how to prepare for the potential mechanisms of acquired resistance, and the development of next-generation selective RET inhibitors.

RET-mediated modulation of tumor microenvironment and immune response in multiple endocrine neoplasia type 2 (MEN2).

Medullary thyroid carcinomas (MTC) arise from thyroid parafollicular, calcitonin-producing C-cells and can occur either as sporadic or as hereditary diseases in the context of familial syndromes, including multiple endocrine neoplasia 2A (MEN2A), multiple endocrine neoplasia 2B (MEN2B) and familial MTC (FMTC). In a large fraction of sporadic cases, and virtually in all inherited cases of MTC, activating point mutations of the RET proto-oncogene are found. RET encodes for a receptor tyrosine kinase protein endowed with transforming potential on thyroid parafollicular cells. As in other cancer types, microenvironmental factors play a critical role in MTC. Tumor-associated extracellular matrix, stromal cells and immune cells interact and influence the behavior of cancer cells both in a tumor-promoting and in a tumor-suppressing manner. Several studies have shown that, besides the neoplastic transformation of thyroid C-cells, a profound modification of tumor microenvironment has been associated to the RET FMTC/MEN2-associated oncoproteins. They influence the surrounding stroma, activating cancer-associated fibroblasts (CAFs), promoting cancer-associated inflammation and suppressing anti-cancer immune response. These mechanisms might be exploited to develop innovative anti-cancer therapies and novel prognostic tools in the context of familial, RET-associated MTC.

The neurotrophic factor receptor RET regulates IL-10 production by in vitro polarised T helper 2 cells.

T helper (Th) cells are critical players in the modulation of immune response outcomes. Activation of Th cells gives rise to various subsets of effector cells that are controlled via specialised regulatory T cells or through self-regulation via production of IL-10. However, the environmental factors that regulate IL-10 production by Th cells remain poorly understood. Here, we show that the neurotrophic factor receptor rearranged during transfection (RET) downregulates IL-10 production by Th cells from C57BL/6 mice. We found that effector Th cells express RET and that RET's neurotrophic factor partners are mainly produced by LN stromal cells, allowing context-dependent Th-cell regulation. Despite being dispensable for Th-cell homeostasis, RET controls IL-10 production in Th2 cells: RET-deficient Th cells exhibited increased IL-10 production, while triggering of Th1/2 cells with neurotrophic factors, namely glial-derived neurotrophic factor and neurturin, decreased the expression of IL-10. In agreement, the important IL-10 transcription factor Maf was upregulated in RET-deficient Th2 cells and down-regulated upon RET signalling activation by glial-derived neurotrophic factor family ligands. Thus, our study uncovers neurotrophic factors as novel regulators of Th-cell function, revealing that Th cells and neurons can be regulated by similar signals in tissue-specific responses.

Inflammatory monocytes are potent antitumor effectors controlled by regulatory CD4+ T cells.

The present study evaluates the impact of immune cell populations on metastatic development in a model of spontaneous melanoma [mice expressing the human RET oncogene under the control of the metallothionein promoter (MT/ret mice)]. In this model, cancer cells disseminate early but remain dormant for several weeks. Then, MT/ret mice develop cutaneous metastases and, finally, distant metastases. A total of 35% of MT/ret mice develop a vitiligo, a skin depigmentation attributable to the lysis of normal melanocytes, associated with a delay in tumor progression. Here, we find that regulatory CD4(+) T cells accumulate in the skin, the spleen, and tumor-draining lymph nodes of MT/ret mice not developing vitiligo. Regulatory T-cell depletion and IL-10 neutralization led to increased occurrence of vitiligo that correlated with a decreased incidence of melanoma metastases. In contrast, inflammatory monocytes/dendritic cells accumulate in the skin of MT/ret mice with active vitiligo. Moreover, they inhibit tumor cell proliferation in vitro through a reactive oxygen species-dependent mechanism, and both their depletion and reactive oxygen species neutralization in vivo increased tumor cell dissemination. Altogether, our data suggest that regulatory CD4(+) T cells favor tumor progression, in part, by inhibiting recruitment and/or differentiation of inflammatory monocytes in the skin.

Evaluation of the diagnostic enigma in Hirschsprung disease.

AIM: We aimed to investigate intraoperative diagnosis rate of aganglionosis with hematoxylin eosin (HE) staining, to review the current diagnostic procedures in Hirschsprung disease (HD), to inquire the validity of enzyme staining in diagnosis of HD and to evaluate the utility of ret oncoprotein (RET) antibody for detecting ganglion cells (GC) in paraffin sections. METHODS: Two hundred and thirty three children who are suspected to have HD were included in this study. A total of 302 surgical procedures related to diagnosis and treatment of HD were performed. One to 19 samples (3.5 ± 2.91) per each case were examined with intraoperative pathological consultation. Although establishing primary diagnoses of HD by frozen sections (FS) examination and performing a one-step approach for treatment have been aimed, consecutive surgical operations were required in 30 cases (12.9%). One hundred and sixty three cases (70%) were male. Seventy eight cases (33.5%) were in neonatal period (mean=13.5 ± 9.7 days). Only 56 cases were older than 1 year. GC were absent in 137 of cases. Presence of GC with FS examination weren't decided and prior colostomies were performed in 18 cases (7.7%). RESULTS: There were no discrepancies between the FS diagnoses and final diagnoses of the cases except these children. Requisition of consecutive surgical procedures interestingly was lower in neonates than others (P=0.01). Because of long duration, technical difficulty and standardization problems; not only immune histochemical stains but also enzyme stains should not be prefer for demonstration of GC during surgery. CONCLUSION: We suppose that if one-step approach is desired, the intraoperative evaluation of HE staining FS by experienced pathologists is still gold standard in the diagnosis of HD.

T cells contribute to tumor progression by favoring pro-tumoral properties of intra-tumoral myeloid cells in a mouse model for spontaneous melanoma.

Tumors affect myelopoeisis and induce the expansion of myeloid cells with immunosuppressive activity. In the MT/ret model of spontaneous metastatic melanoma, myeloid cells are the most abundant tumor infiltrating hematopoietic population and their proportion is highest in the most aggressive cutaneous metastasis. Our data suggest that the tumor microenvironment favors polarization of myeloid cells into type 2 cells characterized by F4/80 expression, a weak capacity to secrete IL-12 and a high production of arginase. Myeloid cells from tumor and spleen of MT/ret mice inhibit T cell proliferation and IFNγ secretion. Interestingly, T cells play a role in type 2 polarization of myeloid cells. Indeed, intra-tumoral myeloid cells from MT/ret mice lacking T cells are not only less suppressive towards T cells than corresponding cells from wild-type MT/ret mice, but they also inhibit more efficiently melanoma cell proliferation. Thus, our data support the existence of a vicious circle, in which T cells may favor cancer development by establishing an environment that is likely to skew myeloid cell immunity toward a tumor promoting response that, in turn, suppresses immune effector cell functions.

Prevention of spontaneous tumor development in a ret transgenic mouse model by ret peptide vaccination with indoleamine 2,3-dioxygenase inhibitor 1-methyl tryptophan.

The present study investigated an immunotherapeutic strategy for rearranged during transfection proto-oncogene (ret)-associated carcinomas in a transgenic MT/ret 304/B6 mouse model in which spontaneous tumors develop due to overexpression of the ret gene. A Ret peptide vaccine comprising an extracellular fragment of Ret protein and Th1-polarized immunoregulator CpG oligonucleotide (1826) induced strong and specific cellular and humoral immune responses in wild-type C57BL/6 mice, showing that the Ret peptide has a strong immunogenic potential as part of an antitumor vaccine. In MT/ret 304/B6 mice, however, the vaccine was only modestly effective as an inducer of the humoral immune response, and it failed to elicit a T-cell response. An immunohistochemical analysis revealed marked indoleamine 2,3-dioxygenase expression after immunization with Ret peptide vaccine in the lymph nodes and spleens of MT/ret 304/B6 mice. The systemic administration of the potent inhibitor of indoleamine 2,3-dioxygenase 1-methyl tryptophan (1MT) along with Ret vaccine produced a significant increase in tumor-specific cytotoxic activity. A delay in spontaneous tumor development was also observed in the MT/ret 304/B6 mice to which the Ret vaccine and 1MT were administered. These results indicate that an improved Ret vaccine composed of Ret peptide plus CpG oligonucleotide plus 1MT is a potential therapeutic strategy for treatment of ret-associated carcinomas.