The pathogenic nature of cancer is attributed, at least in part, to the ability of tumors cells to induce systemic and local mechanisms of immune tolerance. However, we previously reported that tumor-free survival in up to 100% of tolerized HER-2/neu transgenic mice can be achieved by administration of neu-specific mAb concurrently with a HER-2/neu-expressing, GM-CSF-secreting whole cell vaccine. In this report, we show that one mechanism of improved antitumor activity induced by the combination of these 2 neu-targeted interventions was enhanced Fc-mediated activation of APCs. Specifically, in vivo studies demonstrated localization of radiolabeled neu-specific mAb at the vaccine site. Subsequently, increased accumulation of neu-specific mAb at the vaccine-draining lymph node correlated with increased vaccine cell uptake by DCs in vivo. This led to enhancement of CD8 + neu-specific T cell function in terms of proliferation, cytokine production, and central memory development. Thus, the administration of a neu-specific mAb with a neu-targeted GM-CSF-secreting tumor vaccine enhanced induction of neu-specific CD8 + T cells through Fc-mediated activation of DCs. This multimodality attack on the same tumor antigen may have the potential to overcome tolerance to self antigens and weaken the immunosuppressive networks within the tumor microenvironment.

BackgroundCancer vaccines are designed to activate and enhance cancer-antigen-targeted T cells that are suppressed through multiple mechanisms of immune tolerance in cancer-bearing hosts. T regulatory cell (Treg) suppression of tumor-specific T cells is one barrier to effective immunization. A second mechanism is the deletion of high avidity tumor-specific T cells, which leaves a less effective low avidity tumor specific T cell repertoire available for activation by vaccines. Treg depleting agents including low dose cyclophosphamide (Cy) and antibodies that deplete CD25-expressing Tregs have been used with limited success to enhance the potency of tumor-specific vaccines. In addition, few studies have evaluated mechanisms that activate low avidity cancer antigen-specific T cells. Therefore, we developed high and low avidity HER-2/neu-specific TCR transgenic mouse colonies specific for the same HER-2/neu epitope to define the tolerance mechanisms that specifically affect high versus low avidity tumor-specific T cells.Methodology/Principal FindingsHigh and low avidity CD8+ T cell receptor (TCR) transgenic mice specific for the breast cancer antigen HER-2/neu (neu) were developed to provide a purified source of naïve, tumor-specific T cells that can be used to study tolerance mechanisms. Adoptive transfer studies into tolerant FVB/N-derived HER-2/neu transgenic (neu-N) mice demonstrated that high avidity, but not low avidity, neu-specific T cells are inhibited by Tregs as the dominant tolerizing mechanism. High avidity T cells persisted, produced IFNγ, trafficked into tumors, and lysed tumors after adoptive transfer into mice treated with a neu-specific vaccine and low dose Cy to deplete Tregs. Analysis of Treg subsets revealed a Cy-sensitive CD4+Foxp3+CD25low tumor-seeking migratory phenotype, characteristic of effector/memory Tregs, and capable of high avidity T cell suppression.Conclusion/SignificanceDepletion of CD25low Tregs allows activation of tumor-clearing high avidity T cells. Thus, the development of agents that specifically deplete Treg subsets should translate into more effective immunotherapies while avoiding autoimmunity.

Summary Patient-derived cancer organoids hold great potential to accurately model and predict therapeutic responses. Efficient organoid isolation methods that minimize post-collection manipulation of tissues would improve adaptability, accuracy, and applicability to both experimental and real-time clinical settings. Here we present a simple and minimally invasive fine-needle aspiration (FNA)-based organoid culture technique using a variety of tumor types including gastrointestinal, thyroid, melanoma, and kidney. This method isolates organoids directly from patients at the bedside or from resected tissues, requiring minimal tissue processing while preserving the histologic growth patterns and infiltrating immune cells. Finally, we illustrate diverse downstream applications of this technique including in vitro high-throughput chemotherapeutic screens, in situ immune cell characterization, and in vivo patient-derived xenografts. Thus, routine clinical FNA-based collection techniques represent an unappreciated substantial source of material that can be exploited to generate tumor organoids from a variety of tumor types for both discovery and clinical applications.

Patients with MYC/BCL2 double-hit lymphoma (DHL) are known to have an aggressive clinical course and to respond poorly to various therapies including intensive chemotherapy and stem cell transplant. Less is known about high-grade B-cell lymphoma with MYC rearrangement without concomitant BCL2 and BCL6 rearrangement, designated here as single-hit lymphoma (SHL). In this study, we assessed 61 cases of SHL and compared them with 83 cases of DHL, all confirmed by MYC, BCL2, and BCL6 fluorescence in situ hybridization studies. Although many clinicopathologic features overlap between patients with SHL and those with DHL, distinct features were observed in SHL. Patients with SHL had tumors with a higher prevalence of p53 overexpression (P=0.047), less frequent expression of CD10, BCL2, and BCL6 (P<0.05), and less often had a history of low-grade B-cell lymphoma (P=0.01). In addition, MYC was more frequently partnered with IGH in SHL than in DHL (P=0.04). With a median follow-up of 25 months, the overall survival of 61 SHL patients was poor and similar to that of DHL patients (2-y overall survival rate of 41% in SHL vs. 48% in DHL; P=0.35) and significantly worse than patients with diffuse large B-cell lymphoma and B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma, without MYC and BCL2 rearrangements (P<0.0001). In conclusion, patients with SHL have distinct clinicopathologic features but a similar poor prognosis compared with patients with MYC/BCL2 DHL. The poor prognosis of patients with SHL may be partially related to the higher frequency and level of p53 expression in these tumors.

Background The American Thyroid Association guidelines task force currently recommends definitive thyroidectomy or lobectomy after an indeterminate thyroid biopsy in children. This recommendation is based on evidence of a greater incidence and a higher risk of malignancy compared with adults in earlier pediatric studies. Such management may lead to overtreatment and unnecessary surgery for many children in the United States. Methods The objective of the current study was to re‐evaluate pediatric thyroid nodules and assess the overall percentages and malignancy rates for indeterminate thyroid biopsies in children. In total, 302 pediatric thyroid fine‐needle aspirations (FNAs) were analyzed retrospectively (2001‐2018). Distribution percentages and malignancy rates were calculated for each category of The Bethesda System for Reporting Thyroid Cytopathology (TBSRTC). Results Two indeterminate TBSRTC groups (atypia of undetermined significance/follicular lesion of undetermined significance and follicular neoplasm/suspicious for a follicular neoplasm) had much lower distribution percentages and malignancy rates compared with earlier pediatric series and American Thyroid Association guidelines. A meta‐analysis further supported these findings and demonstrated distinctly different malignancy rates for the indeterminate groups (atypia of undetermined significance/follicular lesion of undetermined significance, follicular neoplasm/suspicious for a follicular neoplasm, and suspicious for malignancy), suggesting the need for TBSRTC category‐specific management recommendations rather than a nondiscriminatory, up‐front surgical approach. Conclusions Adult patients with indeterminate preoperative thyroid cytopathology are followed by repeat biopsy and possibly molecular testing before undergoing definitive surgery. However, in children, the guidelines are considerably more aggressive and recommend definitive surgery after the first indeterminate thyroid biopsy. Here, the largest pediatric cohort to date with meta‐analysis is presented, and the authors propose a re‐evaluation of this up‐front approach to pediatric thyroid care.

The prevalence of thyroid carcinoma is increasing and represents the most common endocrine malignancy, with papillary thyroid carcinoma (PTC) being the most frequent subtype. The genetic alterations identified in PTCs fail to distinguish tumors with different clinical behaviors, such as extra-thyroidal extension and lymph node metastasis. We hypothesize that the immune microenvironment may play a critical role in tumor invasion and metastasis. Computational immunogenomic analysis was performed on 568 PTC samples in The Cancer Genome Atlas using CIBERSORT, TIMER and TIDE deconvolution analytic tools for characterizing immune cell composition. Immune cell infiltrates were correlated with histologic type, mutational type, tumor pathologic T stage and lymph node N stage. Dendritic cells (DCs) are highly associated with more locally advanced tumor T stage (T3/T4, odds ratio (OR) = 2.6, CI = 1.4–4.5, P = 5.4 × 10−4). Increased dendritic cells (OR = 3.4, CI = 1.9–6.3, P = 5.5 × 10−5) and neutrophils (OR = 10.5, CI = 2.7–44, P = 8.7 × 10−4) significantly correlate with lymph node metastasis. In addition, dendritic cells positively correlate with tall cell morphology (OR = 4.5, CI = 1.6–13, P = 4.9 × 10−3) and neutrophils negatively correlate with follicular morphology (OR = 1.3 × 10−3, CI = 5.3 × 10−5–0.031, P = 4.1 × 10−5). TIDE analysis indicates an immune-exclusive phenotype that may be mediated by increased galectin-3 found in PTCs. Thus, characterization of the PTC immune microenvironment using three computational platforms shows that specific immune cells correlate with mutational type, histologic type, local tumor extent and lymph node metastasis. Immunologic evaluation of PTCs may provide a better indication of biologic behavior, resulting in the improved diagnosis and treatment of thyroid cancer.

The evaluation of dysplasia can be challenging on Pap tests from transgender patients on androgen therapy. The cohort evaluated had higher rates of unsatisfactory and abnormal Pap tests. Pathologists should be familiar with the distinctive cytomorphological changes in the Pap tests from patients on androgen therapy to evaluate them appropriately.

Key Points Question Are pediatric thyroid nodules amenable to cancer prediction by genomic classification? Findings In this retrospective study of 95 pediatric patients with thyroid nodules, surgical samples underwent next-generation sequencing and genomic classification. Testing defined the unique molecular landscape of pediatric thyroid nodules (which, as opposed to adults, comprised more frequent gene fusions and DICER1 variants) and identified a sensitivity of 96% and specificity of 78% regarding cancer detection. Meaning The study results suggest that although the molecular landscape of pediatric thyroid nodules is different than in adults, it remains amenable to multigene genomic classification, which may help prevent potentially unnecessary diagnostic surgeries.