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Immune checkpoint therapies exhibit impressive efficacy in some patients with melanoma or lung cancer, but the lack of response in most cases presses the question of how general efficacy can be improved. In addressing this question, we generated a preclinical tumor model to study anti-PD-1 resistance by in vivo passaging of Kras-mutated, p53-deficient murine lung cancer cells (p53R172HΔg/+K-rasLA1/+) in a syngeneic host exposed to repetitive dosing with anti-mouse PD-1 antibodies. PD-L1 (CD274) expression did not differ between the resistant and parental tumor cells. However, the expression of important molecules in the antigen presentation pathway, including MHC class I and II, as well as β2-microglobulin, were significantly downregulated in the anti-PD-1–resistant tumors compared with parental tumors. Resistant tumors also contained fewer CD8+ (CD8α) and CD4+ tumor-infiltrating lymphocytes and reduced production of IFNγ. Localized radiotherapy induced IFNβ production, thereby elevating MHC class I expression on both parental and resistant tumor cells and restoring the responsiveness of resistant tumors to anti-PD-1 therapy. Conversely, blockade of type I IFN signaling abolished the effect of radiosensitization in this setting. Collectively, these results identify a mechanism of PD-1 resistance and demonstrate that adjuvant radiotherapy can overcome resistance. These findings have immediate clinical implications for extending the efficacy of anti-PD-1 immune checkpoint therapy in patients. Cancer Res; 77(4); 839–50. ©2016 AACR.

This figure shows that PD-L1 expression is not quantitatively altered on the anti-PD-1 resistant tumor cells, compared to parental tumor cells.

This figure shows that CD11b+Gr-1+ cells from anti-PD-1 resistant tumors also show downregulation of MHC class I molecules

This figure shows that the immune suppressive MDSCs and regulatory T cell percentages in parental and anti-PD-1 resistant tumor models.

This figure addresses the reviewer's questions about the effect of blocking type I IFN on tumor cells and tumor-infiltrating lymphocytes.

All the figure legend for supplementary figures

Although an abundance of evidence support that preschoolers use reciprocity as a reply to others, lesser is known about how they use this strategy in initiating social interactions. Aiming to explore this question, the current study focused on two forms of prospective reciprocity, direct and indirect (downstream) reciprocity. Two studies were conducted in which the chance for prospective reciprocity was implicit (study 1) and explicit (study 2). Specifically, 4- to 6-year-olds were asked to share stickers with a non-shown recipient, a shown recipient, or a non-shown recipient while a witness was observing. In study 1, preschoolers did not know whether the shown recipient/witness would interact with them later. In study 2, they knew the shown recipient/witness would be asked to share with them subsequently. Results revealed that, despite the implicit/explicit chance of prospective reciprocity, preschoolers shared more in the prospective direct reciprocity condition than the control/prospective indirect downstream reciprocity conditions. In addition, comparing the two studies found no difference found between the implicit and explicit situations. Overall, these findings indicate that preschoolers have taken direct reciprocity, rather than indirect (downstream) reciprocity in guiding their initial sharing with others. Implications of these findings are further discussed.

To evaluate the effect of decoction prepared with Yang-activating and stasis-eliminating medicinals from Traditional Chinese Medicine (TCM) on the intestinal mucosal permeability in rats with ulcerative colitis induced by dextran sulfate sodium (DSS).Totally 55 male Wistar rats (body weight of 170-190 g) were randomly divided into the blank group (n = 10) and the model duplication group (n = 45). The blank group was not intervened, while the other was modeled with 5% dextran sulfate sodium by gavaging in a dosage of 4 mL per day to induce ulcerative colitis, a total of 7 days. Then, the model rats were divided into model blank group, mesalazine group and TCM group, and each group was consisted of 15 rats. They were given retention enema 10 min with normal saline, mesalazine enema (0.036 g/mL), and Yang-activating and stasis-eliminating decoction [0.54 g/mL of a decoction boiled by Puhuang (Pollen Typhae), Xiebai (Bulbus Allii Macrostemonis) and Wulingzhi (Faeces Trogopteri)] for 10 days respectively. Afterwards, all of the rats were evaluated by disease activity index (DAI), histological changes of distal colon, expression of occludin protein and ultrastructure of intestinal epithelial cells. Furthermore, ratio of lactulose to mannitol (L/M) discharged in urine was evaluated.Comparing the results between TCM and model control groups, scores of DAI and histological lesions decreased significantly (P = 0.0000.01), ultrastructures of intestinal epithelial cells and tight junctions were more complete. The expression of occludin protein (P = 0.0010.01) increased while the L/M value decreased significantly (P = 0.0000.01) in TCM group. There was no statistical difference between the TCM and mesalazine groups in results of each item (P0.05).The decoction prepared with Yang-activating and stasis-eliminating TCM medicianls can restore intestinal mucosal epithelial cells and tight junctions the model rats with ulcerative colitis; it can reduce histological lesions and protect the permeability of intestinal mucosa barrier in the rats as well.

413 Background: Combination therapy with tyrosine kinase inhibitors and anti-PD-1 antibodies can allow selected patients with initially unresectable HCC to convert to surgical resection. We evaluated conversion therapy with a triple combination of lenvatinib (LEN), anti-PD-1 antibodies and transarterial therapy. Methods: We retrospectively searched medical records from 117 consecutive patients with unresectable/advanced HCC who received triple combination therapy between Dec 2018 and Oct 2020 at Tianjin Medical University Cancer Institute & Hospital. Eligible patients were required to have potentially resectable HCC, defined as meeting ≥1 of the following criteria: 1) estimated future liver remnant after radical (R0) resection of < 40% or < 30% in patients with/without cirrhosis, respectively; 2) R0 resection technically difficult to complete; 3) Child-Pugh score ≥7, ECOG performance status ≥1; 4) tumor thrombus in the main portal vein or inferior vena cava; 5) resectable extrahepatic metastases. The primary endpoint was the proportion of patients who underwent a successful resection (conversion rate). Secondary endpoints included objective response rate (ORR), disease control rate (DCR), 6-month disease-free survival (DFS) and safety. Results: Of 37 patients included in the analysis, all received LEN and anti-PD-1 antibodies with transarterial chemoembolization (TACE, n = 22), hepatic arterial infusion chemotherapy (HAIC, n = 9) or both (n = 6). The conversion rate was 40.5% (15) and the ORR and DCR were 67.7% and 86.5% by RECIST v1.1 and 75.7% and 86.5% by mRECIST, respectively. The median conversion time was 4 months (range: 2-15). After a median postoperative follow-up time of 10 months (95% CI, 6.42-13.58), the 6-month DFS rate was 93%. Overall, 89.2% of patients had ≥1 treatment emergent adverse event (TEAE) and 29.7% experienced a Grade 3/4 TEAE, the most common was hypertension (18.9%, n = 7). Conclusions: A triple combination of LEN, anti-PD-1 antibodies and transarterial therapy was well tolerated and effective at converting potentially resectable HCC to resectable disease. These findings warrant confirmation by future prospective studies.

Pulmonary blastoma (PB) is a rare aggressive lung malignancy with a poor prognosis. Surgical resection is the treatment of choice for localized disease, and there are no standard treatment guidelines for metastatic PB. Due to its rareness, its molecular profile has not been elucidated. We present the first case of classic biphasic pulmonary blastoma (CBPB) with CD74-ROS1 rearrangement in a 44-year-old Asian female with stage IV disease diagnosed using capture-based ultra-deep targeted sequencing. It has been reported that ROS1 rearranged lung adenocarcinoma and squamous cell carcinoma are sensitive to crizotinib, an ALK/MET/ ROS1 multitargeted tyrosine kinase inhibitor. However, its efficacy has not been reported in CBPB patients harboring ROS1 rearrangement. This CBPB patient was given crizotinib and she achieved partial response after 1 month of treatment. We report the first clinical evidence of efficacy shown by crizotinib for targeting CD74-ROS1 fusion in CBPB.

Immune checkpoint therapies exhibit impressive efficacy in some patients with melanoma or lung cancer, but the lack of response in most cases presses the question of how general efficacy can be improved. In addressing this question, we generated a preclinical tumor model to study anti-PD-1 resistance by in vivo passaging of Kras-mutated, p53-deficient murine lung cancer cells (p53R172HΔg/+K-rasLA1/+) in a syngeneic host exposed to repetitive dosing with anti-mouse PD-1 antibodies. PD-L1 (CD274) expression did not differ between the resistant and parental tumor cells. However, the expression of important molecules in the antigen presentation pathway, including MHC class I and II, as well as β2-microglobulin, were significantly downregulated in the anti-PD-1–resistant tumors compared with parental tumors. Resistant tumors also contained fewer CD8+ (CD8α) and CD4+ tumor-infiltrating lymphocytes and reduced production of IFNγ. Localized radiotherapy induced IFNβ production, thereby elevating MHC class I expression on both parental and resistant tumor cells and restoring the responsiveness of resistant tumors to anti-PD-1 therapy. Conversely, blockade of type I IFN signaling abolished the effect of radiosensitization in this setting. Collectively, these results identify a mechanism of PD-1 resistance and demonstrate that adjuvant radiotherapy can overcome resistance. These findings have immediate clinical implications for extending the efficacy of anti-PD-1 immune checkpoint therapy in patients. Cancer Res; 77(4); 839–50. ©2016 AACR.

The oncogenic receptor tyrosine kinase AXL is overexpressed in cancer and plays an important role in carcinomas of multiple organs. However, the mechanisms of AXL overexpression in cancer remain unclear. In this study, using HEK293T, Panc-1, and Panc-28 cells and samples of human pancreatic intraepithelial neoplasia (PanIN), along with several biochemical approaches and immunofluorescence microscopy analyses, we sought to investigate the mechanisms that regulate AXL over-expression in pancreatic ductal adenocarcinoma (PDAC). We found that AXL interacts with hematopoietic progenitor kinase 1 (HPK1) and demonstrate that HPK1 down-regulates AXL and decreases its half-life. The HPK1-mediated AXL degradation was inhibited by the endocytic pathway inhibitors leupeptin, bafilomycin A1, and monensin. HPK1 accelerated the movement of AXL from the plasma membrane to endosomes in pancreatic cancer cells treated with the AXL ligand growth arrest-specific 6 (GAS6). Moreover, HPK1 increased the binding of AXL to the Cbl proto-oncogene (c-Cbl); promoted AXL ubiquitination; decreased AXL-mediated signaling, including phospho-AKT and phospho-ERK signaling; and decreased the invasion capability of PDAC cells. Importantly, we show that AXL expression inversely correlates with HPK1 expression in human PanINs and that patients whose tumors have low HPK1 and high AXL expression levels have shorter survival than those with low AXL or high HPK1 expression (p < 0.001). Our results suggest that HPK1 is a tumor suppressor that targets AXL for degradation via the endocytic pathway. HPK1 loss of function may contribute to AXL overexpression and thereby enhance AXL-dependent downstream signaling and tumor invasion in PDAC.

4050 Background: FLOT is the standard perioperative treatment for resectable gastric /gastroesophageal junction (GEJ) adenocarcinoma. However, patient’s outcome is still poor. Toripalimab, a humanized IgG4 monoclonal antibody against programmed cell death receptor-1 (PD-1), has shown remarkable clinical efficacy in various cancers. This trial evaluates the addition of Toripalimab to FLOT for resectable patients. Methods: This is a prospective, single-arm, investigator-initiated phase II trial. Patients with histologically confirmed, resectable, gastric and GEJ adenocarcinoma (≥cT2 or cN+) were enrolled to receive 4 pre-and post-operative cycles of toripalimab (240mg, q2w) plus FLOT (docetaxel 50 mg/m2; oxaliplatin 85 mg/m2; leucovorin 200 mg/m2; 5-FU 2600 mg/m2, q2w). The primary endpoint was pathological complete response rate (pCR). The secondary endpoints included major pathological (complete and nearly complete) response (MPR), and R0-resection rate, 3-year disease-free survival rate, overall survival, and adverse events. Results: In total, of 36 patients were enrolled from June 2019 through Dec 2020. Male, 66.7%; median age, 60y; cT3 8.3%, T4, 83.3%; cN+ 88.9%; GEJ 47%; MSI-H, 5.6%, Her-2neu-positive, 5.6%, EBER-positive, 5.6%). Two patients refused surgery, six patients have not yet completely neoadjuvant treatment. 100% of patients completed the 4 pre-cycle. Patients who had received gastrectomy after neoadjuvant treatment (n=28) were included in this analysis. 6 (21%) patients had operations involving a thoracic approach (oesophagogastrectomy with two field lymphadenectomy), 21 (75%) gastrectomy with D2 lymphadenectomy. 8 (29%) evaluable patients had Clavien-Dindo grade II post-operative complications and 2 (7%) grade IIIA complications; one patient had an anastomotic leakage that was treated endoscopically. There were no emergency re-operations. All 28 patients achieved R0-resection and were discharged home after a median of 12 days (range:7-63) in hospital. 7 （25%）patients achieved pCR (TRG1a) and 12 (42.9%) patients achieved major pathologic response (MPR, TRG1a/b). Treatment-related adverse events (TRAEs) to any drug were reported in 30 (94%) patients. Mostly TRAEs were grade 1-2, the grade 3 or 4 TRAEs included neutropenia (34%), neutropenia (25%), lymphopenia (3%), Alanine aminotransferase increased (3%), hypokalemia (3%) and anaemia (3%). Conclusions: Perioperative toripalimab in combination with FLOT showed promising efficacy with high pCR and MPR rate and well tolerated safety profile in patients with resectable gastric/GEJ adenocarcinoma. This combination regimen might present a new option for patients with locally advanced, resectable gastric/GEJ adenocarcinoma. Clinical trial information: NCT04354662.

Ultrasound-guided biopsy technique with the large-core needle has widely been applied in the diagnosis of adult abdominopelvic cavity, thyroid, and neck tumors. There are few reports on ultrasound-guided biopsy using large-core needle in pediatric abdominopelvic cavity tumors. This study was to evaluate the ultrasound features and the diagnostic value of ultrasound-guided core needle biopsy for pediatric neuroblastic tumors. The pediatric patients with neuroblastic tumor that underwent ultrasound examination and ultrasound-guided core needle biopsy from January 2009 to November 2015 were reviewed. A minimum of two cores in each case was obtained. The biopsy results were confirmed by subsequent surgical histopathology. The ultrasound features and the diagnostic accuracy of ultrasound-guided core needle biopsy were evaluated. Eighty-three patients were enrolled into the study. Conventional ultrasound examination showed irregular hypoechoic or mixed echo masses and calcification and liquefied necrosis. The diagnostic accuracy of ultrasound-guided core needle biopsy was 96.4% (80/83). Three cases were misdiagnosed because of inadequate tissue sample. No serious complication, infection, or needle track seeding occurred. Ultrasound-guided core needle biopsy seems to be an accurate, minimally invasive, and safe diagnostic method of pediatric neuroblastic tumor.

Ximei Zhang,1 Baozhong Zhang,1 Fenglin Zang,2 Lujun Zhao,1 Zhiyong Yuan,1 Ping Wang1 1Department of Radiation Oncology, 2Department of Pathology, Cancer Institute and Hospital, Tianjin Medical University, Tianjin, People’s Republic of China Objectives: Data on breast squamous cell carcinoma (SCC) are rare. The aim of this study was to analyze the clinical characteristics and to explore the rational treatment of patients with breast SCC. Patients and methods: We conducted a retrospective review of breast SCC cases treated at our center from 1966 to 2014. The majority of these patients received primary surgery followed by adjuvant chemoradiotherapy, whilst four elderly patients had lumpectomy only. Results: Patients with breast SCC were usually women, and large masses, large proportion of early stage disease, low levels of estrogen receptor expression, less frequent axillary lymph nodes involvement, and unfavorable prognosis were common. The 5-year overall survival and progression-free survival of all patients were 67.2% and 57.8%, respectively. Axillary nodal involvement was a significant prognostic factor for survival. Conclusion: The current results indicated that breast SCC is clinically aggressive and the outcomes were poor. Distant metastasis was the main failure pattern. New strategies will be needed because of the poor outcomes. Keywords: breast metaplastic carcinoma, breast SCC, treatment, surgery, prognosis

Objectives MAP4K5 plays an important role in regulating a range of cellular responses and is involved in Wnt signaling in hematopoietic cells. However, its functions in human malignancies have not been studied. The major objectives of this study are to examine the expression, functions and clinical significance of MAP4K5 in pancreatic ductal adenocarcinoma (PDAC). Materials and Methods The expression levels of MAP4K5, E-cadherin, vimentin, and carboxylesterase 2 (CES2) were examined by immunohistochemistry in 105 PDAC and matched non-neoplastic pancreas samples from our institution. The RNA sequencing data of 112 PDAC patients were downloaded from the TCGA data portal. Immunoblotting and RNA sequencing analysis were used to examine the expression of MAP4K5 and E-cadherin in pancreatic cancer cell lines. The effect of knockdown MAP4K5 using siRNA on the expression of CDH1 and vimentin were examined by Real-time RT-PCR in Panc-1 and AsPC-1 cells. Statistical analyses were performed using IBM SPSS Statistics. Results MAP4K5 protein is expressed at high levels specifically in the pancreatic ductal cells of 100% non-neoplastic pancreas samples, but is decreased or lost in 77.1% (81/105) of PDAC samples. MAP4K5-low correlated with the loss of E-cadherin (P = 0.001) and reduced CES2 expression (P = 0.002) in our patient populations. The expression levels of MAP4K5 mRNA directly correlated with the expression levels of CDH1 mRNA (R = 0.2490, P = 0.008) in the second cohort of 112 PDAC patients from The Cancer Genome Atlas (TCGA) RNA-seq dataset. Similar correlations between the expression of MAP4K5 and E-cadherin were observed both at protein and mRNA levels in multiple pancreatic cancer cell lines. Knockdown MAP4K5 led to decreased CDH1 mRNA expression in Panc-1 and AsPC-1 cells. MAP4K5-low correlated significantly with reduced overall survival and was an independent prognosticator in patients with stage II PDAC. Conclusions MAP4K5 expression is decreased or lost in majority of PDACs. The strong associations between low MAP4K5 expression and loss of E-cadherin, reduced CES2 expression and decreased overall survival may suggest an important role of MAP4K5 in epithelial-to-mesenchymal transition, chemotherapy resistance and tumor progression in pancreatic cancer. Targeting impaired MAP4K5 signaling may represent a new therapeutic strategy for pancreatic cancer.

The cellular Fas-associated death domain-like interleukin-1beta-converting enzyme inhibitory protein-long form (c-FLIP(L)) is a key regulator of Fas signaling, although owing a dominant-negative homologue of caspase-8, the role of c-FLIP(L) remains controversial. In the present study, two pairs of small interfering RNA (siRNA) directed against c-FLIP(L) were used to assess the effect of c-FLIP(L) on Fas-mediated apoptosis of colon carcinoma in vitro. HT-29 cell line was selected for overexpression of c-FLIP(L) and Fas with RT-PCR and flow cytometry analyses. After electroporation, the mRNA level of c-FLIP(L) was significantly decreased (control siRNA versus c-FLIP(L) siRNA, 77.97+/-5.61% versus 26.22+/-3.79%) and the maximum interfering efficiency was around 66.49% using semi-quantitative RT-PCR analysis. Knockdown of c-FLIP(L) with the specific siRNA sensitized colon carcinoma cells to Fas-mediated apoptosis (control siRNA versus c-FLIP(L) siRNA, 5.68+/-2.11% versus 29.50+/-2.27%) using DNA content analysis and Annexin V-FITC analysis. In conclusion, our study indicated that c-FLIP(L) might be a suppressor of Fas-mediated apoptosis in Fas antigen expressing colon carcinoma and therefore a potential target for novel anticancer therapies.

To investigate the expression of apoptotic regulator c-FLIP(L) in invasive breast carcinoma tissues, and to evaluate its correlation with molecular subtyping and clinical prognosis.Immunohistochemistry using EnVision staining for c-FLIP(L) was performed in 264 cases of invasive breast carcinomas and matched adjacent normal breast tissue samples from January 1996 to December 1999. ER, PR, HER2, Ki-67, CK5/6 and EGFR were evaluated by immunohistochemistry in order to classify the tumors into five molecular subtypes and the difference of c-FLIP(L) expression in these molecular subtypes was also analyzed. The influence of c-FLIP(L) expression on prognosis was evaluated by Kaplan-Meier curves and multi-factor Cox proportional risk model.High expression of c-FLIP(L) was observed in 84.5% (223/264) of cases of invasive breast carcinomas which were significantly higher than the 45.1% (119/264) of cases in adjacent normal epithelium of breast (χ² = 89.78, P = 0.000). The expression of c-FLIP(L) in luminal B (HER2 positive) and basal-like breast cancers was 78.1% (25/32) and 46.2% (18/39), respectively, with significant difference (P0.05). Moreover, the expression of c-FLIP(L) in luminal B (HER2 positive) was higher than in luminal A cancers (P0.05), and the expression of c-FLIP(L) in HER2 positive cancers was higher than in basal-like cancers (P0.01). C-FLIP(L) showed deep yellow staining in node positive breast cancer with a high-expression rate of 93.1% (134/144); whereas the expression was sporadic and light yellow in node negative breast cancer with a lower high-expressed rate of 72.5% (87/120, P0.01). C-FLIP(L) expression had significant influence on disease-free survival time, with c-FLIP(L)-positive patients showing poor prognosis (P0.01). Multi-factor Cox proportional risk model analysis showed that expression of c-FLIP(L), lymph nodes status and molecular subtypes were independent prognostic factors for invasive breast carcinomas (P0.05).C-FLIP(L) is highly expressed in invasive breast carcinomas, and its expression level is closely related to the molecular subtypes and clinical prognosis of breast cancer patients. Thus, c-FLIP(L) could be used as an important tumor marker for personalized cancer therapy and prognostic prediction.

Breast cancers with HER2 amplification have a poorer prognosis than the luminal phenotypes. TRAIL activates apoptosis upon binding its receptors in some but not all breast cancer cell lines. Herein, we investigated the expression pattern of c-FLIP(L) in a cohort of 251 invasive breast cancer tissues and explored its potential role in TRAIL resistance. C-FLIP(L) was relatively high-expressed in HER2-positive breast cancer in comparison with other molecular subtypes, co-expressed with TRAIL death receptors, and inversely correlated with the apoptosis index. Downregulation of c-FLIP(L) sensitized SKBR3 cells to TRAIL-induced apoptosis in a concentration- and time-dependent manner and enhanced the activities and cleavages of caspase-8 and caspase-3, without altering the surface expression of death receptors. Together, our results indicate that c-FLIP(L) promotes TRAIL resistance and inhibits caspase-3 and caspase-8 activation in HER2-positive breast cancer.

The mutation and reduction of mitochondrial DNA (mtDNA) have been extensively detected in human cancers. The effects of mitochondrial dysfunction are particularly important in breast cancer, because estrogen-mediated metabolites generate large quantities of local reactive oxygen species in the breast, which directly bind to mtDNA and facilitate neoplastic transformation. To further elucidate the molecular roles of mtDNA in breast cancer, we determined the oxidative status of a breast tumor cell line lacking mtDNA (T47D ρ⁰) and analyzed its susceptibility after exposure to various anticancer drugs as well as different proapoptotic signals. Our data showed that T47D ρ⁰ cells generated significantly increased levels of lactate with concomitantly reduced oxygen consumption and ATP production compared with the wild-type (WT). The amount of reactive oxygen species generation in ρ cells was lowered to approximately 12% that of parental cells, as evidenced by the oxidation of redox-sensitive probes. Although mtDNA depletion did not affect the expression of superoxide dismutase or its activity, the activities of antioxidant enzymes, catalase and glutathione peroxidase, were significantly higher in ρ⁰ cells compared with WT cells. In addition, mtDNA-depleted cells displayed a decreased sensitivity and accumulation of chemotherapeutic drugs (doxorubicin, vincristine, and paclitaxel), potentially because of the upregulated expression of multidrug resistance 1 (MDR1) gene and its product P-glycoprotein. When compared with their WT counterparts, T47D ρ⁰ cells were also more resistant to apoptosis induced by varying concentrations of staurosporine and anti-Fas antibody. Altogether, our results indicate the importance of intact mtDNA for maintaining the proper intracellular oxidative status. These data provide evidence for a possible role of mtDNA content reduction in acquiring an apoptosis-resistant phenotype during breast tumor progression and might contribute to effective therapeutic strategies for this common malignancy.