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DDMS diagnostics of thyroid nodules. The table contains post-surgical diagnosis according to participating pathologists, diagnosis based on fine needle aspiration biopsy, presence of thyroiditis and Hurthle cells, cancer risk scores according to DDMS and diagnosis according to DDMS.

PDF file - 3492K, Figure S3. Sanger DNA-sequencing for AT/RT tissue specific point mutations including DBF4 (c.794C>G, p.T265S); PMS1 (c.2524C>A, p.L842I); RASSF4 (c.494A>C, p.H165P); Rap2b (c.240G>C, p.L80I); and Tie2 (c.1249C>A, p.P417T) genes. The upper panel for sequencing of genomic DNA extracted from saliva, the lower panel for AT/RT.

PDF file - 1045K, Figure S1. The comparability of whole exome sequencing for saliva and FFPE AT/RT tissue of the adult patient. The sequencing coverage folds for each chromosome of A. saliva genomic DNA and B. AT/RT genomic DNA.

Table S2 shows sequences of the primers used in RT-qPCR

PDF file, 93K, High expression of SPARCL1 is associated with survival of patients with colon cancers and related to prognosis in both early and late stages.

Figure S3 shows that CCR2 is required for MCP-induced stemness

Purpose: To conduct a phase I trial of a modified vaccinia Ankara (MVA) vaccine delivering wild-type human p53 (p53MVA) in patients with refractory gastrointestinal cancers.Experimental Design: Three patients were vaccinated with 1.0 × 108 plaque-forming unit (pfu) p53MVA followed by nine patients at 5.6 × 108 pfu. Toxicity was classified using the NCI Common Toxicity Criteria and clinical responses were assessed by CT scan. Peripheral blood samples were collected pre- and post-immunization for immunophenotyping, monitoring of p53MVA-induced immune response, and examination of PD1 checkpoint inhibition in vitro.Results: p53MVA immunization was well tolerated at both doses, with no adverse events above grade 2. CD4+ and CD8+ T cells showing enhanced recognition of a p53 overlapping peptide library were detectable after the first immunization, particularly in the CD8+ T-cell compartment (P = 0.03). However, in most patients, this did not expand further with the second and third immunization. The frequency of PD1+ T cells detectable in patients' peripheral blood mononuclear cells (PBMC) was significantly higher than in healthy controls. Furthermore, the frequency of PD1+ CD8+ T cells showed an inverse correlation with the peak CD8+ p53 response (P = 0.02) and antibody blockade of PD1 in vitro increased the p53 immune responses detected after the second or third immunizations. Induction of strong T-cell and antibody responses to the MVA backbone were also apparent.Conclusion: p53MVA was well tolerated and induced robust CD8+ T-cell responses. Combination of p53MVA with immune checkpoint inhibition could help sustain immune responses and lead to enhanced clinical benefit. Clin Cancer Res; 20(17); 4459–70. ©2014 AACR.

Purpose: Therapeutic strategies against hormonal receptor–positive (HR+)/HER2+ breast cancers with poor response to trastuzumab need to be optimized.Experimental Design: Two HR+/HER2+ patient-derived xenograft (PDX) models named as COH-SC1 and COH-SC31 were established to explore targeted therapies for HER2+ breast cancers. RNA sequencing and RPPA (reverse phase protein array) analyses were conducted to decipher molecular features of the two PDXs and define the therapeutic strategy of interest, validated by in vivo drug efficacy examination and in vitro cell proliferation analysis.Results: Estrogen acted as a growth driver of trastuzumab-resistant COH-SC31 tumors but an accelerator in the trastuzumab-sensitive COH-SC1 model. In vivo trastuzumab efficacy examination further confirmed the consistent responses between PDXs and the corresponding tumors. Integrative omics analysis revealed that mammalian target of rapamycin (mTOR) and ERα signaling predominantly regulate tumor growth of the two HR+/HER2+ PDXs. Combination of the dual mTOR complex inhibitor MLN0128 and anti-HER2 trastuzumab strongly suppressed tumor growth of COH-SC1 PDX accompanied by increasing ER-positive cell population in vivo. Instead, MLN0128 in combination with antiestrogen fulvestrant significantly halted the growth of HR+/HER2+ cancer cells in vitro and trastuzumab-resistant COH-SC31 as well as trastuzumab-sensitive COH-SC1 tumors in vivo.Conclusions: Compared with the standard trastuzumab treatment, this study demonstrates alternative therapeutic strategies against HR+/HER2+ tumors through establishment of two PDXs coupled with integrative omics analyses and in vivo drug efficacy examination. This work presents a prototype of future “co-clinical” trials to tailor personalized medicine in clinical practice. Clin Cancer Res; 24(2); 395–406. ©2017 AACR.

PDF file, 67K, Primer sequences in RT-PCR analysis.

Supplementary Figure 1. Frequency of myeloid derived suppressor cells in the peripheral blood in patients pre and post vaccination with p53MVA

Suppl Information Table S1. Clinical characteristics of COH-SC1 and COH-SC31 PDXs

PDF file, 65K, Determine the specificity of SPARCL1 antibody.

PDF file - 382K, Figure S2.The relative whole exome copy number of genomic DNA from FFPE AT/RT tissue (normalized to genomic DNA extracted from saliva).

Figure S1. Characterization of two HR+/HER2+ breast patient-derived xenografts (PDXs), COH-SC1 and COH-SC31. Figure S2. Uterus and tumor weight of mice bearing COH-SC1 and COH-SC31 tumors in respond to estrogen treatment. Figure S3. Observations of body weight in response to drug treatment in vivo. Figure S4: Pathway analysis of up-regulated loci in COH-SC1 and COH-SC31 transcriptomes. Figure S5: Expression patterns of up-regulated proteins in COH-SC1 and COH-SC31 PDXs assayed by RPPA. Figure S6 In vivo efficacy examination of trastuzumab and/or MLN0128 on COH-SC1 PDX.

PDF file - 72K, Table S1. Genetic alterations and protein expression of the SMARCB1 and HMGA2 in AT/RT patients.

Table S2. Highly expressed loci in COH-SC31 in relation to COH-SC1

DDMS locations are frequently co-localized with active enhancers. A. Representative snap-shot of H3K27Ac pattern in two analyzed adjacent thyroid tissues (7N and 10N). The snap-shot indicates the normalized number of reads. Transcriptional start sites and transcript directions are shown. B. Representative snap-shot of co-localization DDMS site with H3K27Ac enrichment at the NFIC gene body in two analyzed adjacent thyroid tissues (7N and 10N). The snap-shot indicates the normalized number of reads. The red dotted line indicates the position of the DDMS location. Transcriptional start site and transcript directions are shown. C. Representative snap-shot of co-localization DDMS site with H3K27Ac enrichment at the EPHA2 gene body in two analyzed adjacent thyroid tissues (7N and 10N). The snap-shot indicates the normalized number of reads. The red dotted line indicates the position of the DDMS location. Transcriptional start site and transcript direction are shown.

Purpose: Atypical teratoid/rhabdoid tumors (AT/RT) are highly aggressive pediatric malignancies characterized by biallelic inactivation of the SMARCB1 tumor suppressor gene. We searched for novel genomic aberrations by investigating the copy number and expression alterations of let-7a3/let-7b microRNA (miRNA) and correlated these with expression of high-mobility group AT-hook 2 (HMGA2) oncoprotein, a target of let-7 miRNA family, in 18 AT/RT samples to elucidate potential roles of HMGA2 in the pathogenesis of AT/RT.Experimental Design: Genomic aberrations, let-7a3/let-7b miRNA and HMGA2 expression in AT/RT tissues were identified using quantitative PCR, reverse transcription PCR (RT-PCR), and immunohistochemistry. The impact of let-7b miRNA on HMGA2 expression and the malignant potential of human rhabdoid tumor cell G401 (SMARCB1−/−) were investigated by antisense inhibition and ectopic overexpression studies.Results: The copy number of let-7a3/let-7b miRNA was substantially decreased in 4 of 11 AT/RT samples. A significantly inverse correlation between let-7a3/let-7b miRNA expression and HMGA2 mRNA expression was observed in AT/RT tissues (R = −0.34; P < 0.05). Immunohistochemistry analysis demonstrated that HMGA2 was highly overexpressed in 83.3% (15 of 18) of AT/RT tissues. Restoration of let-7 miRNA or knockdown of HMGA2 expression significantly suppressed proliferation and colony formation, and almost abolished the invasive potential of G401 cells.Conclusion: Reduction of let-7a3/let-7b miRNA may be one of mechanisms leading to overexpression of HMGA2 in AT/RT tissues. HMGA2 oncoprotein plays critical roles in the pathogenesis of AT/RT development; and reconstitution of let-7 miRNA or knockdown of HMGA2 oncoprotein may provide a novel therapeutic strategy for the treatment of patients with AT/RT. Clin Cancer Res; 20(5); 1179–89. ©2014 AACR.

PDF file - 224K, Figure S4. Impact of let-7 miRNA and HMGA2 on proliferation and colony formation of G401 cells. A. Let-7 inhibitors up-regulated HMGA2 and promoted cell proliferation, and B. Overexpression HMGA2 increased colony formation of G401 cells, *: p < 0.05, compared with G401 cells transfected with control smRNA.

Purpose: Preoperative or neoadjuvant therapy (NT) is increasingly used in patients with locally advanced or inflammatory breast cancer to allow optimal surgery and aim for pathologic response. However, many breast cancers are resistant or relapse after treatment. Here, we investigated conjunctive chemotherapy-triggered events occurring systemically and locally, potentially promoting a cancer stem–like cell (CSC) phenotype and contributing to tumor relapse.Experimental Design: We started by comparing the effect of paired pre- and post-NT patient sera on the CSC properties of breast cancer cells. Using cell lines, patient-derived xenograft models, and primary tumors, we investigated the regulation of CSCs and tumor progression by chemotherapy-induced factors.Results: In human patients and mice, we detected a therapy-induced CSC-stimulatory activity in serum, which was attributed to therapy-associated monocytosis leading to systemic elevation of monocyte chemoattractant proteins (MCP). The post-NT hematopoietic regeneration in the bone marrow highlighted both altered monocyte–macrophage differentiation and biased commitment of stimulated hematopoietic stem cells toward monocytosis. Chemotherapeutic agents also induce monocyte expression of MCPs through a JNK-dependent mechanism. Genetic and pharmacologic inhibitions of the MCP-CCR2 pathway blocked chemotherapy's adverse effect on CSCs. Levels of nuclear Notch and ALDH1 were significantly elevated in primary breast cancers following NT, whereas higher levels of CCR2 in pre-NT tumors were associated with a poor response to NT.Conclusions: Our data establish a mechanism of chemotherapy-induced cancer stemness by linking the cellular events in the bone marrow and tumors, and suggest pharmacologic inhibition of CCR2 as a potential cotreatment during conventional chemotherapy in neoadjuvant and adjuvant settings. Clin Cancer Res; 24(10); 2370–82. ©2018 AACR.

Purpose:Secreted protein acidic and rich in cysteines-like 1 (SPARCL1) is an extracellular matrix glycoprotein with malignancy-suppressing potential. The hypothesis that SPARCL1 reduces cancer invasiveness and predicts better survival in colorectal cancers (CRC) was investigated.Experimental Design: Stable SPARCL1 transfectants, RKO-SPARCL1, and corresponding vector control were constructed and implanted into nude mice to generate a mouse xenograft model of liver metastasis. Also, a retrospective outcome study was conducted on the COH set (222 CRCs) and ZJU set (412 CRCs). The protein expression level of SPARCL1 was determined by immunohistochemistry. The Kaplan–Meier and Cox analyses were used for survival analysis. The association of SPARCL1 with mesenchymal–epithelial transition (MET) was examined by reverse transcription PCR (RT-PCR) and Western blot analysis.Results: The ectopic expression of SPARCL1 significantly reduced the potential for anchorage-independent growth, migration, invasion and induced cell differentiation in RKO and SW620 cells. In mouse xenograft model, the expression of SPARCL1 significantly reduced the liver metastasis (P < 0.01). The patient-based studies revealed that the expression of SPARCL1 was related to better differentiation (P < 0.01), less lymph node involvement [OR, 0.67; 95% confidence interval (CI), 0.45–1.00], and less distant metastasis (OR, 0.38; 95% CI, 0.18–0.79). The Kaplan–Meier and Cox analysis showed that the expression of SPARCL1 was associated with better overall survival (log-rank: P < 0.01; HR, 0.57; 95% CI, 0.39–0.84). Transfection of SPARCL1 induced MET of colon cancer cells.Conclusion:SPARCL1 functions as a tumor suppressor promoting differentiation possibly via MET, which inhibits the aggressiveness of CRCs. Clin Cancer Res; 18(19); 5438–48. ©2012 AACR.

PDF file, 38K, Study design.

Purpose:Thyroid cancer is frequently difficult to diagnose due to an overlap of cytologic features between malignant and benign nodules. This overlap leads to unnecessary removal of the thyroid in patients without cancer. While providing some improvement over cytopathologic diagnostics, molecular methods frequently fail to provide a correct diagnosis for thyroid nodules. These approaches are based on the difference between cancer and adjacent thyroid tissue and assume that adjacent tissues are the same as benign nodules. However, in contrast to adjacent tissues, benign thyroid nodules can contain genetic alterations that can be found in cancer.Experimental Design: For the development of a new molecular diagnostic test for thyroid cancer, we evaluated DNA methylation in 109 thyroid tissues by using genome-wide single-base resolution DNA methylation analysis. The test was validated in a retrospective cohort containing 65 thyroid nodules.Results:By conducting reduced representation bisulfite sequencing in 109 thyroid specimens, we found significant differences between adjacent tissue, benign nodules, and cancer. These tissue-specific signatures are strongly linked to active enhancers and cancer-associated genes. Based on these signatures, we developed a new epigenetic approach for thyroid diagnostics. According to the validation cohort, our test has an estimated specificity of 97% [95% confidence interval (CI), 81–100], sensitivity of 100% (95% CI, 87–100), positive predictive value of 97% (95% CI, 83–100), and negative predictive value of 100% (95% CI, 86–100).Conclusions:These data show that epigenetic testing can provide outstanding diagnostic accuracy for thyroid nodules.See related commentary by Mitmaker et al., p. 457

PDF file, 49K, The quality of CRC database in COH set and ZJU set.

Suppl Figure Legends

PDF file, 379K, Recombinant human SPARCL1 protein reversed malignant phenotypes colon cancer cells in vitro.

Purpose: This study aims to address the hypothesis that the high-mobility group A2 (HMGA2), an oncofetal protein, relates to survivability and serves as a prognostic biomarker for colorectal cancer (CRC).Experimental Design: This is a retroprospective multiple center study. The HMGA2 expression level was determined by performing immunohistochemistry on surgical tissue samples of 89 CRCs from a training set and 191 CRCs from a validation set. The Kaplan–Meier analysis and COX proportional hazard model were employed to analyze the survivability.Results: Multivariate logistic analysis indicated that the expression of HMGA2 significantly correlates with distant metastasis in training set (odds ratio, OR = 3.53, 95% CI: 1.37–9.70) and validation set (OR = 6.38, 95% CI: 1.47–43.95). Survival analysis revealed that the overexpression of HMGA2 is significantly associated with poor survival of CRC patients (P < 0.05). The adjusted HRs for overall survival were 2.38 (95% CI: 1.30–4.34) and 2.14 (95% CI: 1.21–3.79) in training and validation sets, respectively. Further investigation revealed that HMGA2 delays the clearance of γ-H2AX in HCT-116 and SW480 cells post γ-irradiation, which supports our finding that CRC patients with HMAG2-positive staining in primary tumors had augmented the efficacy of adjuvant radiotherapy (HR = 0.18, 95% CI: 0.04–0.63).Conclusion: Overexpression of HMGA2 is associated with metastasis and unequivocally occurred in parallel with reduced survival rates of patients with CRC. Therefore, HMGA2 may potentially serve as a biomarker for predicting aggressive CRC with poor survivability and as an indicator for better response of radiotherapy. Clin Cancer Res; 17(8); 2570–80. ©2011 AACR.

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PDF file, 126K, Uni- and Multivariate COX analysis for SPARCL1 and overall survival of CRCs.

PDF file, 89K, Standard of cytoplasm staining of SPARCL1.

Table S1. qRT-PCR primers. Table S2. CRM1 expression among different cancer types patient sample tissues. Table S3. CRM1 Expression among different cancer types ( cancer cell lines from CCLE database). Table S4. patients clinical infomation and h score sorting. Table S5. Overlaps of lists of genes in EW8 and SKES1 under treatment with KPT-330. Table S6. Overlaps of lists of genes by KPT-330 and EWS-FLI1 regulation.

Supplementary Table 3 from Ribonucleotide Reductase Small Subunit M2B Prognoses Better Survival in Colorectal Cancer

Supplementary Table S4 PDF file 59K, Relative gene copy number determined by qPCR analysis (ratio to adjacent normal tissue) of selected eight genes located on chromosome 12q13-15 of individual UHGPS

PDF file - 111K, Methods: Includes Mammosphere formation assay, Cell transfection and reporter assays, RNAi studies, as well as Western blot analyses and xenograft data. Figures S1-S9 include the following: Marker expression in primary fibroblasts and BC cells (S1); Serial confocal images of representative mammospheres formed by PKH67-labeled BT474 cells in the presence or absence of CCL2 (S2); CCL2 expression in CAF3 cells upon CM treatments (S3); Sustained CCL2 induction in CAFs co-cultured with tumor cells (S4); Expression of CCR2 and CCR4 in BC cells (S5); HES1 and HEY1 reporter assays in BT474 cells (S6); In vitro secretase cleavage assay (S7); Representative IHC images of XP/CAF265922 xenograft tumors treated with PBS, IgG, or anti-CCL2 (S8); Representative IHC images of CCL2 and NOTCH1 in primary BC specimens (S9).

Supplementary Table 1 from Ribonucleotide Reductase Small Subunit M2B Prognoses Better Survival in Colorectal Cancer

Supplementary Figure Legends 1-5 from Ribonucleotide Reductase Small Subunit M2B Prognoses Better Survival in Colorectal Cancer

Ewing sarcoma (EWS) is an aggressive bone malignancy that mainly affects children and young adults. The mechanisms by which EWS (EWSR1) fusion genes drive the disease are not fully understood. CRM1 (XPO1) traffics proteins from the nucleus, including tumor suppressors and growth factors, and is overexpressed in many cancers. A small-molecule inhibitor of CRM1, KPT-330, has shown therapeutic promise, but has yet to be investigated in the context of EWS. In this study, we demonstrate that CRM1 is also highly expressed in EWS. shRNA-mediated or pharmacologic inhibition of CRM1 in EWS cells dramatically decreased cell growth while inducing apoptosis, cell-cycle arrest, and protein expression alterations to several cancer-related factors. Interestingly, silencing of CRM1 markedly reduced EWS–FLI1 fusion protein expression at the posttranscriptional level and upregulated the expression of the well-established EWS-FLI1 target gene, insulin-like growth factor binding protein 3 (IGFBP3), which inhibits IGF-1. Accordingly, KPT-330 treatment attenuated IGF-1–induced activation of the IGF-1R/AKT pathway. Furthermore, knockdown of IGFBP3 increased cell growth and rescued the inhibitory effects on IGF-1 signaling triggered by CRM1 inhibition. Finally, treatment of EWS cells with a combination of KPT-330 and the IGF-1R inhibitor, linsitinib, synergistically decreased cell proliferation both in vitro and in vivo. Taken together, these findings provide a strong rationale for investigating the efficacy of combinatorial inhibition of CRM1 and IGF-1R for the treatment of EWS. Cancer Res; 76(9); 2687–97. ©2016 AACR.

Supplementary Table 2 from Ribonucleotide Reductase Small Subunit M2B Prognoses Better Survival in Colorectal Cancer

Figure legends for supplemental figures S1-S6.

Supplementary Table Legend PDF file 58K, Legend for Supplementary Tables S1-S5