Mixed responses to first-line alectinib in non-small cell lung cancer patients with rare ALK gene fusions: A case series and literature review.

Anaplastic lymphoma kinase (ALK) fusion is a well-defined biomarker for ALK tyrosine kinase inhibitors (TKIs) treatment in non-small cell lung cancer (NSCLC). Alectinib, a second-generation ALK-TKI, has been shown to have significantly longer progression-free survival (PFS) than first-generation ALK inhibitors in untreated ALK-rearranged NSCLC patients. However, its clinical efficacy on rare ALK fusions remains unclear. Herein, two advanced NSCLC patients received first-line alectinib treatment, given their positive ALK fusion status as determined by immunohistochemistry (IHC) testing results. Patients showed limited clinical response (PFS: 4 months) and primary resistance to alectinib respectively. Molecular profiling using next-generation sequencing (NGS) further revealed a striatin (STRN)-ALK fusion in the first patient accompanied by MET amplification, and a LIM domain only protein 7 (LMO7)-ALK fusion in another patient without any other known oncogenic alterations. Both patients demonstrated improved survival after they switched to second-line crizotinib (PFS: 11 months) and ensartinib (PFS: 18 months), respectively, up till the last follow-up assessment. In conclusion, the clinical efficacy of ALK-TKIs including alectinib for lung cancer with uncommon ALK gene fusions is still under evaluation. This study and literature review results showed mixed responses to alectinib in NSCLC patients who harboured rare ALK fusions. Comprehensive molecular profiling of tumour is thus strongly warranted for precise treatment strategies.

Identification of UBE2C as hub gene in driving prostate cancer by integrated bioinformatics analysis.

BACKGROUND: The aim of this study was to identify novel genes in promoting primary prostate cancer (PCa) progression and to explore its role in the prognosis of prostate cancer. METHODS: Four microarray datasets containing primary prostate cancer samples and benign prostate samples were downloaded from Gene Expression Omnibus (GEO), then differentially expressed genes (DEGs) were identified by R software (version 3.6.2). Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were performed to identify the function of DEGs. Using STRING and Cytoscape (version 3.7.1), we constructed a protein-protein interaction (PPI) network and identified the hub gene of prostate cancer. Clinical data on GSE70770 and TCGA was collected to show the role of hub gene in prostate cancer progression. The correlations between hub gene and clinical parameters were also indicated by cox regression analysis. Gene Set Enrichment Analysis (GSEA) was performed to highlight the function of Ubiquitin-conjugating enzyme complex (UBE2C) in prostate cancer. RESULTS: 243 upregulated genes and 298 downregulated genes that changed in at least two microarrays have been identified. GO and KEGG analysis indicated significant changes in the oxidation-reduction process, angiogenesis, TGF-beta signaling pathway. UBE2C, PDZ-binding kinase (PBK), cyclin B1 (CCNB1), Cyclin-dependent kinase inhibitor 3 (CDKN3), topoisomerase II alpha (TOP2A), Aurora kinase A (AURKA) and MKI67 were identified as the candidate hub genes, which were all correlated with prostate cancer patient' disease-free survival in TCGA. In fact, only UBE2C was highly expressed in prostate cancer when compared with benign prostate tissue in TCGA and the expression of UBE2C was also in parallel with the Gleason score of prostate cancer. Cox regression analysis has indicated UBE2C could function as the independent prognostic factor of prostate cancer. GSEA showed UBE2C had played an important role in the pathway of prostate cancer, such as NOTCH signaling pathway, WNT-ß-catenin signaling pathway. CONCLUSIONS: UBE2C was pivotal for the progression of prostate cancer and the level of UBE2C was important to predict the prognosis of patients.

A comparative study of RTK gene status between primary tumors, lymph-node metastases, and Krukenberg tumors.

Krukenberg tumor (KT) refers to a rare ovarian tumor that has metastasized from a primary site. Patients with KTs have a poorer prognosis and worse survival. Thus far, little is known about the frequency of receptor tyrosine kinase (RTK) gene amplification and the concordance of gene amplification between primary tumors, lymph-node metastases, and KTs. Herein, 50 paired samples, including primary cancers, metastatic lymph nodes, and KTs were collected, and RTK gene amplification was tested by fluorescence in situ hybridization (FISH). There were four cases positive for human epidermal growth factor receptor type 2 (HER2) amplification, all of which showed conversion of HER2 status between different lesions. Of the two cases with c-mesenchymal-epithelial transition (c-MET) amplification, the primary tumors and lymph nodes were negative while the right involved ovaries were positive. Inconsistent fibroblast growth factor receptor 2 (FGFR2) status in different lesions was observed in three of the six FGFR2-amplified cases. Co-amplification of RTK genes was identified in only one patient for primary cancer and two for KTs. Collectively, there were 46, 48, 50, and 44 cases negative for HER2, c-MET, EGFR, and FGFR2 amplification in all lesions, respectively. There was no significant difference in overall survival between KTs of gastric origin and colorectal origin. However, of all synchronous cancers, KTs of colorectal origin had a better prognosis than those of gastric origin. In conclusion, the positive rate of RTK gene amplification in KTs was low. Intratumoral heterogeneity was frequent in KTs with RTK gene amplification. A mutually exclusive pattern of RTK gene amplification was dominant in primary cancers, lymph-node metastases, and KTs. There was no survival difference between KTs of gastric origin and colorectal origin. However, of all synchronous cancers, KTs of colorectal origin had a better prognosis than those of gastric origin.

Cytology cell blocks from malignant pleural effusion are good candidates for PD-L1 detection in advanced NSCLC compared with matched histology samples.

BACKGROUND: Detection of programmed cell death ligand-1 (PD-L1) by immunohistochemistry (IHC) has been commonly used to predict the efficacy of treatment with PD-1/PD-L1 inhibitors. However, there is limited literature regarding the reliability of PD-L1 testing using malignant pleural effusion (MPE) cell blocks. Here, we assess PD-L1 expression in sections from MPE cell blocks and evaluate the value of IHC double staining in the interpretation of PD-L1 expression. METHODS: In all, 124 paired formalin-fixed tissues from advanced NSCLC patients, including MPE cell blocks and matched histology samples, were included. PD-L1 expression was assessed using the SP263 assay, and the tumor proportion score (TPS) and the staining intensity were evaluated. PD-L1 staining results were also compared between IHC double and single staining techniques. RESULTS: PD-L1 expression was concordant in most paired cases (86/101, 85.1%) among three TPS cut-offs (<1%, 1-49% and ≥ 50%), with a kappa value of 0.774. Moreover, a significant difference in PD-L1 expression between MPE cell blocks and biopsy samples was observed (p = 0.005). For the 15 discordant pairs, 13 MPE cell block samples showed increased expression of PD-L1. Compared with the standard IHC single PD-L1 assay, double staining with anti-TTF-1 and anti-PD-L1 revealed a negative effect on PD-L1 expression testing and resulted in weaker staining intensity and a lower TPS (p = 0.000). CONCLUSIONS: MPE cell block samples are good candidates for PD-L1 expression detection in advanced NSCLC patients. The mechanism and clinical significance of the higher PD-L1 expression rate in MPE cell blocks compared with small biopsy samples remain to be evaluated prospectively.

Unusual findings of acute myeloid leukemia with inv(3)(q21q26.2) or t(3;3)(q21;q26.2): A multicenter study.

INTRODUCTION: AML with inv(3)(q21.3q26.2) or t(3;3)(q21.3;q26.2) [inv(3)/t(3;3)] was very rare. Currently, most reports of AML-inv(3)/t(3;3) were from Western countries, and few reports were from Asian countries. Racial differences in patients with AML-inv(3)/t(3;3) are still unknown. METHODS: Between January 1996 and April 2018, a total of 37 AML cases with inv(3)/t(3;3) were studied retrospectively. They were collected from 2229 primary AML cases performed with conventional cytogenetic analysis (37/2229, 1.66%). RESULTS: Here, some differences were found by comparing our data with those from Western countries. In our series, AML with inv(3)(q21q26) had a lower incidence than that with t(3;3)(q21;q26) (11 vs 26 cases). Our patients seemed to be more younger (median, 43 years) and have lower hemoglobin concentrations (median, 73 g/L) and higher platelet count (median, 351 × 109 /L). A higher incidence of acute monoblastic and monocytic leukemia (45.9%) was observed in our patients. Immunophenotypic studies showed that CD38 (30.8%) was not so frequently expressed as that in the earlier reports. Mutations analysis showed a high frequency of NRAS mutations (45.0%), followed by SF3B1(15.0%), GATA2(15.0%), FLT3-ITD(10.0%), C-Kit/D816(5.0%), and CEBPA(5.0%), without mutation of NPM1(Exon12)or JAK2V617. CONCLUSION: Ethnic differences do exist between the Chinese and Western patients with AML-inv(3)/t(3;3), and more attention should be paid involving different ethnic populations and geographic regions.

Secondary mixed phenotype acute leukemia following chemotherapy for diffuse large B-cell lymphoma: a case report and review of the literature.

Therapy-related mixed phenotype acute leukemia (MPAL) following non-Hodgkin's lymphoma (NHL) is extremely rare. We present here the case of an elderly man, diagnosed with diffuse large B-cell lymphoma (DLBCL) through a tonsil biopsy. After treatment with seven cycles of CHOP (cyclophosphamide, doxorubicin, vincristine and prednisolone) like regimen, the patient developed to MPAL (B/myeloid) with del(7)(q22), t(6;9)(p23;q34), DEK/NUP214 fusion, as well as EZH2 and TET2 mutations. The patient was successively treated with chemotherapy and allogenetic hematopoietic stem cell transplantation. Until recently he is still alive more than 23 months without relapse.

Clinicopathologic characteristics of prefibrotic-early primary myelofibrosis in Chinese patients.

The clinicopathologic features of patients with prefibrotic-early primary myelofibrosis (PEPMF) are still uncertain, and the characteristics of PEPMF in Asian patients are rarely reported. This study analyzed the clinicopathologic characteristics of 42 Chinese patients with PMF newly diagnosed according to the 2008 World Health Organization criteria. Some clinical and laboratory features of the patients differed significantly from those of the predominantly white patients in Western countries. Chinese patients with PEPMF were more often male (1.28:1) and younger, less likely to have higher median hemoglobin concentrations (126 g/L), less frequently had palpable spleens (35.7%), and had longer median times between prefibrotic-early and classical PEPMF (64 months). On bone marrow trephine sections, Chinese patients were more likely to have increased granulopoiesis (78.6%) and less frequently had balloon-like megakaryocytes (61.9%), giant and staghorn megakaryocytes (35.7%), or megakaryocytes with hyperchromatic and dysplastic nuclei (40.4%). In conclusion, some clinicopathologic characteristics of PEPMF in Chinese patients in China differ substantially from those seen in predominantly white patients in Western countries, and more clinicopathologic studies involving different ethnic populations and geographic regions of the world should help unfold the characteristics of this disease.

Lentiviral miR30-based RNA interference against heparanase suppresses melanoma metastasis with lower liver and lung toxicity.

AIM: To construct short hairpin RNAs (shRNAs) and miR30-based shRNAs against heparanase (HPSE) to compare their safety and their effects on HPSE down-modulation in vitro and in vivo to develop a more ideal therapeutic RNA interference (RNAi) vector targeting HPSE. METHODS: First, we constructed shRNAs and miR30-based shRNAs against HPSE (HPSE-shRNAs and HPSE-miRNAs) and packed them into lentiviral vectors. Next, we observed the effects of the shRNAs on knockdown for HPSE expression, adhesion, migration and invasion abilities in human malignant melanoma A375 cells in vitro. Furthermore, we compared the effects of the shRNAs on melanoma growth, metastasis and safety in xenograft models. RESULTS: Our data showed that these artificial miRNAs targeting HPSE could be effective RNAi agents mediated by Pol II promoters in vitro and in vivo, although these miRNAs were not more potent than the HPSE-shRNAs. It was noted that obvious lung injuries, rarely revealed previously, as well as hepatotoxicity could be caused by lentivirus-mediated shRNAs (LV shRNAs) rather than lentivirus-mediated miRNAs (LV miRNAs) in vivo. Furthermore, enhanced expression of pro-inflammatory cytokines IL-6 and TGF-ß1 and endogenous mmu-miR-21a-5p were detected in lung tissues of shRNAs groups, whereas the expression of mmu-let-7a-5p, mmu-let-7b-5p and mmu-let-7c-5p were down-regulated. CONCLUSION: These findings suggest that artificial miRNAs display an improved safety profile of lowered lung injury or hepatotoxicity relative to shRNAs in vivo. The mechanism of lung injuries caused by shRNAs may be correlated with changes of endogenous miRNAs in the lung. Our data here increase the flexibility of a miRNA-based RNAi system for functional genomic and gene therapy applications.

[The morphology features of bone marrow in the prefibrotic-early primary myelofibrosis].

OBJECTIVE: To compare the morphologic features of bone marrow (BM) between the prefibrotic-early primary myelofibrosis (PMF) and essential thrombocythaemia (ET). METHOD: Seven cases of prefibrotic-early PMF were selected and analyzed. Based on the diagnostic standard of prefibrotic-early PMF by WHO, BM aspirate smears, trephine biopsy sections and imprints of 156 uncertain ET cases conducted simultaneously were recruited into this study, the BM morphologic features between the prefibrotic-early PMF and ET groups were analyzed. The morphological difference in 22 cases of prefibrotic-early PMF and 27 ET were compared between the JAK2V617F mutation positive and negative groups. RESULTS: Of the 156 uncertain ET cases, it was reclassified 61 prefibrotic-early PMF (34 MF-0, 27 MF-1), 12 PMF and 83 ET. The platelet count and LDH level in MF-1 group were obviously higher than that of ET group (P < 0.05). The blast percentage of BM smear in MF-1 group was also higher than that of ET group (P < 0.05). As to BM section, cases with increased nucleated cells (granulocyte), compact megakaryocytic cluster, megakaryocyte near bone trabecula, cloud-like megakaryocyte, small bare nucleus of megakaryocyte and large ball-like megakaryocyte in MF-0 and MF-1 group were significantly higher than that of ET group (all P < 0.05), cases with megakaryocytic cluster of various size in MF-1 group were significantly higher than that of MF-0 and ET groups (P < 0.05). The JAK2V617F mutation rate in prefibrotic-early PMF and ET groups were 54.5% and 48.1%, respectively. Hb level in JAK2V617F mutation positive group was obviously higher than the negative group (P < 0.05), no special change with megakaryocytic morphology was found between the positive and negative groups. CONCLUSION: Morphology of BM section, especially megakaryocytic morphologic characteristics are the main basis in distinguishing prefibrotic-early PMF from ET. The importance of morphologic index were megakaryocytic cluster with various size, cloud-like megakaryocyte, large ball-like megakaryocyte, increased nucleated cells (granulocyte), small bare nucleus, megakaryocyte near bone trabecula and compact megakaryocytic cluster in order. JAK2V617F mutation provides no specific effect on the megakaryocytic morphology.

[Expression of GADD153 in follicular tumors of thyroid and comparison with CK19, Galectin-3 and HBME-1].

OBJECTIVE: To study immunohistochemical expression of GADD153 and assess its usefulness as markers in the differential diagnoses in follicular tumors of the thyroid. METHODS: Immunohistochemical staining was performed on formalin-fixed paraffin-embedded tissue of 34 cases of follicular thyroid adenomas (FTA), 46 cases of follicular thyroid carcinomas (FTC), 29 cases of follicular variant papillary carcinomas (FVPC). RESULTS: (1) GADD153 was expressed in cell nucleus with positive or strong positive expression in FTC, and no or weak expression in FTA and FVPC. The positive expressions of GADD153 were present in 38 of 46(82.6%) in FTC, 11 of 34(32.4%) in FTA and three of 29(10.3%) in FVPC, the positive expression rate in FTC was obviously higher than that in FTA and in FVPC, the differences were statistically significant (χ² = 20.80 and 37.48; P < 0.01). (2) CK19, Galectin-3 (Gal-3) and HBME-1 were all expressed in the cytoplasm, the positive expressions of CK19, Gal-3 and HBME-1 were present in 54.3% (25/46), 67.4% (31/46) and 58.7% (27/46) in FTC; 50.0% (17/34), 29.4% (10/34) and 32.4% (11/34) in FTA; 100% (29/29), 93.1% (27/29) and 89.7% (26/29) in FVPC, the differences were statistically significant as well (χ² = 21.20 and 8.22; P < 0.01). (3) According to the expressions of CK19, Gal-3, HBME-1 and GADD153, we divided the results into low expression group (0 or 1+) and high expression group (2+ or 3+), the sensitivity and the specificity were calculated. in FTA, the sensitivity were 26.5%, 8.8%, 2.9% and 11.8%; the specificity were 50.7%, 52.0%, 54.7% and 58.7%. in FTC, the sensitivity were 19.6%, 26.1%, 23.9% and 65.2%; the specificity were 41.3%, 57.1%, 62.0% and 92.1%. in FVPC, the sensitivity were 96.6%, 82.8%, 79.3% and 3.4%; the specificity were 77.5%, 81.3%, 85.0% and 57.5%. CONCLUSIONS: The sensitivity and the specificity of GADD153 expression are well for diagnosing FTC, and CK19, Gal-3, HBME-1 are well for FVPC. The four markers when used in combination, are better to identify the follicular tumors of the thyroid.