Molecular Detection of FGFR2 Rearrangements in Resected Intrahepatic Cholangiocarcinomas: FISH Could Be An Ideal Method in Patients with Histological Small Duct Subtype.

Background and Aims: Intrahepatic cholangiocarcinoma (ICC) is a subtype of primary liver cancer for which effective therapeutic agents are lacking. Fibroblast growth factor receptor 2 (FGFR2) has become a promising therapeutic target in ICC; however, its incidence and optimum testing method have not been fully assessed. This study investigated the rearrangement of FGFR2 in intrahepatic cholangiocarcinoma using multiple molecular detection methods. Methods: The samples and clinical data of 167 patients who underwent surgical resection of intrahepatic cholangiocarcinoma in Zhongshan hospital, Fudan university were collected. The presence of FGFR2 gene rearrangement was confirmed using fluorescence in situ hybridization (FISH) and targeted next-generation sequencing (NGS). FGFR2 protein expression was determined using immunohistochemistry (IHC). The concordance between the methods was statistically compared. PD-L1 expression was also assessed in this cohort. The clinicopathological characteristics and genomic profile related to FGFR2 rearrangements were also analyzed to assist candidate-screening for targeted therapies. Results: FGFR2 rearrangement was detected in 21 of the 167 ICC cases (12.5%) using FISH. NGS analysis revealed that FGFR2 rearrangement was present in 16 of the 20 FISH-positive cases, which was consistent with the FISH results (kappa value=0.696, p<0.01). IHC showed that 80 of the 167 cases (48%) were positive for FGFR2 expression, which was discordant with both FISH and NGS results. By comparison, FGFR2-positivity tended to correlate with unique clinicopathological subgroups, featuring early clinical stage, histologically small duct subtype, and reduced mucus production (P<0.05), with improved overall survival (p<0.05). FGFR2-positivity was not associated with PD-L1 expression in ICCs. In genome research, we identified eight partner genes fused with FGFR2, among which FGFR2-BICC1 was the most common fusion type. BAP1, CDKN2A, and CDKN2B were the most common concomitant genetic alterations of FGFR2, whereas KRAS and IDH1 mutations were mutually exclusive to FGFR2 rearrangements. Conclusions: FISH achieved satisfactory concordance with NGS, has potential value for FGFR2 screening for targeted therapies. FGFR2 detection should be prioritized for unique clinical subgroups in ICC, which features a histological small duct subtype, early clinical stage, and reduced mucus production.

Prognostic value and characterization of NTRK1 variation by fluorescence in situ hybridization in esophageal squamous cell carcinoma.

PURPOSE: Rearrangement of the neurotrophic tyrosine kinase receptor (NTRK) 1 gene is a target of tropomyosin receptor kinase A (TRKA) inhibitors, and its targeted drug (larotrectinib) has been approved by the US Food and Drug Administration. We investigated the existence and prognostic importance of NTRK1 variation in esophageal squamous cell carcinoma (ESCC). METHODS: Fluorescence in situ hybridization of a NTRK1 rearrangement was conducted on 523 ESCC samples through tissue microarrays. Kaplan-Meier curves with log-rank tests were used to evaluate survival. RESULTS: We identified 8 (1.5%), 35(6.7%) and 109 (20.8%) cases with a NTRK1 rearrangement using 15%, 10% and 5% as cut-off values, respectively. We observed copy number (CN) variation of NTRK1 in some cases: 79 (15.1%) cases had a gain in NTRK1 CN ≥ 3, and 24 (4.6%) cases had NTRK1 CN ≥ 4. A NTRK1 rearrangement at the above-mentioned thresholds was not related to disease-free survival (DFS, P = 0.45, 0.47, 0.87) and overall survival (OS, P = 0.80, 0.74, 0.57), respectively. Gain in NTRK1 CN was associated with a poor prognosis irrespective of whether NTRK1 CN ≥ 4 (DFS, P = 0.015; OS, P = 0.035) or NTRK1 CN ≥ 3 (DFS, P = 0.039; OS, P = 0.025). CONCLUSION: A NTRK1 rearrangement occurred rarely in ESCC. The increased CN of NTRK1 might be a prognostic indicator for DFS and OS in patients with ESCC.

SOX2 amplification and chromosome 3 gain significantly impact prognosis in esophageal squamous cell carcinoma.

BACKGROUND: We aimed to investigate the prevalence and prognostic role of Sex determining region Y-box 2 (SOX2) amplification and expression in surgically resected esophageal squamous cell carcinoma (ESCC). METHODS: We evaluated 450 ESCC samples using fluorescence in-situ hybridization and immunohistochemistry for SOX2 gene amplification and protein expression, respectively. The relationships of gene status with various clinicopathological characteristics and patient survival were statistically analyzed. RESULTS: SOX2 amplifications and chromosome 3 gain were observed in 4.4% and 12.9% of patients with ESCC. SOX2 amplification was associated with later clinical stage, and chromosome 3 gain was associated with earlier clinical stage (P=0.025). Low and high SOX2 expression were found in 28.9% and 24.7% of cases, respectively. SOX2 expression was significantly associated with gene copy number variation (P=0.007). SOX2 amplification was associated with a significantly shorter disease-free survival (DFS) or overall survival (OS). However, chromosome 3 gain was associated with a significantly longer DFS or OS (P<0.001). Multivariate analysis using the Cox proportional hazard model indicated that SOX2 amplification was an independently poorer prognostic factor (DFS, P<0.001, HR 2.638, 95% CI, 1.581-4.403; OS, P<0.001, HR 2.608, 95% CI, 1.562-4.355), along with pathology tumor-node-metastasis (pTNM) stage, whereas chromosome 3 gain was an independently better prognostic factor (DFS, P=0.003, HR 0.486, 95% CI, 0.300-0.789; OS, P=0.003, HR 0.474, 95% CI, 0.289-0.779) for ESCC. CONCLUSIONS: This is the first study wherein SOX2 amplification and chromosome 3 gain in a large cohort of ESCC were evaluated. SOX2 amplification is an independently poorer prognostic factor, whereas chromosome 3 gain is an independently favorable prognostic factor. Our results suggest that SOX2 amplification and chromosome 3 gain are potential biomarkers related to tumor progression and risk stratification in ESCC.

Prognostic value of visual residual tumour cells (VRTC) for patients with esophageal squamous cell carcinomas after neoadjuvant therapy followed by surgery.

BACKGROUND: We assessed visual residual tumour cells (VRTC) with both Becker's tumour regression grading (TRG) system and Japanese TRG system in esophageal squamous cell carcinoma (ESCC) patients treated with neoadjuvant therapy followed by surgery. METHODS: We compared Becker system and Japanese system in 175 ESCC patients treated between 2009 and 2015. RESULTS: According to Becker system, the 5-year DFS/DSS rates were 70.0%/89.3, 53.8%/56.7, 43.0%/49.0, and 42.4%/39.1% for TRG 1a (VRTC 0), TRG 1b (1-10%), TRG 2 (11-50%), and TRG 3 (> 50%). According to Japanese system, the rates were 38.8%/34.1, 49.5%/58.7, 50.2%/49.0 and 70.0%/89.3% for Grade 0-1a (VRTC> 66.6%), Grade 1b (33.3-66.6%), Grade 2 (1-33.3%) and Grade 3 (0). TRG according to two systems significantly discriminate the patients' prognosis. TRG according to Becker system (HR 2.662, 95% CI 1.151-6.157), and lymph node metastasis (HR 2.567, 95% CI 1.442-4.570) were independent parameters of DSS. CONCLUSIONS: Both Becker and Japanese system had their advantage in risk stratification of these ESCC patients. It was speculated that dividing 1-10% VRTC into a group might contribute to independently prognostic significance of Becker's TRG system. Therefore, in addition to TRG of different systems, the percentage of VRTC might be recommended in the pathologic report, which could make the results more comparable among different researches, and more understandable for oncologists in the clinical practice.

Increased MET gene copy number negatively affects the survival of esophageal squamous cell carcinoma patients.

BACKGROUNDS: Since Mesenchymal epithelial transition (MET) amplification has been regarded as a potential treatment target, the knowledge of its prevalence and prognostic importance is crucial. However, its clinical pathologic characteristics are not well known in esophageal squamous cell carcinoma (ESCC). METHODS: We investigated MET gene status with fluorescence in situ hybridization (FISH) assay in 495 ESCC cases using tissue microarrays. Prognostic significance as well as correlations with various clinicopathological parameters was evaluated. RESULTS: Among 495 patients, 28 (5.7%) cases were MET FISH positive, including 5 cases (1%) with true gene amplification. There were no statistically significant associations between MET FISH-positivity and clinicopathologic characteristics. A significantly poorer prognosis was observed in 28 patients with MET FISH-positivity (disease free survival/DFS, P < 0.001 and overall survival/OS, P = 0.001). Multivariate analysis revealed MET FISH-positivity was an independent prognostic factor for DFS (hazard ratio/HR, 1.953; 95% confidence interval/CI, 1.271-2.999; P = 0.002) and OS (HR, 1.926; 95% CI, 1.243-2.983; P = 0.003). MET FISH-positivity was associated with DFS (P = 0.022 and 0.020) and OS (P = 0.046 and 0.024) both in stage I-II ESCC and in stage III-IVa ESCC. No statistical significance (DFS, P = 0.492 and OS, P = 0.344) was detected between stage I-II ESCC with MET FISH-positivity and stage III-IVa ESCC with FISH-negativity. CONCLUSIONS: Increased MET gene copy number is an independent prognostic factor in ESCC, and ESCC might have potentially been up-staged by increased MET gene copy number. The results indicate that increased MET gene copy number is a very promising parameter, in clinical therapy and follow-up plans.

Midostaurin potentiates rituximab antitumor activity in Burkitt's lymphoma by inducing apoptosis.

An intensive short-term chemotherapy regimen has substantially prolonged the overall survival of Burkitt's lymphoma (BL) patients, which has been further improved by addition of rituximab. However, the inevitable development of resistance to rituximab and the toxicity of chemotherapy remain obstacles. We first prepared two BL cell lines resistant to rituximab-mediated CDC. Using a phosphorylation antibody microarray, we revealed that PI3K/AKT pathway contained the most phosphorylated proteins/hits, while apoptosis pathway that may be regulated by PKC displayed the greatest fold enrichment in the resistant cells. The PI3K/AKT inhibitor IPI-145 failed to reverse the resistance. In contrast, the pan-PKC inhibitor midostaurin exhibited potent antitumor activity in both original and resistant cells, alone or in combination with rituximab. Notably, midostaurin promoted apoptosis by reducing the phosphorylation of PKC and consequently of downstream Bad, Bcl-2 and NF-κB. Therefore, midostaurin improved rituximab activity by supplementing pro-apoptotic effects. In vivo, midostaurin alone powerfully prolonged the survival of mice bearing the resistant BL cells compared to rituximab alone treatments. Addition of midostaurin to rituximab led to dramatically improved survival compared to rituximab but not midostaurin monotherapy. Our findings call for further evaluation of midostaurin alone or in combination with rituximab in treating resistant BL in particular.

Poor prognostic impact of FGF4 amplification in patients with esophageal squamous cell carcinoma.

In the present study, we aimed to determine the prognostic impact and clinicopathological feature of FGF4 amplification in patients with esophageal squamous cell carcinoma (ESCC). Fluorescence in situ hybridization with FGF4 probe was analyzed using tissue microarray consisting of representative cores of 267 ESCC cases. FGF4 amplification was observed in 52.8% (141/267) of patients. Patients with FGF4 amplification showed a significantly shorter disease-free survival (DFS) or disease-specific overall survival (OS) compared with those without FGF4 amplification (both P < .05). Moreover, FGF4 amplification was an independent prognostic factor (DFS, P = .036; OS, P = .021) along with clinical stage and lymph node metastasis in multivariate analysis. Among stage I-II or III patients whose DFS was greater than or equal to 24 months (n = 125 or 32), patients with FGF4 amplification showed a significantly worse prognosis (OS, P = .027 or P = .010). Moreover, the survival curve of stage I-II patients with FGF4 amplification was identical to stage III patients without FGF4 amplification (DFS, P = .643; OS, P = .707). Taken together, FGF4 amplification was an independent prognostic factor in ESCC patients, and ESCC might have potentially been upstaged by FGF4 amplification. Therefore, FGF4 amplification in combination with clinical stage could be used as a relatively accurate predictor for the 5-year probability of death and recurrence for ESCC patients.

A case report of nodal CD4-positive T-cell lymphoproliferative disorder with an indolent course.

RATIONALE: Primary nodal CD4-positive T-cell lymophoproliferative disorder with a relatively indolent process is a rare kind of lymphoproliferative disease. Here we report the first case of a 49 year-old man developed indolent nodal CD4-positive T-cell lymophoproliferative disorder. To our knowledge, based on a careful search of PubMed, it is the first case of primary nodal CD4-positive T-cell lymophoproliferative disorder. PATIENT CONCERNS: A 49-year-old Chinese man presented to our hospital with fever, enlargement of multiple superficial lymphonodes more than 14 years and splenomegaly. Clinical and pathological data were collected under treatment. This case was diagnosed based on histologically characteristic, immunohistochemical staining, and lymphoid clonality testing. On immunohistochemical staining, the abnormal T-cells were CD4 positive and CD8 negative. The lymphoid clonality testing showed positive results. The patient also has enlarged spleen. DIAGNOSES: The patient was diagnosed with nodal CD4-positive T-cell lymophoproliferative disorder. INTERVENTIONS: A watch-and-wait stratagem was performed without any chemotherapy or radiation therapy. OUTCOMES: During 17 years of follow-up, this case presented an indolent course without evidence of systemic dissemination. LESSONS: This report presents the first case of indolent nodal CD4-positive T-cell lymophoproliferative disorder. In this case, the proliferated T-cell in the paracortex of lymph node showed T-cell receptor gene rearrangement, which indicated a clonal proliferation. There are several kinds of nodal CD4-positive T-cell lymphoma, which have a relatively aggressive course; however, this case has a relatively indolent course.

The prognostic significance of MCL1 copy number gain in esophageal squamous cell carcinoma.

BACKGROUND: MCL1 copy number variations have been reported to be associated with cancer prognosis in several cancers. However, the role of MCL1 gain has not yet been determined in esophageal squamous cell carcinomas (ESCC). METHODS: Fluorescence in situ hybridization (FISH) for MCL1 was performed on 262 ESCC samples using tissue microarray (TMA). RESULTS: The median age of ESCC patients was 62 years (range 37-83), with frequencies between women (16.4%) and men (83.6%). Of the 262 tumors, 77 tumors (29.4%) had high MCL1 gain. In the multivariate analysis, lymph node metastasis (HR: 3.236, P<0.001 for DFS; HR: 3.501, P<0.001 for OS) and clinical stage (HR: 3.388, P<0.001 for DFS; HR: 3.616, P<0.001 for OS) were identified as independent worse prognostic factors. Interestingly, among patients without lymph node metastasis or stage I-II patients, high MCL1 gain was associated with better DFS (P=0.009 or 0.046) and OS (P=0.014 or 0.069) after disease free survival time was more than or equal to 12 months. Reversely, among patients with lymph node metastasis or stage III-IVa patients, high MCL1 gain was associated with poorer DFS (P=0.007 or 0.021) and OS (P=0.029 or 0.068) after disease free survival time was more than or equal to 29 months. CONCLUSION: We observed that high MCL1 gain had bidirectional prognostic significance in ESCC patients with different lymph node status or clinical stage. These findings might provide the useful way of detailed risk stratification in patients with ESCC, and an insight into pathogenesis and mechanism of progression in ESCC.

Three mutations in Escherichia coli that generate transformable functional flagella.

Hydrodynamics predicts that swimming bacteria generate a propulsion force when a helical flagellum rotates because rotating helices necessarily translate at a low Reynolds number. It is generally believed that the flagella of motile bacteria are semirigid helices with a fixed pitch determined by hydrodynamic principles. Here, we report the characterization of three mutations in laboratory strains of Escherichia coli that produce different steady-state flagella without losing cell motility. E. coli flagella rotate counterclockwise during forward swimming, and the normal form of the flagella is a left-handed helix. A single amino acid exchange A45G and a double mutation of A48S and S110A change the resting flagella to right-handed helices. The stationary flagella of the triple mutant were often straight or slightly curved at neutral pH. Deprotonation facilitates the helix formation of it. The helical and curved flagella can be transformed to the normal form by torsion upon rotation and thus propel the cell. These mutations arose in the long-term laboratory cultivation. However, flagella are under strong selection pressure as extracellular appendages, and similar transformable flagella would be common in natural environments.