Introduction: Epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) mutational analysis in adenocarcinoma lung are growing in importance as these tests are essential in guiding the use of targeted therapy. However, the prevalence of these mutations in various populations in India has not been studied. Furthermore, the correlation of histologic pattern with the mutation is not clear.
Materials and Methods: A total of 64 biopsy-proven lung adenocarcinomas were selected. In 51 of these, EGFR mutational analysis was performed using Qiagen EGFR pyrosequencing kit and in 56 cases of these ALK1 immunohistochemistry (IHC) was done using ALK (D5F3) antibody and the Ventana Benchmark
Results: EGFR mutation was positive in 23 cases (45.10%) and positive ALK1 expression by IHC in 12 cases (21.43%). Both mutations were positive in two cases. Both mutations were seen in well-differentiated and moderately differentiated adenocarcinoma, and the expression was highest in tumors with a predominant acinar pattern.
Conclusions: The incidence of both EGFR and ALK1 mutations is higher in the population studied and seem to correlate with a well differentiated, acinar pattern on morphology.

Homoharringtonine (HHT), a known protein synthesis inhibitor, has an anti-myeloid leukemia effect and potentiates the therapeutic efficacy of anthracycline/cytarabine induction regimens for acute myelogenous leukemia (AML) with favorable and intermediate prognoses, especially in the t(8;21) subtype. Here we provide evidence showing that HHT inhibits the activity of leukemia-initiating cells (Lin

OBJECTIVE: Predictive and prognostic markers have revolutionized personalized therapy in non-small cell lung carcinoma (NSCLC). Crizotinib is now approved for locally advanced or metastatic NSCLC that is anaplastic lymphoma kinase (ALK) positive by either Fluorescence in situ hybridization (FISH) or immunohistochemistry (IHC). The current study aimed to detect the incidence of ALK gene re-arrangement in the Indian population, to compare the various IHC antibodies with FISH as a gold standard, and to analyze the morphology of cases with ALK phenotype.
MATERIAL AND METHOD: A case series of 614 cases of NSCLC were included. IHC for detection of ALK phenotype was compared with FISH using 5A4 clone (Labvision, USA), ALK-1(Dako, Denmark) and D5F3 clone (Ventana, USA).
RESULTS: ALK gene rearrangement was evident in 4.07% of the cases. Cases with ALK phenotype had unique histomorphology with presence of mucin or signet ring cells in association with necrosis, high tumour grade and poor differentiation. Comparison of various antibody clones used in IHC revealed that the sensitivity and specificity using the D5F3 clone (100%, 100%) and 5A4 clone (87.5%, 100%) were similar while the ALK-1 clone had the lowest sensitivity and specificity (50%, 95.5%).
CONCLUSION: The incidence of ALK gene rearrangement in NSCLC in the current Indian study is within the worldwide reported range of 3-5%. This is the first study from the Indian subcontinent to compare various IHC antibodies used for detection of ALK phenotype. IHC using D5F3 clone and 5A4 clone may be considered as a rapid reliable and inexpensive method for detection of ALK gene rearrangement.

BACKGROUND Lung cancer has become a leading disease for the tumor-induced mortality. Non-small cell lung cancer (NSCLC) accounts for approximately 80% of all lung cancers. The present research aimed to evaluate the correlation between the anaplastic lymphoma kinase/oncogene or c-ros oncogene 1 (ALK/ROS1) fusions or mutations of epidermal growth factor receptor (EGFR) and ages or gender of patients. MATERIAL AND METHODS Among 1449 NSCLC patients, 457 patients who were diagnosed as consecutive EGFR mutations or ALK/ROS1 fusions between November 2016 and February 2018 were involved in the present study. EGFR genes or ALK/ROS1 mutations were detected by using DNA sequencing technique and amplification-refractory mutation system (ARMS). The mRNAs of ROS1 and ALK fusion were examined by using polymerase chain reaction technique and fusion gene detection kit. RESULTS Females were more often inflicted by the EGFR mutations, especially for the exon 19 deletion and L858R mutation. There were significantly more ALK/ROS1 fusions in females compared to males (P<0.05) and significantly more ALK/ROS1 fusions in <60 years of age patients compared to patients older than 60 years of age (P<0.05). Exon 21 L858R and L861Q dominantly occurred in patients ≥60 years of age and exon 19 deletion in patients <60 years of age. EML-ALK-1 mainly existed in the female NSCLC patients. CONCLUSIONS EGFR mutations and ALK/ROS1 fusions mainly occurred in the NSCLC female patients who were older than 60 years of age.

Juvenile polyposis syndrome (JPS) may coexist with hereditary hemorrhagic telangiectasia (HHT) due to implication of the SMAD4 gene in a subset of both diseases. To the best of our knowledge, we present the first case in the radiologic literature on the MRI findings in a patient with this rare combined diagnosis undergoing workup for burden of disease.

BACKGROUND: Chemotherapeutic agents such as anthracyclines and taxanes are commonly used in the neoadjuvant setting. Bevacizumab is an antibody which binds to vascular endothelial growth factor A (VEGFA) and inhibits its receptor interaction, thus obstructing the formation of new blood vessels.
METHODS: A phase II randomized clinical trial of 123 patients with Her2-negative breast cancer was conducted, with patients treated with neoadjuvant chemotherapy (fluorouracil (5FU)/epirubicin/cyclophosphamide (FEC) and taxane), with or without bevacizumab. Serial biopsies were obtained at time of diagnosis, after 12 weeks of treatment with FEC ± bevacizumab, and after 25 weeks of treatment with taxane ± bevacizumab. A time course study was designed to investigate the genomic landscape at the three time points when tumor DNA alterations, tumor percentage, genomic instability, and tumor clonality were assessed. Substantial differences were observed with some tumors changing mainly between diagnosis and at 12 weeks, others between 12 and 25 weeks, and still others changing in both time periods.
RESULTS: In both treatment arms, good responders (GR) and non-responders (NR) displayed significant difference in genomic instability index (GII) at time of diagnosis. In the combination arm, copy number alterations at 25 loci at the time of diagnosis were significantly different between the GR and NR. An inverse aberration pattern was also observed between the two extreme response groups at 6p22-p12 for patients in the combination arm. Signs of subclonal reduction were observed, with some aberrations disappearing and others being retained during treatment. Increase in subclonal amplification was observed at 6p21.1, a locus which contains the VEGFA gene for the protein which are targeted by the study drug bevacizumab. Of the 13 pre-treatment samples that had a gain at VEGFA, 12 were responders. Significant decrease of frequency of subclones carrying gains at 17q21.32-q22 was observed at 12 weeks, with the peak occurring at TMEM100, an ALK1 receptor signaling-dependent gene essential for vasculogenesis. This implies that cells bearing amplifications of VEGFA and TMEM100 are particularly sensitive to this treatment regime.
CONCLUSIONS: Taken together, these results suggest that heterogeneity and subclonal architecture influence the response to targeted treatment in combination with chemotherapy, with possible implications for clinical decision-making and monitoring of treatment efficacy.
TRIAL REGISTRATION: NCT00773695 . Registered 15 October 2008.

BACKGROUND: Little exists currently in research about the mechanisms of resistance of ALK inhibitors in inflammatory myofibroblastic sarcoma. It is known, however, that ALK gene rearrangements are common in inflammatory myofibroblastic tumors, similar to non-small cell lung cancer. In roughly 50% of inflammatory myofibroblastic tumors, gene rearrangement has been found to occur on chromosome 2 at band 2p23. In non-small cell lung cancer, it has been shown that about a third of patients who progress on the first generation ALK inhibitor, crizotinib develops mutations in the ALK kinase domain. The remaining two-thirds of patients tend to develop amplification of ALK or activation of alternative signaling pathways. Chromoplexy has also been described as a mechanism of resistance, where multiple closed chain rearrangements cause loss-of-function of tumor suppressor genes and gain-in-function of oncogenic fusions. Partner genes that have been identified in IMTs are tropomyosin 3 (TPM3), tropomyosin 4 (TPM4), clathrin heavy chain (CLTC), Ran-binding protein 2 (RANBP2), cysteinyl-tRNA synthetase (CARS), 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (ATIC), and SEC31L1. All are active promoters for the fusion gene, in response to NPM binding. Several inflammatory myofibroblastic tumor case reports indicated that fusion of ALK and RANBP2 led to a more aggressive clinical course. Although the majority of inflammatory myofibroblastic tumor case reports have utilized first and second generation ALK inhibitors, all generations of ALK inhibitors have demonstrated some ability to impair disease progression and extend life expectancy. However, at some point in the course of therapy with each generation of ALK inhibitor, resistance ultimately developed. In order to better understand the pharmacology and resistance patterns behind three generations of ALK inhibitors, we sought to examine a patient with metastatic anaplastic lymphoma kinase-1-rearranged inflammatory myofibroblastic sarcoma to the brain. We also explored the similarities and differences of this clinical case to other inflammatory myofibroblastic sarcoma case reports involving the use of ALK inhibitors.
CASE REPORT: A rare case of pulmonary IMS with ALK-1 gene rearrangement and multiple brain metastases responded to three generations of ALK inhibitors. However, similar to other case reports, due to the development of resistance and recurrence, the patient eventually succumbed to the disease.
CONCLUSIONS: ALK inhibitors are beneficial in the temporary prevention of progression of disease in patients with inflammatory myofibroblastic tumors. In this case, due to the inability to reveal the fusion partner in this patient via DNA sequencing, it is unknown exactly if that partner was RANBP2 or another ALK partner gene. Brain biopsy tissue was also unobtainable during sequence of ALK due to risk versus benefit, which would have provided insight as which type of ALK resistance mutations the patient was developing. It is likely that this patient had some form of chromoplexy occurring.

Inflammatory myofibroblastic tumors (IMTs) are neoplasms with low malignant potential, and the most common tumor in the lung and orbit. Their occurrence in the uterus is rare. Approximately 50% of IMT patients have anaplastic lymphoma kinase gene ( ALK) rearrangements. Recent studies described novel fusions involving ROS1, platelet-derived growth factor receptor beta ( PDGFR-β), and ETS translocation variant ( ETV6) genes in a subset of ALK-negative patients. We report a 44-year-old woman with anemia and uterine IMT. Ultrasonography and magnetic resonance imaging revealed a myxoid degenerative myoma-like mass, 7.4 cm in maximum diameter, on the left uterine side wall. Hysterectomy was performed as a definitive treatment. Microscopic examination revealed spindle cell proliferation with numerous lymphocytes and plasma cells. Immunohistochemically, the spindle cells were negative for ALK-1, desmin, and smooth muscle actin. The pathological diagnosis was IMT arising from the uterus. Fluorescence in situ hybridization demonstrated an ETV6-neurotrophic tyrosine kinase, receptor, type 3 gene ( NTRK3) translocation but no ALK, ROS1, or PDGFR-β translocations. Lung and abdomen computed tomography at 31 months postoperatively revealed no disease recurrence. This association of an ETV6-NTRK3 fusion oncogene with an ALK-negative uterine IMT increases our understanding of this neoplasm, which may help the development of specific therapies.

BACKGROUND: The signal transducer and activator of transcription (STAT) and transforming growth factor-β (TGF-β) signaling pathways play important roles in epithelial ovarian cancer (EOC). However, the mechanism of crosstalk between two pathways is not completely understood.
METHODS: The expression of STAT1 protein was detected by tissue microarray and immunoblotting (IB). The interaction of STAT1 isoforms with TGF-β receptors was confirmed by immunoprecipitation and IB. The effect of TGF-β signaling on STAT1 activation was examined in EOC and non-tumorous HOSEpiC cells treated with TGF-β1 in the presence or absence of the inhibitor of TGF-β type I receptor. The gain-of-function and loss-of-function approaches were applied for detecting the role of STAT1 on EOC cell behaviours.
RESULTS: The high level of STAT1 was observed in patients with high-grade serous EOC. STAT1 expression was higher in ovarian cancer cells than noncancerous cells. TGF-β1 activated the STAT1 pathway by inducing the phosphorylation of STAT1α on S727 residue. The full-length STAT1α and the truncated STAT1β directly interacted with TGF-β receptors (ALK1/ALK5 and TβRII), which was mediated by TGF-β1. STAT1α and STAT1β blocked the activation of the TGF-β1 signaling pathway in EOC cells by reducing Smad2 phosphorylation. STAT1 overexpression induced EOC cell proliferation, migration, and invasion; whereas its inhibition enhanced TGF-β1-induced phospho-Smad2 and suppressed EOC cell proliferation, migration, and invasion.
CONCLUSIONS: Our data unveil a novel insight into the molecular mechanism of crosstalk between the STAT1 and TGF-β signaling pathways, which affected the cancer cell behavior. Suppression of STAT1 may be a potential therapeutic strategy for targeting ovarian cancer.

BACKGROUND: No biomarkers exist to predict benefit from antiangiogenic therapy in metastatic colorectal cancer patients. ACVRL1 (activin receptor like-protein 1) encodes for ALK1, a member of the transforming growth factor-β receptor family, which directs pathologic angiogenesis. We examined the intratumoral expression of ACVRL1 and other angiogenesis pathway-related genes to identify molecular markers in the TRIBE study.
MATERIALS AND METHODS: Of 503 randomized patients, 228 had sufficient tissue for analysis. Formalin-fixed paraffin-embedded specimens were examined for expression of VEGF-A, VEGF-B, VEGF-C, VEGFR1, VEGFR2, ACVRL1, EphB4, and EGFL7 using reverse transcription polymerase chain reaction. A maximal χ
RESULTS: High ACVRL1 expression was associated with superior OS in both treatment arms (FOLFOXIRI [5-fluorouracil, leucovorin, oxaliplatin, irinotecan]-bevacizumab, 32.7 vs. 13.5 months, hazard ratio [HR], 0.38, P = .023; FOLFIRI [5-fluorouracil, leucovorin, irinotecan]-bevacizumab, 35.1 vs. 22.0 months, HR, 0.36, P = .006) and prolonged PFS (11.7 vs. 5.9 months, multivariate HR, 0.17; P = .001) for patients receiving FOLFOXIRI-bevacizumab on univariate and multivariate analyses. In recursive partitioning analysis, ACVRL1 was the strongest discriminator of the response rate, PFS, and OS in patients receiving FOLFOXIRI-bevacizumab and of OS in patients receiving FOLFIRI-bevacizumab. In silico validation revealed significant associations between ACVRL1 expression, disease recurrence, and 1-year survival (P < .05) among all colorectal cancer stages.
CONCLUSION: ACVRL1 expression could serve as a prognostic biomarker in metastatic colorectal cancer patients receiving chemotherapy and bevacizumab and warrants further evaluation in prospective studies.

INTRODUCTION: Peri-implant breast seroma is a late clinical presentation of reconstructive surgery or augmentation mammoplasty with breast implants. Pre-operative cytological evaluation of the peri-implant breast seroma is a common clinical approach, showing mainly an inflammatory reaction or more rarely a breast implant-associated anaplastic large cell lymphoma. Herein, we reported the role of cytology in the evaluation of peri-implant breast seroma and its critical pre-operative implications.
METHODS: Eight cases of peri-implant breast seroma from files at Luigi Vanvitelli University were identified between January and December 2017. In all cases, seroma was aspirated; cytospins were performed and stained by Papanicolaou stain; finally, in all cases, a cell block was obtained for immunocytochemical evaluation and, in one case, for FISH to detect ALK1-gene translocation.
RESULTS: The median age of patients was 48 years and the mean time between the implant placement and the occurrence of peri-implant breast seroma was 18 months. Microscopic examination showed breast implant-associated anaplastic large cell lymphoma in one case, aspecific inflammatory reaction in six cases and silicon-associated reaction in one case.
CONCLUSIONS: Peri-implant breast seroma may be caused by several pathological conditions with different clinical behaviour. A proper cytological approach to peri-implant breast seroma allows a correct differential diagnosis between inflammatory conditions and breast implant-associated anaplastic large cell lymphoma and an appropriate management of the patient.

Frequent kinase fusions have been reported in spitzoid neoplasms, approximately 10% of which involve ALK rearrangements. Herein, we report a case of atypical Spitz tumor with a novel MLPH-ALK fusion, which has not been previously reported to contribute to cancer development. The tumor was detected in the right arm of a 40-year-old woman. The novel ALK fusion was identified by a 5'-rapid amplification of cDNA ends-based system optimized for formalin-fixed, paraffin-embedded tissue. Initially, ALK expression was detected by immunohistochemistry using 5A4 antibodies for both sensitive and conventional polymer detection methods. However, the anti-ALK1 antibody, which is commonly used for the diagnosis of ALK-positive anaplastic large cell lymphoma, failed to confirm ALK expression. These results indicated that ALK immunohistochemistry results in ALK-rearranged atypical Spitz tumor may differ based on the type of primary antibody clone, which can be a potential diagnostic pitfall.

Previous studies showed that ALK is often positive in epithelioid fibrous histiocytoma (EFH). Two cases of EFH with ALK gene fusions have been recorded. Our objective was to study a series of EFH to present histopathological variations of EFH, identify novel ALK gene fusions, and determine whether there is a correlation between histopathological features and particular gene. We investigated 14 cases of EFH, all ALK immunopositive. The cases were assessed histopathologically as well as for ALK and TFE-3 rearrangements using FISH and ALK gene fusions using next-generation sequencing. The analysis of the sequencing results was performed using the Archer Analysis software (v5; ArcherDX Inc). The study group consisted of 8 female and 6 male patients, ranging in age from 18 to 79 years (mean 42 years; median 37.5 years). All presented with a solitary lesion. Microscopically, most lesions were polypoid and composed of epithelioid cells with ample cytoplasm. In addition, a variable number of bi-, tri-, or multinucleated, spindled, multilobated, cells with eccentric nuclei, cells with nuclear pseudoinclusions, mucinous, and grooved cells were admixed. In 5 cases, the predominant epithelioid cell component consisted of rather small cells, whereas spindled cells dominated in 3 cases. Of these, 2 lesions were composed rather of pale eosinophilic to clear cells, occasioning a resemblance to PEComa or leiomyoma. Immunohistochemically, all cases expressed ALK and 11 were positive for TFE-3. The break apart test for ALK was positive in 11 cases, whereas specimens from the remaining 3 cases were not analyzable. ALK genes fusions were found in all but 3 cases and included SQSTM1-ALK (3), VCL-ALK (3), TMP3-ALK (2), PRKAR2A-ALK (1), MLPH-ALK (1), and EML4-ALK (1). No correlation between histological features and type of ALK fusion was found. TFE-3 break apart test was negative. It is concluded that ALK-immunopositive EFH shows ALK gene fusions that involve various protein-coding genes, implicated in a variety of biological processes. Rare variants of EFH rather consist of spindled "non-epithelioid" cells.

INTRODUCTION: Systemic anaplastic large cell lymphoma (ALCL) accounts for 5%-10% of adult non Hodgkin's lymphoma (NHL) and 10%-30% of childhood NHL. Owing to significant differences in survival and gene expression profile, current WHO classifies ALCL into two distinct entities as anaplastic lymphoma receptor tyrosine kinase (ALK) positive and ALK negative ALCL with ALK expression by tumour as a good prognostic indicator. However, in our institute which is a cancer referral institute, our preliminary experience was that even ALK positive tumours did not fare well as compared to ALK- negative ALCL. So, the current study aims at exploring more clinical and pathological factors impacting survival in ALCL patients.
OBJECTIVE: To study clinical and pathological prognostic factors in cases of ALCL.
METHODS: 102 cases of ALCL were retrieved from pathology database. Pathological features and clinical features of these cases were recorded and factors found to impact overall survival (OAS) and disease-free survival (DFS) curves were identified based on univariate and multivariate analysis.
RESULTS: ALK 1 expression was seen in 71/102 (69.6%) cases and was not found to impact OAS or DFS. The 2 year OAS rate for ALK positive patients was 63.5% and DFS rate was 54.4%, while for ALK negative patients, the OAS was 60.5% and DFS was 43.5%. The Ann Arbor stage, performance status, international prognostic index, histological subtype, and the degree of the background inflammatory infiltrate were found to impact the OAS significantly. Increased reactive inflammatory component also negatively impacted DFS. In the multivariate analysis, only the histologic type emerged as significant for OAS.
CONCLUSION: Though ALK plays a role in prognostication of systemic ALCL, advanced stage disease and an inflammatory milieu may modulate the final outcome. We report a study of clinical and pathologic prognostic features in 102 cases of anaplastic large cell lymphoma (ALCL) from a cancer referral institute in India. Anaplastic lymphoma receptor tyrosine kinase (ALK-1) expression was seen in 71/102 (69.6%) cases and was not found to impact overall survival (OAS) or disease-free survival (DFS). The 2-year OAS rate for ALK-positive patients was 63.5% and DFS rate was 54.4%, while for ALK-negative patients, the OAS was 60.5% and DFS was 43.5%. The Ann Arbor stage, performance status, international prognostic index, histological subtype, and the degree of the background inflammatory infiltrate were found to impact the OAS significantly. Increased reactive inflammatory component also negatively impacted DFS. In the multivariate analysis, only the histologic type emerged as significant for OAS. Thus, though ALK plays a role in prognostication of systemic ALCL, advanced stage disease and an inflammatory milieu may modulate the final outcome.

FLT3 fusions are associated with myeloid and lymphoid neoplasms with eosinophilia. We describe a patient presenting with clinicopathologic features of both chronic eosinophilic leukemia, not otherwise specified (CEL, NOS) and systemic mastocytosis (SM). The bone marrow demonstrated a myeloproliferative neoplasm with eosinophilia and aggregates of atypical mast cells. Cytogenetic analysis revealed a t(13;14)(q12;q32), which was subsequently molecularly characterized as a novel TRIP11-FLT3 rearrangement. A KIT D816V mutation was also identified. The patient rapidly transformed to T-lymphoblastic leukemia/lymphoma and expired shortly after diagnosis. This is the fifth FLT3 fusion gene described in the literature; the presence of both myeloid and lymphoid neoplasms implicates involvement of an early hematopoietic progenitor by rearranged FLT3. We suggest that leukemias and lymphomas with FLT3 fusion genes exhibit similar clinicopathologic features to, and should be included in, the WHO category of "Myeloid and lymphoid neoplasms with eosinophilia and abnormalities of PDGFRA, PDGFRB, or FGFR1, or with PCM1-JAK2."

Endoglin is part of the TGF-β receptor complex and has a crucial role in fibrogenesis and angiogenesis. It is also an important protein for tumor growth, survival, and cancer cell metastasis. In a previous study, we have shown that hepatitis C virus (HCV) infection induces epithelial-mesenchymal transition (EMT) state and cancer stem-like cell (CSC) properties in human hepatocytes. Our array data suggested that endoglin (CD105) mRNA is significantly upregulated in HCV-associated CSCs. In this study, we have observed increased endoglin expression on the cell surface of an HCV core-expressing hepatocellular carcinoma (HepG2) cell line or immortalized human hepatocytes (IHH) and activation of its downstream signaling molecules. The status of phospho-SMAD1/5 and the expression of inhibitor of DNA binding protein 1 (ID1) were upregulated in HCV-infected cells or viral core gene-transfected cells. Additionally, we observed upregulation of endoglin/ID1 mRNA expression in chronic HCV patient liver biopsy samples. CSC generation by HCV core protein was dependent on the endoglin signaling pathway using activin receptor-like kinase 1 (ALK1) Fc blocking peptide and endoglin small interfering RNA (siRNA). Further, follow-up from

BACKGROUND ALK gene rearrangements as oncogenic drivers have been described in many cancers, including inflammatory myofibroblastic sarcoma (IMS). The first-generation ALK inhibitor was limited in its ability to cross the blood-brain-barrier to treat brain metastasis. Drug-resistance invariably develops over time in ALK-rearranged tumors, which leads to disease progression. The newer generations of ALK inhibitors are designed to have higher potency in ALK inhibition and improved CNS penetration. CASE REPORT We report a rare case of pulmonary IMS with ALK-1 gene rearrangement and multiple brain metastases as initial presentation. After the primary lung tumor and the larger brain metastases were resected, control of residual CNS disease and subsequent progression and CNS spread was achieved with favorable clinical response by all three generations of ALK inhibitors. CONCLUSIONS ALK inhibitors may be an effective therapy for this rare and unusual form of ALK-1-rearranged cancer, even in the presence of multifocal CNS metastases with leptomeningeal involvement.

Distinguishing between follicular thyroid cancer (FTC) and follicular thyroid adenoma (FTA) constitutes a long-standing diagnostic problem resulting in equivocal histopathological diagnoses. There is therefore a need for additional molecular markers. To identify molecular differences between FTC and FTA, we analyzed the gene expression microarray data of 52 follicular neoplasms. We also performed a meta-analysis involving 14 studies employing high throughput methods (365 follicular neoplasms analyzed). Based on these two analyses, we selected 18 genes differentially expressed between FTA and FTC. We validated them by quantitative real-time polymerase chain reaction (qRT-PCR) in an independent set of 71 follicular neoplasms from formaldehyde-fixed paraffin embedded (FFPE) tissue material. We confirmed differential expression for 7 genes (

TMPRSS2:ERG (T/E) gene fusions are present in approximately 50% of all prostate cancer (PCa) cases. The expression of fusion mRNAs from distinct T/E variants is associated with clinicopathological parameters, while the underlying molecular processes remain unclear. We characterized the molecular mechanisms and functional implications caused by doxycycline (Dox)-inducible overexpression of the frequent T/E III and VI fusion variants in LNCaP cells. Induction of T/E expression resulted in increased cellular migratory and invasive potential, and reduced proliferation and accumulation in G1 phase. T/E overexpressing cells showed epithelial-to-mesenchymal transition (EMT), as demonstrated by upregulation of TGF-β and WNT pathway genes, mesenchymal markers, and increased phosphorylation of the p38 MAPK. Augmented secretion of TGF-β1 and -β2, and T/E-mediated regulation of ALK1, a member of the TGF-β receptor family, was detected. ALK1 inhibition in T/E overexpressing cells blocked p38 phosphorylation and reduced the expression of the TGF-β target genes VIM, MMP1, CDH2, and SNAI2. We found a T/E variant VI-specific induction of miR-503 associated with reduced expression of SMAD7 and CDH1. Overexpression of miR-503 led to increased levels of VIM and MMP1. Our findings indicate that TGF-β signaling is a major determinant of EMT in T/E overexpressing LNCaP cells. We provide evidence that T/E VI-specific transcriptional modulation by miR-503 accounts for differences in the activation of EMT pathway genes, promoting the aggressive phenotype of tumors expressing T/E variant VI. We suggest that ALK1-mediated TGF-β signaling is a novel oncogenic mechanism in T/E positive PCa.

BACKGROUND: The anti-leukemic mechanism of homoharringtonine (HHT) differs from that of IM, and HHT is one of the most useful agents for use in patients with IM resistance or intolerance. The Bcl-6/p53 pathway has been shown to regulate the sensitivity of tumor cells to antitumor drugs. We tested whether HHT blocked the Bcl-6/p53 pathway in order to promote the apoptosis of IM-resistant cells in vitro and in vivo.
RESULTS: Ph+ acute lymphoblastic leukemia (ALL) cells and IM-resistant chronic myeloid leukemia (CML) cells showed high expression of Bcl-6 protein. Bcl-6 mediated the upregulation of p53, and and Bcl-6 induced growth inhibition of IM-resistant cells as well as its apoptosis by targeting p53. In addition, Bcl-6 was downregulated moderately after HHT treatment in different cells. The Bcl-6 expression was significantly increased in patients with CML when compared with healthy subjects. Furthermore, the expression of Bcl-6 was higher in patients with CML-blastic phase (CML-BP) than in those with CML-chronic phase (CML-CP).
METHODS: The inhibitory effect of drugs on cell growth was detected by Cell Counting Kit-8 (CCK-8), The apoptosis rate and the cell cycle were investigated by flow cytometry. The expression of Bcl-6, p53, Bcl-2, caspase9, and caspase3 proteins was assayed by western blot, Real- Time PCR (qPCR) detect Bcl-6 and p53 mRNA.
CONCLUSIONS: HHT can suppress the growth and induce apoptosis of IM-resistant cells, the mechanism of which is associated with blocking of the Bcl-6/p53 pathway. Our results could offer a theoretical explanation for HHT use in patients with IM resistance or intolerance.

Anaplastic lymphoma kinase ( ALK) gene aberrations-such as mutations, amplifications, and copy number gains-represent a major genetic predisposition to neuroblastoma (NB). This study aimed to evaluate the correlation between ALK gene copy number status, ALK protein expression, and clinicopathological parameters. We retrospectively retrieved 30 cases of poorly differentiated NB and constructed tissue microarrays (TMAs). ALK copy number changes were assessed by fluorescence in situ hybridization (FISH) assays, and ALK immunohistochemistry (IHC) testing was performed using three different antibodies (ALK1, D5F3, and 5A4 clones). ALK amplification and copy number gain were observed in 10% (3/30) and 53.3% (16/30) of the cohort, respectively. There were positive correlations between ALK copy number and IHC-positive rate in ALK1 and 5A4 antibodies ( P < 0.001 and P = 0.019, respectively). ALK1, D5F3, and 5A4 antibodies equally showed 100% sensitivity in detecting ALK amplification. However, the sensitivity for detecting copy number gain differed among the three antibodies, with 75% sensitivity in D5F3 and 0% sensitivity in ALK1. ALK-amplified NBs were correlated with synchronous MYCN amplification and chromosome 1p deletion. ALK IHC positivity was frequently observed in INSS stage IV and high-risk group patients. In conclusion, this study identified that an increase in the ALK copy number is a frequent genetic alteration in poorly differentiated NB. ALK-amplified NBs showed consistent ALK IHC positivity with all kinds of antibodies. In contrast, the detection performance of ALK copy number gain was antibody dependent, with the D5F3 antibody showing the best sensitivity.

Acute myeloid leukemia (AML) is a highly heterogeneous disease and internal tandem duplication mutation in FMS-like tyrosine-kinase-3 (FLT3-ITD) has a negative impact on outcome. Finding effective treatment regimens is desperately needed. In this study, we explored the inhibitory effect and mechanism of homoharringtonine (HHT) in combination with ibrutinib on FLT3-ITD mutant AML cells. Consequently, we observed a synergistic inhibitory effect when ibrutinib was combined with HHT to inhibit cell proliferation, induce apoptosis and arrest cell cycle at G0/G1 phase in MV4-11 and MOLM-13 leukemia cells. Our results indicate that the mechanisms of the combination effect are mainly via regulating the STAT5/Pim-2/C-Myc pathway, AKT pathway and Bcl-2 family, activating p21WAF1/CIP1 and inhibiting CCND/CDK complex protein. Interestingly, synergistic cytotoxicity of ibrutinib and HHT was dependent on both FLT3 and BTK. Here we provide a novel effective therapeutic approach for the treatment of AML patients with FLT3-ITD mutation.

Angiogenesis-related gene expression is associated with the efficacy of anti-VEGF therapy. We tested whether intratumoral mRNA expression levels of genes involved in vascular morphogenesis and early vessel maturation predict response, recurrence-free survival (RFS), and overall survival (OS) in a unique cohort of patients with colorectal liver metastases (CLM) treated with bevacizumab-based chemotherapy followed by curative liver resection. Intratumoral mRNA was isolated from resected bevacizumab-pretreated CLM from 125 patients. In 42 patients, a matching primary tumor sample collected before bevacizumab treatment was available. Relative mRNA levels of 9 genes (ACVRL1, EGFL7, EPHB4, HIF1A, VEGFA, VEGFB, VEGFC, FLT1, and KDR) were analyzed by RT-PCR and evaluated for associations with response, RFS, and OS. P values for the associations between the individual dichotomized expression level and RFS were adjusted for choosing the optimal cut-off value. In CLM, high expression of VEGFB, VEGFC, HIF1A, and KDR and low expression of EGFL7 were associated with favorable RFS in multivariable analysis (P < 0.05). High ACVRL1 levels predicted favorable 3-year OS (P = 0.041) and radiologic response (PR = 1.093, SD = 0.539, P = 0.002). In primary tumors, low VEGFA and high EGFL7 were associated with radiologic and histologic response (P < 0.05). High VEGFA expression predicted shorter RFS (10.1 vs. 22.6 months; HR = 2.83, P = 0.038). High VEGFB (46% vs. 85%; HR = 5.75, P = 0.009) and low FLT1 (55% vs. 100%; P = 0.031) predicted lower 3-year OS rates. Our data suggest that intratumoral mRNA expression of genes involved in vascular morphogenesis and early vessel maturation may be promising predictive and/or prognostic biomarkers. Mol Cancer Ther; 15(11); 2814-21. ©2016 AACR.

INTRODUCTION: We performed a genomic study in lung adenocarcinoma cases with discordant anaplastic lymphoma receptor tyrosine kinase gene (ALK) status by fluorescent in situ hybridization (FISH) and immunohistochemical (IHC) analysis.
METHODS: DNA from formalin-fixed paraffin-embedded tissues of 16 discordant (four FISH-positive/IHC-negative and 12 FISH-negative/IHC-positive) cases by Vysis ALK Break Apart FISH and ALK IHC testing (ALK1 clone) were subjected to whole gene capture and next-generation sequencing (NGS) of nine genes, including ALK, echinoderm microtubule associated protein like 4 gene (EML4), kinesin family member 5B gene (KIF5B), staphylococcal nuclease and tudor domain containing 1 gene (SND1), BRAF, ret proto-oncogene (RET), ezrin gene (EZR), ROS1, and telomerase reverse transcriptase (TERT). All discordant cases (except one FISH-negative/IHC-positive case without sufficient tissue) were analyzed by IHC with D5F3 antibody. In one case with fresh frozen tissue, whole transcriptome sequencing was also performed. Twenty-six concordant (16 FISH-positive/IHC-positive and 10 FISH-negative/IHC-negative) cases were included as controls.
RESULTS: In four ALK FISH-positive/IHC-negative cases, no EML4-ALK fusion gene was observed by NGS, but in one case using fresh frozen tissue, we identified EML4-baculoviral AIP repeat containing 6 gene (BIRC6) and AP2 associated kinase 1 gene (AAK1)-ALK fusion genes. Whole transcriptome sequencing revealed a highly expressed EML4-BIRC6 fusion transcript and a minimally expressed AAK1 transcript. Among the 12 FISH-negative/IHC-positive cases, no evidence of ALK gene rearrangement was detected by NGS. Eleven of 12 FISH-negative/IHC-positive cases detected by ALK1 clone were concordant by repeat ALK IHC with D5F3 antibody (i.e., FISH-negative/IHC-negative by D5F3 clone). Among the 16 ALK FISH-positive/IHC-positive positive controls, whole gene capture identified ALK gene fusion in 15 cases, including in one case with Huntington interacting protein 1 gene (HIP1)-ALK. No ALK fusion gene was observed in any of the 10 FISH-negative/IHC-negative cases. Other fusion genes involving ROS1, EZR, BRAF, and SND1 were also found.
CONCLUSIONS: ALK FISH results appeared to be false-positive in three of four FISH-positive/IHC-negative cases, whereas no false-negative ALK FISH case was identified among 12 ALK FISH-negative/IHC-positive cases by ALK1 clone, which was in keeping with the concordant FISH-negative/IHC-negative status by D5F3 clone. Our targeted whole gene capture approach using formalin-fixed paraffin embedded samples was effective for detecting rearrangements involving ALK and other actionable oncogenes.

Angiogenesis occurs early in tumor development, sustains primary tumor growth and provides a route for metastatic escape. The TGF-β family receptors modulate angiogenesis via endothelial-cell specific pathways. Here we investigate the interaction of two such receptors, ALK1 and endoglin, in pancreatic neuroendocrine tumors (PanNET). Independently, ALK1 and endoglin deficiencies exhibited genetically divergent phenotypes, while both highly correlate to an endothelial metagene in human and mouse PanNETs. A concurrent deficiency of both receptors synergistically decreased tumor burden to a greater extent than either individual knockdown. Furthermore, the knockout of Gdf2 (BMP9), the primary ligand for ALK1 and endoglin, exhibited a mixed phenotype from each of ALK1 and endoglin deficiencies; overall primary tumor burden decreased, but hepatic metastases increased. Tumors lacking BMP9 display a hyperbranching vasculature, and an increase in vascular mesenchymal-marker expression, which may be implicit in the increase in metastases. Taken together, our work cautions against singular blockade of BMP9 and instead demonstrates the utility of dual blockade of ALK1 and endoglin as a strategy for anti-angiogenic therapy in PanNET.

An in vitro drug-screening platform on patient samples was developed and validated to design personalized treatment for relapsed/refractory acute myeloid leukemia (AML). Unbiased clustering and correlation showed that homoharringtonine (HHT), also known as omacetaxine mepesuccinate, exhibited preferential antileukemia effect against AML carrying internal tandem duplication of fms-like tyrosine kinase 3 (FLT3-ITD). It worked synergistically with FLT3 inhibitors to suppress leukemia growth in vitro and in xenograft mouse models. Mechanistically, the effect was mediated by protein synthesis inhibition and reduction of short-lived proteins, including total and phosphorylated forms of FLT3 and its downstream signaling proteins. A phase 2 clinical trial of sorafenib and HHT combination treatment in FLT3-ITD AML patients resulted in complete remission (true or with insufficient hematological recovery) in 20 of 24 patients (83.3%), reduction of ITD allelic burden, and median leukemia-free and overall survivals of 12 and 33 weeks. The regimen has successfully bridged five patients to allogeneic hematopoietic stem cell transplantation and was well tolerated in patients unfit for conventional chemotherapy, including elderly and heavily pretreated patients. This study validated the principle and clinical relevance of in vitro drug testing and identified an improved treatment for FLT3-ITD AML. The results provided the foundation for phase 2/3 clinical trials to ascertain the clinical efficacy of FLT3 inhibitors and HHT in combination.

Anaplastic lymphoma kinase-positive large B-cell lymphoma (ALK+ LBCL) is a rare distinct type of non-Hodgkin's lymphoma that arises in association with alterations of the ALK gene. This distinct disease entity is typically associated with an aggressive clinical course and appears in light microscopic preparations as a monomorphic population of large, immunoblast-like cells. In this report, we describe a case of ALK+ LBCL diagnosed by transgastric endoscopic ultrasound-guided fine needle aspiration (EUS FNA) of splenic hilar lymph nodes. Modified Giemsa stained direct smears from the FNA sample demonstrated large lesional cells with foamy cytoplasm and macronucleoli admixed with small lymphocytes in tigroid backgrounds, mimicking the cytologic appearance of seminoma. Ancillary immunohistochemical studies subsequently confirmed the diagnosis of ALK+ LBCL with the lesional cells being immunoreactive for CD138, VS38c, MUM1, ALK1, and lambda light chain. The cohesiveness of the cells, the cellular morphology, and the tigroid backgrounds were all pitfalls for accurate diagnosis of this rare specific type of lymphoid malignancy by cytology. To our knowledge this is the first case report detailing the diagnosis of ALK+ LBCL by EUS FNA and the first report describing a glycogen-rich tigroid background in direct FNA smears. Establishing a refined diagnosis in cases of this rare form of LBCL is necessary, as therapies targeting ALK may be of value in clinical management. Diagn. Cytopathol. 2017;45:148-155. © 2016 Wiley Periodicals, Inc.

Bone morphogenetic protein (BMP)9 and BMP10 are high affinity ligands for activin receptor-like kinase 1 (ALK1), a type I BMP receptor mainly expressed on vascular endothelial cells (ECs). ALK1-mediated BMP9/BMP10 signalling pathways have emerged as essential in EC biology and in angiogenesis. Several genetic mutations in the genes encoding the ligands and receptors of this pathway have been reported in two cardiovascular diseases, pulmonary arterial hypertension (PAH) and hereditary haemorrhagic telangiectasia (HHT). Administration of recombinant BMP9 reverses experimental PAH in preclinical rodent models. Dalantercept, an Fc-fusion protein of the extracellular domain of ALK1 and a ligand trap for BMP9 and BMP10, is in phase II clinical trials for anti-tumour angiogenesis. Understanding the regulation of BMP9 and BMP10, at both gene and protein levels, under physiological and pathological conditions, will reveal essential information and potential novel prognostic markers for the BMP9/BMP10-targeted therapies.

Inflammatory myofibroblastic tumors (IMTs) are rare mesenchymal tumors initially described in the lung. About half of them exhibit expression of the ALK1 protein, generally resulting from a gene rearrangement. Paranasal sinus IMTs are extremely uncommon, and gene rearrangement of ALK1 is very rare in this localization. A 47-year-old woman presented with rapidly progressive vision loss in her left eye. Clinical and imaging work-up revealed a tumor invading the left ethmoidal and sphenoidal sinuses and extending into the nasal cavity, the orbit and the skull base. Complete tumor resection was performed using an endonasal approach. Pathological examination revealed a paranasal localization of IMT, positive for ALK1 immunostaining. FISH analysis showed an ALK1 gene rearrangement. This case illustrates the local aggressive potential for IMTs. Treatment is primarily surgical, but targeted therapies (crizotinib) might be a solution for ALK1 rearranged cases with a poor prognosis.

Inflammatory myofibroblastic tumor (IMT) is a rare neoplasm. Approximately 50 % of IMTs show an anaplastic lymphoma kinase (ALK) gene fusion resulting in ALK overexpression on immunohistochemistry (IHC). A novel anti-ALK monoclonal antibody (D5F3) has been suggested to be of superior sensitivity to the ALK1 antibody which is currently used. We compared the performance of D5F3 in detecting ALK protein expression in IMTs from various anatomic sites compared to the currently utilized ALK1. We selected 25 IMTs from our surgical pathology files (2005-2015). The novel rabbit monoclonal anti-human CD246 (clone D5F3) and the currently used mouse monoclonal anti-human CD246 (clone ALK1) were used for immunohistochemical staining (IHC) in an automated slide stainer. The percentage of immunoreactive tumor cells (0, <5 %, 5-50 %, >50 %) and cytoplasmic staining intensity (graded 0-3) were assessed and compared between the two antibodies. Fluorescence in situ hybridization (FISH) studies for ALK gene rearrangement were performed on 11 tumors. D5F3 antibody stained 76 % and ALK1 antibody stained 72 % of IMTs (p = 0.747). Compared to staining with ALK1, D5F3 stained a higher proportion of cases extensively (>50 % cells) (76 vs. 28 %, p < 0.001) and with high intensity (grade 3 76 % vs 0; p < 0.001). FISH and IHC findings (for both antibodies) were concordant in 9/10 (90 %) IMTs, in which results were informative. The novel anti-ALK rabbit monoclonal antibody (D5F3 clone) demonstrates superior overall performance in term of intensity and extent of staining of ALK protein in IMT. We found IHC staining with both antibody clones to correlate equally well with FISH results for detection of ALK rearrangement.