Identification of Driving ALK Fusion Genes and Genomic Landscape of Medullary Thyroid Cancer

The genetic landscape of medullary thyroid cancer (MTC) is not yet fully understood, although some oncogenic mutations have been identified. To explore genetic profiles of MTCs, formalin-fixed, paraffin-embedded tumor tissues from MTC patients were assayed on the Ion AmpliSeq Cancer Panel v2. Eighty-four sporadic MTC samples and 36 paired normal thyroid tissues were successfully sequenced. We discovered 101 hotspot mutations in 18 genes in the 84 MTC tissue samples. The most common mutation was in the ret proto-oncogene, which occurred in 47 cases followed by mutations in genes encoding Harvey rat sarcoma viral oncogene homolog (N = 14), serine/threonine kinase 11 (N = 11), v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (N = 6), mutL homolog 1 (N = 4), Kiesten rat sarcoma viral oncogene homolog (N = 3) and MET proto-oncogene (N = 3). We also evaluated anaplastic lymphoma kinase (ALK) rearrangement by immunohistochemistry and break-apart fluorescence in situ hybridization (FISH). Two of 98 screened cases were positive for ALK FISH. To identify the genomic breakpoint and 5’ fusion partner of ALK, customized targeted cancer panel sequencing was performed using DNA from tumor samples of the two patients. Glutamine:fructose-6-phosphate transaminase 1 (GFPT1)-ALK and echinoderm microtubule-associated protein-like 4 (EML4)-ALK fusions were identified. Additional PCR analysis, followed by Sanger sequencing, confirmed the GFPT1-ALK fusion, indicating that the fusion is a result of intra-chromosomal translocation or deletion. Notably, a metastatic MTC case harboring the EML4-ALK fusion showed a dramatic response to an ALK inhibitor, crizotinib. In conclusion, we found several genetic mutations in MTC and are the first to identify ALK fusions in MTC. Our results suggest that the EML4-ALK fusion in MTC may be a potential driver mutation and a valid target of ALK inhibitors. Furthermore, the GFPT1-ALK fusion may be a potential candidate for molecular target therapy.     

Clinicopathological Characteristics of Patients with Non-Small-Cell Lung Cancer Who Harbor EML4-ALK Fusion Gene: A Meta-Analysis

BackgroundA novel fusion gene of echinoderm microtubule-associated protein-like 4 (EML4) and anaplastic lymphoma kinase (ALK) has been recently identified in non-small-cell lung cancers (NSCLCs). Patients with the EML4-ALK fusion gene demonstrate unique clinicopathological and physiological characteristics. Here we present a meta-analysis of large-scale studies to evaluate the clinicopathological characteristics of NSCLC patients harboring the EML4-ALK fusion gene.MethodsBoth English and Chinese databases were systematically used to search the materials of the clinicopathological characteristics of patients with NSCLC harboring the EML4-ALK fusion gene. Pooled relative risk (RR) estimates and the 95% confidence intervals (95% CI) were calculated with the fixed or random effect model. Publication bias and chi-square test were also calculated.Results27 retrospective studies were included in our meta-analysis. These studies included a total of 6950 patients. The incidence rate of EML4-ALK fusion in NSCLC patients was found to be 6.8% (472/6950). The correlation of the EML4-ALK fusion gene and clinicopathological characteristics of NSCLC patients demonstrated a significant difference in smoking status, histological types, stage, and ethnic characteristics. The positive rate of the EML4-ALK fusion gene expression in females were slightly higher than that in males, but not significantly (P = 0.52). In addition, the EML4-ALK fusion gene was mutually exclusive of the EGFR and KRAS mutation genes (P = 0.00).ConclusionOur pooled analysis revealed that the EML4-ALK fusion gene was observed predominantly in adenocarcinoma, non-smoking and NSCLC patients, especially those diagnosed in the advanced clinical stage of NSCLC. Additionally, the EML4-ALK fusion gene was exclusive of the EGFR and KRAS mutation genes. We surmise that IHC assay is a valuable tool for the prescreening of patients with ALK fusion gene in clinical practice, and FISH assay can be performed as a confirmation method. These insights might be helpful in guiding the appropriate molecular target therapy for NSCLC.     

Anaplastic Lymphoma Kinase Rearrangement in Digestive Tract Cancer: Implication for Targeted Therapy in Chinese Population

Background

Anaplastic lymphoma kinase (ALK) rearrangements define a subgroup of lung cancer which is eligible to targeted kinase inhibition. The aim of this study is to observe the incidence rate of ALK fusion in a large cohort of Chinese digestive tract cancer patients.

Patients and Methods

Tissue microarray (TMA) was constructed from 808 digestive tract cancer cases, including 169 esophageal squamous cell carcinoma, 182 gastric cancer and 457 colorectal cancer (CRC) cases. We tested all cases for ALK expression via a fully automated immunohistochemistry (IHC) assay. The IHC-positive cases were subjected to fluorescence in situ hybridization (FISH), real-time polymerase chain reaction (qRT-PCR), target gene enrichment and sequencing for confirmation of ALK gene rearrangement and discovery of novel fusion partner.

Results

Among the tested cases, 2 (0.44%) CRC cases showed positive both by IHC and FISH. By qRT-PCR, EML4–ALK fusion was found in one IHC-positive CRC case. In another IHC-positive CRC case, target gene enrichment and sequencing revealed ALK was fused to a novel partner, spectrin beta non-erythrocytic 1 (SPTBN1). One gastric cancer case showed partially positive IHC result, but no fusion was found by FISH and gene sequencing.

Conclusions

The incidence rate of ALK gene fusion in Chinese CRC patients was 0.44%,but not detectable in gastric and esophageal cancers. The novel SPTBN1 -ALK fusion, together with other ALK fusion genes, may become a potential target for anti-ALK therapy.     

Oral Metronomic Topotecan Sensitizes Crizotinib Antitumor Activity in ALKF1174L Drug-Resistant Neuroblastoma Preclinical Models

BACKGROUND: Anaplastic lymphoma kinase (ALK) inhibitor crizotinib has proven to be effective in the treatment of ALK-mutated neuroblastoma, but crizotinib resistance was commonly observed in patients. We aimed to overcome crizotinib resistance by combining with the MEK inhibitor trametinib or low-dose metronomic (LDM) topotecan in preclinical neuroblastoma models. METHODS: We selected a panel of neuroblastoma cell lines carrying various ALK genetic aberrations to assess the therapeutic efficacy on cell proliferation in vitro. Downstream signals of ALK activation, including phosphorylation of ERK1/2, Akt as well as HIF-1α expression were evaluated under normoxic and hypoxic conditions. Tumor growth inhibition was further assessed in NOD/SCID xenograft mouse models. RESULTS: All NBL cell lines responded to crizotinib treatment but at variable ED50 levels, ranging from 0.25 to 5.58 μM. ALK-mutated cell lines SH-SY5Y, KELLY, LAN-5, and CHLA-20 are more sensitive than ALK wild-type cell lines. In addition, we demonstrated that under hypoxic conditions, all NBL cell lines showed marked decrease of ED50s when compared to normoxia except for KELLY cells. Taking into consideration the hypoxia sensitivity to crizotinib, combined treatment with crizotinib and LDM topotecan demonstrated a synergistic effect in ALK^F1174L-mutated SH-SY5Y cells. In vivo, single-agent crizotinib showed limited antitumor activity in ALK^F1174L-mutated SH-SY5Y and KELLY xenograft models; however, when combined with topotecan, significantly delayed tumor development was achieved in both SH-SY5Y and KELLY tumor models. CONCLUSIONS: Oral metronomic topotecan reversed crizotinib drug resistance in the ALK^F1174L-mutated neuroblastoma preclinical model.     

Downregulation of PDGFRß Signaling Overcomes Crizotinib Resistance in a TYRO3 and ALK Mutated Neuroendocrine-Like Tumor

Patient-derived xenografts provide significant advantages over long-term passage cell lines when investigating efficacy of treatments for solid tumors. Our laboratory encountered a high-grade, metastatic, neuroendocrine-like tumor from a pediatric patient that presented with a unique genetic profile. In particular, mutations in TYRO3 and ALK were identified. We established a human patient-derived xenoline (PDX) of this tumor for use in the current study. We investigated the effect of crizotinib, a chemotherapeutic known to effectively target both TYRO3 and ALK mutations. Crizotinib effectively decreased viability, proliferation, growth, and the metastatic properties of the PDX tumor through downregulation of STAT3 signaling, but expression of PDGFRß was increased. Sunitinib is a small molecule inhibitor of PDGFRß and was studied in this PDX independently and in combination with crizotinib. Sunitinib alone decreased viability, proliferation, and growth in vitro and decreased tumor growth in vivo. In combination, sunitinib was able to overcome potential crizotinib-induced resistance through downregulation of ERK 1/2 activity and PDGFRß receptor expression; consequently, tumor growth was significantly decreased both in vitro and in vivo. Through the use of the PDX, it was possible to identify crizotinib as a less effective therapeutic for this tumor and suggest that targeting PDGFRß would be more effective. These findings may translate to other solid tumors that present with the same genetic mutations.     

In Vitro Drug Sensitivity Tests to Predict Molecular Target Drug Responses in Surgically Resected Lung Cancer

BackgroundEpidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) and anaplastic lymphoma kinase (ALK) inhibitors have dramatically changed the strategy of medical treatment of lung cancer. Patients should be screened for the presence of the EGFR mutation or echinoderm microtubule-associated protein-like 4 (EML4)-ALK fusion gene prior to chemotherapy to predict their clinical response. The succinate dehydrogenase inhibition (SDI) test and collagen gel droplet embedded culture drug sensitivity test (CD-DST) are established in vitro drug sensitivity tests, which may predict the sensitivity of patients to cytotoxic anticancer drugs. We applied in vitro drug sensitivity tests for cyclopedic prediction of clinical responses to different molecular targeting drugs.MethodsThe growth inhibitory effects of erlotinib and crizotinib were confirmed for lung cancer cell lines using SDI and CD-DST. The sensitivity of 35 cases of surgically resected lung cancer to erlotinib was examined using SDI or CD-DST, and compared with EGFR mutation status.ResultsHCC827 (Exon19: E746-A750 del) and H3122 (EML4-ALK) cells were inhibited by lower concentrations of erlotinib and crizotinib, respectively than A549, H460, and H1975 (L858R+T790M) cells were. The viability of the surgically resected lung cancer was 60.0 ± 9.8 and 86.8 ± 13.9% in EGFR-mutants vs. wild types in the SDI (p = 0.0003). The cell viability was 33.5 ± 21.2 and 79.0 ± 18.6% in EGFR mutants vs. wild-type cases (p = 0.026) in CD-DST.ConclusionsIn vitro drug sensitivity evaluated by either SDI or CD-DST correlated with EGFR gene status. Therefore, SDI and CD-DST may be useful predictors of potential clinical responses to the molecular anticancer drugs, cyclopedically.     

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.     

Transmission and selection of macrolide resistant Mycoplasma genitalium infections detected by rapid high resolution melt analysis

Background

Mycoplasma genitalium (MG) causes urethritis, cervicitis and pelvic inflammatory disease. The MG treatment failure rate using 1 g azithromycin at an Australian Sexual Health clinic in 2007–9 was 31% (95%CI 23–40%). We developed a rapid high resolution melt analysis (HRMA) assay targeting resistance mutations in the MG 23S rRNA gene, and validated it against DNA sequencing by examining pre- and post-treatment archived samples from MG-infected patients.

Methodology/Principal Findings

Available MG-positive pre-treatment (n = 82) and post-treatment samples from individuals with clinical treatment failure (n = 20) were screened for 23S rRNA gene mutations. Sixteen (20%) pre-treatment samples possessed resistance mutations (A2058G, A2059G, A2059C), which were significantly more common in patients with symptomatic azithromycin-treatment failure (12/26; 44%) than in those clinically cured (4/56; 7%), p<0.001. All 20 patients experiencing azithromycin-failure had detectable mutations in their post-treatment samples. In 9 of these cases, the same mutational types were present in both pre- and post-treatment samples indicating transmitted resistance, whilst in 11 of these cases (55%), mutations were absent in pre-treatment samples indicating likely selection of resistant isolates have occurred. HRMA was able to detect all mutational changes determined in this study by DNA sequencing. An additional HRMA assay incorporating an unlabelled probe was also developed to detect type 4 single-nucleotide polymorphisms found in other populations, with a slightly lower sensitivity of 90%.

Conclusions/Significance

Treatment failure is associated with the detection of macrolide resistance mutations, which appear to be almost equally due to selection of resistant isolates following exposure to 1 g azithromycin and pre-existing transmitted resistance. The application of a rapid molecular assay to detect resistance at the time of initial detection of infection allows clinicians to shorten the time to initiate effective second line treatment. This has the potential to reduce transmission of resistant strains and to avoid sequelae associated with persistent untreated infection.     

Molecular docking studies shows tivozanib and lapatinib as potential inhibitors of EML4-ALK translocation mediated fusion protein in non small cell lung cancer

Identification of activating mutations in non-small cell lung cancers (NSCLC) has been a focus in recent years. This led to successful evidence of using tyrosine kinase inhibitors (TKIs) over the standard platinum doublet based chemotherapy as the first line treatment in the metastatic setting.The rearrangements of fusion protein EML4-ALK in NSCLC lead to the use of crizotinib for this class of tumors. Preclinical and Phase 1 clinical studies show that ceritinib is more effective against both crizotinib sensitive and resistant tumors. Although robust responses to crizotinib are observed in NSCLC harboring ALK mutations, majority of tumors eventually become resistant, posing a major challenge in treatment course. Thus, there is a need for the identification and development of second-generation of ALK inhibitors. Computer aided molecular docking data show Tivozanib and Lapatinib bind EML4-ALK with high score. Tivozanib is in clinical trials for renal cell cancer and Lapatinib is a known dual tyrosine kinase inhibitor effective in breast cancer patients with HER2 over-expression. Additional data on these compounds for use in EML4-ALK positive NSCLC will provide evidence for use in patients treated with crizotinib. Data shows the importance of computer aided molecular docking in developing candidates with improved activity for further consideration in vitro and in vivo validation.     

Spectrum of oncogenic driver mutations in lung adenocarcinomas from East Asian never smokers

Purpose

We previously showed that 90% (47 of 52; 95% CI, 0.79 to 0.96) of lung adenocarcinomas from East Asian never-smokers harbored well-known oncogenic mutations in just four genes: EGFR, HER2, ALK, and KRAS. Here, we sought to extend these findings to more samples and identify driver alterations in tumors negative for these mutations.

Experimental Design

We have collected and analyzed 202 resected lung adenocarcinomas from never smokers seen at Fudan University Shanghai Cancer Center. Since mutations were mutually exclusive in the first 52 examined, we determined the status of EGFR, KRAS, HER2, ALK, and BRAF in stepwise fashion as previously described. Samples negative for mutations in these 5 genes were subsequently examined for known ROS1 fusions by RT-PCR and direct sequencing.

Results

152 tumors (75.3%) harbored EGFR mutations, 12 (6%) had HER2 mutations, 10 (5%) had ALK fusions all involving EML4 as the 5′ partner, 4 (2%) had KRAS mutations, and 2 (1%) harbored ROS1 fusions. No BRAF mutation were detected.

Conclusion

The vast majority (176 of 202; 87.1%, 95% CI: 0.82 to 0.91) of lung adenocarcinomas from never smokers harbor mutant kinases sensitive to available TKIs. Interestingly, patients with EGFR mutant patients tend to be older than those without EGFR mutations (58.3 Vs 54.3, P = 0.016) and patient without any known oncogenic driver tend to be diagnosed at a younger age (52.3 Vs 57.9, P = 0.013). Collectively, these data indicate that the majority of never smokers with lung adenocarcinoma could benefit from treatment with a specific tyrosine kinase inhibitor.     

Prognosis of ALK-rearranged non-small-cell lung cancer patients carrying TP53 mutations

Non-small-cell lung cancer (NSCLC) is the primary cause of cancer-related death. Gene rearrangements involving the anaplastic lymphoma kinase (ALK) tyrosine kinase identify a clinical and molecular subset of NSCLC patients, who benefit from the monotherapy with ALK tyrosine kinase inhibitors. Nonetheless, responsiveness to TKIs and prognosis of these patients are influenced by several factors, including resistance mechanisms and mutations affecting genes involved in key molecular pathways of cancer cells. In a cohort of 98 NSCLC patients with ALK gene rearrangements, we investigated the role of Tumor Protein (TP53) gene mutations in predicting patients prognosis. TP53 mutations were evaluated in relation to disease control rate (DCR), objective response rate (ORR), progression-free survival (PFS) and overall survival (OS).Results: In patients with available clinical and TP53 mutation information, we found that 13 patients (20.3%) were affected by TP53 mutations. Considered together, even though showing a trend, TP53 mutations were not associated with PFS and OS. Considering the different TP53 mutations by functionality in terms of disruptive and non-disruptive mutations, we observed that TP53 non-disruptive mutations were able to predict worse OS in the overall case series. Moreover, a worse PFS was seen in the subgroup of patients with TP53 non-disruptive mutation, in first-, second-, and third line of treatment. Our results show that mutations affecting TP53 gene, especially non-disruptive mutations, are able to affect prognosis of ALK-rearranged NSCLC patients.     

Will the clinical development of 4th-generation “double mutant active” ALK TKIs (TPX-0131 and NVL-655) change the future treatment paradigm of ALK+ NSCLC?

Our current treatment paradigm of advanced anaplastic lymphoma kinase fusion (ALK+) non-small cell lung cancer (NSCLC) classifies the six currently approved ALK tyrosine kinase inhibitors (TKIs) into three generations. The 2nd-generation (2G) and 3rd-generation (3G) ALK TKIs are all “single mutant active” with varying potencies across a wide spectrum of acquired single ALK resistance mutations. There is a vigorous debate among clinicians which is the best upfront ALK TKI is for the first-line (1L) treatment of ALK+ NSCLC and the subsequent sequencing strategies whether it should be based on the presence of specific on-target ALK resistance mutations or not. Regardless, sequential use of “single mutant active” ALK TKIs will eventually lead to double ALK resistance mutations in cis. This has led to the creation of fourth generation (4G) “double mutant active” ALK TKIs such as TPX-0131 and NVL-655. We discuss the critical properties 4G ALK TKIs must possess to be clinically successful. We proposed conceptual first-line, second-line, and molecularly-based third-line registrational randomized clinical trials designed for these 4G ALK TKIs. How these 4G ALK TKIs would be used in the future will depend on which line of treatment the clinical trial design(s) is adopted provided the trial is positive. If approved, 4G ALK TKIs may usher in a new treatment paradigm for advanced ALK+ NSCLC that is based on classifying ALK TKIs based on the intrinsic functional capabilities (“singe mutant active” versus “double mutant active”) rather than the loosely-defined “generational” (first-, second-,third-,fourth-) classification and avoid the current clinical approaches of seemingly random sequential use of 2G and 3G ALK TKIs.     

Mutation Analysis of BRCA1, BRCA2, PALB2 and BRD7 in a Hospital-Based Series of German Patients with Triple-Negative Breast Cancer

Triple-negative breast cancer (TNBC) is an aggressive form of breast carcinoma with a poor prognosis. Recent evidence suggests that some patients with TNBC harbour germ-line mutations in DNA repair genes which may render their tumours susceptible to novel therapies such as treatment with PARP inhibitors. In the present study, we have investigated a hospital-based series of 40 German patients with TNBC for the presence of germ-line mutations in BRCA1, BRCA2, PALB2, and BRD7 genes. Microfluidic array PCR and next-generation sequencing was used for BRCA1 and BRCA2 analysis while conventional high-resolution melting and Sanger sequencing was applied to study the coding regions of PALB2 and BRD7, respectively. Truncating mutations in BRCA1 were found in six patients, and truncating mutations in BRCA2 and PALB2 were detected in one patient each, whereas no truncating mutation was identified in BRD7. One patient was a double heterozygote for the PALB2 mutation, c.758insT, and a BRCA1 mutation, c.927delA. Our results confirm in a hospital-based setting that a substantial proportion of German TNBC patients (17.5%) harbour germ-line mutations in genes involved in homology-directed DNA repair, with a preponderance of BRCA1 mutations. Triple-negative breast cancer should be considered as an additional criterion for future genetic counselling and diagnostic sequencing.     

High-Resolution Array CGH Profiling Identifies Na/K Transporting ATPase Interacting 2 (NKAIN2) as a Predisposing Candidate Gene in Neuroblastoma

Neuroblastoma (NB), the most common solid cancer in early childhood, usually occurs sporadically but also its familial occurance is known in 1-2% of NB patients. Germline mutations in the ALK and PHOX2B genes have been found in a subset of familial NBs. However, because some individuals harbouring mutations in these genes do not develop this tumor, additional genetic alterations appear to be required for NB pathogenesis. Herein, we studied an Italian family with three NB patients, two siblings and a first cousin, carrying an ALK germline-activating mutation R1192P, that was inherited from their unaffected mothers and with no mutations in the PHOX2B gene. A comparison between somatic and germline DNA copy number changes in the two affected siblings by a high resolution array-based Comparative Genomic Hybridization (CGH) analysis revealed a germline gain at NKAIN2 (Na/K transporting ATPase interacting 2) locus in one of the sibling, that was inherited from the parent who does not carry the ALK mutation. Surprisingly, NKAIN2 was expressed at high levels also in the affected sibling that lacks the genomic gain at this locus, clearly suggesting the existance of other regulatory mechanisms. High levels of NKAIN2 were detected in the MYCN-amplified NB cell lines and in the most aggressive NB lesions as well as in the peripheral blood of a large cohort of NB patients. Consistent with a role of NKAIN2 in NB development, NKAIN2 was down-regulated during all-trans retinoic acid differentiation in two NB cell lines. Taken together, these data indicate a potential role of NKAIN2 gene in NB growth and differentiation.