bluuebunny/crossref_metadata_2025 huggingface dataset:
Rituximab, gemcitabine, cisplatin, and methylprednisolone (R-GEM-P) in treatment of relapsed diffuse large B-cell lymphoma (DLBCL).
Authors: Barton Sarah Rachel , Hawkes Eliza Anne , Cunningham David , Peckitt Clare , Chua Sue , Wotherspoon Andrew , Attygalle Ayoma , Horwich Alan , Dearden Claire E. , Potter Mike , Chau Ian
Published: March 2020

e19543

Background: Patients with DLBCL who relapse after 1st line therapy have poor prognosis. Although there is limited evidence for benefit of rechallenging patients with rituximab, this is considered justified by minimal toxicity and potential outcome improvement. GEM-P+/-R is an accepted salvage regimen, with merits of outpatient delivery and mainly non-overlapping toxicity with 1stline therapy, but there are relatively few reports of its efficacy and tolerability. Methods: We performed a retrospective single centre analysis of all patients meeting the following criteria: age≥18, histologically proven DLBCL, ≥1 FDG-PET, treated between 2001-2011 with GEM-P+/-R (gemcitabine 1000mg/m2 D1, D8, D15, methylprednisolone 1000mg D1-5, cisplatin 100mg/m2 D15 every 28 days +/- rituximab 375mg/m2 D1 & D15) or GEM-CARBO+/-R (cisplatin substituted for carboplatin AUC5). Data were collected on patient and treatment characteristics, response evaluated by modified IWG 2007 criteria (FDG-PET +/- CT), survival, and toxicity (CTCAE v4.0). Results: We identified 74 patients with median age 56 (range 25-79) who received a median of 3 cycles (range 1-6) of either GEM-P (89%) or GEM-CARBO(11%), combined with rituximab in 69%. 41% had transformed indolent lymphoma. Overall response rate (ORR) was 49% (complete response (CR) 16%, partial response (PR) 32%). Among 40/74 patients evaluated with FDG-PET, metabolic ORR was 63% (mCR 23%, mPR 40%). Of 51/74 patients treated in 2ndline, ORR was 45%. Significantly higher ORR was observed in patients treated with R-chemotherapy (61% vs 22%, p=0.002), despite 88% having received prior rituximab. 17 patients proceeded to autologous transplant. With median follow up of 50.5 months, 3 yr overall survival in all patients from start of GEM-P was 39% and 3 yr progression free survival 19%. Predominant Grade≥3 toxicity was haematological; thrombocytopenia 69%, neutropenia 58%, anaemia 15% and febrile neutropenia 8%. Conclusions: R-GEM-P is an effective salvage regimen in DLBCL, with the convenience of outpatient delivery. Despite previous rituximab exposure, patients in our cohort benefited from the addition of rituximab, supporting current practice.
Hypofractionated adjuvant radiation therapy of soft tissue sarcoma in elderly patients.
Authors: Merimsky Ofer , Soyfer Viacheslav , Corn Benjamin W. , Dadia Solomon , Kollender Yehuda
Published: March 2020

e21521

Background: Adjuvant radiation therapy is an essential part of combined limb sparing treatment of STS. The recommended dose of radiation lies in the range of 60 Gy in standard fractionation of 1.8-2 Gy. Elderly or medically unfit patients often have difficulty in completing 6-7w of daily treatment. A prolonged course of radiation may be interrupted by acute side effects, which sometimes demands further extension of the overall course or even discontinuation of treatment. We intended to evaluate the efficacy of a hypofractionated adjuvant approach with radiation therapy for STS in the elderly and debilitated patients. Methods: 21 elderly patients were treated with a short course of adjuvant RT (39 to 48 Gy, 3 Gy per fraction) for STS. The medical records of the patients were retrospectively reviewed for the local or distant recurrence and side effects of RT. Results: Overall, the hypofractionated irradiation regimen of 39-48 Gy in 13-16 fractions was well tolerated with only 3 patients developing Grade 2-3 acute toxicity (mainly dermatitis). Three patients suffered from delayed Grade 2-3 toxicity (chronic pain, skin atrophy, teleangiectasiae) scaled according to CTSC. The mean time from the surgery until the initiation of RT was 65 days (SD 21.6). Mean RT time was 18.4 (SD 3) days. No delay of treatment due to acute toxicity was registered. All patients except for one were able to receive RT in the ambulatory setting. With a mean follow-up of 532 days (SD: 325), three local recurrences (14%) were detected. Three of eight patients with distant metastases died of sarcoma (graph 1). One patient with metastatic disease in the lung received salvage stereo tactic radiation therapy and was still alive 6 month after completion of SBRT with no evidence of disease. At a mean 532 days of follow up three local recurrences (14%) were detected .Eight patients (38%) had lung metastases during the observed period. Three of them died from metastatic disease. The hypofractionated radiation was well tolerated with minimum long term side effects. Conclusions: Hypofractionated adjuvant radiation appears to be an effective treatment in terms of local control in elderly and debilitated patients.
Gene fusions evidence in a KIT/PDGFRA wild-type GIST without mutations in SDH units identified by a whole transcriptome study.
Authors: Urbini Milena , Astolfi Annalisa , Indio Valentina , Saponara Maristella , Nannini Margherita , Lolli Cristian , Mandrioli Anna , Gatto Lidia , Pallotti Maria Caterina , Biasco Guido , Pantaleo Maria A.
Published: March 2020

e21523

Background: A subset of KIT/PDGFRA wild-type GIST (WT) harbour mutations in SDH units. In the majority of the remaining cases of WT GIST no other molecular events are identified.We performed a RNA-seq in a WT GIST without mutations in SDH genes using next generation approach to discover molecular events in this GIST population. Methods: In 2003, a 63-year old woman underwent surgery for an ileal GIST (size 6 cm, MI 6/50HPF).After 6 years, she developed a recurrence with a single hepatic lesion. The KIT and PDGFRA analysis of the lesion did not show mutations. Therefore, she did not receive imatinib but she underwent a surgical removal. The analysis of all SDH units did not show mutations. So paired-end RNA-seq (75X2) was performed with Illumina HiScanSQ platform. After mapping the short reads on the human genome(HG19), SNVs and InDels were called by SNVMix2 with an accurate filtering procedures including predictors of mutations effect at protein level. Gene fusions discovery was done considering the agreement between DeFuse, ChimeraScan and FusionMap tools and validated by SangerSequencing using primers spanning the mRNA breakpoints. Results: Four different gene fusions and 206 non-synonymous SNVs were discovered, of which 62 were called deleterious by at least one predictor, and they are undergoing further validation. SPRED2-NELFCD gene fusion originated from an interchromosomal translocation-inversion between chr 20 and 2. The event involved exon1 of SPRED2 and exon11 of NELFCD, probably leading to inactivation of both genes. NELFCD encodes a component of the NELF complex that negatively regulates transcription elongation by RNA pol II, while SPRED2 is a member of the Sprouty /SPRED family that repress growth factor-induced activation of the MAPK/ERK pathway. The other three events were intrachromosomal aberrations: MARK2-PPFIA1 and PLA2G16-ATL3 on chr 11 and ASCC1-C10orf11 on chr 10. Only the first event led to an in-frame fusion (MARK2 ex1- PPFIA1 ex2) probably dysregulating the expression of the downstream gene. Conclusions: This is the first evidence of gene fusions in GIST. The oncogenetic role and the tumor frequency of these events deserve to be studied.
Salmonella typhimurium A1-R targets chemoresistant stem-like human pancreatic cancer cells.
Authors: Hiroshima Yukihiko , Zhao Ming , Zhang Yong , Maawy Ali , Hassanein Mohamed K. , Suetsugu Atsushi , Chishima Takashi , Tanaka Kuniya , Bouvet Michael , Endo Itaru , Hoffman Robert M.
Published: March 2020

e22011

Background: Salmonella typhimurium A1-R can selectively target tumors. XPA1 human pancreatic cancer cells are dimorphic (spindle and round shape); the spindle-shaped cancer cells have stem-like properties (J Cell Biochem 112, 3549-3554, 2011). Methods: Serial dilution was performed to obtain clones of both cellular morphologies. To determine chemosensitivity, in vitro IC50 values were obtained for both types of XPA1 cells for (1) Cisplatin (CDDP); (2) 5-fluorouracil (5-FU); (3) Gemcitabine (GEM); and (4) S. typhimurium A1-R. The efficacy of these drugs was also determined for both types of XPA1 cells in vivo. Results: IC50 values of spindle-shaped and round XPA1 cells, respectively, were 2.65 ± 0.22 μg/ml and 1.43 ± 0.36 μg/ml for CDDP (p = 0.012); 2.44 ± 0.25 μg/ml and 1.48 ± 0.19 μg/ml for 5-FU (p = 0.007); 3.17 ± 0.15 ng/ml and 2.70 ± 0.29 ng/ml for GEM (p = 0.133); and (19.7 ± 1.46) x 108 cfu/ml and (17.8 ± 9.78) × 108 cfu /ml for S. typhimurium A1-R (p = 0.771). In vivo, 5-FU and A1-R significantly reduced the tumor weight of round cells (5-FU; p = 0.028; S. typhimurium A1-R; p = 0.011). However, only S. typhimurium A1-R significantly reduced the tumor weight of spindle-shaped cells (p = 0.012). Conclusions: The spindle-shaped stem-like XPA1 cells were thus chemo-resistant but sensitive to S. typhimurium A1-R, suggesting S. typhimurium A1-R could be a promising therapy for chemo-resistant human pancreatic cancer stem-like cells.
Salmonella typhimurium A1-R effectively targets human-patient pancreatic tumorgrafts in nude mice.
Authors: Zhao Ming , Katz Matthew H. G. , Fleming Jason B. , Suetsugu Atsushi , Zhang Yong , Maawy Ali , Chishima Takashi , Tanaka Kuniya , Bouvet Michael , Endo Itaru , Hoffman Robert M. , Hiroshima Yukihiko
Published: March 2020

e22012

Background: Genetically-modified Salmonella typhimurium A1-R selectively targets tumors of cancer in mouse models of human cancer. The aim of this study was to determine the efficacy S. typhimurium A1-R on human-patient pancreatic tumorgrafts growing orthothopically in nude mice. Methods: Pancreatic-cancer-patient tumor specimens were initially established subcutaneously in NOD/SCID mice immediately after surgery. The patient tumors were then harvested from NOD/SCID mice and passed orthotopically in transgenic nude mice, expressing fluorescent proteins in order for the tumors to acquire fluorescent stroma for imaging. After confirmation of tumor growth by fluorescence imaging, the nude mice were treated in the following groups: (1) 5-fluorouracil (5-FU) (10 mg/kg, ip); (2) cisplatin (CDDP) (10 mg/kg, ip); (3) gemcitabine (GEM) (150 mg/kg, ip); (4) S. typhimurium A1-R (1.5x108 cfu/body, ip); and (5) PBS (vehicle/control) (ip). Control, 5-FU, CDDP, GEM and S. typhimurium A1-R injections were performed on a weekly basis from day-21 after tumor implantation for 4 weeks. Animals were sacrificed at 7 weeks, and tumors were harvested for analysis. Each treatment arm involved 5 tumor-bearing mice. Results: No significant effects on body weight, morbidity, or severe toxicities were observed in any treatment arm. The tumor weight of each group was as follows: (1) 5-FU, 0.044 ± 0.027g; (2) CDDP, 0.04 ± 0.032g; (3) GEM, 0.058 ± 0.051g; (4) S. typhimurium A1-R, 0.106 ± 0.038g; and (5) PBS control, 0.258 ± 0.209g. S. typhimurium A1-R treatment significantly reduced the tumor weight compared to control treatment (p = 0.011), as did the other treatments including 5-FU (p = 0.005), CDDP (p = 0.004), and GEM (p = 0.001). Conclusions: S. typhimurium A1-R treatment was effective on human pancreatic tumorgrafts and should be tested in combination with chemotherapy.
Effect of p53 gene transfection on human hepatocarcinoma cells' sensitivity to irradiation.
Authors: Tao Zhengchao , Qian Liting
Published: March 2020

e22018

Background: Several studies demonstrated that p53 gene transfection enhanced the anti-tumor effects of radiotherapy. This study is to investigate whether p53 gene transfection can increase the radiosensitivity of liver tumor cells. Methods: Liver tumor cell lines, HepG2 (with wild type p53 genes) and PLC/PRF/5 (with mutant p53 genes), were separately transfected with recombinant adenoviral human p53 gene (rAdp53) and adenoviral enhance green fluorescent protein gene (AdEGFP), forming 4 types of cells: HepG2 transfected with rAdp53, HepG2 with AdEGFP, PLC/PRF/5 with rAdp53, and PLC/PRF/5 with AdEGFP. The transfected and untransfected HepG2 and PLC/PRF/5 cells were irradiated with 6MV-X at doses of 0, A2, A4, A6, A8, A and 10 Gy. After exposition to radiation, cell survival, clonogenic capacity, and apoptosis were analyzed. The effect differences between cell types were analyzed using statistical methods of pairwise group comparisons and mixed effect model. Results: After exposing irradiation, all the cells’ survival rate and clonogenic capacity decreased, and the proportion of apoptotic cell increased. These effects become stronger with increaseing dose of radiation. Between untransfected cells, radiation had a stronger effect on HepG2 cells than PLC/PRF/5 and the differences were statistically significant for all the 3 measures. The rAdp53 transfection, not the AdEGFP, significantly enhanced radiation effects on both cell lines. The enhanced effects on PLC/PRF/5 cells were significantly stronger then the enhanced effects on HepG2 cells. Conclusions: PLC/PRF/5 cells with mutant p53 genes were more resistant to radiation then HepG2 with wide type of p53 genes. The rAd-P53 transfection could enhance radiosensitivity of both cell lines, but the enhanced effect on PLC/PRF/5 cells with mutant p53 gene was stronger than HepG2 with wide type of p53 genes.
Comparison of next-generation sequencing methodologies and direct sequencing for the detection of EGFR mutations in non-small-cell lung carcinoma: Implications for optimal clinical testing.
Authors: Kim Hye Sook , Kim Yeul Hong , Sung Jae Sook , Kim Seung Tae , Park Kyong Hwa , Kim Jung Sun , Choi Yoon Ji
Published: March 2020

e22025

Background: The efficacy of next-generation sequencing (NGS) for the prediction of clinical benefits from EGFR TKIs in non small cell lung cancer(NSCLC) has yet to be fully established. The purpose of the present study was to evaluate the predictive effect of EGFR-evaluating methodologies on the use of EGFR TKIs in patients with NSCLC. Methods: cTumor samples were obtained from patients with NSCLC at 5 institutions in Korea. Study cohort A consisted of surgically resected or biopsy NSCLC samples enriched with adenocarcinoma histology. Study cohort B consisted of patients who received EGFR TKI during the treatment period. EGFR status was tested with direct sequencing, Peptide nucleic acid-locked nucleic acid PCR clamp (PNA-LNA PCR Clamp) method, and Personal genome machine (PGM) sequencing using the Ion AmpliSeq Cancer Panel on the Ion Torrent. Results: EGFR mutations in cohort A were identified in 16 (28.1%), 27 (47.4%), and 29 (50.9%) of 57 patients by direct sequencing, PNA-LNA clamping, and the Ion Torrent PGM, respectively. In 2 cases of EGFR mutant detected by Ion Torrento PGM in contrast to EGFR wild type by PNA-LNA clamping, EGFR mutation was found on novel sequence other than classical EGFR mutations. In total, 33 out of 37 patients in cohort B whose frozen NSCLC tissue was available were examined. Patients with EGFR mutations tested by PNA-LNA clamping did not show a significantly higher response rate than did patients with the wild-type EGFR (P=0.607). Mutational status detected by 2 methods did not significantly predict overall survival and progression free survival. However, curves showed a greater differentiation when the mutation was detected by PNA-LNA clamp as compared to mutations detected by direct sequencing. Conclusions: Though this study was conducted in a small number of cases, the results suggest that both PNA-LNA clamping and the Ion Torrent PGM are highly sensitive procedures relative to direct sequencing, and are useful screening tools for the detection of EGFR mutations in clinical practice.
Levels of circulating natural killer T and natural killer cells in breast cancer patients.
Authors: Thein Mya Sanda , Chumsri Saranya , Eeast James , Li Junxin , Kesmodel Susan , Bao Ting , Feigenberg Steven J. , Tkaczuk Katherine Hanna , Webb Tonya
Published: March 2020

e22034

Background: Invariant natural killer T (iNKT) cells are a unique subset of T lymphocytes that express markers characteristic of both T cells and natural killer (NK) cells. Following activation, iNKT cells rapidly produce a variety of cytokines and activate many other immune cells, thus iNKT cells are key mediators of innate immune responses (IIR) against tumors. The aim of this study was to assess the percentage (%) of iNKT, NKT-like, and NK cells in the peripheral blood of healthy volunteers (HV) and breast cancer (BC) patients. Methods: The % of circulating iNKT (Va24Ja18+CD3+), NKT-like (CD3+CD16CD56+), and NK (CD3-CD16CD56+) cells was analyzed by standardized flow cytometry in 22 newly diagnosed stage 0-3 BC patients and 27 HV. Luminal A BC was defined as ER/PR+≥1%, HER2- Ki67<20%. Differences between groups were assessed using the Mann-Whitney U test for nonparametric data and ANOVA for ≥ 3 groups. Results: iNKT cells % in BC patients (ages: 37-75) was significantly lower compared to HV (ages: 21-64) (Mean: 0.32 vs 0.12, p=0.0036). Among BC patients, NK cells were lower in patients aged ≥50 yrs (p=0.003) and with BMI ≥ 30 (p=0.0036). NK cell % was higher in Asians compared to other races. Patients with luminal A BC, which generally have the best prognosis, have a significantly higher % of NKT-like cells. Conclusions: Interestingly our data shows that older and obese BC patients have lower % of circulating NK cells, while patients with luminal A tumors have a higher % of NKT-like and a lower % of iNKT cells. Further studies on the effects of BMI and aging on IIR as well as functional studies are needed to better understand immunosenescence-related increasing incidence of cancers and IIR-mediated anti-tumor activity in BC patients. [caption]iNKT, NKT-like, and NK cells (% lymphocytes) by age, BMI, race, and pathology in BC patients.[/captions] [Table: see text]
Potential applications of circulating melanoma cells (CMCs) expressing γ-H2AX.
Authors: Rossi Elisabetta , Facchinetti Antonella , Grassi Angela , Rossi Carlo Riccardo , Stragliotto Silvia , Pigozzo Jacopo , Chiarion-Sileni Vanna , Amadori Alberto , Zanovello Paola , Zamarchi Rita
Published: March 2020

e22036

Background: The CMC analysis was recently improved by the CellSearch platform that offers an accurate (and reproducible) assessment of tumor burden, to be used as prognosticator and predictor of treatment response.Moreover, not only decreased total circulating cells numbers but also increased fraction of apoptotic cells may represent response-related markers. To monitor changes in the balance between live and apoptotic Circulating Tumor Cells (CTCs) ( Rossi, Basso et al. 2010 ) during therapy, a novel CTCs assay with a companion algorithm (the ΔAUC) was reported to be a pharmacodynamic marker, as documented by consistent radiological findings in MBC and mRCC patients. Methods: Fluorescent anti γ-H2AX mAb (specific for the phosphorylated form of H2AX) was integrated in the standard CMC assay to assess DNA damage and to measure cell viability and apoptosis in spreading melanoma cells. The integrated test was developed using MCF-7 and SK23mel cells line and tested in melanoma patients. From June-2011 till now, basal blood samples from 64 patients (pts) in stage IV were collected; median age 62 (range: 27-85). Both considering the cost of the CMC assay and the little already known about this new method in melanoma, to evaluate the feasibility of CMC retrieval and ΔAUC calculation an interim analysis was specified in the study protocol and conducted at September 2012 on the first 40 pts. Results: At baseline, in Padova’s cohort 72.6% of pts are CMC-positive. The CMC number ranged from 1 to 94. The γ-H2AX-positive CMCs ranged from 0% to 100% (median 67%). Shorter median Progression-free survival PFS was observed for patients who had > 2 CMCs (n=26, 162 days) compared with < 2 CMCs patients (n=14, 203 days, P value = 0.0270). No significant differences were found with the ΔAUC criterion (i.e. exceeding live vs apoptotic CMCs). Furthermore, a significant association was found between CMC-positive status at baseline and disease sites at progression (visceral and CNS, Chi-square = 8,521 with 3 df, P value = 0.036). Conclusions: Accrual is ongoing. Updated data including evaluations on prognostic value of total and apoptotic CMCs and correlation with clinical stage, tumor burden, metastatic sites and survival will be available and presented at the meeting.
Screening of NSCLC samples from Chinese lung cancer patients for activating rearrangements of the ALK, RET, and ROS1 genes.
Authors: Zheng Li-Mou , Whyte David B. , Ruan Li , Song Roman , Fei Luo , Yang Nancy , Zhou Caicun
Published: March 2020

e22066

Background: The ALK, RET, and ROS1 genes are involved in gene rearrangements in a fraction of non-small cell lung cancers. The resulting oncogenic fusion genes define molecular sub-types of NSCLC with distinct sensitivities to treatment with various kinase inhibitors. We developed real-time reverse transcriptase PCR assays to detect rearrangements of ALK, RET, and ROS1 in FFPE lung cancer tissue. Methods: mRNA from NSCLC FFPE tissue samples was reverse transcribed to cDNA. Multiplex quantitative PCR was performed to detect 9 variants of EML4-ALK fusions, 9 variants of RET fusions and 14 variants of ROS1 fusions. A total of 409 samples were analyzed: 267 were classified as adenocarcinoma, 104 as squamous cell carcinoma and 38 had undetermined histology. EGFR and KRAS mutation status is unknown. The junctions of fusion-positive samples were sequenced by Sanger sequencing. Results: Among the 409 NSCLC specimens tested the frequency was 5.4% (22/409) for EML4-ALK fusions, 1.5% (6/409) for RET fusions, and 2.2% (9/409) for ROS1 fusions. EML4-ALK fusions were more prevalent in patients that were less than 60 years old (9.1% versus 2.0%, p= 0.004). The TNM stage was not correlated with the presence of any of the fusions. The table below lists the frequencies for specific rearrangements as determined by sequencing the real-time PCR products. Conclusions: Real-time PCR assays based on cDNA from FFPE tissue can identify patients with ALK, RET and ROS1 fusion genes. The ALK, RET and ROS1 assays will allow selection of patients most likely to respond to therapies that specifically target these cancer drivers. Further clinical testing of NSCLC patients in the Chinese population will be performed to support SFDA registration of these assays in China. [Table: see text]
Dynamic changes of miRNA and cancer related proteins (CRPs) expression during neoadjuvant chemotherapy (NAC) for locally advanced breast cancer (LABC).
Authors: Al-Khanbashi Manal , Caramuta Stefano , Al-Ajmi Adel , Al-Hadabi Ibrahim , Kumar Shiyam , Dsouza Philomena charlotte , Zahid Khawaja Farhan , Malik Kamran Ahmed , Muhammad Furrukh , Burney Ikram A , Al Bahrani Bassim J. , Lui Weng-onn , Al-Moundhri Mansour S.
Published: March 2020

e22099

Background: NAC is used in LABC to downsize the primary tumor and eradicate metastases. This study assessed the Dynamic changes of miRNA and CRP expression during NAC with aim to identify molecular biomarkers that may predict outcome. Methods: Tissue, blood and serum samples were collected at four time points from LABC patients undergoing NAC, including time of diagnosis (base line) after 1st and 4th cycle of Adriamycin/cyclophosphamide, and after 4th cycle of Docetaxel (end point). We used microRNA-microarray for tissue samples and Proximity Ligation Assay (PLA) for serum samples to quantify global miRNA expression and 92 CRPs in LABC patients. The miRNA and CRPS were correlated with clinico-pathological features including stage, grade and clinical response.Statistical analysis was performed using Significant Analysis of Microarray ( SAM), Student's t-test, and Pearson's correlation test. Significant p value was defined as p<0.05. Results: Tissue miRNA expression profiling (n=14) revealed 58 miRNA species that were differentially expressed between NAC endpoint and the baseline (FDR < 15%). Reduced miR-21 expression was associated with disease recurrence. Using PLA (n=33) we identified serumosteoprotegerin, MCP-1, Prolactin, Carbonic Anhydrase IX, CXCL10, CD30L and TGF-alpha levels were significantly associated with overall clinical response (t-test, p<0.05). Moreover, Prolactin and Erythropoietin showed significant associations with the stage (t-test, p<0.05), while MMP3, ErbB4, Midkine, Cathepsin D and Prostasin proteins were associated with the recurrence. Analysis is ongoing to determine the dynamic changes of these biomarkers during the NAC. Conclusions: Our study identified specific miRNAs and CRPs that were differentially expressed during NAC in LABC patients which may have clinical significance.
Utility of microsieve device for enumeration and miRNA expression profiling of CTCs for personalized cancer management.
Authors: Tucker Steven , Tan Karen Mei Ling , Leong Sai Mun , Wen Chua Hui , Jumaat Delly Fareda , Tan Doreen Chek Yee , Li Mo-Huang , Koay Evelyn S. C.
Published: March 2020

e22107

Background: Although a rare peripheral cell population, circulating tumor cells (CTCs) represent a potential alternative to serial invasive biopsies as a source of tissue in monitoring clinical states of non-hematological malignancies. CTCs harvested from a single patient are a heterogeneous mixture of cells differing in their transcriptome expressional profiles, metastatic potential and drug resistance properties. Mere identification and enumeration of CTCs are unable to provide qualitative information about individual CTC biology. The presence of a few highly malignant, metastasis-capable CTCs and cancer stem cells among the other less aggressive CTCs in the circulating population will not be apparent from CTC counts alone. Methods: We developed a high-throughput size-exclusion microsieve device (CellSievo, Singapore) to isolate CTCs (defined as DAPI-positive, CD45-negative cells), regardless of the cell surface receptors expressed, from 74 breast cancer patients. Using CTCs eluted from the microsieve post-capture, we performed microRNA expression profiling to further characterize breast cancer CTCs. Results: Using CellSievo’s microsieve device, we obtained an average mean CTC count of 21.2 (+/- 31.8; range=0-112.5) per 7.5ml peripheral blood, for breast cancer patients, as opposed to 1.3 (+/-1.6, range 0-5.0) for healthy subjects. Viable CTCs from breast cancer patients were successfully harvested for downstream microRNA profiling. CTCs from breast cancer patients were routinely found to express microRNA profiles mediating epithelial-to-mesenchymal transition, tumor invasion, and regulation of chemotherapeutic drug resistance to tamoxifen, anthracyclines, taxanes, and trastuzumab. Serial monitoring of microRNA expression pattern of CTCs could identify emergence of novel tumor populations with distinct microRNA expression patterns during or after anticancer therapy (detailed microRNA expression data to be presented). Conclusions: Our results suggest that microRNA biomarkers derived from peripheral CTCs can yield highly useful information and offer prognostic and predictive applications for therapeutics and companion diagnostic development.
Clinical importance of circulating tumor cells in lung cancer patients.
Authors: Benedikova Andrea , Srovnal Josef , Klein Jiri , Skalicky Pavel , Szkorupa Marek , Cwiertka Karel , Radova Lenka , Hajduch Marian
Published: March 2020

e22109

Background: Assessment of the circulating tumor cells (CTCs) in lung cancer patients could prevent burdensome surgery in understaged cases. Therefore we have tested the hypothesis that the presence of CTCs is a negative prognostic factor in these patients and correlates with poor survival. Methods: This was a prospective study to test the presence of CTCs in peripheral blood, pulmonary blood and bone marrow in 108 lung cancer patients (75 males, 33 females; mean age 66.2 years, ranging from 29 to 82 years) at the time of surgery using real-time RT-PCR for carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR), lung-specific X protein (LUNX) and hepatocyte growth factor receptor (c-met). Results: We found a statistically significant association between c-met expression level in the pulmonary blood and clinical stage (p<0.044) and lymph node involvement (p<0.0023). C-met marker also showed significantly higher positivity ratio in the peripheral (p<0.034) and pulmonary (p<0.04) blood in patients with histologically proven lymphatic metastases than in patients with negative lymph nodes. The positivity of CTCs in peripheral blood or bone marrow using c-met was independent of clinical stage, age, sex and grading using Cox regression analysis (p=0.006, resp. 0.001). 82 patients followed up for more than 1 year were involved into the overall survival (OS) analysis. 27 of them (32.9 %) died, the OS median was 19.8 months. The patients with the presence of CTCs in the peripheral blood or the bone marrow using c-met had significantly shorter OS and higher hazard ratio (p<0.024; HR=4.39 [95% CI: 1.58-12.22], resp. p<0.008; HR=5.08 [95% CI: 1.79-14.43]). In addition, patients with the presence of CTCs detected in bone marrow using CEA and/or c-met had significantly shorter OS (p<0.025; HR=3.08 [95% CI: 1.26-7.5]). Conclusions: Our study demonstrates that the presence of CTCs is independent negative prognostic factor in lung cancer patients. The analysis of CTCs is clinically feasible, reproducible and further studies focused on early clinical stages should demonstrate its applicability in individualization of adjuvant therapy in lung cancer patients and identification of individuals who would not benefit from extensive lung surgery.
Effect of PARP inhibitors on sensitivity of lung and breast cancer cells to trans-platinum as compared to cis-platinum.
Authors: Jankovic Radmila , Radulovic Sinisa , Arandjelovic Sandra , Filipovic Lana
Published: March 2020

e22115

Background: Poly(ADP-ribose) polymerase inhibitors (PARP-i) represent promising strategy in treatment of cisplatin resistant lung cancer and triple-negative breast cancer. We compared PARP inhibition by 3-aminobenzamide (3-AB) or NU1025, on activity of cis- and trans-platinum on NSCLC cells (A549) and breast cancer cells (MDA-MB-361), and correlated with p53 and ERCC1 status. Methods: Cell cycle distribution was determined by FACS-flow and PI-staining, while morphological changes were followed by light microscopy. ERCC1 siRNA transfection was performed by HiperFect reagent, and mRNA was analyzed by qRT-PCR. Protein expression levels were determined by Western blot. PARP-inhibition was quantified as NAD(P)H depletion using colorimetric XTT assay. Results: Trans-platinum compound with planar ammine ligands, trans-[PtCl2(4-acetylpyridine)2] showed cytotoxic activity comparable to cis-platin and exhibited enhanced action in combination with PARP-i. 3-AB enhanced the activity of trans more efficient comparing to cis-platinum in MDA-MB-361 cells. At equitoxic concentrations corresponding to IC50 values in A549 cells, cis-platin and trans-platinum compound differently affected cell survival in combination with PARP-i. XTT assay confirmed efficient inhibition of PARP activity at 200 μM NU1025 and 2 mM 3-AB in both cell lines. ERCC1 silencing in p53 proficient A549 cells, alone or in combination with NU1025, almost completely diminished resistance to trans-[PtCl2(4-acetylpyridine)2], decreasing cell survival to 50 % following 24 h action and more than 85 % following 48 h treatment, comparing to siRNA/NU1025 treated control. By contrast, sensitivity to cisplatin was much less altered. Simultaneous alteration of ERCC1 and PARP itself did not affect cell cycle progression, but prolonged sub-G1 fraction by trans-platinum as well as G2-M fraction by cis-platin. Conclusions: PARP-inhibition enhanced tumor cell sensitivity to trans-platinum drug, stronger than cis-platinum. Knockdown of ERCC1 further increases sensitivity to platinum drugs, irrelevant to p53 status. Trans-platinum complexes should be further explored as good candidates for combining with PARP-i.
Identification of real-time PCR-negative EGFR mutations by direct sequencing test.
Authors: Wang Tao-Yeuan , Chen Chi-Kuan
Published: March 2020

e22118

Background: High percentage of patients with non-small cell lung cancer (NSCLC) has EGFR activating mutations in Taiwan and responses to EGFR inhibitors such as Tarceva (1, 2, 3). Timely molecular diagnosis is critical and molecular testing should be performed before treatment for the individual patient. It is important to develop an effective molecular diagnostic method for accurately detecting mutations and preventing false positive results. Therefore, we used a universal genetic detecting method (FemtoPath) to increase sensitivity of gene mutation detection and improve the sensitivity limitation (0.1%). Our previous reports indicated that FemtoPath/direct sequencing test is more sensitive, accurate and inexpensive than Cobas KRAS real- time PCR mutation test. Methods: In this study, we compared two molecular testing methods for the detection of EGFR mutation; the direct sequencing and the real-time PCR. Results: FemtoPath EGFR direct sequencing test identified mutations in 57 of the 73 tumors (77.03%) and Cobas EGFR mutation test identifies mutations in 53 of the 73 tumors (72.97%). Six negative results obtained by Real-Time PCR test were re-confirmed to be positive by direct sequencing test. From the results above, FemtoPath/direct sequencing test not only accurately detected all of the mutations, but also identified mutations adjacent toexons 18, 19, 20 and 21 of the EGFR gene. In 52 consensus cases of EGFR mutation obtained from both methods, there were 10 major discrepancies including two S768, four Exon 19 deletion, one L858, five T790, and one exon 20 insertion in this experiment. Conclusions: FemtoPath/direct sequencing test has lower invalid rate than Real-Time PCR test while fewer sample amount was required with increased detection efficiency of unknown mutation site.
The role of apoptosis in colorectal carcinogenesis and its prognostic value.
Authors: Alcaide Julia , Funez Rafael , Rueda Antonio , Pérez-Ruiz Elisabeth , Pereda Teresa , Rodrigo Isabel , Perez Diego , Villatoro Rosa , Nogales Esteban , Redondo Maximino
Published: March 2020

e22131

Background: Most colorectal cancers (CRCs) progress through a multistep process from normal tissue to adenoma to carcinoma. The role of apoptosis during this tumourigenesis sequence has not yet been completely clarified. The aim of our study was to determine the influence of apoptotic variations on the carcinogenesis and prognosis of CRC. Methods: A TUNEL assay was performed on the formalin-fixed, paraffin-embedded tissues from 103 CRCs resected at the Costa del Sol Hospital (after excluding patients previously treated with chemotherapy or radiation therapy), 26 adenomas and 20 samples of normal epithelia. Clinical data were obtained from the tumour registry and hospital charts. The study was approved by Research Ethics Committee. We used the median (m) of apoptotic percentage (1%, range: 0-6%) as a threshold value for survival (sv) studies. Analysis of association between apoptosis and prognostic factors used the X2test for categorical variables and the analysis of variance test for continuous variables (natural log transformed if necessary). We estimated sv using the Kaplan-Meier method, and sv curves were compared with the log rank test. Cox’s proportional hazards sv analysis was used to determine the relative risk (RR) through multivariate analyses. Results: Median follow-up was 50 months (mo). Apoptotic index (AI) was higher in CRCs (1.09 ± 0.13) than in adenomas (0.38 ± 0.23, p=0.059) and significantly greater than in normal epithelium (0.06 ± 0.04, p=0.001). AI was greater in stage IV than in other stages (p=0.017). Higher AI was associated with lower disease-free survival (DFS) and overall survival (OS) than lower AI (Table). In multivariate analysis, AI (RR=2.18, 95% CI 1.08-4.37) and TNM stage (RR=2.41, 95% CI 1.20-4.85) were independent prognostic factors for OS. Conclusions: Our study demonstrates an increase in apoptosis during colorectal carcinogenesis and a correlation between apoptotic rates and survival. Higher AIs are associated with more aggressive tumours and a poorer prognosis in CRC. [Table: see text]
COL1A1 copy number alterations detected in array-based comparative genomic hybridization (aCGH) inmyeloproliferative neoplasms: Implications for molecular pathogenesis and targeted therapy.
Authors: Savona Michael R. , Chandra Pranil , Ma Zeqiang , Liang Shile , Cooper R. Seth , Raefsky Eric , Gian Victor G. , Hockett Richard , Berdeja Jesus G. , Lennon P. Alan
Published: March 2020

e22139

Background: Myeloproliferative neoplasms (MPNs) are a heterogeneous group of tumors marked by clonal proliferation of myeloid cells with variable marrow changes and clinical findings. Though progress has been made since the discovery of the JAK2V617F mutation, few data exist on the genetic distinction amongst these disorders, or pathogenesis of fibrosis seen in MPNs. Methods: Array-based comparative genomic hybridization (aCGH) was performed on genomic DNA extracted from marrow aspirate using an Agilent 180K oligonucleotide array platform in order to discover recurrent genetic aberrations, cooperating mutations, and gain insight into the hierarchy of molecular pathogenesis of fibrosis. BM aspirate from 17 pts were analyzed. Copy number alterations (CNAs) were compared to a reference set and mapped to functional genes. Genes with CNA were subjected to gene ontology, pathway and clustering analysis. Results: aCGH yielded copy number gains or losses in 17 out of 17 cases, 11 of which had normal karyotype and/or FISH. Numerous CNAs were identified within genes found in a variety of cellular pathways. In particular, alterations in COL1A1, NFKb and PDGFRb were implicated in this MPN cohort in 12, 8, and 6, pts, respectively. Interestingly, only patients with COL1A1 CNA had aberrancy within NFKb and PDGFRb. The remaining 5 pts had abnormal aCGH, but normal signals at these 3 genes. Conclusions: aCGH is a valuable tool which may be used to distinguish MPNs, particularly when standard testing does not. The presence of the COL1A1 aberration in 12/17 cases edifies evolving study of the role of TGFB superfamily in development of fibrosis and provides potential targets to exploit in the treatment of MPNs. Though aberrations within COL1A1 have not been previously reported in MPNs, a gene fusion involving COL1A1 and PDGFRb is found in 90% of dermatofibrosarcoma protuberans. The cooperation between these genes is currently under study in a larger cohort of pts with MPNs. [Table: see text]
NSCLC driver mutations in the Quebec population: Epidemiologic and clinical evaluation.
Authors: Saliba Wissam , Tehfe Mustapha Ali , Albadine Roula , Tran-Thanh Danh , Soulieres Denis , Gorska Isabelle , Weng Xiaoduan , Fan Boli , Florescu Marie , Audet Marie-Lise , Gougeon Francois , Blais Normand
Published: March 2020

e22159

Background: Recent advances in lung cancer treatment embrace the recognition of molecular pathways implicated in its pathogenicity, paving the path to personalized therapies. We conducted a retrospective analysis to characterize the molecular features of the population treated for non-squamous non-small cell lung cancer (NS-NSCLC) in the province of Quebec. Methods: 622 patients with NS-NSCLC and adequate tumor blocks, treated at the CHUM between 2006 and 2008, were included. All samples were tested for ALK translocations (by IHC and FISH), EGFR classical exon 19 and 21 mutations by PCR (fragment analysis and qPCR) and for KRAS codon 12 and 13 mutations by mismatch PCR-RFLP. Molecular features were matched to demographic characteristics and clinical outcomes. Results: So far, complete results are available for 153 patients. Considering the amount of tumor tissue available, this population is largely represented by patients with local or loco-regional disease (n= 140, 91.5%). A minority of patients (10.3%) was never or light smokers (< 10 pack-yrs). Only 2 patients (1.3%) were of Asian descent. The following table depicts the outcomes of this cohort of patients segregated according to mutation status and extent of disease. Conclusions: ALK rearrangements were not identified in this unselected NS-NSCLC population characterized by localized disease and strong smoking history. ALK translocation prevalence in different populations is likely to be largely influenced by its tumor stage distribution, tobacco exposure and the use of selection criteria for molecular testing. An expanded cohort of patients will be presented at the meeting. [Table: see text]
Design, validation, and application of a PTEN quantitative reference standard for oncology assay development.
Authors: Frampton Jonathan Mark , Spivey Vicky , Goodall John , Schmitt Karin , Morrill Paul
Published: March 2020

e22164

Background: The phosphatase PTEN is a tumor suppressor whose deletion is associated with tumor progression and results in loss of PTEN protein expression. This results with the activation of both phosphoinositide 3-kinase/AKT and mitogen-activated protein kinase pathways. The PI3K/PTEN pathway is a potential target for molecular therapy and controls multiple cellular functions (cell metabolism, proliferation, apoptosis, migration, and survival). Both PI3K and PTEN are frequently mutated in human cancer. Diagnostic testing for PTEN deletion can be performed using sequencing based methods across the coding region, intron-exon borders and promoter or alternatively using an antibody IHC based assay. Currently, however, reliable and reproducible methods for measuring PTEN expression on formalin-fixed tissue are not standardized. Methods: An isogenic X-Man (gene-X, Mutant And Normal) PTEN heterozygous deletion cell line was developed using our gene editing technology (GENESIS) using a HCT116 human colon carcinoma cell background. A 10kb fragment of PTEN, containing both exon6 and exon7, was deleted to generate a PTEN (Δex6Δex7/+) cell line. The engineering event was confirmed using sequencing, gene expression analysis and digital PCR. Metaphase spreads of the mutant and corresponding matched wild type cell line were generated for CISH and SISH analysis. In addition to this FFPE blocks containing either an allelic frequency of 25% PTEN (Δex6Δex7/+) or 100% PTEN (+/+) were developed for CISH, SISH and IHC analysis. Results: Cell validation results indicated that the PTEN (Δex6Δex7/+) was successfully engineered in a HCT116 background and digital PCR data confirmed the allelic frequency of the PTEN (Δex6Δex7/+) mixed FFPE block at 25%. Preliminary data indicates that the metaphase spreads and FFPE reference slides provide consistent results for CISH, SISH and IHC and are continuing to be used for assay development. Conclusions: Here we show data for the requirement of a Quantitative Reference Standard that can be used to standardize PTEN assay development as well as facilitate the development of potentially new diagnostic assays using SISH and CISH.
The role of macrophages in docetaxel (DTX) resistance in castrate-resistant prostate cancer (CRPC).
Authors: Horvath Lisa , Chatfield Mark D. , Lee-Ng Michelle , Lin Hui-Ming , Castillo Lesley , Breit Samuel N. , Brown David A. , Molloy Mark P. , Marx Gavin M. , Pavlakis Nick , Boyer Michael J. , Stockler Martin R. , Wykes Richard , Henshall Susan M. , Mahon Kate Lynette
Published: March 2020

e22175

Background: DTX improves symptoms and survival in advanced CRPC, however ~ 50% of patients have chemoresistant disease. Given the toxicity of cytotoxic treatment in this population, there is an urgent need for early chemoresistance markers, a greater understanding of chemoresistance mechanisms and the development of new therapies. This study examined whether early changes in cytokine levels predict chemoresistance and explored the role of macrophages in cytokine generation and chemoresistance in vitro. Methods: 28 cytokines were measured pre/post cycle 1 of chemotherapy from 59 men with metastatic CRPC. Cytokine levels were correlated with PSA response and OS. DTX sensitive PC3 and DTX resistant PC3Rx cells were cultured alone and in coculture with U937 monocytes +/- DTX treatment. Conditioned media (CM) was analysed by a 36 cytokine array panel and by ELISA for MIC1. CM from U937 cells exposed to DTX was used to treat PC3 cells combined with escalating DTX doses. Results: Change in the levels of 10 cytokines over 1 cycle of chemotherapy (MIC-1 p=0.003; IL1ra p=0.004; IL1b p=0.01; IL4 p<0.001; IL5 p=0.041; IL6 p=0.003; IL7 p=0.009; IL8 p=0.017; IL12 p=0.004; IFNg p=0.001) was associated with clinical benefit (PR + SD vs PD). The combination of changes in MIC1, IL4 and IL6 best predicted PSA response (ROC AUC 0.88, 95% CI 0.79-0.97). PC3Rx/U937 coculture expressed more cytokines, chiefly markers of alternative macrophage differentiation, compared to PC3/U937 co-culture (IL10 17-fold; IL27 15-fold). DTX treatment enhanced cytokine production by PC3Rx/U937 coculture while reducing cytokine levels in PC3/U937 CM. The IC70 for DTX in PC3 cells incubated with CM from DTX exposed U937 cells was 10 fold greater than with U937 CM without DTX exposure. Conclusions: Early changes in circulating cytokine levels, especially those involved in macrophage activity, were associated with chemoresistance in men with CRPC. In vitro studies revealed a cytokine-mediated interaction between the macrophages and the DTX resistant cells when treated with DTX that was not apparent in the DTX sensitive cell model. When considered together, these data suggest a significant role for host macrophages in the development of DTX resistance in CRPC.
Progressive multifocal leucoencephalopathy (PML): A novel complication of brentuximab vedotin from the Southern Network on Adverse Reactions (SONAR) project.
Authors: Bennett Charles L. , Carson Kenneth Robert , Newsome Scott D. , Kim Ellen J. , Norris LeAnn B. , Jalan Pankaj , Raisch Dennis W. , Major Gene
Published: March 2020

e22181

Background: BV is FDA approved for angio-immunoblastic large cell lymphoma and refractory Hodgkin’s lymphoma (HL). BV-linked PML cases reviewed. Methods: Case 1—Age:47/HIV negative male relapsed stage IIIB mixed cellularity undergoing treatment with BV. Prior therapies: 8 cycles of ABVD, ESHAP, BEAM, autologous stem cell transplant, involved field radiotherapy to the neck and mediastinum in 2008 and GVD ending in 2009. A 2011 relapse treated with BV q 3 weeks. Patient complained of impaired coordination and left greater than right dysdiadokinesis after 2 BV doses. Third BV cycle administered. Patient formed dysarthria, left-sided hemianopsia, hemiparesis. CSF analysis for JC virus DNA by PCR positive. Patient died shortly after. Case 2—Age:38/female revealed cutaneous anaplastic T-cell lymphoma refractory started on BV in mid-2011 led to loss of cutaneous tumors. A day after 2ndBV dose, difficulty finding words and unsteady gait. MRI revealed multifocal white matter lesions in bilateral cerebral hemispheres. Brain biopsy revealed PML. Prednisone initiated. Patient continued mixed non-fluent aphasia, quadriparesis, gait ataxia. MRI showed white matter lesions and contrast enhancement consistent with IRIS. High dose oral corticosteroids continued. Repeat MRI shows decreased lesion load, resolution of enhancement. Patient improving. Case 3—Age:72/female stage IB mycosis fungoides (MF) controlled with topical steroids and nitrogen mustard, phototherapy and interferon alpha, developed painful ulcerated plaques and tumors 4 years after diagnosis. BV administered, clinical response of skin tumors, decreased pain. Two weeks after 3rdBV dose: disoriented, poor short term memory. MRI revealed right frontal white matter. CSF sent for JC virus DNA was negative. Patient died 2 months after last BV dose. Autopsy: demyelination associated JC virus positive cells. Results: Shown. Conclusions: Unexpected PML occurrences after 2 or more BV doses, with IRIS and survival in 1 patient.