bluuebunny/crossref_metadata_2025 huggingface dataset:
Detection of BRAF and KRAS mutations in DNA released by tumors in peripheral blood by an advanced digital droplet PCR.
Authors: Marisi Giorgia , Del Re Marzia , Bianchi Marco Giorgio , Martinetti Antonia , Antoniotti Carlotta , Ulivi Paola , Marconcini Riccardo , Pietrantonio Filippo , Schirripa Marta , Sottotetti Elisa , Loupakis Fotios , Tesei Anna , Zoli Wainer , De Braud Filippo G. , Amadori Dino , Falcone Alfredo , Danesi Romano
Published: March 2020

e22056

Background: Cell-free tumor DNA (cftDNA) is released into the circulation and its recovery from plasma is a non-invasive alternative to tumor biopsy for applications related to molecular profiling. A potential use in therapeutic interventions is the periodic monitoring of cftDNA for the identification of molecular changes associated with resistance to target-specific treatments or when the mutational status in tumor tissue is not available. Methods: Samples (6 ml) of peripheral blood were drawn from patients with colorectal (CRC, n= 19) and thyroid (n= 1) cancers and melanoma (n= 8). CRC tumors were KRAS wild-type (n= 3) or carried the BRAF V600E (n= 6), KRAS G12D (n= 9) and G12V (n= 4) mutations. All melanomas and the thyroid cancer carried the BRAF V600E mutation. DNA was extracted from plasma with QIAamp Circulating Nucleic Acid Kit to recover DNA fragments of ≤1000 bp. PCR amplification was carried out with a QX100 ddPCR System (Bio-Rad) on 20 μL-samples containing cftDNA and TaqMan probes for BRAF V600E (1799T>A), KRAS G12D (35G>A) and G12V (35G>T) labeled with FAM/VIC. Samples were then loaded into a droplet reader, which discriminates the difference in fluorescence amplitudes on the basis of target gene amplification. Results: The concordance between mutations in tumors (T) and plasma (P) for BRAF V600E was: melanomas 8 T vs. 6 P; thyroid cancer 1 T vs. 0 P; CRC 6 T vs 3 P. Concerning KRAS in CRC the results were: G12D 9 T vs. 7 P, G12V 4 T vs. 0 P. Overall, the percentage of concordant samples were: V600E 60%, G12D 77,8%, G12V 0%. Interestingly, in 2 patients with wild-type KRAS tumor, the G12D and G12V mutations were found in plasma. Conclusions: ddPCR is a third-generation PCR technique for highly sensitive detection of DNA fragments. Detection of mutations in cftDNA by advanced technological platforms has important applications in the monitoring of patients for the occurrence of secondary mutations, amplifications and expansion of cell clones that render their tumors resistant to target-specific anticancer agents, unraveling the resistant phenotype before the clinical progression of the disease. Acknowledgments: this study was funded in part by MIUR/PRIN 2011-2012 (Rome, Italy).
Isolation of circulating tumor cells in colon cancer patients by size and chromosomal abnormalities.
Authors: Matos Ignacio , Lozano Rebeca , Fonseca Emilio , Hernandez Jesus MariÂa , Amores Arantxa , Lopez Lina , Lopez Carolina , Hernandez Maria Angeles , Hernandez Juan Luis , Cruz Juan J.
Published: March 2020

e22058

Background: Determining the number of circulating tumor cells (CTCs) in patients with metastatic colorectal cancer (CRC) has shown to be a prognostic factor, recent studies have proven the existence of EpCAM and cytokeratin negative CTCs. These cells present morphological and genetic alterations. Our study is based on an algorithm determined by the selection of the size of the cells and the genetic alterations detected by FISH which enables the isolation of CTCs in CRC patients by an automated system of fluorescence. Methods: Patients with metastatic CRC with normal blood differential and healthy controls between June 2011 and June 2012 were chosen. The objective was to show the differences between the quantification of morphogenetically altered cells in peripheral blood obtained from control and patients. The selected cells were over 7 micra in size and presented two or more genetic alterations. A combination of four probe sets was used: centromeric probe for CEP3, CEP7 and CEP17 and LSI p16INK4A 9p21.3. Fluorescent signals in specimens were quantified using an automated fluorescent system (Metafer) that is capable of scanning and classifying hundreds of cells under fluorescent illumination and allows for detection of cells according to nuclear size > 7 um and FISH pattern based on the algorithm that we have developed. Results: We selected 28 healthy individuals and 45 patients with CRC. In all the cases, 1,000 cells were analyzed in each individual. Only 30 cells >7 micra being found in the controls out of the 28,000 cells analyzed (0.1%). In the case of the patients, 640 cells were found (1.42%), the difference becoming statistically relevant after the T-test (p=0.003). None of the controls presented cells with two or more genetic alterations and over 7 micra in size, whereas 38% of the patients presented these characteristics despite having received treatment. The most frequent chromosomal changes were trisomy 3 and 17 (26% and 18%, respectively). Conclusions: This method allows us to select genetically abnormal circulating cells in a simple and automatic way. This opens the door for future studies to analyze the prognostic factor and the response rate to treatment under this method.
Personalized molecular testing for therapeutic guidance in Taiwan.
Authors: Su Kang-Yi , Yu Sung-Liang
Published: March 2020

e22067

Background: Personalized cancer therapy is based on the precept that detail molecular characterization of tumors as well as tumor microenvironment will enable tailored therapies to improve outcomes and decrease toxicity. The goal of personalized therapy is to target aberrations driving tumor growth and prolongs survival, by administering the right drug combination for the right person. However, several practical and technological challenges including tumor heterogeneity, molecular evolution, costs and morbidity of biopsies as well as technical limitation critical for molecular testing. In addition, development of biomarkers should be considered all aspects of drug development, from discovery through to clinical trials. Methods: In Taiwan, we have established a well-qualified reference laboratory with ISO15189 certification for molecular testing of personalized therapy in clinical practice. Based on our innovation, we utilized DNA mass spectrometry platform to develop high sensitive EGFR mutation identification system. Recently, KRAS, BRAF, HER2 together with EGFR were also combined into multiplex gene testing for biomarker-assessed therapeutic decision. To this end, the National Science Council of Taiwan government had supported this project through NRPB (National Research Program of Biopharmaceutical) at the middle of 2011. This facility performed selected molecular tests for cancer patients from medical centers and regional hospitals. Results: A specific program had also been implemented to anticipate the launch of molecular targeting therapy (MTT) and reduce time-to-assess to MTT drugs and experimental therapies. From Jane 2011 to Dec 2012, more than 2,500 patients with lung cancer in Taiwan benefited from this facility. Conclusions: The Taiwan nationwide initiative for tumor molecular profiling is a tool to fight inequalities in access to molecular testing and target therapy, and demonstrates that molecular stratification of tumor for therapeutic decisions is a cost-effective strategy that can be integrated into the national health-care system.
Impact of family history of cancer on the incidence of mutation in epidermal growth factor receptor gene in non-small cell lung cancer patients.
Authors: He Yayi
Published: March 2020

e22070

Background: Epidermal growth factor receptor (EGFR) activating mutation is an important predictive biomarker of EGFR tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer (NSCLC), while family history of cancer also plays an important role in the carcinogenesis of lung cancer. This study aimed to investigate the association between family history of cancer and EGFR mutation status in NSCLC population. Methods: From February 2008 to May 2012, 538 consecutive NSCLC patients with known EGFR mutation status were included into this study. Amplification refractory mutation system (ARMS) method was used to detect EGFR mutation. The associations between EGFR mutation and family history of cancer were evaluated using logistic regression models. Results: EGFR activating mutation was found in 220 patients and 117 patients had family cancer histories among first-degree relatives. EGFR mutation was more frequently detected in adenocarcinoma patients (p<0.001), never-smoker (p<0.001) and with family history of cancer (p=0.031), especially who had family history of lung cancer (p=0.008). In multivariate analysis, the association of EGFR mutation with family history of cancer also existed (p=0.039). Conclusions: NSCLC Patients with family history of cancer, especially family history of lung cancer, might have a significantly higher incidence of EGFR activating mutation.
Pathologic complete response and changes related to T-infiltrating lymphocytes and regulatory T cells in tissue and peripheral blood after neoadjuvant chemotherapy in breast carcinoma.
Authors: De La Cruz-Merino Luis , Barco Sanchez Antonio , Henao Carrasco Fernan , Ibanez Martinez Jose , Nogales Esteban , Vallejo BenÃƒÂtez Ana , Brugal Molina Javier , Sanchez Margalet V. , Lobo Acosta M. , Nieto Garcia A.
Published: March 2020

e22073

Background: Some clinical trials in breast cancer have reported imppressive outcomes related to laboratory immune findings in the neoadjuvant setting. In this context, tumor infiltrating lymphocytes (TILs) and regulatory T cells (Tregs) in tissue specimens and in peripheral blood are being tested as two emerging prognostic and predictive factors. We designed a protocol to analyze specifically TILs before, during and after neoadjuvant chemotherapy (CT) in breast cancer in blood and tissue, and their eventual relation with pathological complete response. Methods: From March 2011 to January 2013, 48 patients (18 HER2+/ 30 HER2-) with T2-4 N0-3 breast carcinoma treated with neoadjuvant chemotherapy in the Breast Cancer Unit of the Hospital Universitario Virgen Macarena (Seville, Spain) were included in the study protocol. CD3+, CD8+, CD8-16-56+ and Foxp3+ cell infiltrates were detected by immunohistochemistry before and after the end of neoadjuvant chemotherapy in tissue specimens. Blood samples were collected in EDTA-K3 tubes before every cycle of CT to determine the immunophenotype and regulatory cell profile. Cell populations were determined by flow cytometry analysis of whole blood. Results: By January 2013, 35 patients (11 HER2+/ 24 HER2-) were operated. Pathological complete responses (pCR) or near pCR (grade 4/5 Myller and Payne) were attained in 13 patients. pCR was achieved in 63.63% (7 of 11) of tumors overexpressing HER2, but in only a 25% (6 of 24) of HER2-negative tumors. Overall Tregs diminished in the pCR and non pCR groups. Disappearance of Tregs (Black grading system) was more frequent in the pCR group (69.23 vs 54.54%) and HER2+ population (63.63 vs 58.53%), although differences were not statistically significant. CD8+ infiltrates remain stable during treatment. Conclusions: Neoadjuvant CT decreases Tregs immunosuppressive infiltrates with no changes in CD8+ cytotoxic lymphocytes. Changes observed in lymphocytic infiltrates during neoadjuvant treatment indicate that Tregs may represent an interesting therapeutic target in breast carcinoma.
Detection of DNA stool mutations in colorectal cancer patients.
Authors: Forones Nora Manoukian , Lima Jacqueline Miranda , Teixeira Yolanda , Pimenta Celia Aparecida Marques , Murray Helena , Donizetti Tiago , Felipe Aledson Vitor , Oliveira Juliana , Vidigal Veronica , Libera Ermelindo Della , Saad Sidney Sahran
Published: March 2020

e22074

Background: Colorectal cancer (CRC) is the third leading cause of cancer in the world. Noninvasive screening tests have higher compliance. Changes in tumoral cells are also found in the stools after cancer cell exfoliation. Aim: to detect mutations in the stools of CRC patients and compare to mutations found in CRC tissue Methods: Patients referred for colonoscopy were divided into 3 groups: control, cancer, and polyp. The DNA was extracted from tissue and stool and it was analysed by two microarrays, the KRAS/BRAF/PIK3CA Array and the RanplexCRC Array (Randox Laboratories Ltd). Both based on the simultaneous detection of 20 mutations in KRAS, BRAF and PIK3CA and 28 mutations in KRAS, BRAF, TP53 and APC respectively. Positive tissue sample mutations were confirmed by Sanger sequencing. Results: 90 stools and 112 tissue biopsies were studied. Analysis of tissue samples was initially performed using the KRAS/BRAF/PIK3CA Array. In total mutations were detected in 16(55%) CRC, 12(45%) polyp and 2(6%) control samples. Mutations were confirmed via Sanger sequencing (76%). In the stool the frequency were 17(35%) in CRC, 10(71%) in polyp and 1(4%) in the control. Inconclusive tests were higher among the controls (32.5%) and polyps (22%), being less frequent in the CRC patients (6%). Excluding the inconclusive tests, WT genes were common in the controls (96%). The most common mutation was within KRAS codon 12. Stools samples from 48 subjects were compared to the results obtained by the RanplexCRC Array and mutations were found in 50% of CRC. KRAS/BRAF/PIK3CA Array stool results had a correlation with RanplexCRC Array in 64% of CRC patients and in 47% of polyp patients. The average human DNA in the stools was 15ng/ul in CRC patients and 0.46ng/ul in the controls (p=0.0001). Conclusions: Both arrays had a good correlation and a sensitivity between 30-50% with a high specificity (95-100%). The KRAS/BRAF/PIK3CA Array is currently optimized and recommended for assessment of tissue samples. This initial set of data however does indicate its potential applicability to stool specimens. Inconclusive tests are likely as a result of limited human DNA content within stool control samples. FAPESP (Sao Paulo Research Foundation) project 09/16618-8.
Detection of circulating tumor cells (CTCs) in stage T3-4 and/or N positive rectal cancer (RC) patients undergoing neoadjuvant therapy followed by curative surgery.
Authors: Zampino M. Giulia , Magni Elena , Ravenda Simona Paola , Botteri Edoardo , Bertani Emilio , Chiappa Antonio , Valvo Manuela , Zorzino Laura , Adamoli Laura , Nole Franco , Sandri Maria Teresa
Published: March 2020

e22078

Background: CTCs count at baseline and during treatment is an independent prognostic factor in metastatic colorectal cancer, while its role in early stages is still an open issue. No data are available in RC patients suitable for neoadjuvant chemoradiotherapy (CT-RT). Aim of the study: To investigate the role of CTCs in patients with locally advancedRC undergoing neo-adjuvant CT-RT. Methods: In a prospective single Institution study, cT3-4 and/or N+RC staged by rectal EUS and/or pelvic MRI and chest-abdomen CT scan, received capecitabine (825 mg/mq, orally, tid) with concurrent pelvic radiotherapy (50.4 Gy/28 fractions), followed by two cycles of intermittent capecitabine (1250 mg/mq, tid 14/21 days) and by low anterior resection or abdominopelvic resection with TME. Primary endpoints are evaluation of CTCs at baseline (t0), after CT-RT (t1), within 7 days after surgery (t2), and at 6-month from surgery (t3) and its correlation with main patient/tumor characteristics, CEA and response to neoadjuvant therapy. CTCs are enumerated with CellSearch System in 22.5 ml peripheral blood at over-mentioned time-points. A repeated measure analysis for binary outcome was used to evaluate changes in time of the percentage of patients with CTCs>0. Results: 85/90 patients are evaluable: 52M/33F, median age 63 yrs (range 37-82); median follow up 25 months (range 6-52). At baseline (t0) 13 pts had 1 CTC (15.3%), two had 2 CTCs (2.4%) and one had 27 CTCs(1.2%) while in 69 cases (81.2%) no CTCs were detected. Information on CTCs was available for 67 patients at t1, 68 patients at t2 and 62 at t3. CTCs>0 was reported on 16 (18.8%) at t0, 5 (7.5%) at t1, 6 (8.8%) at t2 and 3 (4.8% ) at t3 (P-value for trend: 0.039). CTCs at t0 was not statistically associated with any patient/tumor characteristics except for ultrasound T-stage that showed a trend CTCs (0% in uT2, 18.9% in uT3 and 40% in uT4, p-value 0.093), while no correlation with pCR was reported. Conclusions: CTCs count ≥ 1 was observed in 18.8% of patients with trend reduction over time probably due to therapy. Statistical correlation will be planned between CTCs and outcome.
Biomarker testing methods in breast, gastric, and lung cancers: A benchmarking survey of NCI cancer centers.
Authors: Schink Julian C. , Trosman Julia R. , Weldon Christine B. , Siziopikou Kalliopi P. , Gradishar William John , Tsongalis Gregory J. , Patel Jyoti D. , Benson Al Bowen , Perez Edith A.
Published: March 2020

e22093

Background: Biomarkers are an integral part of cancer care. A variety of testing methods exist, allowing institutional selection of which technologies to use and in what order to run them. This descriptive study provides a benchmark on methods used in practice for biomarker testing in breast (BC), gastric/esophageal (GC), and non-small cell lung cancers (NSCLC). Methods: We conducted an IRB-approved web survey of the 58 NCI designated cancer centers (pathologists and oncologists) providing adult care in BC, GC, and NSCLC. We developed a survey instrument with 14 BC, 5 GC, and 10 NSCLC items asking about methods used, order of testing, turn around time (TAT) and if tests are internally developed (ID). Results: For BC, 32% (18/57) of sites run concurrent rather than reflex HER2 FISH and IHC testing, citing quality assurance reasons. Average TAT for concurrent vs reflex testing is 6.5 and 9.5 days. Rates of ID tests are 14% (8/57) for PgR, 12% (7/57) for ER and 7% (4/57) for HER2. For GC, 39% (21/54) of sites run concurrent HER2 FISH and IHC tests, citing reasons of quality assurance and evolving guidelines. TAT for concurrent vs reflex testing is 6.3 and 9.5 days. 19% (10/54) of sites use ID HER2 tests. All reporting sites use EGFR and ALK testing in NSCLC and 96% use KRAS. 24% (13/55) of sites run them concurrently with an average TAT of 7.6 days, while the average TAT for sites with sequential testing is 22.8 days. CAP recommends a 10 day TAT for NSCLC tests, and 92% (12/13) of sites with concurrent testing meet this guideline vs only 11% (4/38) of sites with sequential testing (p<.0001). Rates of ID tests are 57% (30/53) for KRAS, 47% (26/55) for EGFR and 35% (19/55) for ALK, for which a kit is listed on the FDA drug label. There is a difference in the use of ID tests between BC and NSCLC, p<.0148. Conclusions: Concurrent testing for BC and GC HER2, and for NSCLC markers is a common practice at NCI cancer centers; and it improves TAT. Concurrent NSCLC testing allows meeting CAP TAT guideline, but sequential testing does not. New markers (GC HER2, EGFR, KRAS and ALK) are commonly adopted, with a higher rate of internally developed tests than for BC HER2. [Table: see text]
An open-label trial of SMK treatment of advanced metastatic cancer.
Authors: Hoffman Steve , Bruckner Howard , Del Priore Giuseppe , Gurell Daniel , Mull Ruslan , Stega Damian , Goodwin Gail , Demurjian Mike , Malanowska-Stega Jeanetta
Published: March 2020

e22095

Background: SMK is a novel therapy that creates alteration in defenses to oxidative stress and increases free radical availability to the cancer cell. SMK is designed to penetrate the living cancer cells and introduce multiple mechanisms to kill the cell. Inducing transfer of electrons in the cancer cells allow catalyzed external free radicals to react and stress the cell. SMK is a combination of low dose agents used for non-cancer treatment. Methods: IRB approved study for metastatic cancer. No additional chemotherapy allowed. First 30 subjects meeting criteria were consented. SMK given orally and SQ, 5 days/wk, 6 weeks (1 cycle). Eligible subjects had cycles 2 and 3. Results: Average age 57.5 (30-81); 70% female; 30% Male. 90% Caucasian, 3.3% each- Asian, Hispanic, Native American. Cancer type: 43% breast, 20% lung, 10% pancreatic, 6.6% each: bile duct and prostate with bone metastasis, 3.3% each: colon, tongue, appendix and thyroid. 100% Breast cancer subjects had metastasis: 15% each: bone, lung, bone/lymph 8% each: lung, lymph, bone/brain/lung, bone/brain/spine, bone/liver, bone/brain, liver/bones/lymph. 10% treated for 1 cycle, 90% 2 -3 cycles. 13.3% maintained same ECOG rating. 76.6% had 1 pt, 6.6% had 2 pt, and 3.3% had 3 pt improvement. 13.3% had 1 pt decrease, 23.3% maintained the same, 63.3% had 1-3 pt improvement in health on EORTC (1-7) rating. 13.3% had a 1 pt decrease, 20% maintained the same, 66.7% had 1-4 pt improvement in quality of life on EORTC (1-7) rating. 56.6% gained 1-13lbs. 16.6% same weight, 26.6% lost 1-4 lbs. 73.3% had 1-8 pt. pain reduction (scale 1-10). 19.9% had minimal or moderate pain and maintained. 6.6% had minimal pain and had a 1 pt increase. 46.6% were on pain medication. 43% no longer needed pain medication after study. 100% Health and quality of life significantly improved: 16.6% show no uptake on a PT scan; 16.6% have significant reduction in quantity and/or size of the tumor; 26.8% have reduction in quantity/size of the tumor; 40% have a stable disease; 90% -Alive, median survival 257 days to date. 10% died, median survival 163 days.Total median survival 247 days (from first day of treatment). 100% had hyperpigmentation. No SMK related adverse events reported. Conclusions: SMK is a very promising treatment for metastatic cancer.
Is tryptase a novel serum bio-marker predictive of radical surgery in colo-rectal cancer patients?
Authors: Ammendola Michele , Sacco Rosario , Donato Giuseppe , ZuccalÃ Valeria , Goffredo Veronica , Patruno Rosa , Brandi Mario , Sammarco Giuseppe , Ruggieri Eustachio , Montemurro Severino , Gadaleta Cosmo Damiano , Ranieri Girolamo
Published: March 2020

e22104

Background: Data from experimental tumour models suggest that mast cells (MCs) accumulate near tumour cells before the onset of angiogenesis. Tryptase is a serin protease stored in mast cell granules that plays a role in tumour angiogenesis. Mast cells (MCs) can release tryptase following c-Kit receptor activation. Here we aims to assess tryptase serum levels in CRC patients before and after radical surgery resection and c-Kit expressing cells in primary tumour tissue. Methods: In this study patients with stage B and C CRC (according to Astler e Coller staging system) were selected. Samples of blood were taken from CRC patients between 7 and 9 a.m. 1 day before and 1 day after tumour surgical resection. Venous blood was dispensed into a tube for serum (Becton Dickinson Hemogard Vacutainer Systems, Plymouth, UK). Serum blood samples were centrifuged at 1,500g for 10 minutes and then aliquoted and frozen at -80 °C. Tryptase levels were measured using the UniCAP Tryptase Fluoroenzymeimmunoassay (Pharmacia,Uppsala, Sweden). In addition primary tissue section were immunostained with a primary anti c-Kit antibody (A4502; Dako, Glostrup, Denmark) by mean of immunohistochemistry. Results: Mean ± s.d. tryptase level pre-tumour surgical resection was 6.38 ± 4.49 μg/L, and mean ± s.d. tryptase level post tumour surgical resection was 5.11 ± 3.81 μg/L. A statistically significant difference between pre-tumour surgical resection and post-tumour surgical resection tryptase level concentrations was found: p=0.000 by t-test. A strong correlation between pre-tumour surgical tryptase level and c-Kit expressing cells was also found (r=0.81, p=0.000). Conclusions: This is the first report that analyzes the possible significance of serum tryptase levels changes in CRC patients who underwent radical surgical resection. Our results demonstrated higher serum tryptase levels CRC patients suggesting the release of tryptase from c-Kit expressing cells CRC tissue. We suggest that tryptase may play a role as a novel serum tumour bio-marker predictive of radical surgery in CRC patients. In this context tryptase inhibitors might be evaluated in adjuvant clinical trials as a new antiangiogenetic approach.
Brightness analysis in contrast-enhanced ultrasound using sonazoid for breast cancer.
Authors: Masumoto Norio , Kadoya Takayuki , Akimoto Etsushi , Kajitani Keiko , Emi Akiko , Shigematsu Hideo , Haruta Rumi , Kataoka Tsuyoshi , Okada Morihito
Published: March 2020

e22113

Background: We evaluated the contrast effects of Sonazoid in tumor tissue according to brightness intensity, in cases pathologically diagnosed with breast cancer. Methods: Six cases of ductal carcinoma in situ (DCIS) and seven cases of invasive ductal carcinoma (IDC) diagnosed with needle or Mammotome biopsy between September and December 2012 were examined. The mean ages of the DCIS and IDC cases were 51.7 ± 11.8 years and 62.5 ± 15.7 years respectively. In the IDC cases, tumor diameter was 6-10 mm in three cases, 11-20 mm in three cases and over 50 mm in one case. Two IDC cases were of the luminal A subtype, three cases were luminal B and two cases were triple negative. The ultrasound equipment used was HI VISION ASCENDUS (Hitachi Medical Corporation). The contrast agent, Sonazoid, was administered intravenously at 0.01ml/kg, and for approximately 50 seconds, the tumor sections were fixated and underwent imaging. Sonazoid brightness in the tumor was digitized and a time intensity curve was created based on brightness intensity and time changes. Early and peak contrast brightness was then compared in order to objectively examine changes in contrast effects. Results: Peak brightness was 1.6 ± 0.3 times and 4.8 ± 3.2 times the early brightness in DCIS and IDC cases respectively, indicating a more significant increase in IDC cases (P < 0.05). Comparison according to subtype in IDC cases also revealed a strong increasing trend in triple negative cases, with results showing that peak brightness compared to early brightness was 9.1 times greater in triple negative cases, 2.7 times greater in luminal A cases and 3.3 times greater in luminal B cases. Classification of IDC cases according to nuclear grade (NG) found that peak brightness was 6.2 times greater than early brightness in NG3 cases, 5.7 times greater in NG2 cases and 1.7 times greater in NG1 cases. Conclusions: Brightness intensity, when digitized and analyzed, could be applied to the differential diagnosis of DCIS and IDC. Brightness intensity may also exhibit correlations with subtypes and NG, suggesting that it could be applied to malignancy grading, as well as the prediction of effects of drug therapy and effect measurement.
Impact of deviated balance of regulatory and cytotoxic T cells on release and reseeding of circulating tumor cells in hepatocellular carcinoma.
Authors: Yang Xin-Rong , Sun Yun-Fan , Xu Yang , Jian Zhou , Fan Jia
Published: March 2020

e22133

Background: Our previous data showed that a preoperative CTC ≥2 was significantly related with relapse of hepatocellular carcinoma (HCC) after surgery. However, how CTCs managed to evade immune surveillance, invade into circulation and reseed in the original organ still remains largely unclear. In this study, we illustrated the pro-tumor effect of Treg infiltration in the context of CTC releasing and reseeding. Methods: Using CellSearch system, 115 HCC patients were tested for CTCs prior to resection. Foxp3+, CD8+ and Granzyme B-positive (GrB+) tumor -infiltrating lymphocytes were evaluated by immunohistochemistry (IHC) in tissue microarrays. For IHC markers, the cutoff for stratification subsets was the median value. The pro-invasiveness properties of Tregs were analyzed in both in vitro and vivo assays. Results: Twenty-two patients with high Tregs and low activated CTLs in tumoral tissues had increased number of CTCs compared to 22 patients with low Tregs and high activated CTLs (6.68 vs. 0.91, P<0.001). In 77 patients with detectable CTC, high Tregs and low activated CTLs in peritumoral tissues was significantly associated with an increased incidence of HCC recurrence (80.5% vs. 19.4%, P<0.001), and was an independent prognostic factor for HCC early recurrence (P=0.007). Further in vitro study showed Tregs-derived TGF-β promoted HCC cell migration and invasion; down-regulated E-cadherin and up-regulated N-cadherin, vimentin, fibronectin and snail. The data in human tumor sample also showed that the TGF-β level had positive relationships with Treg count, N-cadherin level and CTC count; and had inverse relationship with E-cadherin (all P values <0.05). In mice, depletion of Tregs by neutralizing antibody abolished intrahepatic colonization and lung metastasis. Conclusions: Local immune contexture in HCC orchestrates with biological behavior of CTCs. High intratumoral Tregs may promote the release of CTCs through inducing tumor cells EMT transition via TGF-β secretion. High peritumoral Tregs created an immune-subversive microenvironment by suppressing CTL to favor colonization of CTCs, which led to recurrence after HCC resection.
Bevacizumab as maintenance therapy (mBev) in metastatic breast cancer (MBC).
Authors: Militello Loredana , Carli Paolo , Di Lauro Vincenzo , Spazzapan Simon , Scalone Simona , Lombardi Davide , Tuzi Alessandro , Talamini Renato , Quitadamo Daniela , Turchet Elisa , Crivellari Diana
Published: March 2020

e22149

Background: Preclinical models suggest that the anti-VEGF may improve the efficacy of anti-estrogen therapies in Hormonal Receptor positive (HR+) breast cancer, but there is lack of informations about the maintenance therapy with Bev (mBev) and Hormonal Therapy (HT). Methods: sixty-one pts with HER-2 negative MBC were treated, at our institution from 2007, with Bev+Taxanes (BT) as 1stline chemotherapy and with HT in HR+ pts as maintenance therapy. Primary endpoints were the evaluation of Progression Free Survival (PFS) and Overall Survival (OS). Secondary endpoint was the safety with HT in HR+ pts. Results: Hormonal status was positive in 52/61 (85%). Antracyclines were administered as adjuvant therapy in 26 pts (42%), antra+tax in 26 (42%) pts, no adjuvant therapy in 9 pts (14%). At the time of first relapse, median age was 50 y/o (range 33-72). First line HT was given to 12 pts. Metastatic sites were only bone in 20 pts (32%), visceral in 15 pts (25%), bone + visceral in 19 pts (31%), lymph nodes in 7 pts (11%). ECOG PS was 0 in 56 pts and 1 in 5. Median number of cycles of BT was 7 (1-14). All pts were evaluated for PFS and OS and 45 pts were evaluated for objective response: complete response (CR) was achieved in 5/45 pts (11%) (duration 12 months), partial response (PR) in 34/45 pts (75%) (duration 6-7 months), stable disease (SD) in 6/45 pts (14%) (6 months). After the assessment of the response, 34/61 pts received maintenance Bev (mBev); among this group, 24/34 pts with HR+ were also treated with HT until disease progression. The median number of cycles of mBev was 8 (1-42). Median PFS was 13.5 months (95%CI: 10.2-18.2) and median OS was 36 months (95%CI: 22-51). The BT regimen was well tolerated: 2 pts experienced cardiotoxicity with a reduction in left ventricular ejection fraction (LVEF); the most common side effects were hypertension (grade 1 in 11 pts e grade 2 in 16 pts), bleeding in 8 pts, proteinuria in 7 pts (grade 1 in 5 pts and grade 2 in 2 pts). Conclusions: Hormonalmaintenance and Bev can extend the overall benefit of therapy and it is well tolerated and associated with long-term clinical outcome. Our results are encouraging for prolonging Bev in association with HT as maintenance therapy until disease progression.
The claudin expression signature of BRCA1-deficient breast cancer.
Authors: Heerma van Voss Marise R. , van der Groep Petra , Smolders Yvonne H. C. , Bart Joost , Van Der Wall Elsken , van Diest Paul J.
Published: March 2020

e22174

Background: Unraveling the biology of BRCA1 related breast cancers may help to improve early detection methods in mutation carriers and patient tailored therapy. Claudins constitute a family of integral membrane proteins, forming the backbone of tight junctions. Altered claudin expression has been linked to oncogenesis in several ways. Decreased cellular polarity increases nutrient and growth factor supply to the rapid expanding tumor cells and decreased intercellular attachments might increase metastatic potential. The discovery of a claudin-low intrinsic subtype of breast cancer has further increased the interest in claudin expression. This study searched for distinct claudin expression profiles that characterize BRCA1 related breast cancers. Methods: 40 BRCA1 related breast cancers and 40 age-matched sporadic breast cancers were immunohistochemically stained for claudin 3, claudin 4, claudin 6 and claudin 7. Cytoplasmic and membranous expression were scored. In addition, a score was given for the overall expression (cytoplasmic and membranous combined) in comparison to the surrounding normal breast tissue. Results: Distinct claudin expression profiles were observed in BRCA1 deficient breast cancers. The hereditary cases had significantly more overexpression of claudin 3, when compared to the tumor surrounding tissue (45.7% vs 17.9%, p=0.03). Membranous claudin 6 expression was seen in 28% in BRCA1 related, compared to only 2.9% in sporadic cases (p=0.007; OR 13.2 95% CI 1.50-116). Claudin 6 was also more often overexpressed, when compared to the normal surrounding tissue in BRCA1 cases (62.5% vs. 19.4%). For claudin 7 an interesting pattern was observed. Membranous expression was observed more often in sporadic (80.0%) than hereditary (42.0%) carcinomas (p=0.001; OR 0.19 95% CI 0.07-0.50), whereas cytoplasmic expression was more abundant in BRCA1 related tumors (85.0% vs. 66.7%; p=0.06; OR 2.83 95% CI 0.95-8.46). Conclusions: Distinct claudin expression profiles characterize breast cancer in BRCA1 mutation carriers. Therefore, claudins may serve as biomarkers for diagnosis of BRCA1 related breast cancer. The mechanism of cytoplasmic relocalization of claudin 7 in BRCA1 deficient tumors deserves to be further studied.
Approaching individualized care with a clinical tissue proteomics methodology.
Authors: Johann Donald Joseph , Mukherjee Sumana , Prieto DaRue , Blonder Josip
Published: March 2020

e22180

Background: The vast majority of therapy plans for cancer patients are derived categorically. Currently, large and time lengthy clinical trials supply the oncology community with aggregate population and survival statistics. Although this approach has been successful there are known shortfalls. More specifically, since the inherent molecular heterogeneity of the solid tumor under study has not been examined, it is not surprising that results may vary widely across individual patients. Therefore, what is needed is a rationally-based molecular profiling of an individual patient's tumor, in a timely manner. The aim is to link the best drug(s) to the individual patient's cancer based on objective molecular data, thus maximizing therapeutic efficacy and minimizing toxicity. Methods: Presented is a new form of a protein-based molecular profiling approach developed utilizing LC/MS-MS. Importantly, this method has the same tissue and time requirements as IHC. A total of 30 breast tumors and 10 normal breast samples were analyzed. Tumors consisted of 10 ER+, 10 HER2+, and 10 triple negative. Custom software was developed. First, to better illustrate the molecular circuitry of the tumor, proteomic data was rendered as a virtual wiring diagram. This was done on a per tumor basis thus graphically illustrating the molecular traffic intensities. Second, computationally deriving a numeric similarity score to allow for easier comparison of molecular circuitry. Results: Biologically relevant proteins (p53, ER, HER2, Ki67) including driver oncoproteins were found. Effective molecular profiling of breast cancer at the protein level was achieved and the established clinical tumor subtypes were discriminated by unsupervised clustering. A visualized simulation of an estrogen carcinogenesis network was also developed. Experimentally identified proteins and expression levels were mapped and objectified onto known cancer pathways contained in the network. Conclusions: Since the tissue quantity and time requirements are the same as IHC the approach is clinically practical. The approach is discovery based and complementary to IHC but is not dependent on antibodies. This method shows promise as a research tool in a clinical trial.
Quantitative test of mutant EGFR and its effect on efficacy of EGFR TKI in advanced NSCLC.
Authors: Zhou Caicun , Li Xuefei , Ren Shengxiang , Yang Guohua , He Wei
Published: March 2020

e22186

Background: It is reported that abundance of EGFR mutation is related with efficacy of EGFR TKI in advanced NSCLC patients with positive EGFR mutations. This study was designed to investigate influence of EGFR mutations and their abundance on efficacy of EGFR TKI. Methods: 141 advanced NSCLC treated with EGFR TKI and available tissue for EGFR mutations were enrolled into the study. EGFR mutation was detected with the kit of AmoyDx ARMS and concentration of mutant EGFR was detected with the method of a quantitative competitive allele specific Taqman PCR technology (qCAST). In this assay, copies from EGFR mutants were calibrated by standard curve respectively, and the mutation rates were estimated through normalizing by copies of a conserved sequence in EGFR exon2. The relationship between abundance of EGFR mutations and efficacy of EGFR TKI was analyzed. Results: The median age of patients was 59 years old, and in which 54.3% were male, 71.7% ex-somkers. Among all of the patients, adenocarcinoma accounted for 57.7%, squamous cell carcinoma 27.2%, adeno-squamous cell carcinoma 7.6%, and others 7.6%. 46.7% of patients harbored EGFR mutations, and in which 48.7% existed more than 20% abundance of EGFR mutations. Overall response rate was 31.4% and progression free survival was 5.0 months. The final analysis data will be reported at the conference. Conclusions: The abundance of EGFR mutations might affect the efficacy of EGFR-TKI, and quantitation of mutant EGFR may better predict for efficacy of EGFR TKI in advanced NSCLC.
Assessing factors for predictive response to ADC targets in clinical tissue for companion diagnostic development.
Authors: Potts Steven James , Krueger Joseph , Lange Holger , Eberhard David , Young George David
Published: March 2020

e22187

Background: One premise of antibody-drug conjugates (ADC) is that the bound mAb-antigen complex on the cell surface will internalize and be metabolized by lysosomal proteases to release the free drug. Thus, the efficacy of an ADC is dependent not only on the presence of cell surface antigens, but also intact delivery of the conjugated drug. In most cases, the biological mechanisms behind these processes have not been elucidated. Furthermore, the extracellular stability of the ADC may be affected by the activity of the target proteases in the tumor microenvironment outside of the lysozome. These concepts are especially important for CDx approaches, which would ideally account not only for the degree of cell surface expression of the target, but also receptor internalization and potential effects of tumor microenvironment. Although in vitro approaches exist to measure these attributes, there are no clinically amenable approaches to measure these critical parameters. Methods: Flagship Biosciences has invented several proprietary approaches for measuring critical properties of the therapeutic target on the cell surface or inside the cell, as well as properties of the TME which could be used to predict efficacy to an ADC using FFPE biopsies. These image analysis approaches have been designed specifically in context of ADC CDx programs to: 1) Accurately define cell surface target expression independent of cytoplasmic expression; 2) Assess critical factors in the TME which may affect delivery of the drug to the intracellular target; and 3) Multiplex these evaluations for an integrative answer to be derived from a typical clinical biopsy. Results: The measurement of cytoplasm/membrane localization was evaluated on several hundred tissue sections, and the methodology was found to be highly reproducible, with coefficients of variation lower than that observed by manual pathologist accessment. Conclusions: Our approach discretely measures endpoints of cell surface biomarker prevalence, biomarker membrane/cytoplasmic ratio, heterogeneity, stromal contribution, inflammatory environment, and other important cell-by-cell outputs from a single FFPE slide in the context of typical drug discovery.
Using a program that computes tumor decay (d) and regrowth (g) rates and the fraction (Φ) of tumor killed to assist in clinical trials and improve patient survival.
Authors: Wilkerson Julia , Bates Susan Elaine , Stein Wilfred Donald , Fojo Tito
Published: March 2020

e22213

Background: Clinical trials collect much data on tumor quantity as therapy proceeds. From this data progression endpoints and response rates are reported and used to evaluate efficacy. But there is much information contained therein that allows us to understand mechanisms of drug action and how to best administer therapies. Methods: We have developed mathematical equations that enable extraction of the rates of tumor decay (d) and regrowth (g) and also the fraction (Φ) of tumor killed. Results: We have analyzed data from >3,000 cases of various tumor histologies including prostate, breast, renal, and medullary thyroid cancers as well as multiple myeloma (MM). These malignancies have been treated with “cytotoxic” and “targeted” therapeutics as well as vaccines. Across all treatment modalities, overall survival does not correlate with the rate of decay, d, but correlates strongly with the growth rate, g. In some drug/tumor combinations, Φ can also be extracted and its role in the drug’s action ascertained. Most therapies developed in metastatic cancers such as ixabepilone in breast cancer and sunitinib in renal cancer are “g therapies”. Such therapies reduce g, impact survival favorably and could further impact survival if administered beyond conventional endpoints for progression. In contrast, “Φ therapies”, such as bortezomib in MM and ATTP in prostate cancer kill a larger fraction of tumor and are proposed to be effective as adjuvant and neo-adjuvant treatments, with little benefit from extending treatment in the metastatic setting. Furthermore, the data suggest resistance in most is likely intrinsic since tumor growth rates on therapy do not change appreciably over time. Conclusions: We have developed an analytical method that characterizes a tumor’s response to a therapy and enables extraction of tumor decay (d) and regrowth (g) rates and the fraction (Φ) of tumor killed. Both g and Φ, but not d, determine the effectiveness of a therapy. Use of these parameters should allow for the conduct of smaller trials and may also help improve patient survival. A stand-alone computer program has been built, is available to the research community and will be demonstrated.
Radiotherapy or surgery of the axilla after a positive sentinel node in breast cancer patients: Final analysis of the EORTC AMAROS trial (10981/22023).
Authors: Rutgers Emiel J. , Donker Mila , Straver Marieke Evelien , Meijnen Philip , Van De Velde Cornelis J. H. , Mansel Robert E. , Westenberg Helen , Orzalesi Lorenzo , Bouma Willem H. , van der Mijle Huub , Nieuwenhuijzen Grard A. P. , Veltkamp Sanne C. , Slaets Leen , Messina Carlo G. M. , Duez Nicole J. , Hurkmans Coen , Bogaerts Jan , van Tienhoven Geertjan
Published: March 2020

LBA1001

The full, final text of this abstract will be available at abstract.asco.org at 7:30 AM (EDT) on Monday, June, 3, 2013, and in the Annual Meeting Proceedings online supplement to the June 20, 2013, issue of Journal of Clinical Oncology. Onsite at the Meeting, this abstract will be printed in the Monday edition of ASCO Daily News.
Bevacizumab, irinotecan, and radiotherapy versus standard temozolomide and radiotherapy in newly diagnosed, MGMT-non-methylated glioblastoma patients: First results from the randomized multicenter GLARIUS trial.
Authors: Herrlinger Ulrich , Schaefer Niklas , Steinbach Joachim Peter , Weyerbrock Astrid , Hau Peter , Goldbrunner Roland , Friedrich Franziska , Stockhammer Florian , Ringel Florian , Braun Christian , Kohnen Ralf , Leutgeb Barbara , Belka Claus , Urbach Horst , Stummer Walter , Glas Martin
Published: March 2020

LBA2000

The full, final text of this abstract will be available at abstract.asco.org at 7:30 AM (EDT) on Saturday, June, 1, 2013, and in the Annual Meeting Proceedings online supplement to the June 20, 2013, issue of Journal of Clinical Oncology. Onsite at the Meeting, this abstract will be printed in the Saturday edition of ASCO Daily News.
Randomized, double-blind, phase III trial of pazopanib versus placebo in women who have not progressed after first-line chemotherapy for advanced epithelial ovarian, Fallopian tube, or primary peritoneal cancer (AOC): Results of an international intergroup trial (AGO-OVAR16).
Authors: Du Bois Andreas , Floquet Anne , Kim Jae Weon , Rau Jörn , Del Campo Jose Maria , Friedlander Michael , Pignata Sandro , Fujiwara Keiichi , Vergote Ignace , Colombo Nicoletta , Mirza Mansoor Raza , Monk Bradley J. , Wimberger Pauline , Ray-Coquard Isabelle , Zang Rongyu , Diaz-Padilla Ivan , Baumann Klaus H. , Kim Jae Hoon , Harter Philipp ,   on behalf of an Intergroup consortium
Published: March 2020

LBA5503

The full, final text of this abstract will be available at abstract.asco.org at 7:30 AM (EDT) on Saturday, June 1, 2013, and in the Annual Meeting Proceedings online supplement to the June 20, 2013, issue of Journal of Clinical Oncology. Onsite at the Meeting, this abstract will be printed in the Saturday edition of ASCO Daily News.
Pilot study of redirected autologous T cells engineered to contain anti-CD19 attached to TCRζ and 4-1BB signaling domains in patients with chemotherapy-resistant or -refractory CD19+ leukemia and lymphoma.
Authors: Grupp Stephan A. , Porter David L. , Levine Bruce , Kalos Michael , Strait Christine , Rheingold Susan R. , Aplenc Richard , June Carl H.
Published: March 2020

TPS10072

Background: Outcomes remain poor for patients (pts) with relapsed or refractory (r/r) B-cell malignancies such as acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia (CLL). CD19 is an attractive therapeutic target because it is widely expressed on normal and malignant B cells throughout B-cell maturation but not on pluripotent stem cells or non–B-cell tissues. We have developed chimeric antigen receptor T cells to target CD19+ cells (CART-19 or CTL019). This approach involves patient-derived T cells that are genetically modified via lentiviral transduction to express a CD19 antigen recognition domain attached to intracellular signaling domains (TCRζ and 4-1BB) that mediate T-cell activation. A recent study in CLL showed that CART-19 therapy had potent activity with responses in 5/8 evaluable pts (3 CR, 2 PR). Pts achieving CR (two > 24 months and one > 5 months) remain in CR with detectable CART-19 cells (Porter et al. ASH 2012). Here we describe a study of CART-19 therapy in pediatric pts with r/r leukemia and lymphoma (NCT01626495). Methods: Pts eligible for this single-arm, open-label study are aged 1 to 24 years with r/r CD19+ B-cell malignancies, without a transplant option or having relapsed after allogeneic stem cell transplantation (ASCT). Pts will undergo leukapheresis to obtain T cells, which will be stimulated, expanded, and transduced ex vivo to express the chimeric antigen receptor. Pts may receive lymphodepleting chemotherapy prior to CART-19 infusion. Study objectives: determine the safety and feasibility of administering CART-19 therapy to pediatric pts, assess duration of in vivo survival of CART-19 cells, and measure antitumor response. There are 2 cohorts in the study: Cohort 1 includes pts who have not undergone ASCT and are not currently eligible for a transplant, and cohort 2 includes pts who relapsed after ASCT. Cells are collected from the recipient, not the donor, which allows for this approach to be used in pts s/p cord blood transplant, and which we hypothesize will reduce the risk of GVHD as a result of toleration of the T cells in the recipient. To date, 7 pts have been enrolled. Clinical trial information: NCT01626495.