bluuebunny/crossref_metadata_2025 huggingface dataset:
Cell-free circulating DNA as a promising marker of colorectal cancer detection and progression
Authors: Pucciarelli S. , Enzo M. , Agostini M. , Pizzini S. , Del Bianco P. , Lonardi S. , Friso M. , Mescoli C. , Urso E. , Nitti D.
Published: August 2020

11059

Background: Since the pathologic stage is the most powerful prognostic factor for colorectal cancer (CRC), there is a strong need of non-invasive methods for early detection. Cell-free circulating DNA (cfDNA) released from cancer cells varies in size. It has been suggested that cfDNA (ALU repeats of 115 bp, representative of total DNA; ALU repeats of 247 DNA, representative of tumor DNA) may be associated with presence of tumor. Aim of this study was then to investigate whether the cfDNA may have a role as marker of CRC detection and progression. Methods: cfDNA was extracted from plasma samples from 136 patients with primary CRC at different stages [median age 64 yrs; male/female 78/58; stages I-II, 61; stages III-IV, 75], and from 24 patients with adenomas [median age 67 yrs; male/female 17/7)] and from 55 clean-colon healthy subjects [median age 56 yrs; male/female 13/43). cfDNA was assessed by quantitative real-time PCR (qPCR) of ALU repeats with 2 sets of primers (115 and 247 bp) amplifying different lengths of DNA. The levels of cfDNA (ALU-115, ALU-247) of CRC patients (stages I-II and stages III-IV) were compared with those of healthy subjects and patients with adenoma. Results: The median concentrations of total cfDNA (ALU115) in the plasma samples from patients with stages III-IV and stages I-II CRC, adenoma and normal controls were 52,4, 11.9; 1.9, and 1.7 ng/ml, respectively (p<.0001). The corresponding figures for tumor-related cfDNA (ALU247) were 48.8, 4.7, 2.2, and 0.7 ng/ml, respectively. (p<.0001). With a cut-off of 4.86 ng/ml, total DNA (ALU115) showed a sensitivity of 78.52 (95% CI 70.6–85.1) and a specificity of 86.08 (95% CI 76.4–92.8) in distinguishing patients with CRC from non-CRC [AUC: 0.860 (95% CI 0.81–0,90), p-value=.0001]. With a cut-off of 3.04, cfDNA tumor-related (ALU247) showed a sensitivity of 77.94 (95% CI 70.0–84.6) and a specificity of 82.28 (95% CI 72.1–90.0) in distinguishing patients with CRC from non-CRC [AUC: 0.864 (95% CI 0.81–0,91), p-value=.0001]. Conclusions: Both ALU115 and ALU 247 fragments of circulating cfDNA seem promising non-invasive molecular markers of detection and progression of CRC. The findings of the current study require to be confirmed on larger cohorts of patients with CRC and colonic adenoma.

No significant financial relationships to disclose.
Correlation of quantitative total HER2 expression and HER2 homodimers with histopathologic characteristics of breast cancers in the FinHer study
Authors: Huang W. , Weidler J. , Lie Y. , Whitcomb J. , Leinonen M. , Bono P. , Isola J. , Kellokumpu-Lehtinen P. , Bates M. , Joensuu H.
Published: August 2020

11061

Background: We recently reported that the HERmark assay (Monogram Biosciences) accurately measures continua of total HER2 expression (H2T) and HER2 homodimers (H2D) over a wide (∼3 logs) dynamic range, and that a higher concordance was observed between H2T and HER2 status with more stringent central tests as compared with IHC tests performed locally (Joensuu et al, 2008 SABCS,abstract 2071). H2D/H2T ratio was reported as a marker of activated HER2 and a prognosticator of disease progression in HER2+ patients not treated with trastuzumab in the adjuvant setting (Bates et al, 2008 SABCS,abstract 1074). In this follow-up analysis, H2T, H2D, and H2D/H2T ratio were correlated with histopathologic characteristics of breast cancers in the FinHer study. Methods: The HERmark assay was used to measure H2T and H2D in 899 formalin-fixed, paraffin-embedded FinHer specimens. The results were correlated with histopathologic characteristics of breast cancers in the FinHer study (Joensuu et al, N Engl J Med2006;354), including estrogen receptor/progesterone receptor (ER/PR), tumor grade, tumor size, lymph node metastasis, and stage. Results: Higher H2T and H2D levels correlated with ER/PR negativity and high tumor grade (P<0.0001). 42% (102/244) of ER- and 37% (137/374) of PR- cases were HERmark Positive; while 17% (110/655) of ER+ and 14% (75/524) of PR+ cases were HERmark Positive. 10% (13/136) of grade 1, 18% (65/353) of grade 2, and 35% (131/375) of grade 3 tumors were HERmark Positive. No significant association was found between H2T or H2D and tumor size, lymph node metastasis or stage. ER/PR negative and poorly differentiated cancers had higher H2D/H2T ratios (P=0.013), and H2D/H2T ratios >0.6 were associated with smaller primary tumor diameters at the time of cancer detection (P=0.009). Conclusions: The quantitative H2T measurement confirms the known correlations between HER2 expression and histopathologic characteristics of breast cancer. The novel H2D measurement and H2D/H2T ratio may provide further insights into HER2 activation and better diagnostic tests for targeted HER2 therapy.

[Table: see text]
EGFR and KRAS mutation status of lung adenocarcinomas in African Americans
Authors: Reinersman M. , Riely G. J. , Nicastri A. , Soff G. A. , Getinet A. , Schwartz A. G. , Zakowski M. F. , Rusch V. W. , Kris M. G. , Ladanyi M.
Published: August 2020

11065

Background: The 2004 discovery of the tyrosine kinase inhibitor-sensitizing mutations in the epidermal growth factor receptor (EGFR) represents a major advance in the study and management of non-small cell lung cancer. Conversely, KRAS mutations in these cancers confer resistance to the EGFR tyrosine kinase inhibitors gefitinib and erlotinib. EGFR mutations occur almost exclusively in adenocarcinoma, and are more common in never smokers, women, and people born in East Asian (compared to Whites). No comprehensive studies exist of EGFR and KRAS mutations in lung cancers from African-American patients. Methods: We collected formalin-fixed paraffin-embedded material from 121 resected lung adenocarcinomas from African-American patients for DNA extraction. EGFR exon 19 deletions and exon 21 L858R point mutations were detected by sensitive mutation-specific PCR-based methods. KRAS codon 12 and 13 mutation testing was performed by mass-spectrometry (Sequenom)-based genotyping and direct sequencing. These data were compared to Memorial Sloan-Kettering data for EGFR and KRAS mutations in all resected adenocarcinomas in white patients. Results: EGFR mutations were detected in 23 of 121 cases (19%), while KRAS mutations were found in 21 (17%). Exon 19 deletions accounted for 18 of 23 of the EGFR mutations compared to 5 EGFR L858R mutations. KRAS mutations were primarily the transversion type mutations (17 of 21). When compared to data from Memorial Sloan-Kettering for White patients (81/273, 30%), the 17% rate of KRAS mutations in lung adenocarcinomas from African-Americans was significantly lower (p=0.01). EGFR mutation status was similar between African-Americans and Whites (19% vs 18%, p=0.9) and the proportion of exon 19 deletions and L858R mutations was comparable as well. Conclusions: This is the first large series reporting results of mutation testing in lung adenocarcinoma specimens from African-Americans. African-American patients are less likely than Whites to harbor KRAS mutations in their lung adenocarcinomas. There was no significant difference in the prevalence of EGFR mutations. Since biological characteristics underlie clinical factors, these differences may help explain differences in outcomes comparing African-Americans to other groups.

No significant financial relationships to disclose.
Characterization of AZD8931, a potent reversible small molecule inhibitor against epidermal growth factor receptor (EGFR), erythroblastic leukemia viral oncogene homolog 2 (HER2) and 3 (HER3) with a unique and balanced pharmacological profile
Authors: Speake G. , Klinowska T. , Hickinson M. , Marshall G. , Smith P. , Vincent J. , Anderton J. , Gray N. , Smith I. , Ogilvie D.
Published: August 2020

11072

Background: Successful combined pharmacological inhibition of EGFR, HER2, and HER3 signaling is currently under investigation in the clinic. HERs (erbB receptors including EGFR, erbB2, erbB3, erbB4) undergo various types of alteration and in many cancers HER ligands are produced either by the tumor cells themselves or surrounding stromal cells. These mechanisms of receptor activation all lead to constitutive proliferative and/or survival signaling driven by homo- and/or heterodimerization of the HER family. Characterization of a novel tyrosine kinase inhibitor with a potent and balanced profile against EGFR, HER2 (erbB2), and HER3 (erbB3) has been carried out. Methods: A range of assays has been developed to assess the detailed pharmacology of AZD8931 and understand how the profile of AZD8931 compares with other HER family inhibitors, such as gefitinib and lapatinib. These assays have provided unique insights into the pharmacology of these drugs that result from the varying levels of HER and their associated ligands. Results: Across a number of cell systems, AZD8931 has been shown to be a potent inhibitor of tumor cell growth. This effect is through the ability of AZD8931 to inhibit potently the phosphorylation of EGFR (0.004 μM; 95% CIR: 1.377), HER2 (0.003 μM; 95% CIR: 1.817) and HER3 (0.004 μM; 95% CIR: 1.890) in a balanced manner. Furthermore, when compared to control AZD8931 has significant and dramatic effects on the downstream signaling pathways (pAKT [p=0.002] & pMAK [NS]), apoptotic (M30 [p=0.004]), and proliferative (Ki67 [p<0.0005]) endpoints. The novel agent AZD8931 displays a distinct pharmacological profile compared to both gefitinib and lapatinib. Conclusions: Based on our data as well as published literature, the combined pharmacological inhibition of EGFR, HER2, and HER3 signaling has not yet been tested in the clinic. AZD8931 offers an agent to test the hypothesis that combined inhibition of HER signaling could provide additional clinical benefit in cancer, particularly in the majority of solid tumors that do not overexpress HER2.

[Table: see text]
Use of combined biomarkers analysis to predict response to chemotherapy in colorectal cancer: A single-institution feasibility study
Authors: Rodriguez-Pascual J. , Garcia E. , Lopez-Rios F. , Cubillo A. , Diaz-Padilla I. , Hernando O. , Ugidos L. , Calvo I. , Duran I. , Hidalgo M.
Published: August 2020

11074

Background: Treatment options for patients (pts) with colorectal cancer (CRC) have increased in the last years. However, there are no validated prospective molecular markers in CRC to select which agents are better to treat any individual case. The aim of this study was to determine the feasibility of developing an implementing a biomarker panel to guide treatment selection in this setting. Methods: Colorectal cancer tumors were prospectively analyzed with a predefined set of 11 molecular targets, including: KRas and PI3K mutations, EGFR amplification (FISH), and ERCC-1, TS, TP expression by IHC. Clinical characteristics and response to chemotherapy were registered. To establish the utility of this panel, we determine as congruent-treatment if the panel predict the best treatment in patients with more of 1 chemotherapy line and no-congruent-treatment if do not. Results: A total of 84 patients were studied. Only 6 % required a repeated biopsy to obtain sufficient tumor for marker analysis. In 81 % of patients was feasible to study almost 8/11 targets. There were 29 pts (39%) with KRas mutant CRC; 3 pts with PI3K mutations (4%, all of them with KRas mutation); and 2 patients with EGFR amplification. ERCC-1 was positive in 5/78 (6.4%) and TS was positive in 47%. None of 54 pts had TP positivity. Clinical floow up was available in 66 pts (44 males, median age 59, 93% ECOG 0–1). Nineteen patients had early CRC; 23 with metastatic CRC treated with a first line chemotherapy and 24 with advanced CRC treated with 2 or more prior regimens. In this last group for whom the response to multiple agents is known, the panel predictive the most effective treatment in 14 of 24 cases. Conclusions: This targeted-therapy-panel is feasible to implement and should be explore to predict treatment response to CRC .

No significant financial relationships to disclose.
SWOG S0342 and S0536: Expression of EGFR protein and markers of epithelial-mesenchymal transformation (EMT) in cetuximab/chemotherapy-treated non-small cell lung cancer (NSCLC)
Authors: Franklin W. A. , Gandara D. R. , Kim E. S. , Herbst R. S. , Moon J. , Redman M. W. , Olsen C. , Hirsch F. R. , Mack P. , Kelly K.
Published: August 2020

11076

Background: High EGFR gene copy number is associated with efficacy in NSCLC patients (pts) receiving combined chemotherapy and cetuximab (S0342). EGFR protein is typically overexpressed in tumors with high copy number but no consistent association has yet been demonstrated between EGFR protein expression and outcome in pts treated with chemotherapy plus cetuximab. EMT is associated with aggressive biological behavior and resistance to anti-EGFR TKI therapy in NSCLC. Our objective was to identify any association between EGFR protein and EMT and to correlate findings with pt outcomes from cetuximab/chemotherapy in SWOG trials S0342 (paclitaxel [P]-carboplatin [CB] plus sequential or concurrent cetuximab [CX]) and S0536 (P-CB-CX + bevacizumab). Methods: Paraffin sections were stained by immunoperoxidase methods using monoclonal antibodies against EGFR and the EMT markers vimentin, E-cadherin and Zeb1. Sections were scored on continuous scale ranging from 0–300 based on the H score (sum of % positive at each intensity from 0–3). Results were compared to outcome by Kaplan-Meier plot. Results: 79 samples from S0342 were evaluated for EGFR and EMT markers. 67 samples from S0536 were assessed for EGFR only. Mean EGFR H score was 153 and 137 for S0342 and S0536 respectively. At all cut points tested (scores 0, <100, <300) no association between EGFR H score and response or progression-free survival (PFS) was detected in either trial. There was a trend for overall survival and EGFR level at each cutpoint in S0536 but the results did not achieve statistical significance (15 vs 11 mos, p=0.14; 15 vs 11 mos, p=0.20 and 14 mos vs not reached, p=0.10, respectively). Vimentin (6 positive pts) was associated with a shorter PFS, HR=2.60 (1.10–6.14), p=0.03. ECAD and Vimentin were significantly inversely correlated with one another (Spearman p-value<0.01) Conclusions: EGFR protein level by IHC does not significantly correlate with efficacy parameters in chemotherapy/cetuximab-treated NSCLC pts. Patients with vimentin producing tumors (and possible other EMT markers) had shorter PFS, suggesting possible relative resistance to EGFR blockade from cetuximab.

[Table: see text]
Use of serum proteins identified by proteomics to predict prognosis in patients with metastatic renal cell cancer (mRCC) and comparison to the currently used MSKCC survival model
Authors: Vermaat J. , van der Tweel I. , Mehra N. , Sleijfer S. , Engwegen J. Y. , Korse C. M. , Schellens J. H. , Haanen J. B. , Beijnen J. H. , Voest E. E.
Published: August 2020

11078

Background: In mRCC the Memorial Sloan-Kettering Cancer Center (MSKCC) risk model (Motzer et al.,JCO2002) is widely used for clinical trial design and patient management. To improve this model, we searched for serum proteins associated with overall survival (OS) using proteomics and determined their potential contribution to the composition of better prognostic models. Methods: Sera from 125 mRCC patients, mostly interferon-treated, collected between 2001–2006 in three Dutch Cancer Centers, were screened by SELDI-TOF mass spectrometry (MS). Identified proteins were analyzed for their association with OS by Cox regression analysis and Kaplan-Meier curves. Three proteins were subsequently validated with conventional ELISAs and immunoturbidimetry. Computed Cox PH regression models were individually compared with the Akaike's Information Criteria (AIC). The final model was statistically bootstrapped to strengthen its validity. Results: SELDI-TOF MS successfully detected eleven prognostic proteins associated with OS. The strongest prognostic proteins, apolipoprotein A-II (ApoA2), serum amyloid alpha (SAA), and transthyretin were validated by alternative methods and confirmed for their association with OS (p=7.4×10-9, p=3.2×10-8 and p=0.0002, respectively). Stepwise multivariable Cox regression analysis provided an optimal combination of prognostic factors; ApoA2, SAA, LDH, performance status and number of metastasis sites. Subsequent categorization of patients into three risk groups generated a novel survival model that robustly predicts patient prognosis (AIC=684 and p=5.8×10-15) with increased accuracy compared to the MSKCC model (AIC=719, p=2.0×10-7). Bootstrapping our model resulted in an optimism correction of 0.04 (R2=0.45). Initiating systemic therapy according to novel proposed model instead of the MSKCC model would result in a change of treatment in 14% of the patients. Conclusions: Using proteomics, serum proteins associated with OS in mRCC patients can be identified. Incorporating some of these factors together with traditional risk factors yields a prognostic model outperforming the MSKCC risk model thereby further improving patients’ management.

No significant financial relationships to disclose.
Effect of pattern of lymphocyte infiltration in NSCLC on outcome
Authors: O'Callaghan D. , Rexhepaj E. , Gately K. , Gallagher W. M. , Delaney D. , Kay E. , O'Byrne K.
Published: August 2020

11079

Background: The role played by the immune system in determining survival in non-small-cell lung cancer (NSCLC) is unclear. The aim of this study was to investigate the prognostic significance of T-lymphocyte infiltration in NSCLC focusing on CD3+, cytotoxic CD8+ and FOXP3+ T regulatory (Treg) cells. Methods: Immunohistochemistry was used to detect CD3+, CD8+ and FOXP3+ lymphocytes in the tumor islets and tumor stroma in 186 patients with surgically resected NSCLC with a minimum follow-up of 3 years. Quantification of immune infiltration was performed using the Aperio automated image analysis system incorporating the Genie software tool. The median of tumor:stroma CD3+, CD8+ and FOXP3+ infiltration ratios were used as thresholds to dichotomise patients to either high or low infiltration rates. Prognostic variables were identified using univariate and Cox multivariate analysis. Kaplan-Meier analysis and the log-rank test were used to illustrate differences in overall survival. Results: Patients with a higher intratumoral CD3+ and CD8+ lymphocyte infiltration ratio had significantly better survival compared to those with a low tumour/stroma infiltration ratio (p=0.023 & <0.001 respectively). Conversely high intratumoral T-regulatory FOXP3+ positive lymphocyte infiltration rates were associated with a particularly poor prognosis independent of surgical stage (p<0.001). Conclusions: Microlocalization of infiltrating T-lymphocytes, in particular Treg cells, is a powerful predictor of outcome for surgically resected NSCLC and compares favourably with recently published prognostic genomic approaches. Assessment of inflammatory cell infiltrates may help determine which patients should receive adjuvant chemotherapy and, in the future, in predicting benefit from novel adjuvant vaccine/immunotherapies.

No significant financial relationships to disclose.
Prognostic impact of body mass index upon cancer recurrence and survival in stage III colon cancer patients from adjuvant chemotherapy trials
Authors: Sinicrope F. , Foster N. R. , Sargent D. J. , Alberts S. R. , O'Connell M. J.
Published: August 2020

11088

Background: Obesity is associated with an increased risk of colon cancer. However, the influence of body mass index (BMI) upon the prognosis of patients with established colon cancer remains unknown. Methods: We conducted a retrospective study of 1,803 patients with surgically resected stage III colon cancer who were enrolled in five randomized trials of 5-fluorouracil-based adjuvant chemotherapy conducted by the North Central Cancer Treatment Group. Patient height and weight were recorded at study entry and BMI (kg/m2) was calculated and categorized. Cancer recurrence or death were monitored during 5 years of follow-up. The score and likelihood ratio p-values were determined from univariate and multivariate Cox regression models respectively, after stratifying by study. Results: Among stage III colon cancer patients, 19% were obese (BMI 30 kg/m2), 37% were overweight (BMI, 25 to 29.9 kg/m2), 38% were of normal-weight (BMI, 20 to 24.9 kg/m2), and 6% were underweight (BMI < 20 kg/m2). Obese versus normal-weight patients showed higher rates of lymph node (LN) metastasis (>3 LNs; 38% vs. 29%, p <0.01) and tumor site was more likely to be distal versus proximal (52% vs. 45%, p= 0.03). No differences in age, gender, or histologic grade were found. In a univariate analysis, obese patients had significantly worse disease-free survival (DFS) compared with normal-weight patients (hazard ratio 1.25 (95% CI: 1.04 -1.51; p= 0.02). The 5 year DFS rates were 49% in obese patients versus 57% in normal weight subjects. Furthermore, poorer DFS was observed for obese patients after adjusting for age, sex, histologic grade, and tumor site (p= 0.03). Neither overweight nor underweight patients (vs. normal-weight) had significantly different DFS. Analysis of the predictive impact of BMI for 5-FU-based adjuvant therapy is in progress. Conclusions: Obesity (BMI 30 kg/m2) was associated with a greater number of metastatic lymph nodes and poorer disease-free survival in patients with stage III colon cancer, suggesting that obesity influences tumor progression.

No significant financial relationships to disclose.
Validation of the web-based tool Adjuvant! in 5,381 Dutch breast cancer patients
Authors: Mook S. , Schmidt M. K. , van de Velde A. O. , Visser O. , Rutgers E. J. , van 't Veer L. J. , Ravdin P. M.
Published: August 2020

11090

Background: Adjuvant! is a web-based tool that calculates individualized 10-year survival probability and estimates the benefit of adjuvant systemic therapy based on age, co-morbidity, tumor size, tumor grade, number of involved lymph nodes and estrogen-receptor status. The Adjuvant! model was constructed using observed overall survival (OS) for women diagnosed with breast cancer between 1988 and 1992 recorded in the US SEER registry (surveillance, epidemiology and end results) and has not yet been validated in any European series. It is of interest to see whether intrinsic characteristics of tumors and survival of Dutch patients may differ from those in the US. Methods: For this validation all patients who were treated at the Netherlands Cancer Institute for primary breast cancer between 1987 and 1998 were selected according to the following criteria: T1–3 tumor, N0–3, M0, definitive primary surgery, complete axillary staging, and no prior malignancies. Follow-up for OS was complete for >99% of the patients until at least February 2007 (median follow-up 11.7 years). Clinicopathological characteristics as mentioned above and adjuvant treatment data were entered into Adjuvant! version 8.0 for each individual, blinded to the clinical outcome, to calculate predicted 10-year outcomes. Results: In this hospital-based cohort of 5381 patients, all known prognostic factors were significant predictors of overall survival. Across all patients, the 10-year OS was excellently predicted by Adjuvant! (predicted OS - observed OS=0.2%; p=n.s.). Also, for most relevant clinicopathological subgroups the differences between predicted and observed OS were within 2%. Adjuvant underestimated the OS in patients with tubular type of breast cancer (predicted - observed = -6.8; p<0.01); presently, histology is not incorporated in Adjuvant!. Conclusions: Adjuvant! accurately predicts 10-year OS for common tumor types in this first large scale European validation study and is of use for adjuvant treatment-decision making.

[Table: see text]
Loss of p27 expression and response to neoadjuvant chemotherapy: Results from the I-SPY Trial (CALGB150007/150012, ACRIN6657)
Authors: DeMichele A. , Dunn L. , Gimotty P. , Livasy C. , Perou C. , Carey L. , Esserman L.
Published: August 2020

11091

Background: Loss of expression of the cyclin-dependent kinase (cdk) inhibitor p27 has been associated with decreased survival among patients receiving adjuvant chemotherapy. The I-SPY Trial is a multicenter study of locally advanced breast cancer in which pretreatment tumor biopsies were obtained prior to the administration of anthracycline and taxane-based neoadjuvant therapy, and serial MRIs were performed to assess response to treatment. The aim of this study was to determine if loss of tumor p27 expression was associated with response to cytotoxic therapy. Methods: Eligible patients had biopsy-confirmed primary breast cancer measuring at least 3 cm. Immunohistochemistry for p27 was performed centrally on pretreatment core samples using rabbit anti-p27 (Upstate Cell Signaling #06–445). Nuclear p27 was classified as absent (no staining), low (1–50% staining) or normal (>50% staining). Following completion of chemotherapy, post-surgical excision/mastectomy cases were reviewed centrally to determine response to therapy including residual cancer burden (RCB) and pathologic complete response (pCR) rate. Exact Chi2 was used to compare proportions. Results: Pretreatment samples from 164 of 237 enrolled patients demonstrated that p27 expression was absent in 55 (33%), low in 96 (58%) and normal in 14 (8%). P27 loss was not associated with patient age. Compared to normal p27 expression, absent p27 was significantly associated with ER negativity (48% vs. 14%; p=0.03), PR negativity (59% vs. 21%; p=0.03), Her2 overexpression (28% vs. 14%; p=0.017) and basal molecular subtype (29% vs. 13%). The proportion of patients with p27 loss (absent or low) was higher for those with high Ki-67 expression compared to low/mod expression (98% compare to 88%) and lower for grade 1 (72% vs 92%) compared to grade 2/3 tumors (p=0.071 and p=0.090, respectively). Low or absent p27 was not associated with response to therapy as measured by pCR, RCB or change in tumor diameter by MRI. Conclusions: p27 loss in this neoadjuvant patient population was associated with aggressive tumor features, but not predictive of response to cytotoxic therapy. These findings suggest that the poor prognosis associated with loss of p27 is not due to relative chemotherapy resistance.

No significant financial relationships to disclose.
Use of pretreatment serum CA9 (carbonic anhydrase 9) to predict PFS and survival in trastuzumab-treated metastatic breast cancer
Authors: Leitzel K. , Hou H. Y. , Shrivastava V. , Anyanwu U. , Ali S. M. , Koestler W. , Fuchs E. , Brown-Shimer S. , Carney W. , Lipton A.
Published: August 2020

11092

Background: Approximately half of HER2-positive breast cancer patients will respond to first-line trastuzumab-containing therapy. However, in those patients with an initial trastuzumab response, most will progress within a year with acquired resistance. Since trastuzumab treatment is also now used in the HER2-positive adjuvant breast cancer setting, trastuzumab resistance will continue to be a vexing clinical problem, and better predictive and prognostic biomarkers are urgently needed. Methods: Serum HER2, tissue inhibitor of metalloproteinase-1 (TIMP-1), urokinase-type plasminogen activator (uPA), CA9, VEGF-165, and endoglin were measured using ELISA assays in 66 metastatic breast cancer patients before starting first-line trastuzumab-containing therapy. The HER2, TIMP-1, uPA, CA9, and VEGF-165 ELISAs were from Oncogene Science/Siemens Healthcare Diagnostics, Cambridge, MA; and the endoglin ELISA was from R&D Systems, Minneapolis, MN. Progression-free (PFS) and overall survival (OS) were analyzed using the Kaplan-Meier method and Cox modeling with continuous pretreatment serum biomarker variables. Results: Pretreatment serum HER2 (p= 0.005), TIMP-1 (p< 0.0001), uPA (p= 0.006), endoglin (p= 0.008), and CA9 (p <0.0001) were all significant as univariate continuous biomarkers for predicting PFS to first-line trastuzumab-containing therapy, but VEGF was not. In multivariate analysis for PFS with all six biomarkers, only serum CA9 (p= 0.002) was a significant independent covariate. For OS, pretreatment serum HER2 (p= 0.018), TIMP-1 (p< 0.0001), uPA (p< 0.0001), endoglin (p= 0.002), and CA9 (p< 0.0001) were all significant as univariate continuous biomarkers for prognosis, but serum VEGF was not. In multivariate analysis for OS with all six biomarkers, only serum CA9 was a significant independent prognostic covariate (p= 0.001). Conclusions: Elevated pretreatment serum CA9 (a marker of hypoxia) predicts reduced progression-free survival and overall survival in metastatic breast cancer patients treated with first-line trastuzumab-containing therapy. These serum biomarkers deserve further study in larger trials of HER2-targeted breast cancer treatment. Supported by a grant from Komen for the Cure.

[Table: see text]
Cytokeratin-19 (CK19) and mammaglobin (MGB1) gene expression in circulating tumor cells (CTCs) from metastatic breast cancer patients enrolled in the NCCTG trials, N0436 and N0437
Authors: Reinholz M. M. , Kitzmann K. A. , Hobday T. J. , Northfelt D. W. , LaPlant B. , Allred J. B. , Dueck A. C. , Stella P. J. , Lingle W. L. , Perez E. A.
Published: August 2020

11095

Background: Biologic characterization of CTCs is increasingly important in determining metastatic breast cancer (MBC) patient (pt) prognosis and treatment prediction. Combined preliminary results from two earlier metastatic BC NCCTG trials, N0234 & N0336, suggested that the change in CTC mammaglobin (MGB1) gene expression between baseline and two cycles of chemotherapy predicted tumor response (p=0.04). The objectives of this study were to 1) determine CTC gene expression of CK19 and MGB1 before, during, and after treatment in N0436 & N0437 and 2) determine associations between baseline and post-treatment gene expression and treatment response. Methods: CTCs were enriched using CD45-depletion from ∼10ml EDTA blood obtained from metastatic BC pts before, after two cycles, and at end of treatment with either first/second-line irinotecan plus cetuximab (N0436) or first-line paclitaxel poliglumex and capecitabine (N0437). CK19 and MGB1 mRNA levels were determined using quantitative RT-PCR in baseline and serial CTC samples of up to 19 pts from N0436 and 40 pts from N0437. The relative gene expressions were normalized to β2-microglobulin and calibrated to healthy blood using the 2-ΔΔCt algorithm; a value of 2 was defined as positive for the respective marker. Results: CK19+ mRNA was detected in 58% of baseline samples from N0436 (11/19) and N0437 (23/40). MGB1+ mRNA was detected in 32% (6/19) and 38% (15/40) of N0436 and N0437 baseline samples, respectively. CK19+ mRNA was detected in 50% (7/14) and 56% (29/52) of N0436 and N0437 serial CTC samples, respectively. MGB1+ mRNA was detected in 29% (4/14) and 27% (14/52) of N0436 and N0437 serial CTC samples, respectively. Of the 66 serial samples, 27% of samples (18/66) had turned positive from baseline for CK19 or MGB1. CK19 mRNA was detected in 85% (33/39) of MGB1+ mRNA samples but their baseline mRNA levels were not correlated. Conclusions: CK19 mRNA was detected in MBC pts with similar frequencies to the CellSearch imaging system. CK19 was detected at a higher frequency than MGB1. In the majority of cases, MGB1 was co-expressed with CK19. Associations between gene expression and treatment response using Chi-Squared analyses and Cox regression models will be presented.

No significant financial relationships to disclose.
XPG D1104 single-nucleotide polymorphisms and the prognosis of melanoma
Authors: Becker J. C. , Schneider M. , Scherer D. , Ugurel S. , Zapatka M. , Schadendorf D. , Kumar R. , Schrama D.
Published: August 2020

11096

Background: Sunlight is a major risk factor for melanoma. Since UV radiation causes DNA damage, it is not surprisingly, that genetic variants in DNA repair enzymes contribute to the susceptibility to cutaneous melanoma. Methods: Presence of common non-synonymous single-nucleotide polymorphism in different DNA repair enzymes were established and correlated with overall survival of melanoma patients. To this end, the SNPs of 6 different DNA repair enzymes were evaluated in a cohort of 742 melanoma patients. The impact of these polymorphisms on overall survival was subsequently calculated by the cox hazard model. Results: This analysis demonstrated that after adjustment to gender and primary tumor T classification XPG 1104 His/His as well as XPD 751 Lys/Lys genotypes were significantly associated with improved survival. Cox hazard coefficients were 0.744 for XPG 1104 His/His (p = 0.0059) and 0.651 for XPD 751 Lys/Lys (p = 0.017). Importantly, bootstrapping confirmed theses results for subpopulations. Furthermore, multivariate analysis demonstrated that XPG 1104 His/His is an independent factor affecting overall survival (cox coefficient 0.95788; p = 0.0011). Conclusions: XPG codon 1104 polymorphism may be predictive of survival outcome in patients with cutaneous melanoma.

No significant financial relationships to disclose.
Measurement of gene expression biomarkers by qNPA from archived NSCLC FFPE: Prognosis of 5-year survival
Authors: Joshi M. M. , Seligmann B. , Sabalos C. , Harpole D. H.
Published: August 2020

11098

Background: There are vast archives of formalin fixed paraffin-embedded (FFPE) tissue samples that are clinically annotated offering great research potential. However, the technology currently available to assess gene expression is limited to fresh, frozen, tissue. Recently, a method for measuring gene expression from FFPE using the quantitative Nuclease Protection Assay (qNPA) has been published in a model of diffuse large B-cell lymphoma. It was demonstrated that identical results were obtained from small amounts of FFPE as from matched frozen tissue, or from freshly fixed versus 18 year archived FFPE. Methods: This study used the qNPA assay to measure gene expression in archived FFPE primary tumor samples of patients with stage 1 NSCLC for whom the survival outcomes are known (n=86). HTG lysis buffer is added to the sample; nuclease protection probes that are complementary to the mRNA of interest are then added to the solution. The probes hybridize to all RNA, soluble and cross-linked. After hybridization, S1 nuclease was added and destroys all nonspecific, single strand nucleic acid, producing a stoichiometric amount of target-mRNA to probe duplexes. Base hydrolysis releases the probe from these duplexes. Probes were transferred to a programmed ArrayPlate, detection linker added, and both probes and detection linkers were captured onto the array. The ArrayPlate was washed, HRP-labeled detection probe added, incubated, washed, and chemiluminescent substrate was added. Finally, the ArrayPlate was imaged, to measure the expression of each gene in all the wells. Results: Mantel-Cox analysis indicates that the detection of increased expression of G-CSF (p = 0.07; H.R. =1.904; 95% CI=0.9003–4.028) and Leptin (p=0.09; H.R. =1.910; 95% CI=0.9299–3.924) individually suggest an improved survival. Age, gender and T size were found to not be significant in this data set. Conclusions: These results suggest an improved survival advantage in patients with an elevated native GCSF level in stage 1 NSCLC that is consistent with the survival benefits associated with the prophylactic treatment of GCSF for chemosensitivity in stage III or IV NSCLC patients. These results are currently being assessed using a larger cohort.

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[11C]Choline-PET imaging of breast cancer
Authors: Kenny L. M. , Coombes R. C. , Contractor K. , Stebbing J. , Al-Nahhas A. , Palmieri C. , Shousha S. , Lowdell C. , Aboagye E.
Published: August 2020

1110

Background: Molecular imaging techniques are increasingly being used in cancer diagnosis, staging, and assessment of response to treatment. This study sought to evaluate, for the first time, [11C]choline-PET in patients with breast cancer. The potential of [11C]choline-PET for differentiating tumours from normal tissue, correlation with molecular markers, determine its normal variability range, and finally the effect of trastuzumab on [11C]choline uptake in patients with breast cancer was investigated. Methods: 21 patients with newly diagnosed and recurrent breast cancer AJCC stage II-IV were enrolled in the study, all of whom had a baseline dynamic [11C]choline-PET scan with arterial sampling. 14 patients had 2 [11C]choline-PET scans to examine reproducibility, and 7 had a scan after trastuzumab. Analysis of [11C]choline uptake was measured using SUV, Ki (irreversible retention), and IRF@60min (retention using spectral analysis). Results: Breast tumour lesions were visualised by [11C]choline PET in all patients. The difference in tumour and non-tumour uptake were significant for SUV, Ki, and IRF@60 min (Wilcoxon p < 0.0001 for all parameters). [11C]choline uptake was reproducible in breast tumour lesions (r2 = 0.945 for SUV, 0.894 for Ki, and 0.799 for IRF60). The metabolism analysis of arterial plasma samples in 19 patients showed that [11C]choline decreased rapidly post-injection such that at 60 mins the mean radioactivity in arterial plasma due to choline was 15.15 ± 2.16%.Early responses to trastuzumab were determined to be significant in 5 lesions which corresponded with 3 clinical responses. Conclusions: [11C]choline-PET is a promising imaging modality in breast cancer, and could play an important role for determining response to novel treatment strategies in vivo.

No significant financial relationships to disclose.
Mechanisms of Plk4 haploinsufficiency in hepatocellular carcinogenesis
Authors: Wu P. , Rosario C. , Paderova J. , Gladdy R. , Ko M. , Squire J. , Dennis J. , Swallow C.
Published: August 2020

11100

Background: The pathogenesis of hepatocellular carcinoma (HCC) is multifactorial and includes genetic predisposition, though the precise nature of the latter is as yet poorly understood. Mice heterozygous for the polo like kinase Plk4 develop spontaneous HCC and human HCC cases show a high rate of LOH (loss of heterozygosity) at the Plk4 locus. We hypothesize that Plk4 is a haploinsufficient tumor suppressor in man. The purpose of this study was to investigate the mechanisms by which a 50% reduction of Plk4 leads to carcinogenesis. Methods: We established Plk4± and Plk4+/+ murine embryonic fibroblast cell lines (MEFs) in vitro. 5 of 5 Plk4+/+ MEF lines senesced at passage 7–8 as expected, while 9 of 9 Plk4±MEF lines immortalized in culture under NIH 3T3 protocol. The latter were used at passage 15–20 for in vivo tumorigenesis studies. Results: Plk4± MEFs demonstrated increased ploidy with increased passage number. By passage 4, Plk4± MEFs showed a near tetraploid karyotype as well as multiple chromosomal rearrangements and deletions, as assessed by Spectral Karyotype (SKY) Analysis. 5 out of 9 late passage Plk4± MEF lines injected into NOD-SCID mice grew tumors, with a latency of 3 to 12 weeks. By contrast, injection of Plk4+/+ MEFs yielded no tumors. Tumors generated from injection of Plk4± MEFs were harvested, cultured and submitted to SKY analysis; this showed increased clonal rearrangements and deletions compared to the parent cell lines. Conclusion: A 50% reduction in Plk4 expression results in immortalization, aneuploidy and chromosomal instability in vitro, and tumorigenicity in vivo. These results demonstrate that insufficient levels of Plk4, whether due to mutation or LOH, can contribute to the pathogenesis of HCC.

No significant financial relationships to disclose.
Role of selectins in the pathogenesis of multiple myeloma
Authors: Azab A. , Azab F. , Runnels J. , Roccaro A. M. , Magnani J. L. , Sarkar A. , Anderson K. C. , Lin C. P. , Ghobrial I. M.
Published: August 2020

11103

Background: Multiple myeloma (MM) is characterized by the disseminated involvement of the bone-marrow (BM), and its progression involves a continuous circulation of the MM cells (MMCs) in the peripheral blood and homing back to the BM. Selectins are adhesion molecules involved in the primary interaction of lymphocytes with the endothelial cells (ECs) of blood vessels. In this study we studied the role selectins in the pathogenesis of MM. Methods: We have characterized the expression of E, L and P-selectins and their ligands on MM cell lines, patient sample and plasma cells from normal subjects (NPCs). We have tested the effect of blockade of each of the selectins and selectin-ligands on the interaction of MMCs with ECs. Moreover, we tested the effect of a pan selectin inhibitor on MMCs adhesion to ECs, and trans-well (through filter) and trans-endothelial SDF1-induced migration in vitro, and characterized its effect on cytoskeletal signaling induced by the interaction of MMCs and ECs. Moreover, we have tested the effect of the inhibitor on homing of MMCs to the BM in mice using in vivo flow cytometry to detect the number of circulating cells, and in vivo confocal microscopy to directly visualize the homing. Results: All MM cell lines and patient samples had low expression of all selectins and high expression of L and P, but not E, selectin ligands. While NPCs showed low expression of all selectins and ligands. Blockade of L and P-selectin ligands reduced the interaction of MMCs with ECs in vitro, while blockade of E-selectin ligand or any of selectins did not show any effect. The pan-selectin inhibitor reduced the interaction of MMCs with ECs in vitro, did not alter their SDF1-induced migration through filter, but reduced significantly the migration through ECs. The inhibitor inhibited the activation of FAK and ERK induced by interaction of MMCs and ECs. Moreover, the selectin inhibitor extending the circulation time of MM cells in mice, and reduced the homing of MMCs. Conclusions: We found that L and P selectin ligands are highly expressed in MMCs compared to NPCs, and that those play a major role in homing of MMCs to the BM. Moreover, the pan-selectin inhibitor prevented the homing of MMCs to the BM. This provides a basis for testing the effect of the inhibitor on MM tumor progression and initiation.

No significant financial relationships to disclose.
Marrow cell genetic phenotype change induced by human lung cancer cells
Authors: Quesenberry P. J. , Del Tatto M. , Berz D. , Miner T. , Ng T. , Winer E. S. , Aliotta J. , Colvin G. , Dooner M. , Dooner G. , Fontaine J. P.
Published: August 2020

11108

Background: Murine lung-derived microvesicles are capable of inducing lung-specific mRNA in marrow cells, when co-cultured across from these cells, but separated from them by a cell-impermeable (0.4 micron) membrane. These converted murine marrow cells showed mRNA elevations, lung-specific protein production and enhanced capacity to convert to lung epithelial cells after in vivo transplantation into irradiated mice. We examine here whether fresh tissue from lung cancer patients would have the same capacity to genetically alter co-cultured human marrow cells. Methods: Lung cancer samples were collected from 5 patients undergoing surgery. Minced tumor tissue at 50–100 mg was co-cultured in a semi-permeable culture plate insert opposite 3.0 ×106 human marrow cells. The marrow cells were harvested after 2–7 days of co-culture. Marrow cell RNA was analyzed for lung specific mRNA using real time RT-PCR. Relative levels of gene expression was expressed a fold increase compared to level in controls. Results: Lung cancers studied were adenocarcinoma, endobronchial alveolar carcinoma, bronchioloalveolar carcinoma, non-small cell carcinoma and squamous cell carcinoma. mRNAs for aquaporin 1–5, specific for type I pneumocytes and surfactant A-D, specific for type II pneumocytes, were measured. Aquaporin I was elevated in marrow cells from co culture with all lung cancers; elevations ranging from 2.15 to 56.7 fold (mean 23 fold). Similarly surfactant B mRNA was induced in marrow cells by all lung cancers with fold elevations ranging from 7.9 to 2164 (mean fold elevation 668). More variable elevations were also seen with aquaporin 3, 4, and 5, surfactant A, surfactant C, and surfactant D. Ultracentrifugation (28,000 g) of conditioned media from these cancers revealed the presence of microvesicles with diameters of 100–180 nm. Conclusions: These observations indicate that the genetic phenotype of cells in the vicinity of lung cancer cells can be altered and that these alterations might be mediated by microvesicle transfer of genetic information.

No significant financial relationships to disclose.
Trastuzumab plus capecitabine and docetaxel as first-line therapy for metastatic breast cancer: Phase II results
Authors: Gennatas C. G. , Michalaki V. , Gennatas S.
Published: August 2020

1111

Background: In human epidermal growth factor 2 (HER-2)-positive advanced breast cancer, taxanes plus trastuzumab are among the most widely applied options in the first-line setting. The addition of capecitabine to docetaxel significantly improves overall survival in anthracycline-pretreated metastatic breast cancer. We evaluated the efficacy and tolerability of trastuzumab plus capecitabine and docetaxel regimen as first-line therapy. Methods: Patients who had received adjuvant anthracyclines received docetaxel 75 mg/m2 day 1 and capecitabine 950 mg/m2 twice daily, days 1–14, every 3 weeks until disease progression or unacceptable toxicity. Trastuzumab was administered at a dose of 6 mg/kg every 3 weeks. Time to progression (TTP) was defined as primary end point. Results: Twenty eight patients were evaluable (median age 52 years, range 34–70). The regimen achieved objective responses in 11 patients (39%), including complete response in three patients (11%) and partial response in eight patients (28.5%). The median overall survival time was 25.5 months, and the median progression-free survival time was 7.8 months. The safety profile of the combination was favorable and predictable, with a low incidence of grade 3/4 adverse events. The most common adverse events were hand-foot syndrome, and GI toxicities. Severe myelosuppression was rare and cardiac toxicity did not occur. Conclusions: These data confirm that the combination of trastuzumab plus capecitabine and docetaxel is highly active in patients with HER-2-overexpressing anthracycline-pretreated breast cancer, and is well tolerated.

No significant financial relationships to disclose.