Pockaj, Barbara A.

Abstract Introduction: Amplification of chromosome 9p24.1 encoding PDL1, PDL2, and JAK2 (the PDJ amplicon) has been detected in 5-25% of triplenegative breast cancers (TNBC), and is associated with poor outcome and activation of the JAK/STAT pathway. Here, we have developed a robust multiplexed fluorescence in situ hybridization (FISH) assay for detection of 9p24.1 amplification, and assessed the frequency and cellular distribution of the PDJ amplicon in a cohort of African American (AA) women with TNBC. Methods: The FISH assay combines three chromosome 9 probes to simultaneously target JAK2 and PD-L1 (9p24.1, PDJ), chromosome 9q22, and the 9q34.1 regions. This enables discrimination of PDJ amplicons from whole chromosome 9p arm gains and from chromosome 9 ploidies that occur in the background of genomic instability in aneuploid cancer genomes. Fifty cells were scored for each probe and the average copy number was obtained. We defined PDJ amplification as PDJ>4, PDJ/9q22 ratio≥2, and/or PDJ/9q34.1 ratio≥2. The assay was benchmarked to array CGH of flow sorted TNBC tissue from Mayo Clinic Arizona (n=14). Tissue microarrays from TNBC PDX tumors (n=20) and from African American women with TNBC (n=87) were assessed. Results: Using selected TNBC tissue with known CGH data, the FISH assay tightly correlated with PDJ copy number in 13/14 cases. The discordant case was negative by CGH on a biopsy with low tumor content, but FISH positive on tissue section (PDJ >7.5 and a PDJ/9q22 ratio=4). Of 20 TNBC PDX tumors, 5 were amplified by FISH. Twelve of 87 (14%) of tumors had PDJ amplification by FISH, of which ten were from breast tissue and two were from lymph node. Of these 12, PDJ>4 (n=0), PDJ/9q22 ratio≥2 (n=6), and PDJ/9q34.1 ratio≥2 (n=10). Significant intratumoral variation was observed with the presence of single cells with >10 PDJ copies in 8/17 (47%) amplified and 0/90 non-amplified total cases. Conclusion: 9p24.1 (PDJ) amplification encoding JAK2, PD-L1, and PD-L2 is detected by three-color FISH in 14% of TNBC from African American women. Individual cells with ≥10 copies of PDJ was seen exclusively in 8/17 amplified cases. We identified 5 PDX tumors which may provide valuable preclinical models for the amplicon. The association of this biomarker with clinical outcome will be presented. Citation Format: Karen S. Anderson, Tammey Naab, Lori K Koslosky, Megan Anderson, Michael Lewis, Yasmine Kanaan, Ali Afsari, Luisel Ricks-Santi, Barbara A. Pockaj, Michael T Barrett. Tissue prevalence and cellular heterogeneity of 9p24.1 amplification in TNBC in African American women [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P2-10-17.

9034 Background: Routine imaging for staging of early stage cutaneous melanoma is not recommended by National Comprehensive Cancer Network (NCCN) guidelines. Besides the low probability of finding metastatic disease, detrimental aspects include false-positives and additive cost. We sought to investigate the use of imaging for staging of cutaneous melanoma in the United States. Methods: Patients with newly diagnosed clinically node negative cutaneous melanoma between 2000-2007 were identified from the Surveillance Epidemiology End Results-Medicare registry. Any imaging performed within 90 days following diagnosis was considered a staging study. Patients with metastatic disease were excluded. Results: A total of 25,643 patients were identified, of whom 10,775 (42%) underwent imaging. The mean age was 76.1 years, with the majority being male (61.8%) and Caucasian (98.4%). Breakdown by T classification of the primary was as follows: T1 (63%), T2 (17%), T3 (12%), and T4 (8%). A chest Xray was performed for 9,737 (38.0%), while 3,176 (12.4%) underwent advanced staging imaging studies; PET (7.2%), CT (5.9%), MRI (0.6%), and Ultrasound (0.4%). The use of advanced imaging steadily increased over the period of our study from 9.0% in 2000 to 16.3% in 2007 (p<0.001). When stratified by T classification, advanced imaging was used for 8.9% of T1, 14.5% of T2, 18.8% of T3 and 27.0% of T4 tumors (p<0.001). Similarly, node positive patients (4.7%) underwent advanced imaging 33.4% of the time compared to 11.3% for node negative patients (p<0.001). On multivariate analysis, factors predictive of advanced imaging include higher T classification (OR 3.12 T4 vs. T1, CI 2.77-3.52, p<0.001), node positivity (OR 2.70, CI 2.36-3.09, p<0.001), more recent year of diagnosis (OR 2.01 2007 vs. 2000, CI 1.71-2.37, p=0.006), high school education (OR 1.62, CI 1.43-1.83, p<0.001), non-Caucasian race (OR 1.37, CI 1.05-1.77, p=0.018), and male gender (OR 1.12, CI 1.03-1.21, p=0.006). Conclusions: Contrary to current recommendations, performance of advanced imaging for staging of early stage cutaneous melanoma is increasing in the Medicare population. Further research is needed to identify factors driving this increase.

1037 Background: Folate receptor alpha (FRA the product of the FOLR1 gene) has been identified as a potential prognostic and therapeutic target in a number of cancers. A correlation has been shown between intense expression of FRA in breast tumors and poor prognosis, yet little is known about FOLR1/FRA expression across clinically relevant breast cancer subtypes. Methods: 131 breast cancer tumors including 4 benign, 33 ER+, 26 HER2+, and 68 triple negative (TN) were constructed into tissue microarrays (TMAs). FRA expression was analyzed by immunohistochemistry (IHC) using a high affinity FRA antibody. Tumor membrane staining intensity was scored by a pathologist as negative (0), weak (1+), moderate (2+) and strong (3+). The percent of cells within each tissue core stained at each intensity was recorded to calculate an H-score. The H-score is a weighted score that captured both the proportion of positive staining and intensity for each tumor. H-score values can range from zero (no membrane staining) to a maximum of 300 (100% membrane staining at 3+). H-scores for each patient sample were averaged over 3 TMA cores. The mean H-scores for each tumor subtype and the percentage of 3+ staining in >30% of tumor cells were compared by a Mann-Whitney test. The distribution of FOLR1 mRNA was completed using a TCGA RNA-seq dataset from 691 breast tumors classified as ER+, HER2+ and TN. FOLR1 levels of TN versus ER+ and HER2+ were compared by a Mann-Whitney test. Results: The mean H-score for the benign tumors was 0, ER+ (13.31), HER2+ (39.36), TN (119.02). The median H-score for the benign tumors was 0, ER+ (0), HER2+ (7.5), TN (127.5). The TN tumors mean and median H scores were significantly higher than benign, ER+ or HER2+ (p<0.001). The largest percentage of 3+ staining in >30% of tumor cells was observed in TN tumors (36.7%) and lowest in ER+ tumors (0%) (p<0.001). TN tumors had significantly higher levels of FOLR1 mRNA compared to ER+ and HER2+ subtypes (p<0.0001). Conclusions: Our data indicate that expression of FRA is highly prevalent in TN tumors and is supported by FOLR1 mRNA levels. Anti-FRA therapy may represent an important therapeutic intervention in TNBC who to this point have no active targeted treatment options.

8530 Background: RAD-001 (Everolimus) is an oral inhibitor of mammalian target of rapamycin (mTOR). Interim analysis results from a phase II study of RAD-001 in patients with metastatic melanoma (MM) were presented at ASCO 2005. This study was re-opened using a higher dose based on an improvement in the 16 week progression free survival (PFS) rate and good tolerability. Methods: A two- stage, phase II multi-institutional trial was conducted in patients with MM to assess that 16 weeks PFS rate was at least 50%. Inclusion criteria: measurable disease, ECOG performance score of 0–2. Exclusion criteria: presence of intracranial metastases, concurrent use of inducers of cytochrome 3A4 and abnormal organ function. The dose of RAD-001 in the second cohort was increased to 10 mg daily (increased from 30 mg weekly) based on evidence of safety of the higher dose. Results: Twenty-nine patients were enrolled; baseline information is available on 27. Median age was 63 yrs; 15 (56%) had >2 sites of metastatic disease. Most (48%) had stage M1c disease. PS was 0, 1 and 2 in 58%, 38% and 4%. All but 4 (15%) had received prior therapy. Grade 3 adverse events included stomatitis and fatigue (2 each), leukopenia, neutropenia, diarrhea, anorexia, dehydration, dyspnea, hyperglycemia, and hypersensitivity (1 each). Planned interim analysis was done after 20 patients were enrolled. 14 (70%) had progressed 16 weeks, failing to meet the decision rule needed (PFS >30%) to restart accrual. The median PFS for all 29 patients was 56 days. The median overall survival (OS) has not been reached. For the entire cohort of 53 pts treated on this study (at both dose levels), the median PFS, median OS were 59 and 286 days respectively. Conclusion: Interim analysis after enrollment of 20 patients at a higher dose of RAD-001 demonstrated significantly more toxicity and no added clinical efficacy. The 16 week PFS rate target was not reached, and accrual was suspended. No significant financial relationships to disclose.

612 Background: The adoption of new breast surgical techniques has been rapid for early stage breast cancer. The utilization of these techniques in patients with high risk operable breast cancer has not been thoroughly investigated. Methods: Retrospective surgical data was summarized for patients entered into N9831 “Phase III Trial of Doxorubicin and Cyclophosphamide Followed by Weekly Paclitaxel With or Without Trastuzumab as Adjuvant Treatment for Women With HER-2 Over-expressing or Amplified Node Positive or High-Risk Node Negative Breast Cancer.” Results: 3,505 patients were entered into N9831 from 2000–2005 following primary surgery for breast cancer. The median age was 50 and 43% were premenopausal. 93% of patients underwent a diagnostic biopsy prior to their definitive surgical procedure. Whether HER2-neu status was available at time of definitive surgery is unknown. 61% underwent a mastectomy whereas 39% underwent breast conserving surgery (BCS). The rate of BCS was relative stable over the course of the trial. Patients with T1 tumors had a higher rate of BCS (49%) compared to T2 tumors (37%) and T3 tumors (6%). Sentinel lymph node (SLN) biopsy was performed in 54% of the cases. The rate of SLN biopsy increased from 40% in 2000 to 66% in 2005. Patients who underwent mastectomy had a lower rate of SLN biopsy than patients who underwent BCS (44% versus 71%, respectively). Tumor size was also associated with the rate of SLN biopsy: T1 (60%), T2 (53%), and T3 (36%). Data on local and regional recurrences are still maturing. Conclusions: Surprisingly, low numbers of patients on this trial underwent BCS whereas the adoption of the SLN biopsy procedure among the same group of surgeons was relatively high. The relatively low rate of BCS in this study population needs further investigation to determine what surgeon and patient factors impacted the surgical decision. No significant financial relationships to disclose.

The breast is a rare site for metastases, and their molecular characteristics have not been studied yet. Intrinsic molecular genetics, cancer characteristics, and breast tissue immune responses in diverse metastases to the breast have not been previously studied. We identified 64 patients with cancers metastatic to the breast: 51 carcinomas and 13 melanomas. Programmed death ligand 1 (PD-L1), steroid receptors, and HER2/neu expressions were evaluated using immunohistochemistry. Gene sequencing, copy number alterations, microsatellite instability, and tumor mutational burden were performed using next-generation sequencing platforms. The 3 most common primary sites for metastatic carcinomas were lung (37%), ovary (29%), and fallopian tubes/peritoneum (14%). TP53 mutations were commonly (50%) observed among the carcinoma cases, while other mutations were characteristic for the primary cancers (VHL in renal, BRCA1 in the fallopian tube, and BRAF in melanomas). High tumor mutational burden was detected in 5/14 carcinomas and 3/7 melanomas. Tumor cell PD-L1 expression was detected in 6 carcinomas, but not in any of the melanomas, whereas immune cells’ expression of PD-L1 was seen in 17 carcinomas and 6 melanomas. Estrogen receptor status was positive in 13/49 carcinomas including 12 adenocarcinomas originating from the ovary and fallopian tube or peritoneum and 1 duodenal neuroendocrine carcinoma. No carcinoma was HER2/neu positive. Intrinsic genetic characteristics of the metastases to the breast followed the pattern commonly seen in primary tumors. Biomarkers of potential benefit to immune checkpoint inhibition therapy were limited to PD-L1-positive non–small cell lung cancer. No common characteristics of the heterogeneous group of tumor metastases to this organ were identified.

A robust personalized ctDNA test, TARDIS, achieves high accuracy for residual disease after completion of neoadjuvant therapy.

Background: Histologic subtypes of appendiceal cancer vary in their propensity for metastases to regional lymph nodes (LN). A predictive model would help direct subsequent surgical therapy. Methods: The National Cancer Database was queried for patients with appendiceal cancer undergoing surgery between 1998 and 2012. Multivariable logistic regression was used to develop a predictive model of LN metastases which was internally validated using Brier score and Area under the Curve (AUC). Results: A total of 21,647 patients were identified, of whom 9079 (41.9%) had node negative disease, 4575 (21.1%) node positive disease, and 7993 (36.9%) unknown LN status. The strongest predictors of LN positivity were histology (carcinoid tumors OR 12.78, 95% CI 9.01–18.12), increasing T Stage (T3 OR 3.36, 95% CI 2.52–4.50, T4 OR 6.30, 95% CI 4.71–8.42), and tumor grade (G3 OR 5.55, 95% CI 4.78–6.45, G4 OR 5.98, 95% CI 4.30–8.31). The coefficients from the regression analysis were used to construct a calculator that generated predicted probabilities of LN metastases given certain inputs. Internal validation of the overall model showed an AUC of 0.75 (95% CI 0.74–0.76) and Brier score of 0.188. Histology-specific predictive models were also constructed with an AUC that varied from 0.669 for signet cell to 0.75 for goblet cell tumors. Conclusions: The risk for nodal metastases in patients with appendiceal cancers can be quantified with reasonable accuracy using a predictive model incorporating patient age, sex, tumor histology, T-stage, and grade. This can help inform clinical decision making regarding the need for a right hemicolectomy following appendectomy.

Abstract Tumor–stroma interactions significantly influence cancer cell metastasis and disease progression. These interactions are partly comprised of the cross-talk between tumor and stromal fibroblasts, but the key molecular mechanisms within the cross-talk that govern cancer invasion are still unclear. Here, we adapted our previously developed microfluidic device as a 3D in vitro organotypic model to mechanistically study tumor–stroma interactions by mimicking the spatial organization of the tumor microenvironment on a chip. We cocultured breast cancer and patient-derived fibroblast cells in 3D tumor and stroma regions, respectively, and combined functional assessments, including cancer cell migration, with transcriptome profiling to unveil the molecular influence of tumor–stroma cross-talk on invasion. This led to the observation that cancer-associated fibroblasts (CAF) enhanced invasion in 3D by inducing expression of a novel gene of interest, glycoprotein nonmetastatic B (GPNMB), in breast cancer cells, resulting in increased migration speed. Importantly, knockdown of GPNMB blunted the influence of CAF on enhanced cancer invasion. Overall, these results demonstrate the ability of our model to recapitulate patient-specific tumor microenvironments to investigate the cellular and molecular consequences of tumor–stroma interactions. Significance: An organotypic model of tumor–stroma interactions on a microfluidic chip reveals that CAFs promote invasion by enhancing expression of GPNMB in breast cancer cells.