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Homologous Recombination Function Predicts Treatment Response in Head and Neck Squamous Cell Carcinoma
Volume 96 Number 2S Supplement 2016 pathology groups on univariate and multivariate analyses (both P < 0.05). Hierarchical clustering within the training cohort produced 8 pathologybased radiomics signatures associated with OS. Of the 4 signatures that retained significance within the validation cohort, we focused on a signature comprised of highly reproducible features. Four radiomics features comprised this model: CT intensity global mean, standard deviation, and uniformity and gray level co-occurrence matrix homogeneity. On multivariate analysis adjusting for stage and adjuvant therapy, this model was significantly associated with OS in both cohorts (both P < 0.05). Conclusion: We present a NSCLC radiomics signature based on the local immune environment that may predictive implications especially with immunotherapy. We are currently working on application of this model to select patients for combined radiotherapy with checkpoint inhibitors. Author Disclosure: C. Tang: None. A. Amer: None. B. Hobbs: None. X. Li: None. C. Behrens: None. E. Para Cuentas: None. J. Rodriguez Canales: None. J.Y. Chang: None. D. Hong: Research Grant; Novartis, Genentech, Eisai, AstraZeneca, Pfizer, miRNA. J.W. Welsh: Research Grant; BMS, Merck, Varian, GSK, Mirnatherapeutics, Inc., Merck. Stock; Helios, Molecular Match. Stock Options; Oncoresponse, Reflexion Medical, Checkmate Pharmaceuticals. I. Wistuba: None. E.J. Koay: Research Grant; Phillips. Honoraria; Phillips.
95 Mre11 Dysfunction Is Associated With Triple-Negative Breast Cancer and Confers Sensitivity to DNA Damaging Therapy G.P. Gupta,1 A.Y. Ho,2 W. Feng,1 C. Fan,1 M. Akram,2 E. Brogi,2 S.N. Powell,2 C.M. Perou,1 Y.H. Wen,2 and J.H.J. Petrini2; 1University of North Carolina, Chapel Hill, NC, 2Memorial Sloan Kettering Cancer Center, New York, NY Purpose/Objective(s): Understanding the molecular basis for DNA damage response perturbations in human cancers is vital to improving the molecular precision of radiotherapy and chemotherapy. We have previously demonstrated that the DNA damage sensor Mre11 is a suppressor of breast tumorigenesis and is underexpressed in a subset of triple negative breast cancers (TNBC) that exhibits excellent clinical outcomes. The goals of this study were to develop a transcriptional signature associated with Mre11 dysfunction and evaluate its correlation with response to neoadjuvant chemotherapy (NAC) in clinical cohorts of breast cancer. Materials/Methods: RNA-seq of murine wild-type and Mre11 mutant breast cancers was performed, and differentially expressed genes were identified using Significance Analysis of Microarrays (SAM). For patient studies, immunohistochemistry (IHC) against human Mre11 was performed on tissue microarrays that contained triplicate cores of non-metastatic TNBC (ER/PR <1%; HER2 0/1+, or HER2 2+/FISH not amplified) from 271 patients. Tumors were classified as Mre11 low if <10% of cancer cells had detectable nuclear protein expression. A subset of Mre11 high and Mre11 low TNBC were confirmed by staining whole tissue sections and subject to RNA-seq analysis. SAM was performed to derive a gene signature associated with Mre11 low status, and intersected with the gene list obtained from the mouse model. This cross-species gene signature was incorporated into a cumulative Z-score based classifier and applied to a published cohort of gene expression data (GSE25066) representing 462 breast cancers with known response to NAC. Fisher’s exact test was used to calculate two-sided P values in 2 x 2 contingency table analyses. Results: Breast cancers that expressed the Mre11 low signature in the GSE25066 dataset were highly enriched for TNBC (16/25 vs. 170/462, P < 0.01), consistent with prior findings using Mre11 IHC. Sixteen out of 186 TNBCs (8.6%) in GSE25066 expressed the Mre11 low gene signature, which is highly concordant with the proportion of Mre11 low expressers identified in the IHC cohort (9.0%). Breast cancers that expressed the Mre11 low signature were significantly more likely to achieve pathological complete response (pCR) to NAC (10/24 vs. 81/238, P Z 0.014). Treatment of Mre11 mutant murine breast cancers also demonstrated hypersensitivity to specific DNA damaging agents relative to Mre11 wild type tumors.
Oral Scientific Sessions
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Conclusion: A gene signature associated with Mre11 dysfunction identifies a subset of breast cancers with increased rates of pCR to NAC. Together with results obtained from our Mre11 mutant breast cancer mouse model, these findings provide a mechanistic explanation for improved clinical outcomes in Mre11 low TNBC, and represent an opportunity for personalization of DNA damage-based therapy. Author Disclosure: G.P. Gupta: None. A.Y. Ho: None. W. Feng: None. C. Fan: None. M. Akram: None. E. Brogi: None. S.N. Powell: None. C.M. Perou: None. Y.H. Wen: None. J.H. Petrini: None.
96 Homologous Recombination Function Predicts Treatment Response in Head and Neck Squamous Cell Carcinoma S.A. Bhide,1 K. Thway,2 J. Lee,3 K.H. Wong,4 D.M. Gujral,5 S.H. Zaidi,6 K. Newbold,7 C.M. Nutting,4 and K. Harrington8; 1The Royal Marsden NHS Foundation Trust, Sutton and London, United Kingdom, 2Royal Marsden Hospital, London, United Kingdom, 3Institute of Cancer Research, London, United Kingdom, 4The Royal Marsden NHS Foundation Trust, Sutton, United Kingdom, 5Imperial College Healthcare NHS Trust, London, United Kingdom, 6The Royal Marsden Hospital, London, United Kingdom, 7Royal Marsden Hospital, Sutton, United Kingdom, 8The Institute of Cancer Research, Sutton, United Kingdom Purpose/Objective(s): Homologous recombination (HR) is a part of a complex DNA damage response pathway (DDR), functional assessment of which in tumors could help predict treatment sensitivity. The aim of this study was to investigate if defective repair of DNA double strand break (DSB) by HR pathway in HNSCC could be used as an early predictor of response following radical treatment. Materials/Methods: Punch biopsy of the tumor in patients with Stage III and IV SCC of the oropharynx was obtained 24 hours following induction chemotherapy (IC). Formalin-fixed paraffin embedded tumor blocks (FFPE) prepared from this sample and pre-treatment biopsies were stained for RAD51 and geminin (S-phase marker) for immunofluorescence and images obtained on confocal microscope. The percentage of gemininpositive cells that were also positive for RAD51 was calculated as the RAD51 score for both specimens. Difference between the RAD51 score for the two specimens was then computed to reflect the HR status. Patients with a percentage difference of 10% between the two samples were deemed to have repaired IC-induced DSBs, and were classified as HR proficient. Foci analyses were blinded to the time-point of the sample and all clinical data. Response at 3 months post-treatment and human papilloma virus (HPV) status (p16 IHC and RT-PCR, E6 mRNA) were assessed. Results: Samples from 13/15 patients recruited were available for analyses. The difference in in RAD51 score between pre and posteIC was statistically significant (median 12% and 50%, P < 0.001). Three samples were classified as HR proficient and 10 as HR deficient at 24 hours postIC. All of the 3 patients classified as HR proficient had partial response or progressive disease and the ten patients deemed HR deficient had a CR
Abstract 96; Table 1: Describes the HPV status, HR functional status, and treatment response. HPV status ve +ve ve ve +ve +ve +ve +ve +ve +ve ve ve +ve
Difference in RAD51 foci pre and post-IC (%)
HR status
Treatment response
31 25 7 3 62 44 71 74 27 72 3 27 57
Deficient Deficient Proficient Proficient Deficient Deficient Deficient Deficient Deficient Deficient Proficient Deficient Deficient
CR CR PR PR CR CR CR CR CR CR PR CR CR
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(Table 1). Two of the five (40%) HPV-negative patients and 100% of the HPV-positive patient were HR deficient and had a complete response. Conclusion: We have demonstrated that HR function following platinum induced DNA damage predicts response following radical treatment for HNSCC. This has future potential as a biomarker for patient selection in trials of DDR pathway modulation, in HPV-negative patients. In addition, this study provides clinical proof that impaired DSB DNA repair may underlie enhanced treatment sensitivity of HPV positive HNSCC. Author Disclosure: S.A. Bhide: None. K. Thway: None. J. Lee: None. K.H. Wong: None. D.M. Gujral: None. S.H. Zaidi: None. K. Newbold: None. C.M. Nutting: None. K. Harrington: None.
Remaining candidate biomarkers were not statistically significantly associated with resection after SBRT. Conclusion: Serum protein expression was variable among LAPC patients who did and did not undergo pancreatectomy in this select population, with high expression of PDGFRa and VEGFD being inversely associated with resection. After further exploration and validation, PLA may help identify LAPC patients who may convert to surgical candidates after chemotherapy and SBRT. Author Disclosure: L.M. Rosati: None. Y. Liu: None. A.D. Rao: None. C.C. Hsu: None. A. Parekh: None. K. Ng: None. A. Hacker-Prietz, PAC: None. L. Zheng: None. D.A. Laheru: None. E.M. Jaffee: None. D.T. Le: None. A. De Jesus-Acosta: None. R.H. Hruban: None. T.M. Pawlik: None. M.J. Weiss: None. C.L. Wolfgang: None. D.T. Chang: None. J.M. Herman: Research Grant; Nucletron. Consultant; Merrimack, Oncosil. A.C. Koong: None.
97 Serum Protein Expression and Associations With Conversion to Resectable Status Following Chemotherapy and Stereotactic Body Radiation Therapy in Locally Advanced Pancreatic Adenocarcinoma L.M. Rosati,1 Y. Liu,2 A.D. Rao,1 C.C. Hsu,3 A. Parekh,1 K. Ng,4 A. Hacker-Prietz, PA-C,1 L. Zheng,5 D.A. Laheru,5 E.M. Jaffee,5 D.T. Le,5 A. De Jesus-Acosta,5 R.H. Hruban,6 T.M. Pawlik,7 M.J. Weiss,7 C.L. Wolfgang,7 D.T. Chang,4 J.M. Herman,1 and A.C. Koong2; 1Johns Hopkins University School of Medicine, Department of Radiation Oncology & Molecular Radiation Sciences, Baltimore, MD, 2Department of Radiation Oncology, Stanford University School of Medicine, Stanford, CA, 3University of Arizona Department of Radiation Oncology, Tucson, AZ, 4Stanford University School of Medicine, Department of Radiation Oncology, Stanford, CA, 5Johns Hopkins University School of Medicine, Department of Medical Oncology, Baltimore, MD, 6Johns Hopkins University School of Medicine, Department of Pathology, Baltimore, MD, 7 Johns Hopkins University School of Medicine, Department of Surgery, Baltimore, MD Purpose/Objective(s): Patients with locally advanced pancreatic cancer (LAPC) are rarely converted to resectable status. However, preliminary data show that select patients may be converted and have long-term benefits following maximal systemic and local therapy. Proximal ligation assay (PLA) was performed to develop a panel of pancreatic-specific blood-based biomarkers and hypoxia-regulated proteins/inflammatory cytokines with the goal of informing treatment decisions. The purpose of this study was to identify candidate markers that are associated with eventual surgical resection for LAPC patients receiving induction chemotherapy and stereotactic body radiation therapy (SBRT). Materials/Methods: Serum of LAPC patients (N Z 68) from two prospective clinical trials investigating fractionated SBRT (6.6 Gy x 5) was collected pre-SBRT. The PLA panel comprised 30 candidate biomarkers including Axl, BMP2, CA 19-9, CA-125, CEA, CXCL1, CXCL6, CXCL9, CXCL10, EGFR, Gas6, IGFBP-2, IGFBP-7, IL-7, IL-8, IL-12, mesothelin, MMP1, MMP2, MMP3, MMP7, osteopontin, PDGFRa, PDK1, PF4, SPARC, TGFB, VEGFa, VEGFD, and YKL40. Expression levels were quantified from 20 mL of plasma and dichotomized using median cutoff. Univariate (UVA) and multivariate (MVA) logistic regression models adjusting for age, sex, performance status, and tumor location were used to assess if high vs. low protein expression was associated with surgery after SBRT, in comparison with SBRT alone. Results: The rate of successful conversion to surgery after SBRT was 25%, with a 76% margin-negative and 59% node-negative resection rate. At median follow-up of 11.3 months from SBRT, 53% of surgical patients and 14% of non-surgical patients are alive. Sixty percent of patients were males, while 53% were >65 years of age and 69% had head of pancreas tumors. The majority (88%) completed 1 cycle of pre-SBRT chemotherapy and 31% completed >3 cycles. High PDGFRa was inversely associated with surgery on UVA (OR Z 0.3, 95% CI Z 0.08-0.9, P Z 0.03) and MVA (OR Z 0.2, 95% CI Z 0.07-0.9, P Z 0.04). High VEGFD was also inversely associated on both UVA (OR Z 0.3, 95% CI Z 0.090.9, P Z 0.05) and MVA (OR Z 0.2, 95% CI Z 0.07-0.9, P Z 0.04). Those with high CA 19-9 expression reached borderline significance for inverse association with resection on MVA (OR Z 0.3, P Z 0.07).
98 Prognostic Implication of Tumor-Infiltrating Lymphocytes in Patients With HER2-Positive Breast Cancer E.J. Yoshida, Q. Li, A. Chung, B. Knudsen, M. Burnison, A.J. Mirhadi, Z.S. Zumsteg, A.E. Giuliano, F.F. Amersi, and S.L. Shiao; Cedars-Sinai Medical Center, Los Angeles, CA Purpose/Objective(s): Despite significant improvements in disease-free survival with HER2-targeted therapies, 20-30% of patients with HER2positive breast cancer will have local and/or distant recurrences. The etiology of these failures remains unknown; however, recent evidence has suggested an association between tumor-infiltrating lymphocytes (TILS) and disease-free survival among women with HER2-positive breast cancer receiving trastuzumab-based chemotherapy. The purpose of this study is to objectively quantify and characterize the TILS located within and around tumors, and to correlate these quantities with survival and prognostic variables. Materials/Methods: Paraffin-embedded sections were generated and clinical records were reviewed for 67 patients, age 18 years, with pathologically-proven HER2/Neu-positive breast cancer treated at a single institution from January 2001 to December 2013. Single-color immunohistochemical staining was performed and scored by a breast pathologist for CK5/6, CK14, and EGFR. Using a multicolor immunohistochemical approach, adjacent sections were stained for TILS expressing CD4, CD8, and CD68. A pan-cytokeratin antibody was utilized to identify the tumor. Slides were subsequently scanned using the Vectra Automated Quantitative Pathology Imaging System and analyzed using an in-house algorithm to quantify immune cells within the tumor, at the tumor margin and within the stroma. We correlated various combinations of CD4+, CD8+, and CD68+ immune cells with overall survival, clinical prognostic variables (tumor size, nodal involvement, tumor grade), as well as expression of CK5/6, CK14, and EGFR. These correlations were conducted for TILs located within the tumor, at the margin and within the stroma. Results: Variable densities of CD4+, CD8+, and CD68+ cells were observed within each tumor, at the margin and in the stroma. Initial analysis revealed increased presence of CD68+ cells within the stroma predicted for worse overall survival. However, the increased presence of CD68+ cells, combined with increased presence of CD4+ cells and reduction in CD8+ cells, was a stronger predictor of worse survival (P Z 0.0299). No correlation was found between this TIL combination and tumor size (P Z 0.1423), nodal involvement (P Z 0.8185), tumor grade (P Z 0.5372), or expression of CK5/6 (P Z 0.6951), CK14 (P Z 0.2425), and EGFR (P Z 0.6630). No association between overall survival and presence/absence of immune cells at the margin or within the tumor was identified. Conclusion: The combined presence of CD4+ and CD68+ immune cells as well as concurrent absence of CD8+ immune cells within the stroma suggests that a poor anti-tumor cytotoxic immune response leads to worse survival. Our findings suggest that inflammation-related biomarker patterns may serve as predictors of survival in patients undergoing trastuzumabbased therapies for HER2-positive breast cancer.
95 Mre11 Dysfunction Is Associated With Triple-Negative Breast Cancer and Confers Sensitivity to DNA Damaging Therapy G.P. Gupta,1 A.Y. Ho,2 W. Feng,1 C. Fan,1 M. Akram,2 E. Brogi,2 S.N. Powell,2 C.M. Perou,1 Y.H. Wen,2 and J.H.J. Petrini2; 1University of North Carolina, Chapel Hill, NC, 2Memorial Sloan Kettering Cancer Center, New York, NY Purpose/Objective(s): Understanding the molecular basis for DNA damage response perturbations in human cancers is vital to improving the molecular precision of radiotherapy and chemotherapy. We have previously demonstrated that the DNA damage sensor Mre11 is a suppressor of breast tumorigenesis and is underexpressed in a subset of triple negative breast cancers (TNBC) that exhibits excellent clinical outcomes. The goals of this study were to develop a transcriptional signature associated with Mre11 dysfunction and evaluate its correlation with response to neoadjuvant chemotherapy (NAC) in clinical cohorts of breast cancer. Materials/Methods: RNA-seq of murine wild-type and Mre11 mutant breast cancers was performed, and differentially expressed genes were identified using Significance Analysis of Microarrays (SAM). For patient studies, immunohistochemistry (IHC) against human Mre11 was performed on tissue microarrays that contained triplicate cores of non-metastatic TNBC (ER/PR <1%; HER2 0/1+, or HER2 2+/FISH not amplified) from 271 patients. Tumors were classified as Mre11 low if <10% of cancer cells had detectable nuclear protein expression. A subset of Mre11 high and Mre11 low TNBC were confirmed by staining whole tissue sections and subject to RNA-seq analysis. SAM was performed to derive a gene signature associated with Mre11 low status, and intersected with the gene list obtained from the mouse model. This cross-species gene signature was incorporated into a cumulative Z-score based classifier and applied to a published cohort of gene expression data (GSE25066) representing 462 breast cancers with known response to NAC. Fisher’s exact test was used to calculate two-sided P values in 2 x 2 contingency table analyses. Results: Breast cancers that expressed the Mre11 low signature in the GSE25066 dataset were highly enriched for TNBC (16/25 vs. 170/462, P < 0.01), consistent with prior findings using Mre11 IHC. Sixteen out of 186 TNBCs (8.6%) in GSE25066 expressed the Mre11 low gene signature, which is highly concordant with the proportion of Mre11 low expressers identified in the IHC cohort (9.0%). Breast cancers that expressed the Mre11 low signature were significantly more likely to achieve pathological complete response (pCR) to NAC (10/24 vs. 81/238, P Z 0.014). Treatment of Mre11 mutant murine breast cancers also demonstrated hypersensitivity to specific DNA damaging agents relative to Mre11 wild type tumors.
Oral Scientific Sessions
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Conclusion: A gene signature associated with Mre11 dysfunction identifies a subset of breast cancers with increased rates of pCR to NAC. Together with results obtained from our Mre11 mutant breast cancer mouse model, these findings provide a mechanistic explanation for improved clinical outcomes in Mre11 low TNBC, and represent an opportunity for personalization of DNA damage-based therapy. Author Disclosure: G.P. Gupta: None. A.Y. Ho: None. W. Feng: None. C. Fan: None. M. Akram: None. E. Brogi: None. S.N. Powell: None. C.M. Perou: None. Y.H. Wen: None. J.H. Petrini: None.
96 Homologous Recombination Function Predicts Treatment Response in Head and Neck Squamous Cell Carcinoma S.A. Bhide,1 K. Thway,2 J. Lee,3 K.H. Wong,4 D.M. Gujral,5 S.H. Zaidi,6 K. Newbold,7 C.M. Nutting,4 and K. Harrington8; 1The Royal Marsden NHS Foundation Trust, Sutton and London, United Kingdom, 2Royal Marsden Hospital, London, United Kingdom, 3Institute of Cancer Research, London, United Kingdom, 4The Royal Marsden NHS Foundation Trust, Sutton, United Kingdom, 5Imperial College Healthcare NHS Trust, London, United Kingdom, 6The Royal Marsden Hospital, London, United Kingdom, 7Royal Marsden Hospital, Sutton, United Kingdom, 8The Institute of Cancer Research, Sutton, United Kingdom Purpose/Objective(s): Homologous recombination (HR) is a part of a complex DNA damage response pathway (DDR), functional assessment of which in tumors could help predict treatment sensitivity. The aim of this study was to investigate if defective repair of DNA double strand break (DSB) by HR pathway in HNSCC could be used as an early predictor of response following radical treatment. Materials/Methods: Punch biopsy of the tumor in patients with Stage III and IV SCC of the oropharynx was obtained 24 hours following induction chemotherapy (IC). Formalin-fixed paraffin embedded tumor blocks (FFPE) prepared from this sample and pre-treatment biopsies were stained for RAD51 and geminin (S-phase marker) for immunofluorescence and images obtained on confocal microscope. The percentage of gemininpositive cells that were also positive for RAD51 was calculated as the RAD51 score for both specimens. Difference between the RAD51 score for the two specimens was then computed to reflect the HR status. Patients with a percentage difference of 10% between the two samples were deemed to have repaired IC-induced DSBs, and were classified as HR proficient. Foci analyses were blinded to the time-point of the sample and all clinical data. Response at 3 months post-treatment and human papilloma virus (HPV) status (p16 IHC and RT-PCR, E6 mRNA) were assessed. Results: Samples from 13/15 patients recruited were available for analyses. The difference in in RAD51 score between pre and posteIC was statistically significant (median 12% and 50%, P < 0.001). Three samples were classified as HR proficient and 10 as HR deficient at 24 hours postIC. All of the 3 patients classified as HR proficient had partial response or progressive disease and the ten patients deemed HR deficient had a CR
Abstract 96; Table 1: Describes the HPV status, HR functional status, and treatment response. HPV status ve +ve ve ve +ve +ve +ve +ve +ve +ve ve ve +ve
Difference in RAD51 foci pre and post-IC (%)
HR status
Treatment response
31 25 7 3 62 44 71 74 27 72 3 27 57
Deficient Deficient Proficient Proficient Deficient Deficient Deficient Deficient Deficient Deficient Proficient Deficient Deficient
CR CR PR PR CR CR CR CR CR CR PR CR CR
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(Table 1). Two of the five (40%) HPV-negative patients and 100% of the HPV-positive patient were HR deficient and had a complete response. Conclusion: We have demonstrated that HR function following platinum induced DNA damage predicts response following radical treatment for HNSCC. This has future potential as a biomarker for patient selection in trials of DDR pathway modulation, in HPV-negative patients. In addition, this study provides clinical proof that impaired DSB DNA repair may underlie enhanced treatment sensitivity of HPV positive HNSCC. Author Disclosure: S.A. Bhide: None. K. Thway: None. J. Lee: None. K.H. Wong: None. D.M. Gujral: None. S.H. Zaidi: None. K. Newbold: None. C.M. Nutting: None. K. Harrington: None.
Remaining candidate biomarkers were not statistically significantly associated with resection after SBRT. Conclusion: Serum protein expression was variable among LAPC patients who did and did not undergo pancreatectomy in this select population, with high expression of PDGFRa and VEGFD being inversely associated with resection. After further exploration and validation, PLA may help identify LAPC patients who may convert to surgical candidates after chemotherapy and SBRT. Author Disclosure: L.M. Rosati: None. Y. Liu: None. A.D. Rao: None. C.C. Hsu: None. A. Parekh: None. K. Ng: None. A. Hacker-Prietz, PAC: None. L. Zheng: None. D.A. Laheru: None. E.M. Jaffee: None. D.T. Le: None. A. De Jesus-Acosta: None. R.H. Hruban: None. T.M. Pawlik: None. M.J. Weiss: None. C.L. Wolfgang: None. D.T. Chang: None. J.M. Herman: Research Grant; Nucletron. Consultant; Merrimack, Oncosil. A.C. Koong: None.
97 Serum Protein Expression and Associations With Conversion to Resectable Status Following Chemotherapy and Stereotactic Body Radiation Therapy in Locally Advanced Pancreatic Adenocarcinoma L.M. Rosati,1 Y. Liu,2 A.D. Rao,1 C.C. Hsu,3 A. Parekh,1 K. Ng,4 A. Hacker-Prietz, PA-C,1 L. Zheng,5 D.A. Laheru,5 E.M. Jaffee,5 D.T. Le,5 A. De Jesus-Acosta,5 R.H. Hruban,6 T.M. Pawlik,7 M.J. Weiss,7 C.L. Wolfgang,7 D.T. Chang,4 J.M. Herman,1 and A.C. Koong2; 1Johns Hopkins University School of Medicine, Department of Radiation Oncology & Molecular Radiation Sciences, Baltimore, MD, 2Department of Radiation Oncology, Stanford University School of Medicine, Stanford, CA, 3University of Arizona Department of Radiation Oncology, Tucson, AZ, 4Stanford University School of Medicine, Department of Radiation Oncology, Stanford, CA, 5Johns Hopkins University School of Medicine, Department of Medical Oncology, Baltimore, MD, 6Johns Hopkins University School of Medicine, Department of Pathology, Baltimore, MD, 7 Johns Hopkins University School of Medicine, Department of Surgery, Baltimore, MD Purpose/Objective(s): Patients with locally advanced pancreatic cancer (LAPC) are rarely converted to resectable status. However, preliminary data show that select patients may be converted and have long-term benefits following maximal systemic and local therapy. Proximal ligation assay (PLA) was performed to develop a panel of pancreatic-specific blood-based biomarkers and hypoxia-regulated proteins/inflammatory cytokines with the goal of informing treatment decisions. The purpose of this study was to identify candidate markers that are associated with eventual surgical resection for LAPC patients receiving induction chemotherapy and stereotactic body radiation therapy (SBRT). Materials/Methods: Serum of LAPC patients (N Z 68) from two prospective clinical trials investigating fractionated SBRT (6.6 Gy x 5) was collected pre-SBRT. The PLA panel comprised 30 candidate biomarkers including Axl, BMP2, CA 19-9, CA-125, CEA, CXCL1, CXCL6, CXCL9, CXCL10, EGFR, Gas6, IGFBP-2, IGFBP-7, IL-7, IL-8, IL-12, mesothelin, MMP1, MMP2, MMP3, MMP7, osteopontin, PDGFRa, PDK1, PF4, SPARC, TGFB, VEGFa, VEGFD, and YKL40. Expression levels were quantified from 20 mL of plasma and dichotomized using median cutoff. Univariate (UVA) and multivariate (MVA) logistic regression models adjusting for age, sex, performance status, and tumor location were used to assess if high vs. low protein expression was associated with surgery after SBRT, in comparison with SBRT alone. Results: The rate of successful conversion to surgery after SBRT was 25%, with a 76% margin-negative and 59% node-negative resection rate. At median follow-up of 11.3 months from SBRT, 53% of surgical patients and 14% of non-surgical patients are alive. Sixty percent of patients were males, while 53% were >65 years of age and 69% had head of pancreas tumors. The majority (88%) completed 1 cycle of pre-SBRT chemotherapy and 31% completed >3 cycles. High PDGFRa was inversely associated with surgery on UVA (OR Z 0.3, 95% CI Z 0.08-0.9, P Z 0.03) and MVA (OR Z 0.2, 95% CI Z 0.07-0.9, P Z 0.04). High VEGFD was also inversely associated on both UVA (OR Z 0.3, 95% CI Z 0.090.9, P Z 0.05) and MVA (OR Z 0.2, 95% CI Z 0.07-0.9, P Z 0.04). Those with high CA 19-9 expression reached borderline significance for inverse association with resection on MVA (OR Z 0.3, P Z 0.07).
98 Prognostic Implication of Tumor-Infiltrating Lymphocytes in Patients With HER2-Positive Breast Cancer E.J. Yoshida, Q. Li, A. Chung, B. Knudsen, M. Burnison, A.J. Mirhadi, Z.S. Zumsteg, A.E. Giuliano, F.F. Amersi, and S.L. Shiao; Cedars-Sinai Medical Center, Los Angeles, CA Purpose/Objective(s): Despite significant improvements in disease-free survival with HER2-targeted therapies, 20-30% of patients with HER2positive breast cancer will have local and/or distant recurrences. The etiology of these failures remains unknown; however, recent evidence has suggested an association between tumor-infiltrating lymphocytes (TILS) and disease-free survival among women with HER2-positive breast cancer receiving trastuzumab-based chemotherapy. The purpose of this study is to objectively quantify and characterize the TILS located within and around tumors, and to correlate these quantities with survival and prognostic variables. Materials/Methods: Paraffin-embedded sections were generated and clinical records were reviewed for 67 patients, age 18 years, with pathologically-proven HER2/Neu-positive breast cancer treated at a single institution from January 2001 to December 2013. Single-color immunohistochemical staining was performed and scored by a breast pathologist for CK5/6, CK14, and EGFR. Using a multicolor immunohistochemical approach, adjacent sections were stained for TILS expressing CD4, CD8, and CD68. A pan-cytokeratin antibody was utilized to identify the tumor. Slides were subsequently scanned using the Vectra Automated Quantitative Pathology Imaging System and analyzed using an in-house algorithm to quantify immune cells within the tumor, at the tumor margin and within the stroma. We correlated various combinations of CD4+, CD8+, and CD68+ immune cells with overall survival, clinical prognostic variables (tumor size, nodal involvement, tumor grade), as well as expression of CK5/6, CK14, and EGFR. These correlations were conducted for TILs located within the tumor, at the margin and within the stroma. Results: Variable densities of CD4+, CD8+, and CD68+ cells were observed within each tumor, at the margin and in the stroma. Initial analysis revealed increased presence of CD68+ cells within the stroma predicted for worse overall survival. However, the increased presence of CD68+ cells, combined with increased presence of CD4+ cells and reduction in CD8+ cells, was a stronger predictor of worse survival (P Z 0.0299). No correlation was found between this TIL combination and tumor size (P Z 0.1423), nodal involvement (P Z 0.8185), tumor grade (P Z 0.5372), or expression of CK5/6 (P Z 0.6951), CK14 (P Z 0.2425), and EGFR (P Z 0.6630). No association between overall survival and presence/absence of immune cells at the margin or within the tumor was identified. Conclusion: The combined presence of CD4+ and CD68+ immune cells as well as concurrent absence of CD8+ immune cells within the stroma suggests that a poor anti-tumor cytotoxic immune response leads to worse survival. Our findings suggest that inflammation-related biomarker patterns may serve as predictors of survival in patients undergoing trastuzumabbased therapies for HER2-positive breast cancer.