Citations

Assessment of tumor-infiltrating lymphocytes (TILs) in histopathologic specimens can provide important prognostic information in diverse solid tumor types, and may also be of value in predicting response to treatments. However, implementation as a routine clinical biomarker has not yet been achieved. As successful use of immune checkpoint inhibitors and other forms of immunotherapy become a clinical reality, the need for widely applicable, accessible, and reliable immunooncology biomarkers is clear. In part 1 of this review we briefly discuss the host immune response to tumors and different approaches to TIL assessment. We propose a standardized methodology to assess TILs in solid tumors on hematoxylin and eosin sections, in both primary and metastatic settings, based on the International Immuno-Oncology Biomarker Working Group guidelines for TIL assessment in invasive breast carcinoma. A review of the literature regarding the value of TIL assessment in different solid tumor types follows in part 2. The method we propose is reproducible, affordable, easily applied, and has demonstrated prognostic and predictive significance in invasive breast carcinoma. This standardized methodology may be used as a reference against which other methods are compared, and should be evaluated for clinical validity and utility. Standardization of TIL assessment will help to improve consistency and reproducibility in this field, enrich both the quality and quantity of comparable evidence, and help to thoroughly evaluate the utility of TILs assessment in this era of immunotherapy.

Assessment of the immune response to tumors is growing in importance as the prognostic implications of this response are increasingly recognized, and as immunotherapies are evaluated and implemented in different tumor types. However, many different approaches can be used to assess and describe the immune response, which limits efforts at implementation as a routine clinical biomarker. In part 1 of this review, we have proposed a standardized methodology to assess tumor-infiltrating lymphocytes (TILs) in solid tumors, based on the International Immuno-Oncology Biomarkers Working Group guidelines for invasive breast carcinoma. In part 2 of this review, we discuss the available evidence for the prognostic and predictive value of TILs in common solid tumors, including carcinomas of the lung, gastrointestinal tract, genitourinary system, gynecologic system, and head and neck, as well as primary brain tumors, mesothelioma and melanoma. The particularities and different emphases in TIL assessment in different tumor types are discussed. The standardized methodology we propose can be adapted to different tumor types and may be used as a standard against which other approaches can be compared. Standardization of TIL assessment will help clinicians, researchers and pathologists to conclusively evaluate the utility of this simple biomarker in the current era of immunotherapy.

Abstract Introduction: MutHER is a phase II trial that demonstrated the anti-tumor activity of the pan-HER inhibitor neratinib in HER2mut, non-amplified MBC. The major challenges to accrue to this trial were the large number of pts to screen for the 2-3% HER2mut population and the high rate (24%) of poor quality tumor DNA for sequencing. The goals of this ctDNA study were: 1) the concordance of HER2mut detected by ctDNA versus tumor testing; 2) the incidence of ctDNA HER2mut in HER2 non-amplified MBC; 3) changes in HER2mut variant allele frequency (VAF) on neratinib therapy. Methods: A sample size of 30 negative (neg) controls was needed to ensure 90% confidence if ctDNA testing has >90% specificity in detecting HER2mut. Thus, plasma from MBC pts obtained at screening for MutHER trial (Neg control: 40 pts without HER2mut on tumor testing; Positive (pos) control: 14 pts with known HER2mut who received neratinib) were subjected to Guardant360 ctDNA 70-gene panel sequencing (all exons of HER2 included). ctDNA from the 14 neratinib treated pts were also analyzed at week (wk) 4 and upon progression. ctDNA data from MBC pts clinically tested at Guardant Health were interrogated for HER2mut incidence. Results: Among the 14 pts with tumor pos for HER2mut, ctDNA sequencing identified the same HER2mut in 11, discrepant HER2mut in 1, and neg in 2. The 2 pts with ctDNA neg for HER2mut had progressive disease (PD) and stable disease (SD > 6 months) on neratinib, respectively. Among the 40 neg controls, 8 were not evaluable (no detectable ctDNA or assay unsuccessful) and all 32 successfully sequenced cases were neg for HER2mut. The sensitivity and specificity of ctDNA for HER2mut detection was 11/14 (79%, 90% CI: 53-94%) and 32/32 (100%, 90% CI: 91-100%), respectively. Among the 11 paired baseline and wk 4 samples, 9 (82%) had lower HER2mut VAFs at wk 4 than at baseline, with 1 complete response (CR), 1 partial response (PR), 5 SD, and 4 PD at wk 8 as best tumor response. Two pts had higher wk 4 ctDNA HER2mut VAFs and both had radiographic PD at wk 8. The absolute HER2mut VAF levels at wk 4 were significantly associated with TTP (Spearman rho=-0.69, p=0.02) and tumor size change (rho=0.67, p=0.05). The HER2mut VAFs were significantly higher at progression than wk 4 in all pts (p<0.01). One pt acquired a new HER2mut T798I, which is analogous to the gate-keeper mutation EGFR T790M. The incidence of HER2mut without amplification in unselected consecutive MBC clinically tested by Guardant360 was 3% (48/1,584), with mutation pattern similar to published tumor testing data. Conclusions: ctDNA sequencing is sensitive and highly specific in detecting HER2mut, offering a non-invasive method to identify pts for trials of HER2mut-targeted therapy. Decreased HER2mut VAFs at wk 4 was observed in 82% of cases, consistent with the on-target effect of neratinib. Increased HER2mut VAFs at wk 4 is a potential early marker of progression. Citation Format: Cynthia Ma, Ron Bose, Feng Gao, Rachel Freedman, Melinda Telli, Gretchen Kimmick, Eric Winer, Michael Naughton, Matthew Goetz, Christy Russell, Debu Tripathy, Melody Cobleigh, Andres Forero, Timothy Pluard, Carey Anders, Shana Thomas, Jill Anderson, Caroline Bumb, Kimberly Banks, Richard Lanman, Richard Bryce, Alshad Lalani, John Pfeifer, Daniel Hays, Mark Pegram, Kimberly Blackwell, Philippe Bedard, Hussam Al-Kateb, Matthew Ellis. Circulating tumor DNA (ctDNA) sequencing for HER2 mutation (HER2mut) screening and response monitoring to neratinib in metastatic breast cancer (MBC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr CT011. doi:10.1158/1538-7445.AM2017-CT011

Abstract Archival tumor samples present a rich resource of annotated specimens for genomics research. However, standard variant calling approaches require a matched normal sample from the same individual, which is often not available with archival tissue. Without a matched normal sample, it is very difficult to distinguish between true somatic variants and germline variants that are private to the individual. Archival FFPE sections often contain adjacent normal tissue, but this normal tissue is often contaminated with infiltrating tumor cells. Comparative somatic variant callers are designed to exclude variants present in the normal sample, so a novel approach is required to leverage sequencing of adjacent normal tissue. Here we present LumosVar 2.0, a software package designed to jointly analyze multiple samples from the same patient. The approach is based on the concept that the allelic fraction of somatic variants, but not germline variants, would be diluted by the presence of normal tissue. Given the allele specific copy number state and expected tumor fraction of each sample, one can model the expected allelic fractions of somatic and germline variants. LumosVar estimates allele specific copy number and tumor sample fractions from the data, and uses the model to call somatic and germline variants. LumosVar is also able to detect sub clonal events and spatial heterogeneity between samples. To evaluate this approach, we used a glioblastoma dataset, where enhancing (high tumor content) and non-enhancing (low tumor content) samples were available for each patient, as well as the matched normal sample to define the true somatic variants. We show that both sensitivity and positive predictive value are improved by analyzing the high tumor and low tumor jointly compared to analyzing the samples individually or in silico pooling of the two samples. Finally, we apply this approach to a set of breast and prostate archival FFPE tumor samples where normal samples were not available for germline sequencing. Citation Format: Rebecca F. Halperin, Winnie S. Liang, Erica Tassone, Jonathan Adkins, Megan Russell, Nhan Tran, Nicole Hank, James Newell, Sandeep Gupta, Michael Berens, Ronald Korn, David W. Craig, Sara A. Byron. Leveraging spatial heterogeneity in tumor purity for improved somatic variant calling of archival tumor-only samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-007. doi:10.1158/1538-7445.AM2017-LB-007

Abstract Aims: Androgen-targeted therapies (ATTs) are the mainstay treatment for metastatic prostate cancer (PCa). However, ATTs promote adaptation of tumour cells and lead to castration resistant disease (CRPC). We have recently identified the cell surface receptor, Neuropilin-1 (NRP1) as increased during EMT and in CRPC. However, the role of NRP1 in the prostate epithelium is poorly understood. This study aims to determine whether the inhibition of NRP1 will be a feasible therapeutic strategy for blocking PCa metastasis and therapy resistance. Methods: qPCR and western blotting were used to assess NRP1 expression in PCa cell lines. NRP1 expression in CRPC was assessed using a murine LNCaP xenograft model of castration. NRP1 was knocked down with shRNA sequences from the pLKO.1 lentiviral construct. For metastasis assays, PC3 cells were microinjected into the zebrafish yolk sac and metastatic dissemination imaged 5 days later. NRP1 expression in radical prostatectomy (RP) samples from Mayo Clinic (545 patients) and Johns Hopkins Medical Institutions (JHMI; 188 patients) cohorts was quantified by Affymetrix exon arrays and multivariable analysis performed. Wound scratch migration and invasion assays were performed with the WoundMaker™ tool and IncuCyte™ FLR imaging systems. Results: NRP1 levels were elevated in humanCRPC xenografts, metastatic and castrate resistant clinical PCa samples (p <0.0001), and PCa cell lines. NRP1 suppression significantly reduced metastasis of human xenografts in zebrafish and the migratory and invasive behaviour of metastatic PCa cells (p=0.0002). Multivariable analysis identified NRP1 as a significant independent prognostic indicator of metastasis and prostate cancer specific mortality in two large clinical cohorts (Mayo Clinic and JHMI; p=0.008/0.048 and 0.013/0,034 respectively). We show that NRP1 knockdown promotes E-Cadherin expression and loss of vimentin in mesenchymal PCa cells. Conclusion: These results will provide the preclinical data necessary to rationalise the use of anti-NRP1 directed adjuvant therapies for clinical use in PCa patients receiving ATTs, and will pave the way for larger scale preclinical and clinical trials in the PCa setting. Citation Format: Marianna Volpert, Brian Tse, Ellca Ratther, Nataly Stylianou, Mannan Nouri, Melanie Lehman, Stephen McPherson, Mani Roshan-Moniri, Mandeep Takhar, Nicholas Erho, Mohamed Alshalafa, Elai Davicioni, Robert Jenkins, Ashley Ross, Jeffrey Karnes, Robert Den, Ladan Fazli, Martin Gleave, Elizabeth Williams, Paul Rennie, Ralph Buttyan, Pamela Russell, Colleen Nelson, Brett Hollier. Neuropilin-1 is up-regulated in the adaptive response of prostate tumors to androgen targeted therapies and is prognostic of metastatic progression and patient mortality [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4908. doi:10.1158/1538-7445.AM2017-4908

Abstract Background: Esophageal cancer is the 6th leading cause of cancer death globally, with geographical high-risk areas in Asia, the Middle East, and eastern and southern Africa. Esophageal squamous cell carcinoma (ESCC) is the more common variant in Africa. In Kenya, its incidence is 2nd in men after prostate cancer and 3rd in women after breast and cervix-uteri cancers. Late presentation is a common occurrence in developing countries and is multifactorial due to challenges in access to health care, low socioeconomic status and delayed or missed diagnosis. A large percentage of these tumors are thus unresectable and are only eligible for palliative care via stenting. Our hospital is a 300-bed referral center in southwestern Kenya, which is a hotspot for ESCC, and we see over 400 cases of ESCC annually. Methods: We have developed a technique for placement of esophageal self-expanding metallic stents (SEMS) without fluoroscopy that is safe and easily reproducible. This is an outpatient procedure, with the majority done under conscious sedation, and routine followup is not necessary. The tumor margins are noted at time of video endoscopy, a guidewire is placed, and dilation done with Savary dilators as required. The SEMS are then loaded on the stent delivery device and deployed into the proper position based on measurements, and placement is subsequently confirmed via endoscopic visualization. Results: A total of 3000 SEMS have been placed to date at our hospital, without using fluoroscopy. The male: female ratio has been 1.5:1, and the average age has been 60.4 years. The distribution of tumor locations was 67% in the middle and distal third. The most common complications were tumor overgrowth with obstruction and stent migration. Procedure related mortality was 0.3%. Post-procedure improvement in dysphagia score was seen in over 80% , and patient satisfaction was high. Initial data puts our post stent survival time around 8 months/ 250 days Conclusions: Placement of SEMS for ESCC, without fluoroscopy, is a safe and reproducible procedure which has a low rate of adverse events. This procedure results in effective palliation of a difficult disease and can easily be done in resource-limited settings which have endoscopy capabilities. Current efforts are ongoing to increase opportunities for training endoscopists in this procedure and for provision of affordable stents in Africa. Citation Format: Michael M. Mwachiro, Robert K. Parker, Stephen Burgert, Justus Lando, Sinkeet Rankeeti, Robert Chepkwony, Emmanuel Kiniga, Sanford Dawsey, Mark Topazian, Russell E. White. Esophageal stenting in resource-limited settings [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2766. doi:10.1158/1538-7445.AM2017-2766

Background Donor safety and the impact on quality of life continue to concern transplant centers as living liver transplantation increases. Methods Living liver donors participated in a focus group to discuss their perceptions of the donation process. Results The decision to donate was a personal choice made before the request by the recipient or transplant service. Spouses verified their support, but indicated that they were not included in the process. No donors felt coerced. The explanation of the procedure was thorough but excessive. Hospital care was good; however, donors admitted being unprepared for the pain and side effects of pain medications. Ongoing incisional discomfort continued beyond 6 weeks and prevented donors from feeling normal. Donors felt conflicted about competing with the “true” patient for healthcare. Conclusions Donors were positive about the need and willingness to donate. Sensitivity to the order of the disclosure process is identified.