Case report: Radiographic complete response of radiation-induced glioblastoma to front-line radiotherapy: A report and molecular characterization of two unique cases.

Radiation-induced gliomas (RIGs) are an uncommon disease type and a known long-term complication of prior central nervous system radiation exposure, often during childhood. Given the rarity of this malignancy subtype, no clinical trials have explored optimal therapy for these patients, and the literature is primarily limited to reports of patient cases and series. Indeed, the genomic profiles of RIGs have only recently been explored in limited numbers, categorizing these gliomas into a unique subset. Here, we describe two cases of RIG diagnosed as glioblastoma (GB), IDH-wildtype, in adults who had previously received central nervous system radiation for childhood cancers. Both patients demonstrated a surprising complete radiographic response of the postoperative residual disease to front-line therapy, a phenomenon rarely observed in the management of any GB and never previously reported for the radiation-induced subgroup. Both tumors were characterized by next-generation sequencing and chromosomal microarray to identify potential etiologies for this response as well as to further add to the limited literature about the unique molecular profile of RIGs, showing signatures more consistent with diffuse pediatric-type high-grade glioma, H3-wildtype, and IDH-wildtype, WHO grade 4. Ultimately, we demonstrate that treatment utilizing a radiation-based regimen for GB in a previously radiated tissue can be highly successful despite historical limitations in the management of this disease.

Clinical utility of a regional precision medicine molecular tumor board and challenges to implementation.

PURPOSE: Molecular tumor boards provide precision treatment recommendations based on cancer genomic profile. However, practical barriers limit their benefits. We studied the clinical utility of the precision medicine molecular tumor board (PMMTB) and described challenges with PMMTB implementation. METHODS: An observational cohort study included patients reviewed by the PMMTB between September 2015 to December 2017. Patients who had consented to the registry study were included. The primary endpoint of this study was time on treatment (ToT) ratio. Clinical utility was established if the primary endpoint had least 15% of patients achieving a ToT ratio of ≥1.3. RESULTS: Overall, 278 patients were presented to the PMMTB and 113 cases were included in the final analysis. The PMMTB identified at least one nonstandard of care (SOC) clinically actionable mutation for 69.0% (78/113) of cases. In patients who received non-SOC treatment, 43.8% (7/16) achieved a ToT ratio of 1.3 or more (p < 0.001). Fifty-nine patients did not receive non-SOC recommendations. Reasons for not pursuing treatment included 35.6% having response to current treatment, 20.3% died prior to starting or considering PMMTB recommendations, 13.6% pursued other treatment options based on clinician discretion, another 10.2% pursued other treatment options because clinical trials recommended were not geographically accessible, 8.5% had rapid decline of performance status, 6.8% lacked of financial support for treatment, and 5.1% were excluded from clinical trials due to abnormal laboratory values. CONCLUSION: The regional PMMTB non-SOC recommendations benefitted a majority of patients and additional processes were implemented to assist with non-SOC treatment accessibility.

Treatment of Metastatic Colorectal Cancer: ASCO Guideline.

PURPOSE: To develop recommendations for treatment of patients with metastatic colorectal cancer (mCRC). METHODS: ASCO convened an Expert Panel to conduct a systematic review of relevant studies and develop recommendations for clinical practice. RESULTS: Five systematic reviews and 10 randomized controlled trials met the systematic review inclusion criteria. RECOMMENDATIONS: Doublet chemotherapy should be offered, or triplet therapy may be offered to patients with previously untreated, initially unresectable mCRC, on the basis of included studies of chemotherapy in combination with anti-vascular endothelial growth factor antibodies. In the first-line setting, pembrolizumab is recommended for patients with mCRC and microsatellite instability-high or deficient mismatch repair tumors; chemotherapy and anti-epidermal growth factor receptor therapy is recommended for microsatellite stable or proficient mismatch repair left-sided treatment-naive RAS wild-type mCRC; chemotherapy and anti-vascular endothelial growth factor therapy is recommended for microsatellite stable or proficient mismatch repair RAS wild-type right-sided mCRC. Encorafenib plus cetuximab is recommended for patients with previously treated BRAF V600E-mutant mCRC that has progressed after at least one previous line of therapy. Cytoreductive surgery plus systemic chemotherapy may be recommended for selected patients with colorectal peritoneal metastases; however, the addition of hyperthermic intraperitoneal chemotherapy is not recommended. Stereotactic body radiation therapy may be recommended following systemic therapy for patients with oligometastases of the liver who are not considered candidates for resection. Selective internal radiation therapy is not routinely recommended for patients with unilobar or bilobar metastases of the liver. Perioperative chemotherapy or surgery alone should be offered to patients with mCRC who are candidates for potentially curative resection of liver metastases. Multidisciplinary team management and shared decision making are recommended. Qualifying statements with further details related to implementation of guideline recommendations are also included.Additional information is available at www.asco.org/gastrointestinal-cancer-guidelines.

Stromal remodeling regulates dendritic cell abundance and activity in the tumor microenvironment.

Stimulatory type 1 conventional dendritic cells (cDC1s) engage in productive interactions with CD8+ effectors along tumor-stroma boundaries. The paradoxical accumulation of "poised" cDC1s within stromal sheets is unlikely to simply reflect passive exclusion from tumor cores. Drawing parallels with embryonic morphogenesis, we hypothesized that invasive margin stromal remodeling generates developmentally conserved cell fate cues that regulate cDC1 behavior. We find that, in human T cell-inflamed tumors, CD8+ T cells penetrate tumor nests, whereas cDC1s are confined within adjacent stroma that recurrently displays site-specific proteolysis of the matrix proteoglycan versican (VCAN), an essential organ-sculpting modification in development. VCAN is necessary, and its proteolytic fragment (matrikine) versikine is sufficient for cDC1 accumulation. Versikine does not influence tumor-seeding pre-DC differentiation; rather, it orchestrates a distinctive cDC1 activation program conferring exquisite sensitivity to DNA sensing, supported by atypical innate lymphoid cells. Thus, peritumoral stroma mimicking embryonic provisional matrix remodeling regulates cDC1 abundance and activity to elicit T cell-inflamed tumor microenvironments.

A randomized phase 2 trial of nivolumab, gemcitabine, and cisplatin or nivolumab and ipilimumab in previously untreated advanced biliary cancer: BilT-01.

BACKGROUND: Gemcitabine and cisplatin has limited benefit as treatment for advanced biliary tract cancer (BTC). The addition of an anti-programmed death receptor (PD-1)/PD-ligand (L1) antibody to either systemic chemotherapy or anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA4) antibody has shown benefit in multiple solid tumors. METHODS: In this phase 2 trial, patients 18 years or older with advanced BTC without prior systemic therapy and Eastern Cooperative Oncology Group Performance Status 0-1 were randomized across six academic centers. Patients in Arm A received nivolumab (360 mg) on day 1 along with gemcitabine and cisplatin on days 1 and 8 every 3 weeks for 6 months followed by nivolumab (240 mg) every 2 weeks. Patients in Arm B received nivolumab (240 mg) every 2 weeks and ipilimumab (1 mg/kg) every 6 weeks. RESULTS: Of 75 randomized patients, 68 received therapy (Arm A = 35, Arm B = 33); 51.5% women with a median age of 62.5 years. The observed primary outcome of 6-month progression-free survival (PFS) rates in the evaluable population was 59.4% in Arm A and 21.2% in Arm B. The median PFS and overall survival (OS) in Arm A were 6.6 and 10.6 months, and in Arm B 3.9 and 8.2 months, respectively, in patients who received any treatment. The most common treatment-related grade 3 or higher hematologic adverse event was neutropenia in 34.3% (Arm A) and nonhematologic adverse events were fatigue (8.6% Arm A) and elevated transaminases (9.1% Arm B). CONCLUSIONS: The addition of nivolumab to chemotherapy or ipilimumab did not improve 6-month PFS. Although median OS was less than 12 months in both arms, the high OS rate at 2 years in Arm A suggests benefit in a small cohort of patients.

Inhibition of B-cell lymphoma 2 family proteins alters optical redox ratio, mitochondrial polarization, and cell energetics independent of cell state.

SIGNIFICANCE: The optical redox ratio (ORR) [autofluorescence intensity of the reduced form of nicotinamide adenine dinucleotide (phosphate) (NAD(P)H)/flavin adenine dinucleotide (FAD)] provides a label-free method to quantify cellular metabolism. However, it is unclear whether changes in the ORR with B-cell lymphoma 2 (Bcl-2) family protein inhibition are due to metabolic stress alone or compromised cell viability. AIM: Determine whether ABT-263 (navitoclax, Bcl-2 family inhibitor) changes the ORR due to changes in mitochondrial function that are independent of changes in cell viability. APPROACH: SW48 colon cancer cells were used to investigate changes in ORR, mitochondrial membrane potential, oxygen consumption rates, and cell state (cell growth, viability, proliferation, apoptosis, autophagy, and senescence) with ABT-263, TAK-228 [sapanisertib, mammalian target of rapamycin complex 1/2 (mTORC 1/2) inhibitor], and their combination at 24 h. RESULTS: Changes in the ORR with Bcl-2 inhibition are driven by increases in both NAD(P)H and FAD autofluorescence, corresponding with increased basal metabolic rate and increased mitochondrial polarization. ABT-263 treatment does not change cell viability or induce autophagy but does induce a senescent phenotype. The metabolic changes seen with ABT-263 treatment are mitigated by combination with mTORC1/2 inhibition. CONCLUSIONS: The ORR is sensitive to increases in mitochondrial polarization, energetic state, and cell senescence, which can change independently from cell viability.

Impact of baseline culture conditions of cancer organoids when determining therapeutic response and tumor heterogeneity.

Representative models are needed to screen new therapies for patients with cancer. Cancer organoids are a leap forward as a culture model that faithfully represents the disease. Mouse-derived cancer organoids (MDCOs) are becoming increasingly popular, however there has yet to be a standardized method to assess therapeutic response and identify subpopulation heterogeneity. There are multiple factors unique to organoid culture that could affect how therapeutic response and MDCO heterogeneity are assessed. Here we describe an analysis of nearly 3500 individual MDCOs where individual organoid morphologic tracking was performed. Change in MDCO diameter was assessed in the presence of control media or targeted therapies. Individual organoid tracking was identified to be more sensitive to treatment response than well-level assessment. The impact of different generations of mice of the same genotype, different regions of the colon, and organoid specific characteristics including baseline size, passage number, plating density, and location within the matrix were examined. Only the starting size of the MDCO altered the subsequent growth. These results were corroborated using ~ 1700 patient-derived cancer organoids (PDCOs) isolated from 19 patients. Here we establish organoid culture parameters for individual organoid morphologic tracking to determine therapeutic response and growth/response heterogeneity for translational studies.

Technologies to Assess Drug Response and Heterogeneity in Patient-Derived Cancer Organoids.

Patient-derived cancer organoids (PDCOs) are organotypic 3D cultures grown from patient tumor samples. PDCOs provide an exciting opportunity to study drug response and heterogeneity within and between patients. This research can guide new drug development and inform clinical treatment planning. We review technologies to assess PDCO drug response and heterogeneity, discuss best practices for clinically relevant drug screens, and assert the importance of quantifying single-cell and organoid heterogeneity to characterize response. Autofluorescence imaging of PDCO growth and metabolic activity is highlighted as a compelling method to monitor single-cell and single-organoid response robustly and reproducibly. We also speculate on the future of PDCOs in clinical practice and drug discovery.Future development will require standardization of assessment methods for both morphology and function in PDCOs, increased throughput for new drug development, prospective validation with patient outcomes, and robust classification algorithms.

Phase II Study of Copanlisib in Patients With Tumors With PIK3CA Mutations: Results From the NCI-MATCH ECOG-ACRIN Trial (EAY131) Subprotocol Z1F.

PURPOSE: Activating mutations in PIK3CA are observed across multiple tumor types. The NCI-MATCH (EAY131) is a tumor-agnostic platform trial that enrolls patients to targeted therapies on the basis of matching genomic alterations. Arm Z1F evaluated copanlisib, an α and δ isoform-specific phosphoinositide 3-kinase (PI3K) inhibitor, in patients with PIK3CA mutations (with or without PTEN loss). PATIENTS AND METHODS: Patients received copanlisib (60 mg intravenous) once weekly on days 1, 8, and 15 in 28-day cycles until progression or toxicity. Patients with KRAS mutations, human epidermal growth factor receptor 2-positive breast cancers, and lymphomas were excluded. The primary end point was centrally assessed objective response rate (ORR); secondary end points included progression-free survival, 6-month progression-free survival, and overall survival. RESULTS: Thirty-five patients were enrolled, and 25 patients were included in the primary efficacy analysis as prespecified in the Protocol. Multiple histologies were enrolled, with gynecologic (n = 6) and gastrointestinal (n = 6) being the most common. Sixty-eight percent of patients had ≥ 3 lines of prior therapy. The ORR was 16% (4 of 25, 90% CI, 6 to 33) with P = .0341 against a null rate of 5%. The most common reason for protocol discontinuation was disease progression (n = 17, 68%). Grade 3/4 toxicities observed were consistent with reported toxicities for PI3K pathway inhibition. Sixteen patients (53%) had grade 3 toxicities, and one patient (3%) had grade 4 toxicity (CTCAE v5.0). Most common toxicities include hyperglycemia (n = 19), fatigue (n = 12), diarrhea (n = 11), hypertension (n = 10), and nausea (n = 10). CONCLUSION: The study met its primary end point with an ORR of 16% (P = .0341) with copanlisib showing clinical activity in select tumors with PIK3CA mutation in the refractory setting.

Multi-ancestral origin of intestinal tumors: Impact on growth, progression, and drug efficacy.

BACKGROUND: Data are steadily accruing that demonstrate that intestinal tumors are frequently derived from multiple founding cells, resulting in tumors comprised of distinct ancestral clones that might cooperate or alternatively compete, thereby potentially impacting different phases of the disease process. AIM: We sought to determine whether tumors with a multi-ancestral architecture involving at least two distinct clones show increased tumor number, growth, progression, or resistance to drug intervention. METHODS: Mice carrying the Min allele of Apc were generated that were mosaic with only a subset of cells in the intestinal epithelium expressing an activated form of PI3K, a key regulatory kinase affecting several important cellular processes. These cells were identifiable as they fluoresced green, whereas all other cells fluoresced red. RESULTS: Cell lineage tracing revealed that many intestinal tumors from our mouse model were derived from at least two founding cells, those expressing the activated PI3K (green) and those which did not (red). Heterotypic tumors with a multi-ancestral architecture as evidenced by a mixture of green and red cells exhibited increased tumor growth and invasiveness. Clonal architecture also had an impact on tumor response to low-dose aspirin. Aspirin treatment resulted in a greater reduction of heterotypic tumors derived from multiple founding cells as compared to tumors derived from a single founding cell. CONCLUSION: These data indicate that genetically distinct tumor-founding cells can contribute to early intratumoral heterogeneity. The coevolution of the founding cells and their progeny enhances colon tumor progression and impacts the response to aspirin. These findings are important to a more complete understanding of tumorigenesis with consequences for several distinct models of tumor evolution. They also have practical implications to the clinic. Mouse models with heterogenous tumors are likely better for predicting drug efficacy as compared to models in which the tumors are highly homogeneous. Moreover, understanding how interactions among different populations in a single heterotypic tumor with a multi-ancestral architecture impact response to a single agent and combination therapies are necessary to fully develop personalized medicine.

Validation of genetic classifiers derived from mouse and human tumors to identify molecular subtypes of colorectal cancer.

Colorectal cancer (CRC) is a leading cause of cancer death in the United States. Standard treatment for advanced-stage CRC for decades has included 5-fluorouracil-based chemotherapy. More recently, targeted therapies for metastatic CRC are being used based on the individual cancer's molecular profile. In the past few years, several different molecular subtype schemes for human CRC have been developed. The molecular subtypes can be distinguished by gene expression signatures and have the potential to be used to guide treatment decisions. However, many subtyping classification methods were developed using mRNA expression levels of hundreds to thousands of genes, making them impractical for clinical use. In this study, we assessed whether an immunohistochemical approach could be used for molecular subtyping of CRCs. We validated two previously published, independent sets of immunohistochemistry classifiers and modified the published methods to improve the accuracy of the scoring methods. In addition, we evaluated whether protein and genetic signatures identified originally in the mouse were linked to clinical outcomes of patients with CRC. We found that low DDAH1 or low GAL3ST2 protein levels in human CRCs correlate with poor patient outcomes. The results of this study have the potential to impact methods for determining the prognosis and therapy selection for patients with CRC.

Breast cancer immunotherapy: current biomarkers and the potential of in vitro assays.

Breakthroughs in metastatic breast cancer care require new model systems that can identify the unique features and vulnerabilities of each cancer. Primary tumor cultures are proposed to efficiently screen multiple treatment options in a patient-specific strategy to maximize therapeutic benefit, minimize toxicity, and enable mechanistic insights that inspire future biomarkers for patient selection. To realize the potential of patient-specific cultures, new tools are needed to capture cell-by-cell variability in behavior and dynamic response to treatments in living 3D specimens. Potential bioengineering tools that can achieve this include optical microscopy to image single-cell dynamics and microphysiological in vitro systems to evaluate cell-cell interactions and immunotherapies.

Novel Murine Pancreatic Tumor Model Demonstrates Immunotherapeutic Control of Tumor Progression by a Toxoplasma gondii Protein.

Pancreatic ductal adenocarcinoma is the fourth leading cause of cancer-related death in the United States, with few effective treatments available and only 10% of those diagnosed surviving 5 years. Although immunotherapeutics is a growing field of study in cancer biology, there has been little progress in its use for the treatment of pancreatic cancer. Pancreatic cancer is considered a nonimmunogenic tumor because the tumor microenvironment does not easily allow for the immune system, even when stimulated, to attack the cancer. Infection with the protozoan parasite Toxoplasma gondii has been shown to enhance the immune response to clear cancer tumors. A subset of T. gondii proteins called soluble Toxoplasma antigen (STAg) contains an immunodominant protein called profilin. Both STAg and profilin have been shown to stimulate an immune response that reduces viral, bacterial, and parasitic burdens. Here, we use STAg and profilin to treat pancreatic cancer in a KPC mouse-derived allograft murine model. These mice exhibit pancreatic cancer with both Kras and P53 mutations as subcutaneous tumors. Pancreatic cancer tumors in C57BL/6J mice with a wild-type background showed a significant response to treatment with either profilin or STAg, exhibiting a decrease in tumor volume accompanied by an influx of CD4+ and CD8+ T cells into the tumors. Both IFN-γ-/- mice and Batf3-/- mice, which lack conventional dendritic cells, failed to show significant decreases in tumor volumes when treated. These results indicate that gamma interferon (IFN-γ) and dendritic cells may play critical roles in the immune response necessary to treat pancreatic cancer.

Live cell molecular analysis of primary prostate cancer organoids identifies persistent androgen receptor signaling.

Prostate Cancer (PC) is a disease with remarkable tumor heterogeneity that often manifests in significant intra-patient variability with regards to clinical outcomes and treatment response. Commonly available PC cell lines do not accurately reflect the complexity of this disease and there is critical need for development of new models to recapitulate the intricate hierarchy of tumor pathogenesis. In current study, we established ex vivo primary patient-derived cancer organoid (PDCO) cultures from prostatectomy specimens of patients with locally advanced PC. We then performed a comprehensive multi-parameter characterization of the cellular composition utilizing a novel approach for live-cell staining and direct imaging in the integrated microfluidic Stacks device. Using orthogonal flow cytometry analysis, we demonstrate that primary PDCOs maintain distinct subsets of epithelial cells throughout culture and that these cells conserve expression of androgen receptor (AR)-related elements. Furthermore, to confirm the tumor-origin of the PDCOs we have analyzed the expression of PC-associated epigenetic biomarkers including promoter methylation of the GSTP1, RASSF1 and APC and RARb genes by employing a novel microfluidic rare-event screening protocol. These results demonstrate that this ex vivo PDCO model recapitulates the complexity of the epithelial tumor microenvironment of multifocal PC using orthogonal analyses. Furthermore, we propose to leverage the Stacks microfluidic device as a high-throughput, translational platform to interrogate phenotypic and molecular endpoints with the capacity to incorporate a complex tumor microenvironment.

Volumetric growth tracking of patient-derived cancer organoids using optical coherence tomography.

Patient-derived cancer organoids (PCOs) are in vitro organotypic models that reflect in vivo drug response, thus PCOs are an accessible model for cancer drug screening in a clinically relevant timeframe. However, current methods to assess the response of PCOs are limited. Here, a custom swept-source optical coherence tomography (OCT) system was used to rapidly evaluate volumetric growth and drug response in PCOs. This system was optimized for an inverted imaging geometry to enable high-throughput imaging of PCOs. An automated image analysis framework was developed to perform 3D single-organoid tracking of PCOs across multiple time points over 48 hours. Metabolic inhibitors and cancer therapies decreased PCOs volumetric growth rate compared to control PCOs. Single-organoid tracking improved sensitivity to drug treatment compared to a pooled analysis of changes in organoid volume. OCT provided a more accurate assessment of organoid volume compared to a volume estimation method based on 2D projections. Single-organoid tracking with OCT also identified heterogeneity in drug response between solid and hollow PCOs. This work demonstrates that OCT and 3D single-organoid tracking are attractive tools to monitor volumetric growth and drug response in PCOs, providing rapid, non-destructive methods to quantify heterogeneity in PCOs.

Autofluorescence Imaging of Treatment Response in Neuroendocrine Tumor Organoids.

Gastroenteropancreatic neuroendocrine tumors (GEP-NET) account for roughly 60% of all neuroendocrine tumors. Low/intermediate grade human GEP-NETs have relatively low proliferation rates that animal models and cell lines fail to recapitulate. Short-term patient-derived cancer organoids (PDCOs) are a 3D model system that holds great promise for recapitulating well-differentiated human GEP-NETs. However, traditional measurements of drug response (i.e., growth, proliferation) are not effective in GEP-NET PDCOs due to the small volume of tissue and low proliferation rates that are characteristic of the disease. Here, we test a label-free, non-destructive optical metabolic imaging (OMI) method to measure drug response in live GEP-NET PDCOs. OMI captures the fluorescence lifetime and intensity of endogenous metabolic cofactors NAD(P)H and FAD. OMI has previously provided accurate predictions of drug response on a single cell level in other cancer types, but this is the first study to apply OMI to GEP-NETs. OMI tested the response to novel drug combination on GEP-NET PDCOs, specifically ABT263 (navitoclax), a Bcl-2 family inhibitor, and everolimus, a standard GEP-NET treatment that inhibits mTOR. Treatment response to ABT263, everolimus, and the combination were tested in GEP-NET PDCO lines derived from seven patients, using two-photon OMI. OMI measured a response to the combination treatment in 5 PDCO lines, at 72 h post-treatment. In one of the non-responsive PDCO lines, heterogeneous response was identified with two distinct subpopulations of cell metabolism. Overall, this work shows that OMI provides single-cell metabolic measurements of drug response in PDCOs to guide drug development for GEP-NET patients.

ctDNA applications and integration in colorectal cancer: an NCI Colon and Rectal-Anal Task Forces whitepaper.

An increasing number of studies are describing potential uses of circulating tumour DNA (ctDNA) in the care of patients with colorectal cancer. Owing to this rapidly developing area of research, the Colon and Rectal-Anal Task Forces of the United States National Cancer Institute convened a panel of multidisciplinary experts to summarize current data on the utility of ctDNA in the management of colorectal cancer and to provide guidance in promoting the efficient development and integration of this technology into clinical care. The panel focused on four key areas in which ctDNA has the potential to change clinical practice, including the detection of minimal residual disease, the management of patients with rectal cancer, monitoring responses to therapy, and tracking clonal dynamics in response to targeted therapies and other systemic treatments. The panel also provides general guidelines with relevance for ctDNA-related research efforts, irrespective of indication.

Label-free redox imaging of patient-derived organoids using selective plane illumination microscopy.

High-throughput drug screening of patient-derived organoids offers an attractive platform to determine cancer treatment efficacy. Here, selective plane illumination microscopy (SPIM) was used to determine treatment response in organoids with endogenous fluorescence from the metabolic coenzymes NAD(P)H and FAD. Rapid 3-D autofluorescence imaging of colorectal cancer organoids was achieved. A quantitative image analysis approach was developed to segment each organoid and quantify changes in endogenous fluorescence caused by treatment. Quantitative analysis of SPIM volumes confirmed the sensitivity of patient-derived organoids to standard therapies. This proof-of-principle study demonstrates that SPIM is a powerful tool for high-throughput screening of organoid treatment response.