Genetic Variants and Tumor Immune Microenvironment: Clues for Targeted Therapies in Inflammatory Breast Cancer (IBC).

To better understand the etiology of inflammatory breast cancer (IBC) and identify potential therapies, we studied genomic alterations in IBC patients. Targeted, next-generation sequencing (NGS) was performed on cell-free DNA (cfDNA) (n = 33) and paired DNA from tumor tissues (n = 29) from 32 IBC patients. We confirmed complementarity between cfDNA and tumor tissue genetic profiles. We found a high incidence of germline variants in IBC patients that could be associated with an increased risk of developing the disease. Furthermore, 31% of IBC patients showed deficiencies in the homologous recombination repair (HRR) pathway (BRCA1, BRCA2, PALB2, RAD51C, ATM, BARD1) making them sensitive to poly (ADP-ribose) polymerase (PARP) inhibitors. We also characterized the tumor-infiltrating lymphocytes (TILs) in tumor tissue biopsies by studying several markers (CD4, CD8, FoxP3, CD20, PD-1, and PD-L1) through immunohistochemistry (IHC) staining. In 7 of 24 (29%) patients, tumor biopsies were positive for PD-L1 and PD-1 expression on TILs, making them sensitive to PD-1/PD-L1 blocking therapies. Our results provide a rationale for considering PARP inhibitors and PD-1/PDL1 blocking immunotherapy in qualifying IBC patients.

Molecular Profiling of Exceptional Responders to Cancer Therapy.

BACKGROUND: The vast majority of metastatic cancers cannot be cured. Palliative treatment may relieve disease symptoms by stopping or slowing cancer growth and may prolong patients' lives, but almost all patients will inevitably develop disease progression after initial response. However, for reasons that are not fully understood, a very few patients will have extraordinary durable responses to standard anticancer treatments. MATERIALS AND METHODS: We analyzed exceptional responders treated at Fox Chase Cancer Center between September 2009 and November 2017. An exceptional response was defined as a complete response lasting more than 1 year or a partial response or stable disease for more than 2 years. Tumor samples were analyzed using an Ambry Genetics test kit with a 142-gene panel. Messenger RNA expression was evaluated using NanoString's nCounter PanCancer Pathways Panel and Immune Profiling Panel and compared with matched controls for gender, age, and cancer type. RESULTS: Twenty-six exceptional responders with metastatic bladder, kidney, breast, lung, ovarian, uterine, and colon cancers were enrolled. Mutations were identified in 45 genes. The most common mutation was an EPHA5 nonsynonymous mutation detected in 87.5% of patients. Mutations in DNA damage repair pathway genes were also frequent, suggesting increased genome instability. We also found varying expression of 73 genes in the Pathways panel and 85 genes in the Immune Profiling panel, many of them responsible for improvement in tumor recognition and antitumor immune response. CONCLUSIONS: The genomic instability detected in our exceptional responders, plus treatment with DNA damage compounds combined with favorable anticancer immunity, may have contributed to exceptional responses to standard anticancer therapies in the patients studied. IMPLICATIONS FOR PRACTICE: With recent advances in the treatment of cancer, there is increased emphasis on the importance of identifying molecular markers to predict treatment outcomes, thereby allowing precision oncology. In this study, it was hypothesized that there is a "specific biologic signature" in the biology of the cancer in long-term survivors that allows sensitivity to systemic therapy and durability of response. Results showed that DNA damage repair pathway alterations, combined with favorable anticancer immunity, may have contributed to exceptional responses. It is very likely that an in-depth examination of outlier responses will become a standard component of drug development in the future.

Autologous Lymphocyte Infusion Supports Tumor Antigen Vaccine-Induced Immunity in Autologous Stem Cell Transplant for Multiple Myeloma.

Autologous stem cell transplant (autoSCT), the standard consolidation therapy for multiple myeloma, improves disease-free survival, but is not curative. This could be an ideal setting for immunologic therapy. However, the immune milieu is impaired after autoSCT. We hypothesized that autologous lymphocyte infusion would restore immune competence, allowing immunotherapies such as cancer vaccines to elicit tumor antigen-specific immunity in the setting of autoSCT. In this pilot study (NCT01380145), we investigated safety, immunologic, and clinical outcomes of autologous lymphocyte infusion combined with peri-autoSCT immunotherapy with recombinant MAGE-A3 (a multiple myeloma-associated antigen) and adjuvant. Thirteen patients with multiple myeloma undergoing autoSCT were enrolled. Autologous lymphocyte infusion and MAGE vaccination were well tolerated. Combination immunotherapy resulted in high-titer humoral immunity and robust, antigen-specific CD4+ T-cell responses in all subjects, and the responses persisted at least one year post-autoSCT. CD4+ T cells were polyfunctional and Th1-biased. CD8+ T-cell responses were elicited in 3 of 13 subjects. These cells recognized naturally processed MAGE-A3 antigen. Median progression-free survival was 27 months, and median overall survival was not reached, suggesting no differences from standard-of-care. In 4 of 8 subjects tested, MAGE-A protein expression was not detected by IHC in multiple myeloma cells at relapse, suggesting therapy-induced immunologic selection against antigen-expressing clones. These results demonstrated that autologous lymphocyte infusion augmentation of autoSCT confers a favorable milieu for immunotherapies such as tumor vaccines. This strategy does not require ex vivo manipulation of autologous lymphocyte products and is an applicable platform for further investigation into combination immunotherapies to treat multiple myeloma.

Association of Circulating Tumor Cells With Late Recurrence of Estrogen Receptor-Positive Breast Cancer: A Secondary Analysis of a Randomized Clinical Trial.

Importance: Late recurrence 5 or more years after diagnosis accounts for at least one-half of all cases of recurrent hormone receptor-positive breast cancer. Objective: To determine whether the presence of circulating tumor cells (CTCs) in a peripheral blood sample obtained approximately 5 years after diagnosis was associated with late clinical recurrence of operable human epidermal growth factor receptor 2-negative breast cancer. Design, Setting, and Participants: This per-protocol secondary analysis of the Double-Blind Phase III Trial of Doxorubicin and Cyclophosphamide Followed by Paclitaxel With Bevacizumab or Placebo in Patients With Lymph Node Positive and High Risk Lymph Node Negative Breast Cancer enrolled patients from 2007 to 2011 who were without clinical evidence of recurrence between 4.5 and 7.5 years after primary surgical treatment of human epidermal growth factor receptor 2-negative stage II-III breast cancer followed by adjuvant systemic therapy. Patients were enrolled in a subprotocol for secondary analysis from February 25, 2013, to July 29, 2016, after signing consent for the subprotocol. The analysis was performed in April 2018. Interventions: A blood sample was obtained for identification and enumeration of CTCs. Main Outcome and Measures: The association between a positive CTC assay result (at least 1 CTC per 7.5 mL of blood) and clinical recurrence. Results: Among 547 women included in this analysis, the results of the CTC assay were positive for 18 of 353 with hormone receptor-positive disease (5.1% [95% CI, 3.0%-7.9%]); 23 of 353 patients (6.5% [95% CI, 4.2%-9.6%]) had a clinical recurrence. The recurrence rates per person-year of follow-up in the CTC-positive and CTC-negative groups were 21.4% (7 recurrences per 32.7 person-years) and 2.0% (16 recurrences per 796.3 person-years), respectively. In multivariate models including clinical covariates, a positive CTC assay result was associated with a 13.1-fold higher risk of recurrence (hazard ratio point estimate, 13.1; 95% CI, 4.7-36.3). Seven of 23 patients (30.4% [95% CI, 13.2%-52.9%]) with recurrence had a positive CTC assay result at a median of 2.8 years (range, 0.1-2.8 years) before clinical recurrence. The CTC assay result was also positive for 8 of 193 patients (4.1% [95% CI, 1.8%-8.0%]) with hormone receptor-negative disease, although only 1 patient (0.5% [95% CI, 0%-2.9%]) experienced disease recurrence (this patient was CTC negative). Conclusions and Relevance: A single positive CTC assay result 5 years after diagnosis of hormone receptor-positive breast cancer provided independent prognostic information for late clinical recurrence, which provides proof of concept that liquid-based biomarkers may be used to risk stratify for late recurrence and guide therapy. Trial Registration: ClinicalTrials.gov identifier: NCT00433511.

Defects in DNA Repair Genes Predict Response to Neoadjuvant Cisplatin-based Chemotherapy in Muscle-invasive Bladder Cancer.

BACKGROUND: Cisplatin-based neoadjuvant chemotherapy (NAC) before cystectomy is the standard of care for muscle-invasive bladder cancer (MIBC), with 25-50% of patients expected to achieve a pathologic response. Validated biomarkers predictive of response are currently lacking. OBJECTIVE: To discover and validate biomarkers predictive of response to NAC for MIBC. DESIGN, SETTING, AND PARTICIPANTS: Pretreatment MIBC samples prospectively collected from patients treated in two separate clinical trials of cisplatin-based NAC provided the discovery and validation sets. DNA from pretreatment tumor tissue was sequenced for all coding exons of 287 cancer-related genes and was analyzed for base substitutions, indels, copy number alterations, and selected rearrangements in a Clinical Laboratory Improvements Amendments-certified laboratory. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The mean number of variants and variant status for each gene were correlated with response. Variant data from the discovery cohort were used to create a classification tree to discriminate responders from nonresponders. The resulting decision rule was then tested in the independent validation set. RESULTS AND LIMITATIONS: Patients with a pathologic complete response had more alterations than those with residual tumor in both the discovery (p=0.024) and validation (p=0.018) sets. In the discovery set, alteration in one or more of the three DNA repair genes ATM, RB1, and FANCC predicted pathologic response (p<0.001; 87% sensitivity, 100% specificity) and better overall survival (p=0.007). This test remained predictive for pathologic response in the validation set (p=0.033), with a trend towards better overall survival (p=0.055). These results require further validation in additional sample sets. CONCLUSIONS: Genomic alterations in the DNA repair-associated genes ATM, RB1, and FANCC predict response and clinical benefit after cisplatin-based chemotherapy for MIBC. The results suggest that defective DNA repair renders tumors sensitive to cisplatin. PATIENT SUMMARY: Chemotherapy given before bladder removal (cystectomy) improves the chance of cure for some but not all patients with muscle-invasive bladder cancer. We found a set of genetic mutations that when present in tumor tissue predict benefit from neoadjuvant chemotherapy, suggesting that testing before chemotherapy may help in selecting patients for whom this approach is recommended.

TP53 mutations detected in circulating tumor cells present in the blood of metastatic triple negative breast cancer patients.

INTRODUCTION: Circulating tumor cells (CTCs) are tumor cells shed from either primary tumors or its metastases that circulate in the peripheral blood of patients with metastatic cancers. The molecular characterization of the CTCs is critical to identifying the key drivers of cancer metastasis and devising therapeutic approaches. However, the molecular characterization of CTCs is difficult to achieve because their isolation is a major technological challenge. METHODS: CTCs from two triple negative breast cancer patients were enriched using CellSearch and single cells selected by DEPArray™. A TP53 R110 fs*13 mutation identified by next generation sequencing in the breast and chest skin biopsies of both patients was studied in single CTCs. RESULTS: From 6 single CTC isolated from one patient, 1 CTC had TP53 R110 delC, 1 CTC showed the TP53 R110 delG mutation, and the remaining 4 single CTCs showed the wild type p53 sequence; a pool of 14 CTCs isolated from the same patient also showed TP53 R110 delC mutation. In the tumor breast tissue of this patient, only the TP53 R110 delG mutation was detected. In the second patient a TP53 R110 delC mutation was detected in the chest wall skin biopsy; from the peripheral blood of this patient, 5 single CTC and 6 clusters of 2 to 6 CTCs were isolated; 3 of the 5 single CTCs showed the TP53 R110 delC mutation and 2 CTCs showed the wild type TP53 allele; from the clusters, 5 showed the TP53 R110 delC mutation, and 1 cluster the wild type TP53 allele. Single white blood cells isolated as controls from both patients only showed the wild type TP53 allele. CONCLUSIONS: We are able to isolate uncontaminated CTCs and achieve single cell molecular analysis. Our studies showed the presence of different CTC sub-clones in patients with metastatic breast cancer. Some CTCs had the same TP53 mutation as their matching tumor samples although others showed either a different TP53 mutation or the wild type allele. Our results indicate that CTCs could represent a non-invasive source of cancer cells from which to determine genetic markers of the disease progression and potential therapeutic targets.

EZH2 knockdown suppresses the growth and invasion of human inflammatory breast cancer cells.

INTRODUCTION: Inflammatory breast cancer (IBC) is the most metastatic variant of breast cancer with the poorest survival in all types of breast cancer patients and presently therapeutic targets for IBC are very limited. Enhancer of zeste homolog 2 (EZH2) is frequently expressed in human IBC and its expression positively correlates with worse clinical outcome. However, the molecular basis for EZH2 promoting IBC has not been explored. Here, we investigated the functional role of EZH2 in IBC cells by examining the effects of its knockdown on the formation of tumor spheroids and invasion of these cells in vitro and in vivo in an orthotopic xenograft model. METHODS: SUM149 and a new IBC cell line-FC-IBC-02 derived from pleural effusion fluid of an IBC patient were used in this study. Specific knockdown of EZH2 was performed using short hairpin RNA (shRNA) specific to the human EZH2 gene. Cell growth and the formation of tumor spheroids were examined in vitro. The effects of EZH2 knockdown on IBC cell migration and invasion were examined by a Boyden chamber assay. For the in vivo tumor growth studies, IBC cells were orthotopically transplanted into the mammary fat pads of immunodeficient mice. RESULTS: The results showed that EZH2 is expressed at higher levels in human IBC cell lines compared with normal human mammary epithelial cells, and the knockdown of EZH2 expression significantly suppressed cell growth and tumor spheroid formation of human IBC cells in vitro. In addition, EZH2 knockdown inhibited the migration and invasion of IBC cells. Significantly, EZH2 knockdown suppressed the angiogenesis and tumor growth of IBC cells in vivo. CONCLUSIONS: Our results provide direct evidence that EZH2 is critical for the formation of tumor spheroids and invasion of human IBC cells and could be a potential target for developing novel therapeutic strategies for human IBC.

Presence of anaplastic lymphoma kinase in inflammatory breast cancer.

Although Inflammatory Breast Cancer (IBC) is recognized as the most metastatic variant of locally advanced breast cancer, the molecular basis for the distinct clinical presentation and accelerated program of metastasis of IBC is unknown. Reverse phase protein arrays revealed activation of the receptor tyrosine kinase, anaplastic lymphoma kinase (ALK) and biochemically-linked downstream signaling molecules including JAK1/STAT3, AKT, mTor, PDK1, and AMPKß in pre-clinical models of IBC. To evaluate the clinical relevance of ALK in IBC, analysis of 25 IBC patient tumors using the FDA approved diagnostic test for ALK genetic abnormalities was performed. These studies revealed that 20/25 (80%) had either increased ALK copy number, low level ALK gene amplification, or ALK gene expression, with a prevalence of ALK alterations in basal-like IBC. One of 25 patients was identified as having an EML4-ALK translocation. The generality of gains in ALK copy number in basal-like breast tumors with IBC characteristics was demonstrated by analysis of 479 breast tumors using the TGCA data-base and our newly developed 79 IBC-like gene signature. The small molecule dual tyrosine kinase cMET/ALK inhibitor, Crizotinib (PF-02341066/Xalkori®, Pfizer Inc), induced both cytotoxicity (IC50 = 0.89 µM) and apoptosis, with abrogation of pALK signaling in IBC tumor cells and in FC-IBC01 tumor xenograft model, a new IBC model derived from pleural effusion cells isolated from an ALK(+) IBC patient. Based on these studies, IBC patients are currently being evaluated for the presence of ALK genetic abnormalities and when eligible, are being enrolled into clinical trials evaluating ALK targeted therapeutics.

Circulating tumor cells (CTCs) in breast cancer: a diagnostic tool for prognosis and molecular analysis.

Metastatic breast cancer (MBC) is characterized by a combination of tumor growth, proliferation and metastatic progression and is typically managed with palliative intent. The benefit of standard systemic therapies is relatively limited and the disease is considered incurable suggesting the need to investigate the biological drivers of the various phases of the metastatic process in order to improve the selection of molecularly driven therapies. The detection, enumeration and molecular analysis of circulating tumor cells (CTCs) provide an intriguing opportunity to advance this knowledge. CTCs enumerated by the Food and Drugs Administration-cleared CellSearch(®) system are an independent prognostic factor of progression-free survival (PFS) and overall survival (OS) in MBC patients. Several published papers demonstrated the poor prognosis for MBC patients that presented basal CTC count ≥5 in 7.5 mL of blood. Therefore, the enumeration of CTCs during treatment for MBC provides a tool with the ability to predict progression of disease earlier than standard timing of anatomical assessment using conventional radiological tests. During the metastatic process cancer cells exhibit morphological and phenotypic plasticity undergoing epithelial-mesenchymal transition (EMT). This important phenomenon is associated with down regulation of epithelial marker (e.g., EpCAM) with potential limitations in the applicability of current CTCs enrichment methods. Such observations translated in a number of investigations aimed at improving our capabilities to enumerate and perform molecular characterization of CTCs. Theoretically, the phenotypic analysis of CTCs can represent a "liquid" biopsy of breast tumor that is able to identify a new potential target against the metastatic disease and advanced the development and monitoring of personalized therapies.

Biodistribution and radiation dosimetry of the hypoxia marker 18F-HX4 in monkeys and humans determined by using whole-body PET/CT.

OBJECTIVES: F-HX4 is a novel positron emission tomography (PET) tracer for imaging hypoxia. The purpose of this study was to determine the biodistribution and estimate the radiation dose of F-HX4 using whole-body PET/computed tomography (CT) scans in monkeys and humans. METHODS: Successive whole-body PET/CT scans were done after the injection of F-HX4 in four healthy humans (422±142 MBq) and in three rhesus monkeys (189±3 MBq). Biodistribution was determined from PET images and organ doses were estimated using OLINDA/EXM software. RESULTS: The bladder, liver, and kidneys showed the highest percentage of the injected radioactivity for humans and monkeys. For humans, approximately 45% of the activity is eliminated by bladder voiding in 3.6 h, and for monkeys 60% is in the bladder content after 3 h. The critical organ is the urinary bladder wall with the highest absorbed radiation dose of 415±18 (monkeys) and 299±38 µGy/MBq (humans), in the 4.8-h bladder voiding interval model. The average value of effective dose for the adult male was estimated at 42±4.2 µSv/MBq from monkey data and 27±2 µSv/MBq from human data. CONCLUSION: Bladder, kidneys, and liver have the highest uptake of injected F-HX4 activity for both monkeys and humans. The urinary bladder wall receives the highest dose of F-HX4 and is the critical organ. Thus, patients should be encouraged to maintain adequate hydration and void frequently. The effective dose of F-HX4 is comparable with that of other F-based imaging agents.

Circulating tumor cells: evolving evidence and future challenges.

Circulating tumor cells (CTCs) are rare malignant cells found in the peripheral blood that originate from the primary tumor or metastatic sites. New techniques have been developed to isolate and characterize these cells. CTC enumeration has been incorporated into different fields of oncology as a prognostic marker, a tool to monitor therapy response, and a method to understand basic tumor characteristics. This review covers the different techniques available for isolation of CTCs, the clinical utility of CTCs in breast, prostate, and colon cancer, and future directions in this field.

Phase I dose escalation, pharmacokinetic and pharmacodynamic study of naptumomab estafenatox alone in patients with advanced cancer and with docetaxel in patients with advanced non-small-cell lung cancer.

PURPOSE: Two phase I studies were conducted of ABR-217620 alone or in combination with docetaxel. This is a recombinant fusion protein consisting of a mutated variant of the superantigen staphylococcal enterotoxin E (SEA/E-120) linked to fragment antigen binding moiety of a monoclonal antibody recognizing the tumor-associated antigen 5T4. PATIENTS AND METHODS: Patients with non-small-cell lung cancer (NSCLC), pancreatic cancer (PC), and renal cell cancer (RCC) received 5 daily boluses of ABR-217620 (3-month cycles) in escalating doses to determine the maximum-tolerated dose (MTD; ABR-217620 dose escalation monotherapy [MONO] study). Doses were selected based on individual patient anti-SEA/E-120 titers pretreatment. Patients with NSCLC received 4 daily, escalating doses of ABR-217620 followed by docetaxel in 21-day cycles (ABR-217620 dose escalation combination with docetaxel [COMBO] study). RESULTS: Thirty-nine patients were enrolled in the MONO study and 13 were enrolled in the COMBO study. The monotherapy MTD was 26 microg/kg (NSCLC and PC) and 15 microg/kg (RCC). Dose-limiting toxicities (DLTs) in the MONO study were fever, hypotension, acute liver toxicity, and vascular leak syndrome. In the COMBO study, the MTD was 22 microg/kg (neutropenic sepsis). Adverse events included grade 1 to 2 fever, hypotension, nausea, and chills. Treatment caused a systemic increase of inflammatory cytokines and selective expansion of SEA/E-120 reactive T-cells. Tumor biopsies demonstrated T-cell infiltration after therapy. Fourteen patients (36%) had stable disease (SD) on day 56 of the MONO study. Two patients (15%) in the COMBO study had partial responses, one in a patient with progressive disease on prior docetaxel, and five patients (38%) had SD on day 56. CONCLUSION: ABR-217620 was well tolerated with evidence of immunological activity and antitumor activity.