Anna-Maria A. De Costa

Defining a role for sphingosine kinase 1 in p53-dependent tumors.

p53 is a crucial tumor suppressor that is mutated or deleted in a majority of cancers. Exactly how p53 prevents tumor progression has proved elusive for many years; however, this information is crucial to define targets for chemotherapeutic development that can effectively restore p53 function. Bioactive sphingolipids have recently emerged as important regulators of proliferative, apoptotic and senescent cellular processes. In this study, we demonstrate that the enzyme sphingosine kinase 1 (SK1), a critical enzyme in the regulation of the key bioactive sphingolipids ceramide, sphingosine and sphingosine-1-phosphate (S1P), serves as a key downstream target for p53 action. Our results show that SK1 is proteolysed in response to genotoxic stress in a p53-dependent manner. p53 null mice display elevation of SK1 levels and a tumor-promoting dysregulation of bioactive sphingolipids in which the anti-growth sphingolipid ceramide is decreased and the pro-growth sphingolipid S1P is increased. Importantly, deletion of SK1 in p53 null mice completely abrogated thymic lymphomas in these mice and prolonged their life span by ∼30%. Deletion of SK1 also significantly attenuated the formation of other cancers in p53 heterozygote mice. The mechanism of p53 tumor suppression by loss of SK1 is mediated by elevations of sphingosine and ceramide, which in turn were accompanied by increased expression of cell cycle inhibitors and tumor cell senescence. Thus, targeting SK1 may restore sphingolipid homeostasis in p53-dependent tumors and provide insights into novel therapeutic approaches to cancer.

Characterization of the evolution of immune phenotype during the development and progression of squamous cell carcinoma of the head and neck

While studies have indicated that squamous cell carcinoma of the head and neck (HNSCC) is associated with immune suppression, these studies did not analyze the immune response at the dysplastic stage. The present study utilized a mouse model of 4-nitroquinoline 1-oxide-induced oral carcinogenesis to examine the alterations in immune phenotype at the premalignant and malignant stages of HNSCC. Cervical lymph nodes of HNSCC-bearing mice were found to contain a greater number of cells, including a greater number of conventional (Tconv) and regulatory (Treg) T cells, compared to cervical lymph nodes of control and premalignant lesion-bearing mice, though the Tconv cells appear to be less proliferative and the Treg cells appear to be less suppressive at the HNSCC stage. Premalignant lesion-bearing mouse lymph nodes consist of a greater percentage of Tconv cells expressing markers for activation, memory, and exhaustion compared to both control and HNSCC-bearing mice. Also, lymph nodes’ cells from both premalignant lesion-bearing and HNSCC-bearing mice include increased levels of Th1, Tc1, and Th17 cells, with no differences in levels of Th2 cells, compared to control mice. The data show that while there is the expected increase in immunosuppressive Tregs in lymph nodes when HNSCC is present, there is also an unexpected increase in immune populations usually associated with a beneficial antitumor response, including Tconv cells and Th1 and Tc1 cells. In addition, the results demonstrate that the premalignant stage of HNSCC development is associated with a robust immune response involving an increase in inflammatory Th1, Tc1, and Th17 cells.

An Inflammatory Cytokine Milieu is Prominent in Premalignant Oral Lesions, but Subsides when Lesions Progress to Squamous Cell Carcinoma

While head and neck squamous cell carcinomas (HNSCC) are associated with profound immune suppression, less is known about the immunological milieu of premalignant oral lesions. The present study shows dynamic shifts in the immune milieu within premalignant oral lesions and when they have progressed to HNSCC. Specifically, this study showed that the premalignant lesion environment consists of inflammatory mediators and IL-17, but this inflammatory phenotype declines when premalignant oral lesions have progressed to HNSCC. The cytokine profiles of human tissues did not correspond with plasma cytokine profiles. A murine carcinogen-induced premalignant lesion model that progresses to HNSCC was used to examine cytokine profiles released from tissues as well as regional lymph nodes. As in human tissues, murine premalignant lesions and regional lymph nodes released high levels of inflammatory cytokines and, very prominently, IL-17. Also similar to human tissues, release of inflammatory cytokines declined in HNSCC tissues of mice and in the regional lymph nodes of mice with HNSCC. Studies focusing on IL-17 showed that mediators from premalignant lesions stimulated normal spleen cells to produce increased levels of IL-17, while mediators from HNSCC were less stimulatory toward IL-17 production. IL-17 production by Th17-skewed CD4+ cells was strongly inhibited by normal oral epithelium as well as HNSCC. In contrast, premalignant lesion-derived mediators further increased IL-17 production by Th17-skewed cells. The stimulation of IL-17 production by premalignant lesions was dependent on IL-23, which premalignant lesions released in higher amounts than control tissues or HNSCC. HNSCC tissues instead produced increased levels of TGF-β compared to premalignant lesions, and skewed normal spleen cells toward the Treg phenotype. This skewing was blocked by supplementation with IL-23. These studies suggest IL-23 to be a significant contributor to the inflammatory IL-17 phenotype in premalignant oral lesions and suggest the decline in IL-23 in HNSCC leads to a decline in Th17 cells.

Immunological modulation by 1α,25-dihydroxyvitamin D3 in patients with squamous cell carcinoma of the head and neck

Prior studies showing that treatment of head and neck squamous cell carcinoma (HNSCC) patients with 1α,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] stimulated intratumoral immune infiltration were extended to analysis of cytokine profiles in the periphery and in oral tissues. Most prominent was the disparity between cytokine levels in plasma and in either pathologically normal oral tissue or HNSCC tissue from patients that were untreated or treated with 1,25(OH)(2)D(3). Levels of IL-6 and IL-10, but not IL-2, IFN-γ or TNF-α, tended to be increased in the plasma of HNSCC patients and 1,25(OH)(2)D(3) further increased plasma levels of all of these cytokines. While these cytokines tended to be increased in HNSCC tissue, 1,25(OH)(2)D(3) resulted in variable cytokine responses that showed a general tendency toward further increased levels. Levels of IL-8 and VEGF were increased in plasma and tissue of untreated HNSCC patients, and were further increased in plasma, but not in tissues, of patients treated with 1,25(OH)(2)D(3). Levels of IL-1α and IL-1β were similar in plasma of controls and HNSCC patients, but were increased in HNSCC tissues. In contrast to that seen in plasma where 1,25(OH)(2)D(3) increased levels of IL-1α and IL-1β, this was not seen in tissue following 1,25(OH)(2)D(3) treatment. These results show a discordant relationship between systemic and intratumoral cytokine profiles and suggest a tendency of 1,25(OH)(2)D(3)to increase a multitude of cytokines within tumor tissue.

Immunotherapy for head and neck cancer: advances and deficiencies

The concept of immunotherapy as a treatment for cancer patients has been in existence for decades. However, more recent immune therapeutic approaches have involved targeting of tumor-specific antigens. Although improvements have been made in using such immune stimulatory treatment strategies for a variety of solid cancers, the use of these strategies for patients with head and neck squamous cell carcinoma (HNSCC) is lagging behind. Immunotherapeutic approaches for HNSCC are particularly complicated by the profound immune suppression that is induced by HNSCC, which potentially decreases the effectiveness of immune stimulatory efforts. Trials involving patients with various solid cancers have shown the enhanced effectiveness of combining various immunotherapeutic approaches or combining immunotherapy with chemotherapy or radiation therapy. Treatment of HNSCC with such combination approaches has not been extensively investigated and has the added challenge of the need to overcome the HNSCC-induced immune suppression. This study focuses on clinical trials that have tested immunotherapeutic approaches for HNSCC patients and the challenges associated with such approaches. In addition, it will call attention to immunotherapeutic strategies that have been shown to be successful in the treatment of other solid cancers to identify potential strategies that may apply to the treatment of HNSCC.

Administration of a vaccine composed of dendritic cells pulsed with premalignant oral lesion lysate to mice bearing carcinogen-induced premalignant oral lesions stimulates a protective immune response

The use of dendritic cell (DC) vaccines as treatment for malignancy is complicated by immune evasion tactics often employed by carcinomas such as head and neck squamous cell carcinoma (HNSCC). The present study aims to determine if an immune response can be elicited by administering a DC vaccine during the premalignant stages of HNSCC, prior to development of immune escape. Mice treated with the carcinogen 4-nitroquinoline 1-oxide (4NQO) in drinking water develop premalignant oral lesions that progress to HNSCC. As previous studies demonstrated that premalignant lesions and HNSCC overexpress common tumor antigens, bone marrow-derived DCs were pulsed with premalignant lesion lysate (DCpm) and administered to 4NQO-treated mice exhibiting premalignant lesions. Lesion progression was tracked through endoscopy, which revealed that DCpm vaccination and control vaccination with dendritic cells pulsed with normal tongue epithelium lysate (DCnt) significantly decreased lesion burden at 8weeks. Analysis of lymph node cells revealed that while DCnt vaccination resulted in a rapid increase in total lymphocyte count, levels of activated conventional CD4(+) T cells and Th1, Tc1, Th17, Tc17, and Th2 cells, DCpm vaccination results in a delayed, yet substantial, increase in these immune effector mechanisms. This suggests that dendritic cell vaccination may have a beneficial effect on clinical outcome regardless of type of antigenic stimulation. Also, pulsing DCs with premalignant lysate rather than normal tongue epithelium lysate affects the dendritic cells in a way that delays the immune effector response upon vaccination of premalignant lesion-bearing mice.

Modulation of therapeutic sensitivity by human papillomavirus.

Human papillomaviruses (HPVs) are small double-stranded DNA viruses that pose significant public health concerns as the causative agent of approximately 5% of worldwide cancers. The HPV oncogenes E6 and E7 play key roles in carcinogenesis. In the last 15years there has been a significant increase in the incidence of HPV-related head and neck cancers arising primarily in the oropharynx. Patients with HPV-positive head and neck cancers (HNCs) have a significantly improved prognosis compared to those with HPV-negative disease. In this review we will discuss data suggesting how HPV oncogenes modulate both the intrinsic radiation sensitivity of HNCs and also have important effects upon the tumor microenvironment. Together, these findings contribute to the improved outcomes seen in patients with HPV-positive HNC.

Novel use of ViewRay MRI guidance for high-dose-rate brachytherapy in the treatment of cervical cancer

PURPOSEnTo characterize image quality and feasibility of using ViewRay MRI (VR)-guided brachytherapy planning for cervical cancer.nnnMETHODS AND MATERIALSnCervical cancer patients receiving intracavitary brachytherapy with tandem and ovoids, planned using 0.35T VR MRI at our institution, were included in this series. The high-risk clinical target volume (HR-CTV), visible gross tumor volume, bladder, sigmoid, bowel, and rectum contours for each fraction of brachytherapy were evaluated for dosimetric parameters. Typically, five brachytherapy treatments were planned using the T2 sequence on diagnostic MRI for the first and third fractions, and a noncontrast true fast imaging with steady-state precession sequence on VR or CT scan for the remaining fractions. Most patients received 5.5xa0Gy × 5 fractions using high-dose-rate Ir-192 following 45xa0Gy of whole-pelvis radiotherapy. The plan was initiated at 5.5xa0Gy to point A and subsequently optimized and prescribed to the HR-CTV. The goal equivalent dose in 2 Gy fractions for the combined external beam and brachytherapy dose was 85xa0Gy. Soft-tissue visualization using contrast-to-noise ratios to distinguish normal tissues from tumor at their interface was compared between diagnostic MRI, CT, and VR.nnnRESULTSnOne hundred and forty-two fractions of intracavitary brachytherapy were performed from April 2015 to January 2017 on 29 cervical cancer patients, ranging from stages IB1 to IVA. The median HR-CTV was 27.78xa0cc, with median D90 HR-CTV of 6.1xa0Gy. The median time from instrument placement to start of treatment using VR was 65xa0min (scan time 2xa0min), compared to 105xa0min using diagnostic MRI (scan time 11xa0min) (t-test, pxa0<xa00.01). The contrast-to-noise ratio of tumor to cervix in both diagnostic MRI and VR had significantly higher values compared to CT (ANOVA and t-tests, pxa0<xa00.01).nnnCONCLUSIONSnWe report the first clinical use of VR-guided brachytherapy. Time to treatment using this approach was shorter compared to diagnostic MRI. VR also provided significant advantage in visualizing the tumor and cervix compared to CT. This presents a feasible and reliable manner to image and plan gynecologic brachytherapy.

Clinical implementation of a novel Double-Balloon single-entry breast brachytherapy applicator

PURPOSEnThe purpose of the study was to describe the clinical utilization of a novel Double-Balloon applicator for accelerated partial breast irradiation (APBI).nnnMETHODS AND MATERIALSnThe Double-Balloon single-entry breast applicator contains a single central treatment catheter, as well as four peripheral catheters that can be differentially loaded to customize radiation dose coverage. An inner balloon is filled with up to 7-30xa0cm3 of saline to increase separation between the peripheral catheters, and an outer balloon is filled with up to 37-115xa0cm3 of saline to displace breast tissue from the peripheral catheters. Treatment planning objectives include coverage of the breast planning target volume to a minimum of V90xa0>xa090%, limiting dose heterogeneity such that V200xa0<xa010xa0cm3 and V150xa0<xa050xa0cm3, and limiting maximum dose to skin (<100% of prescription dose) and ribs (<145% of prescription dose).nnnRESULTSnHigh-dose-rate APBI was delivered to 11 women using this device (34xa0Gy in 10 twice daily fractions). The mean V90 was 98.2% (range 94.2-99.4%). The mean skin Dmax with the Double-Balloon applicator was 83.3% (range 75.6-99.5%). The mean breast V200 was 5.8xa0cm3 (range 2.3-10.2xa0cm3), and the mean breast V150 was 32.9xa0cm3 (range 25.0-41.7xa0cm3). Pretreatment quality assurance was performed using CT prior to each morning fraction and ultrasound prior to each afternoon fraction.nnnCONCLUSIONSnThe Double-Balloon applicator can be easily introduced into a previously existing brachytherapy program. APBI plans created with this applicator achieve excellent planning target volume coverage, while limiting skin dose and maintaining breast V200xa0<xa010xa0cm3.

HPV and Radiation Sensitivity

Human papillomavirus (HPV) is a growing cause of cancers arising in the oropharynx. Patients with HPV-positive head and neck cancers significantly improved outcomes compared to those with traditional tobacco/alcohol associated head and neck cancers. These improved outcomes are likely multifactorial. In this chapter, we review the evidence for increased sensitivity to radiation playing an important role in the improved outcomes of these patients. We discuss how the HPV oncoproteins modulate intrinsic sensitivity to radiation, how differences in the tumor microenvironment may contribute to increased sensitivity to radiation, and how the immune system may play a key role in the response to radiation. Finally, we discuss current efforts aimed at therapeutic de-escalation for this group of patients and highlight future research directions.