3D-conformal-intensity modulated radiotherapy with compensators for head and neck cancer: clinical results of normal tissue sparing

BACKGROUND: To investigate the potential of parotic gland sparing of intensity modulated radiotherapy (3D-c-IMRT) performed with metallic compensators for head and neck cancer in a clinical series by analysis of dose distributions and clinical measures. MATERIALS AND METHODS: 39 patients with squamous cell cancer of the head and neck irradiated using 3D-c-IMRT were evaluable for dose distribution within PTVs and at one parotid gland and 38 patients for toxicity analysis. 10 patients were treated primarily, 29 postoperatively, 19 received concomittant cis-platin based chemotherapy, 20 3D-c-IMRT alone. Initially the dose distribution was calculated with Helax (® )and photon fluence was modulated using metallic compensators made of tin-granulate (n = 22). Later the dose distribution was calculated with KonRad (® )and fluence was modified by MCP 96 alloy compensators (n = 17). Gross tumor/tumor bed (PTV 1) was irradiated up to 60–70 Gy, [5 fractions/week, single fraction dose: 2.0–2.2 (simultaneously integrated boost)], adjuvantly irradiated bilateral cervical lymph nodes (PTV 2) with 48–54 Gy [single dose: 1.5–1.8]). Toxicity was scored according the RTOG scale and patient-reported xerostomia questionnaire (XQ). RESULTS: Mean of the median doses at the parotid glands to be spared was 25.9 (16.3–46.8) Gy, for tin graulate 26 Gy, for MCP alloy 24.2 Gy. Tin-granulate compensators resulted in a median parotid dose above 26 Gy in 10/22, MCP 96 alloy in 0/17 patients. Following acute toxicities were seen (°0–2/3): xerostomia: 87%/13%, dysphagia: 84%/16%, mucositis: 89%/11%, dermatitis: 100%/0%. No grade 4 reaction was encountered. During therapy the XQ forms showed °0–2/3): 88%/12%. 6 months postRT chronic xerostomia °0–2/3 was observed in 85%/15% of patients, none with °4 xerostomia. CONCLUSION: 3D-c-IMRT using metallic compensators along with inverse calculation algorithm achieves sufficient parotid gland sparing in virtually all advanced head and neck cancers. Since the concept of lower single (and total) doses in the adjuvantly treated volumes reduces acute morbidity 3D-c-IMRT nicely meets demands of concurrent chemotherapy protocols

Mutational Alteration of Human Immunodeficiency Virus Type 1 Vif Allows for Functional Interaction with Nonhuman Primate APOBEC3G

Human APOBEC3F (hA3F) and APOBEC3G (hA3G) are antiretroviral cytidine deaminases that can be encapsidated during virus assembly to catalyze C→U deamination of the viral reverse transcripts in the next round of infection. Lentiviruses such as human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) have evolved the accessory protein Vif to induce their degradation before packaging. HIV type 1 (HIV-1) Vif counteracts hA3G but not rhesus macaque APOBEC3G (rhA3G) or African green monkey (AGM) APOBEC3G (agmA3G) because of a failure to bind the nonhuman primate proteins. The species specificity of the interaction is controlled by amino acid 128, which is aspartate in hA3G and lysine in rhA3G. With the objective of overcoming this species restriction, mutations were introduced into HIV-1 Vif at amino acid positions that differed in charge between HIV-1 Vif and SIV Vif. The mutant proteins were tested for the ability to counteract hA3G, rhA3G, and agmA3G. Alteration of the conserved sequence at positions 14 to 17 from DRMR to SERQ, which is the sequence in AGM Vif, caused HIV-1 Vif to functionally interact with rhA3G and agmA3G. Mutation of three residues to the sequence SEMQ allowed interaction with rhA3G. SEMQ Vif also counteracted D128K mutant hA3G and wild-type hA3G. Introduction of the sequence into an infectious molecular HIV-1 clone allowed the virus to replicate productively in human cells that expressed rhA3G or hA3G. These findings provide insight into the interaction of Vif with A3G and are a step toward the development of a novel primate model for AIDS

Identification of B-Cell Epitopes on Virus-Like Particles of Cutaneous Alpha-Human Papillomaviruses ▿ †

Human papillomavirus (PV) (HPV) types 2, 27, and 57 are closely related and, hence, represent a promising model system to study the correlation of phylogenetic relationship and immunological distinctiveness of PVs. These HPV types cause a large fraction of cutaneous warts occurring in immunocompromised patients. Therefore, they constitute a target for the development of virus-like particle (VLP)-based vaccines. However, the immunogenic structure of HPV type 2, 27, and 57 capsids has not been studied yet. Here we provide, for the first time, a characterization of the B-cell epitopes on VLPs of cutaneous alpha-HPVs using a panel of 94 monoclonal antibodies (MAbs) generated upon immunization with capsids from HPV types 2, 27, and 57. The MAbs generated were characterized regarding their reactivities with glutathione S-transferase-L1 fusion proteins from 18 different PV types, the nature of their recognized epitopes, their isotypes, and their ability to neutralize HPV type 2, 27, 57, or 16. In total, 33 of the 94 MAbs (35%) showed type-specific reactivity. All type-specific MAbs recognize linear epitopes, most of which map to the hypervariable surface loop regions of the L1 amino acid sequence. Four of the generated MAbs neutralized pseudovirions of the inoculated HPV type efficiently. All four MAbs recognized epitopes within the BC loop, which is required and sufficient for their neutralizing activity. Our data highlight the immunological distinctiveness of individual HPV types, even in comparison to their closest relatives, and they provide a basis for the development of VLP-based vaccines against cutaneous alpha-HPVs

Design of a highly effective therapeutic HPV16 E6/E7-specific DNA vaccine: optimization by different ways of sequence rearrangements (shuffling).

Persistent infection with the high-risk Human Papillomavirus type 16 (HPV 16) is the causative event for the development of cervical cancer and other malignant tumors of the anogenital tract and of the head and neck. Despite many attempts to develop therapeutic vaccines no candidate has entered late clinical trials. An interesting approach is a DNA based vaccine encompassing the nucleotide sequence of the E6 and E7 viral oncoproteins. Because both proteins are consistently expressed in HPV infected cells they represent excellent targets for immune therapy. Here we report the development of 8 DNA vaccine candidates consisting of differently rearranged HPV-16 E6 and E7 sequences within one molecule providing all naturally occurring epitopes but supposedly lacking transforming activity. The HPV sequences were fused to the J-domain and the SV40 enhancer in order to increase immune responses. We demonstrate that one out of the 8 vaccine candidates induces very strong cellular E6- and E7- specific cellular immune responses in mice and, as shown in regression experiments, efficiently controls growth of HPV 16 positive syngeneic tumors. This data demonstrates the potential of this vaccine candidate to control persistent HPV 16 infection that may lead to malignant disease. It also suggests that different sequence rearrangements influence the immunogenecity by an as yet unknown mechanism

3D-conformal-intensity modulated radiotherapy with compensators for head and neck cancer: clinical results of normal tissue sparing

Abstract Background To investigate the potential of parotic gland sparing of intensity modulated radiotherapy (3D-c-IMRT) performed with metallic compensators for head and neck cancer in a clinical series by analysis of dose distributions and clinical measures. Materials and methods 39 patients with squamous cell cancer of the head and neck irradiated using 3D-c-IMRT were evaluable for dose distribution within PTVs and at one parotid gland and 38 patients for toxicity analysis. 10 patients were treated primarily, 29 postoperatively, 19 received concomittant cis-platin based chemotherapy, 20 3D-c-IMRT alone. Initially the dose distribution was calculated with Helax ® and photon fluence was modulated using metallic compensators made of tin-granulate (n = 22). Later the dose distribution was calculated with KonRad ® and fluence was modified by MCP 96 alloy compensators (n = 17). Gross tumor/tumor bed (PTV 1) was irradiated up to 60–70 Gy, [5 fractions/week, single fraction dose: 2.0–2.2 (simultaneously integrated boost)], adjuvantly irradiated bilateral cervical lymph nodes (PTV 2) with 48–54 Gy [single dose: 1.5–1.8]). Toxicity was scored according the RTOG scale and patient-reported xerostomia questionnaire (XQ). Results Mean of the median doses at the parotid glands to be spared was 25.9 (16.3–46.8) Gy, for tin graulate 26 Gy, for MCP alloy 24.2 Gy. Tin-granulate compensators resulted in a median parotid dose above 26 Gy in 10/22, MCP 96 alloy in 0/17 patients. Following acute toxicities were seen (°0–2/3): xerostomia: 87%/13%, dysphagia: 84%/16%, mucositis: 89%/11%, dermatitis: 100%/0%. No grade 4 reaction was encountered. During therapy the XQ forms showed °0–2/3): 88%/12%. 6 months postRT chronic xerostomia °0–2/3 was observed in 85%/15% of patients, none with °4 xerostomia. Conclusion 3D-c-IMRT using metallic compensators along with inverse calculation algorithm achieves sufficient parotid gland sparing in virtually all advanced head and neck cancers. Since the concept of lower single (and total) doses in the adjuvantly treated volumes reduces acute morbidity 3D-c-IMRT nicely meets demands of concurrent chemotherapy protocols.</p

Growth of C3 tumors in mice after immunization with HPV-encoding vectors.

<p>Mice (n = 10/group) received tumor cells and were immunized twice with DNA (empty vector pTHkan, HPV-encoding vector plasmids as indicated) when the tumors were clearly palpable and surface tumor sizes were measured over time. Data gives the average tumor sizes at day 0 (day of first immunization) and at day 39 when the experiment was terminated ± SD, respectively. One of two experiment yielding very similar results is shown.</p

Representation of the different versions of HPV-16 E6/E7 shuffled genes.

<p>HPV-16 E6 and E7 wild-type protein genes were dissected at the transformation sites (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113461#pone.0113461.s001" target="_blank">Materials and Methods S1</a>) and rearranged to disrupt their activities. To avoid the loss of T-cell epitopes, the original sequences were added as appendices. Eight constructs with combined E6 and E7 shuffled genes were designed as shown. Kozak consensus sequence and J-Domain were added at the 5′-end and SV40 translocation sequence was added at the 3′-end.</p

<i>Ex vivo</i> IFN-γ Elispot responses after DNA immunization.

<p>Four mice per group were immunized once i.m. with 100 µg empty vectors (pTHkan) or with 100 µg HPV-16-encoding vectors as indicated. The bars show the mean numbers of IFN-γ secreting cells/1×10⁴ splenocytes ± SD. One of two experiment yielding very similar results is shown.</p