Direct and Repeated Clinical Measurement of pO

2

for Enhancing Cancer Therapy

2014 Norris Cotton Cancer Center Comprehensive Thoracic Oncology Program (CTOP) Retreat

Benjamin B. Williams, Ph.D.

The Geisel School of Medicine at Dartmouth, Departments of Medicine (Radiation Oncology) and Radiology

May 22, 2014

Clinical EPR at Dartmouth

Clinical problem Cancer Potential exposure to clinically significant doses of radiation Peripheral vascular disease Radiation Induced Fibrosis Wound healing Parameter to be measured

pO 2 in tumors Radiation induced EPR signals in teeth (magnitude is proportional to dose)

Status of measurements in human subjects

• Numerous measurements performed in patients with superficial tumors.

• FDA IDE submitted for application of implantable oxygen sensors for application at increased depth and with increased sensitivity • NCI PPG proposal in review • Established collaborations with external academic institutions • Underway in unirradiated volunteers and patients receiving significant doses to teeth from radiation therapy. • Measurements at remote locations possible using transportable device.

• Established collaborations with external academic institutions Oxygen at sites of likely pathologies • Measurements underway in normal volunteers.

pO 2 in irradiated tumor beds and peripheral normal tissue pO 2 near wounds and in transplanted tissues • Recruitment and measurements underway • Conceptual stages, with established pre-clinical investigations

Rationale for using in vivo EPR

The response of tumors to cytotoxic therapy, especially ionizing radiation, is critically dependent on pO 2 . Anti-tumor therapies are given repeatedly and often change pO 2 . Knowledge of the changes in individual patients would significantly optimize the timing of the therapy Exposures may occur from terrorism, war, or accident. In vivo EPR is the only physical method capable of making measurements ‘after-the-fact The pO 2 in the tissues is the most significant pathophysiological variable; no other method available to make such direct measurements. Radiation-induced hypoxia may play a critical role in the signaling of pro-inflammatory, pro-fibrotic, and pro-angiogenic growth factors and cytokines that lead to tissue fibrosis.

The pO 2 is a critical variable for successful healing of wounds. Direct measurements would identify patients likely to have poor healing and follow responses to therapy

EPR: Background Fundamentals

• EPR is a form of magnetic resonance spectroscopy that measures the absorption of RF energy by unpaired electrons in a magnetic field.

• The physics is similar to that of MRI, but unpaired e are detected. • Features and considerations:        Non-ionizing modality Spectroscopic data available Sensitive to the magnetic environment In most cases exogenous spin probes must be introduced.

Lower magnetic fields Higher RF frequencies, shallower penetration Rapid relaxation of electron spins affects detection strategies (a) Clinical EPR spectrometer (b) Subject positioned for oximetry using India ink as reporter

Unique or Exceptional Capabilities of In Vivo EPR

In vivo EPR is sensitive to a wide array of physiologic parameters.

This information is observed through simple changes in the shapes of the detected spectra.

• Partial pressure of oxygen (pO 2 ; using oxygen sensitive paramagnetic materials) • Free radicals observed directly or by spin trapping, including oxygen-, carbon-, and sulfur centered radicals • Nitric Oxide (spin trapping) • Redox status (using metabolism of nitroxides) • Thiol groups (using specific nitroxides) • pH (using specific nitroxides) • Perfusion (using washout of paramagnetic tracers) • Metal ions (in paramagnetic states such as chromium, manganese) • Absorbed dose of radiation (in teeth, nails, or bones)

EPR Sensitivities : Oximetry

Collisions between the spin probe and molecular O

2

promote relaxation and broaden the linewidth of the observed spectrum.

2500 a 2000 1500 1000 500 0 -500 (air) (nitrogen) -1000 412.0

LiPc

412.5

413.0

Magnetic field, Gauss 413.5

414.0

80 60 40 20 0 160 b 140 120 100 0.0

0.2

0.4

LW, G 0.6

0.8

1.0

Significance of Hypoxia for Cancer Therapy

• Hypoxia plays crucial roles in tumor development and treatment.

– The response of tumors to cytotoxic therapies, especially ionizing radiation, is critically dependent on pO that are more resistant to therapy.

2 .

– Hypoxia may promote metastatic growth and tumor phenotypes – Hypoxia is dynamic: diffusion, perfusion (incl. cycling), anemic Tatum JL, Kelloff GJ, Gillies RJ, et al. Hypoxia: Importance in tumor Biology, noninvasive measurement by imaging, and value of its measurement in the management of cancer therapy. Int J Radiat Biol. 2006 Oct;82(10):699-757.

Significance of Hypoxia for Cancer Therapy

• Hypoxia plays crucial roles in tumor development and treatment.

– The response of tumors to cytotoxic therapies, especially ionizing radiation, is critically dependent on pO that are more resistant to therapy.

2 .

– Hypoxia may promote metastatic growth and tumor phenotypes – Hypoxia is dynamic: diffusion, perfusion (incl. cycling), anemic Eric J. Hall, Amato J. Giaccia. Radiobiology for the radiologist. Lippincott Williams & Wilkins, 2006; 546p.

Hockel M, Schelnger K, Aral B, Mitze M, Schaffer U, Vaupel P. Association between Tumor Hypoxia and malignant Progression in Advanced Cancer of the Uterine Cervix. Can Res. 1996 Oct;56:4509-4515.

Overall goal



To establish EPR oximetry as a clinical tool for noninvasive monitoring of tumor oxygen in human subjects, under conditions that are fully compatible with clinical practice Optimize Delivery

Timing of dose

Initiation of intervention Design

* Select patients Application of pO 2 modifiers Combination therapies *

Therapeutic Cycle

Tumor profiling pO 2 heterogeneity *

Assessment

pO 2 *

Points of pO 2 measurement

redistribution Vascular remodeling Monitor normal tissues

Capabilities of EPR Oximetry

EPR oximetry can provide repeated and direct measurements of absolute pO

2

in tumors and other tissues.

– Following introduction of the reporters (injection or implantation), the measurements are non-invasive – Measurements are made in the tissue at the site of interest – Measurements can be made continuously or repeatedly as needed, often indefinitely, at the same site – Sub-mm resolution can be achieved – The measurements are especially sensitive and accurate at the low levels of pO 2 (sensitivity to D < 1 mmHg) – Compatibility with complementary techniques for imaging and measurement – Immediate clinical applicability using India ink, advanced techniques in stages of regulatory approval

EPR Oximetry with India Ink

• Patient preparation - Injection of India ink

– India ink formulated with Printex-U carbon black (200mg/ml) in 0.9% NaCl and 1.25% CMC – Sterilized via autoclave prior to injection – 20-50 m L injected into tissue of interest using 22 gauge needle – 1-10mm depth

• Measurements

– Clinical whole-body spectrometer with temperature control – EPR data collection (10s/scan, 1-30 min/set) – Measurements repeated over the course of treatment as desired PU ink new calibration 200701 30 F = y 0 +a*x/(b+x) y0 = 1.1229 a = 51.2401 b = 120.4903

25 20 15 10 5 0 0 Ink injection and calibrated pO 2 response 20 40 60

PO 2 mmHg

regression PO 2 80 100

Preliminary Measurements in Humans

• Tumor pO • Tumor pO 2 has been measured in 14 volunteers.

• Various tumor types of superficial tumors • Measurement sites have ranged from the feet to the scalp.

2 varied among the patients studied and over the course of treatment.

• Tumors were tested for response to changes of inhaled oxygen gas.

• Large differences among tumors were found showing potentially very important implications for improving personalized therapy based on the oxygen levels in that person’s tumor.

pO

2

Variability across Serial Measurements

12 10 8 6 4 Scalp Pre-treatment Scalp Post-treatment Neck Pre-treatment Neck Post-treatment 2 0 3/3/2008 3/5/2008 3/7/2008 3/9/2008 3/11/2008 3/13/2008 3/15/2008 Measurement Day

Tumor pO 2 was monitored in melanoma metastases at two sites, in the scalp and neck, during the course of radiation treatment (6 were recorded immediately before and after each fraction while the patient inspired room air. These results indicate hypoxic environments that vary on a day-to-day basis, but show little acute response in D pO 2  6 Gy). Spectra due to radiation.

Oximetry with Implantable Resonators

The development of implantable resonators will extend applicability of EPR oximetry • • Implantable resonators extend the range of EPR oximetry to deeper tissues.

‒ Improved SNR at all depths ‒ Improved spatial localization Ability to use optimal materials for added sensitivity ‒ Biocompatible coating contains paramagnetic oximetric material ‒ Ability to remove implants following use

Installation for deep tissue measurements of pO 2 a pig in the flank of The coupling loop of the implantable resonator is placed subcutaneously and the detection loop(s) are inserted into the tissue of interest.

The implantable resonators can be fabricated with great flexibility in configuration.

pO

2

Assessment following Sub-lobar Resection with Adjuvant SABR

• NCCC-ACS-IRG funded effort – PI: Philip Schaner (Rad. Onc), Co-I: Erkmen, Williams, Hou, Black • Proposes that local control of early-stage NSCLC following sub lobar resection could be improved with adjuvant SABR – Efficacy of radiation is related to tissue pO 2 , which may be diminished following surgical disruption. – Tissue pO 2 will allow a more complete understanding of the potential of adjuvant SABR in the post-operative setting.

• Primary Objective: to evaluate the technical feasibility of adjuvant stereotactic ablative radiotherapy (SABR) after sub-lobar resection, and obtain preliminary safety data, in a large animal model (pig) • Secondary Objective: to evaluate oxygenation using a novel application of EPR in the pulmonary parenchyma, both after sub-lobar resection and post-operative SABR.

• Status: Entering in vivo stage, following procedural evaluation and refinements with cadaveric animals

PPG Layout: Projects & Cores

PROJECT 1: Oxygen measurements in human tumors using EPR oximetry with carbon-based sensors (PI: Gallez) PROJECT 2: Monitoring of pO 2 in human tumors using EPR oximetry with implantable oxygen-sensing probe, OxyChip (PI: Kuppusamy) PROJECT 3: Oxygen measurements in deep-seated human tumors using EPR oximetry with implantable deep tissue oxygen sensors (PI: Swartz) CORE A: Administrative Core

PROJECT 1

CORE B: CORE C: Instrument/Resource Core Biostatistics Core

PROJECT 2 OxyChip

Primary: India ink OxyChip ImOS

CORE S A,B,C

India ink OxyChip ImOS

PROJECT 3 Deep-tissue Sensor

Synopsis of Project 1

Oxygen measurements in human tumors using EPR oximetry with carbon based sensors (PI: Gallez; Sites: Dartmouth, Brussels, Emory, UPenn)

• • • Make use of already approved materials (India ink in USA and charcoal in Europe) for human measurements • pO 2 measurements in superficial tumors, up to 10 mm depth Dartmouth has performed initial clinical measurements in human tumors Studies will be performed to establish:  Intra-tumor and inter-tumor variations of pO 2 levels using repeated measurements over a period of weeks in the same tumor   Tumor response to hyperoxygenation treatments Comparison of EPR pO 2 data with those obtained using indirect methods such as PET, MRI, and hypoxia gene signatures   Monitor pO 2 dynamics in tumors and normal tissue following radiation therapy Role of oxygen in radiation-induced fibrosis

Tumors: Head & Neck (oral), Cervical, Breast, Mycosis Fungoides

Synopsis of Project 2

Monitoring of pO 2 in human tumors using EPR oximetry with an implantable oxygen sensing probe (OxyChip) (PI: Kuppusamy; Sites: Dartmouth, Emory)

• • • • • Will use OxyChip – a newly developed, high-sensitive, PDMS coated implantable/retrievable sensor for EPR oximetry Awaiting FDA approval for Investigational Device Exemption The sensor has been tested and validated in preclinical animal models Repeated pO 2 measurements will be made in superficial tumors (up to 10-mm depth) Studies will be performed to establish:       Safety and efficacy for repeated measurements of pO 2 in human subjects Monitor temporal changes in tumor pO 2 over a period of weeks to months Tumor response to hyperoxygenation treatments Comparison of EPR pO 2 data with hypoxia gene signatures Monitor pO 2 dynamics in tumor and normal tissue following radiation therapy Role of oxygen in neo-adjuvant trastuzumab therapy for HER2+ breast cancer

Tumors: Head & Neck (oral), Cervical, Breast (HER2+), Mycosis Fungoides

Synopsis of Project 3

• • • • •

Oxygen measurements in human tumors using EPR oximetry with implantable deep tissue oxygen sensors (PI: Swartz; Sites: Dartmouth, Emory)

Will use implantable deep-tissue oxygen sensors for repeated measurement of pO 2 in deep-sited tumors

External surface-loop resonator of the EPR spectrometer

Seek FDA approval for Investigational Device Exemption

Skin Biological tissue Transmission lines

The sensor has been tested and validated in animal models

Coupling loops of the implantable oxygen sensor

pO 2 measurements will be made in tumors deeper than 1 cm

Sensory tips

Studies will be performed to establish:

B Tissue of interest

 Safety and efficacy for repeated measurements of pO 2 in human subjects  Monitor changes in tumor pO 2 over a period of weeks to months  Tumor response to hyperoxygenation treatments  Comparison of EPR pO 2 data with hypoxia gene signatures  Monitor pO 2 dynamics during concurrent treatment with radiation and oxygen-modifying interventions

Tumors: Head & Neck (lymph node), high-grade sarcoma, glioblastoma

Clinical EPR Oximetry Summary

• In vivo EPR oximetry has been used successfully in the clinical setting to make repeated non-invasive direct measurements of tissue pO 2 .

– pO 2 in peripheral tissues can be measured, including responses to changes in FIO – In most tumors, baseline pO increases in tumor pO – Tumor pO 2 2 and perfusion (>10mmHg) and the application of inhaled oxygen led to dramatic courses of treatment 2 . 2 values were observed to be quite low values have varied among the patients studied and over the – Different responses of tumor pO 2 are observed.

to increased fractions of inhaled oxygen • Implantable probes and resonators and are being developed to extend the clinical applicability of EPR oximetry to include deep tumor sites. • Based on the measurements to date, we believe that it is feasible that in vivo EPR oximetry could be used to monitor tumor pO 2 in the clinical setting and guide the optimal application of therapies.

Acknowledgements

NIH, Norris Cotton Cancer Center, ACS Institutional Research Grants, Dartmouth Center for Clinical and Translational Science (DCCTS) Department of Radiology, Section of Radiation Oncology

Investigators

Harold Swartz Periannan Kuppusamy Lesley Jarvis Phil Schaner Nadeem Khan Huagang Hou Ann Flood Eunice Chen Alan Eastman Eugene Demidenko Bernard Gallez (UCL, Brussels, Belguim)