1. Introduction

Pancreatic adenocarcinoma has a five-year survival rate of only 6%, making it the 4th leading cause of cancer death in the United States (US) [1]. By 2020, pancreatic cancer is projected to become the 2nd leading cause of cancer death in the US [1]. Current diagnostic procedures are unable to accurately detect early stage disease [2], successfully diagnosing only 7% of early-stage pancreatic cancers [1]. As a result, only 20% of discovered pancreatic cancers are treatable with surgery. The challenges to accurate detection and characterization of pancreatic neoplasia include the relative inaccessibility of the organ, as well as the non-specific nature of symptoms. In particular, a stromal reaction with intense fibrosis is associated with both adenocarcinoma and chronic pancreatitis (inflammation). As a result, the sensitivity for diagnosing cancer in the setting of chronic pancreatitis (inflammation) has been reported to be only 54% when using the diagnostic procedure-of-choice for tissue acquisition in suspect pancreatic cancer, endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) [3].

Several studies have employed optical techniques for minimally invasive detection of cancer [4] in tissues including breast [5], cervix [6], colon [7, 8], and esophagus [9]. Further, optical spectroscopy is compatible with clinical EUS-FNA procedures [10]. Optical techniques investigated in pancreatic tissues include optical coherence tomography [11, 12], needle-based confocal laser endomicroscopy [13], near-infrared spectroscopy [14, 15], non-linear optical imaging [16], optical field effect analysis from duodenal tissues [17, 18], diffuse optical tomography [19], and our own studies employing optical spectroscopy [20–24]. Advantages to our optical spectroscopy technology include addressing the primary medical need: improved and minimally invasive detection of pancreatic cancer in the presence of overall tissue inflammation.

Our approach examines the feasibility of optical spectroscopy for clinical pancreatic cancer diagnostics in four stages: (1) identify key features in human tissue spectra using an in vivo murine model (human pancreatic tumor xenograft), (2) perform ex vivo pilot study to optically detect human pancreatic malignancy, (3) verify and validate accuracy of a photon-tissue interaction (PTI) model, and (4) perform a human pilot study to assess feasibility in vivo. In Stage 1, measurements from ex vivo human adenocarcinoma tissues were shown to correspond well to in vivo measurements from a tumor xenograft [20]. In Stage 2, accurate detection of normal, chronic pancreatitis (inflamed), and adenocarcinoma tissues was achieved [21–23]. Malignant tissues were distinguished from benign tissues with sensitivities and specificities of 85% and 86%, respectively [21], and with statistical significance [22] in the setting of chronic pancreatitis. In Stage 3, a PTI model was employed to detect a pancreatic cancer precursor [24]. Here, we address Stage 4 by reporting the in vivo feasibility of optical spectroscopy to detect malignant tissues.

2. Clinical optical spectroscopy measurements of in vivo human pancreatic tissues

2.1. Reflectance and Fluorescence Lifetime Spectrometer (RFLS)

A Reflectance and Fluorescence Lifetime Spectrometer (RFLS) [20, 25] was employed to collect optical data from human pancreatic tissues in vivo (Fig. 1).

 

Fig. 1 Pancreatic tissue optical measurement protocol, designed to mimic fine-needle aspiration (FNA) procedures by introducing the optical probe via a hollow angiocatheter. (1) In vivo: at this stage of surgery, tissue was still perfused with some blood and was near body temperature. The sterilized fiber-optic probe (6 m length) extended from the RFLS in the non-sterile field to the patient in the sterile field. At each selected site, the surgeon inserted a 14 gauge angiocatheter (B Braun Medical) ~1 cm into the tissue, removed the stylet from the angiocatheter, and inserted the fiber-optic probe. Data acquisition for each site was < 45 s, with each modality acquired in < 1 s. (2) In vivo and ex vivo measurements were acquired at the same tissue site by marking the site prior to resection. (2, right) Each optical measurement was estimated to interrogate ~1 mm³ of tissue. Repeatability was tested by collecting two successive optical measurements at each site, with up to two tissue sites measured per patient. Tissue sites were biopsied for histopathologic analysis.

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The RFLS sequentially collected steady-state reflectance and fluorescence with a hand-held three-channel fiber-optic probe [20]. Measured spectral data were background subtracted and corrected for the spectral instrument response [20–24]. Reflectance measurements were acquired by delivering broadband white light from a CW tungsten halogen lamp (HL 2000FHSA, Ocean Optics) to tissue. An 850 nm short-pass filter (Semrock) protected human tissue from infrared light. Reflected photons were collected with the common detection fiber and detected by a spectrograph (MS 125, Oriel Instruments) coupled to an intensified charge-coupled device (ICCD 2063, Andor Technology).

Fluorescence measurements were acquired by delivering 355 nm pulsed laser excitation (PNV001525-140, JDS Uniphase) to the tissue. Laser energy delivered to the tissue was < 12 μJ. The fluorescence emission was collected with the common detection fiber and measured with the spectrograph-coupled ICCD.

2.2. In vivo and ex vivo data collection protocol

Optical measurements followed a study protocol approved by the University of Michigan (U-M) Institutional Review Board. Patients enrolled in the study provided written informed consent. Optical fiber probes were sterilized with an ethylene oxide protocol developed in accordance with U-M Hospital Central Sterile Supply. Figure 1 outlines the optical measurement protocol for human pancreatic tissues.

In vivo and ex vivo optical measurements were collected from the same tissue sites (freshly-excised tissues within 30 minutes of resection [20–22]). Ideally, a fixed angiocatheter would mark the tissue site for in vivo and ex vivo measurements. Due to surgical complications, the angiocatheter remained in place for only 1 patient. For the other 5 patients, the point-of-entry was uniquely marked with surgical thread. The surgeon collected optical data in vivo. An expert pathologist oversaw ex vivo data collection and performed a tissue biopsy at each site immediately after optical measurements. Biopsy specimens were sent for analysis via histopathology.

2.3. In vivo optical data set

In total, 10 pancreatic tissue sites were measured from 6 patients with histopathological classification of pancreatic adenocarcinoma (5 sites) and normal (5 sites). Exclusion criteria were employed to remove tissue sites with excessive amounts of absorption (e.g., R_{550 nm}/R_{650 nm} < 0.1) or low signal-to-noise ratio (e.g., < 5). After exclusion, the in vivo data set included steady-state reflectance measurements from 8 (3 normal from 2 patients, 5 adenocarcinoma from 3 patients) tissue sites and steady-state fluorescence measurements from 4 (1 normal from 1 patient, 3 adenocarcinoma from 2 patients) tissue sites. Thus, steady-state reflectance measurements were analyzed quantitatively, as they constituted the largest usable data set and as previously reported results [20–23] showed that reflectance analysis alone can improve diagnosis of pancreatic malignancy.

3. Steady-state reflectance spectroscopy analysis

A reflectance spectral ratio classifier was developed to quickly characterize the measured tissue sites. In human pancreatic tissues, the ratiometric classifier R_{470 nm}/R_{650 nm} was shown in prior work to vary with pancreatic disease [20].

Previously, for an ex vivo human pancreatic tissues data set, we reported a quantitative analysis algorithm that directly extracted optical tissue parameters [22, 23]. The photon-tissue interaction (PTI) model that was fit to the optical spectra was obtained by employing a semi-empirical reflectance model [26] to scale the “canonical normal” reflectance (average of all measured normal reflectance spectra) to a model for each unknown reflectance spectrum. Here, we employed a canonical normal obtained from our in vivo data set (2 of 3 normal sites with comparable hemoglobin content) to model measured in vivo spectra from 450 - 530 nm [22, 23]. This in vivo PTI model extended the maximum hemoglobin concentration from 25 [23] to 60 μM. Model fits to data with more than 15% error between 450 – 530 nm were excluded, including one adenocarcinoma and one normal site.

4. Feasibility of optical spectroscopy on human pancreatic tissues in vivo

For the first time, to our knowledge, tissue optical reflectance and fluorescence measurements were recorded from human pancreatic tissues in vivo. Figure 2 shows reflectance and fluorescence spectra from a tissue site measured in vivo and ex vivo on the same patient. Measurements were recorded rapidly (< 1 s per modality) and were remarkably consistent, especially considering the anticipated large variability in tissue blood content in vivo.

 

Fig. 2 Optical measurements from a tissue site in vivo and ex vivo for (A) steady-state reflectance and (B) steady-state fluorescence. Measurements of the same tissue site contained similar features, attributed to similar tissue composition and morphology [22], and differed only slightly between 400 and 475 nm due to the anticipated blood content variations. The site was histopathologically confirmed to be adenocarcinoma.

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5. Optical reflectance differences between normal and adenocarcinoma tissues in vivo

Figure 3(A) shows that for human tissues measured in vivo, there are significant differences between the optical reflectance spectra of normal pancreas and adenocarcinoma, notably in the shaded wavelength range 455-525 nm. This result is consistent with ex vivo studies [20–24] and is attributed to differences in scattering between tissue types [22]. Indeed, in vivo reflectance measurements on human pancreatic cancer xenografts grown in mice [20] (Fig. 3(B)) clearly show the pronounced reflectance peak in this region.

 

Fig. 3 Representative in vivo reflectance measurements from human pancreatic adenocarcinoma (blue solid: A, B), (A) normal human tissue (green dashed), and (B) human pancreatic cancer tumor xenograft in a non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mouse (red dashed) [20]. In the diagnostically important wavelength range between 455 and 525 nm (shaded), adenocarcinoma tissues have greater relative reflectance than normal tissues, consistent with extensive ex vivo studies [20–24].

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6. Quantitative reflectance analysis with spectral ratios classifier and PTI model

Figure 4 summarizes the results of quantitative analyses applied to in vivo human pancreatic tissue reflectance data. In vivo reflectance data was consistent with ex vivo reflectance data (both for the ex vivo results obtained here and for the ex vivo results obtained on an extensive data set [20–24]) and was able to distinguish between normal human pancreas and pancreatic adenocarcinoma.

 

Fig. 4 Quantitative analysis of in vivo reflectance data is consistent with ex vivo results and can distinguish between normal human pancreas and pancreatic adenocarcinoma. (A) The R_{470 nm}/R_{650 nm} ratio for in vivo data (gray box plot) analyzed the pronounced spectral feature at 470 nm which clearly distinguished adenocarcinoma from normal tissues. The blue and green overlays show the analysis for an extensive ex vivo data set [22], which shows the same trend as that observed in vivo. Minor differences between ex vivo and in vivo results were attributed to the limited experimental control of local blood content during in vivo measurements. (B) PTI model best fit (red, dotted) to the in vivo adenocarcinoma data in Fig. 2 (blue) (4% fit error between 450 – 530 nm). (C) A PTI-extracted morphological parameter (L/L0, related to changes in cellular nuclear size and shape) from in vivo measurements clearly distinguished adenocarcinoma from normal pancreas, consistent with results from an extensive ex vivo data set [22]. In vivo measurements from adenocarcinoma tissues modeled with an ex vivo “canonical normal” (from [22]) showed comparable results to employing an in vivo “canonical normal.” Error bars represent standard error.

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7. Discussion and conclusions

Pancreatic adenocarcinoma has a five-year survival rate of less than 6%, a fact that has not changed in nearly forty years [1]. This exceptionally low survival rate is attributed to the lack of accurate detection methods, which limits diagnosis to advanced stages of disease development [2, 3].

Here, we presented an in vivo pilot study to assess the feasibility of optical spectroscopy to improve the clinical detection of pancreatic adenocarcinoma. This in vivo feasibility study was performed with limited experimental control over blood content using a protocol that mimicked fine-needle aspiration, the diagnostic procedure-of-choice for tissue acquisition in suspect pancreatic cancer. Our promising results demonstrate the robustness of both the data collection and the analysis methods employed to characterize measured optical data in the presence of varying blood.

From optical measurements acquired during open surgery on 6 patients, we verified the ability to collect spectrally-resolved reflectance and fluorescence rapidly (< 45 s) and repeatedly in vivo. From site-matched data, we examined the consistency between in vivo and ex vivo measurements and found both qualitative and quantitative (via known ratiometric [20, 21] and photon-tissue interaction [22–24] models) agreement between the two. Further, quantified differences between adenocarcinoma and normal tissues measured in vivo were consistent with differences established previously using an extensive ex vivo data set [20–24].

Overall, these results suggest that optical spectroscopy is a promising method for the improved diagnosis of pancreatic cancer in vivo.