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Objective To test whether multiphoton microscopy (MPM) might allow identification of prostatic and periprostatic structures with magnification and resolution similar to gold standard histopathology. Material and Methods The present study included 95 robotic radical prostatectomy patients who consented to participate in an Institutional Review Board-approved study starting in 2007. The types of specimens used for imaging were excised surgical margins and biopsies, and sections obtained from the excised prostate. The specimens were imaged with a custom-built MPM system. All images were compared with haematoxylin/eosin histopathology of the same specimen. Results MPM of freshly excised, unprocessed and unstained tissue can identify all relevant prostatic and periprostatic structures, such as nerves, blood vessels, capsule, underlying acini and also pathological changes, including prostate cancer. Histological confirmation and correlation of these structures and pathologies have validated the findings of MPM. Conclusions MPM shows great promise as a tool for real-time intra-surgical histopathology without needing excision or administration of contrast agents. The results will, however, need to be confirmed in true surgical settings using a miniaturized MPM microendoscope. Keywords: robotic prostatectomy, multiphoton microscopy, prostate cancer, nerve sparing, histopathology, real-time imaging Introduction Prostate cancer is the most common male cancer, claiming almost 27 000 lives annually [1]. When confined to the gland, it can be successfully treated by surgical removal of the organ, a procedure termed radical prostatectomy, which is performed in 70 000 patients in the US every year. The success of this surgery is judged by three measures: complete removal of the cancer-harbouring glands; preservation of nerves that control sexual function; and preservation of sphincteric structures, which maintain urinary control. Anatomically, the prostate gland is surrounded by a fibrous capsule, with a thickness that varies substantially between different parts of the gland. This incomplete capsule is surrounded, in turn, by a multi-layered, anatomically complex fascia that contains fat cells intermingled with loose areolar tissue, autonomic ganglia, a neural plexus, nerve trunks, arteries and veins. This fascia adheres to the capsule and is pierced by vessels and nerves. Both the erectile nerve, which has a variable branching pattern, and the sphincteric sling, are intermingled with the fascial fat, connective tissue and blood vessels [2]. Cancer cells sometimes migrate beyond the gland and either involve the surrounding nerves or grow into the sphincter – a phenomenon termed extra-prostatic extension (EPE). Since these nerves and any clusters of cancer cells are too small to be visualized by eye or using the ×10–12 magnification of the stereoscope of a surgical robot, the competing goals of cancer extirpation versus preservation of potency and continence have to be balanced during surgery [2]. Surgeons make judgments based on information that includes pre-surgical PSA levels, MRI results, DRE results and intra-surgical subjective cues such as colour changes, fibrosis and ease of separating the different fascial layers. One option for more detailed feedback, intraoperative frozen sections, takes time, could damage the delicate structures one is trying to save, and suffers from sampling errors. As a result, the current methods for discriminating between EPE and the tissues that need to be preserved during surgery are imprecise and have an impact on the outcomes of this surgery. Indeed, this inability to identify cancerous cells and their association with nerves can result in incomplete removal of cancer, resulting in positive surgical margins (10–40% occurrence) [3–6], postoperative impotence due to damage to, or excision of, these nerves (25–70%) [7–11], or both positive margins and an impotent patient. It has been estimated that half the patients who undergo radical prostatectomy require postoperative treatment for erectile dysfunction. In addition to causing psychological and quality-of-life compromises, these complications exact a staggering financial cost on the US health care system. At a rate of US $10 000–20 000 per patient per year spent on doctors' office visits, oral medications, vacuum pumps, intra-cavernous injections and secondary surgeries, this costs the US health care system $350–700 million every year. In addition, certain patients are excluded as candidates for nerve-sparing surgery based on the less-than-optimal preoperative assessments, and thus could be denied a chance for a better postoperative quality of life. In summary, access to high-resolution, high-contrast live imaging of the prostatic capsule, apex, sphincter and the surrounding nerves and tissues would improve surgical decision-making and patient outcomes, and reduce the emotional and financial impact of post-surgical complications. One potential solution is a novel non-linear optical imaging technology: multiphoton microscopy (MPM) [12–14]. MPM relies on the simultaneous absorption of two (or three) low-energy (near-infrared) photons to cause a non-linear excitation, which greatly reduces the potential for cellular damage. Excitation only occurs where there is sufficient photon density (at the point of laser focus), providing intrinsic optical sectioning with a resolution equivalent to traditional confocal microscopy. Tissue penetration is greater than standard confocal microscopy because absorption and scattering are greatly reduced at near-infrared wavelengths compared with the visible or ultraviolet spectrum. Most importantly, by utilizing two-photon excitation in the 700–800 nm range, MPM enables the imaging of fresh, living, unprocessed and unstained tissue by utilizing intrinsic tissue emissions (ITEs). The ITE signal is composed of two components: tissue autofluorescence, in part from nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) in cells, elastin in the connective tissue, and lipofuscin in fat and other cells; and second harmonic generation (SHG), which arises from non-centrosymmetric structures such as tissue collagen [12–14]. We and others have shown that MPM/ITE imaging is capable of generating distinctive optical signals that enable imaging of animal [13] and human [15–17] tissues at sub-micron resolution in three dimensions to a depth of up to 0.5 mm; at image acquisition rates of ≈ one image/second, with >105 pixels per image. While the miniaturization of MPM for integration with robotic surgical equipment is currently in progress, we have carried out a prospective study to assess the feasibility of using MPM/ITE for structure identification in excised prostatic and periprostatic tissue. In all cases, the MPM findings were confirmed by comparison with gold standard haematoxylin/eosin (H&E)-stained histopathology. Specimens were obtained from consenting subjects undergoing robotic radical prostatectomy at Weill Cornell Medical College with Dr Tewari, in a protocol approved by the Institutional Review Board. Samples included periprostatic tissue, specifically surgical margins containing the lateral pelvic fascia (LPF), as well as the prostatic acini, in the form of either biopsies or sections of the gland. In LPF specimens, we confirm our ability to identify its components, such as nerves, arteries, fat and connective tissue, as well as sites of local inflammation. These structures are the ones that must be identified during surgery. In the excised specimens (biopsies or sections), we identify the prostatic acini, the surrounding stroma, and the prostatic capsule. Architectural changes in malignant transformation are used during histopathology to determine tumour grade (Gleason grade), and hence the aggressiveness, of the cancer. We show here that MPM/ITE can provide such architectural information. |
