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Analyzing and effectively communicating the efficacy and toxicity of treatment is the basis of risk benefit analysis (RBA). More efficient and objective tools are needed. We apply Chauhan Weighted Trajectory Analysis (CWTA) to perform RBA with superior objectivity, power, and clarity. We used CWTA to perform 1000-fold simulations of RCTs using ordinal endpoints for both treatment efficacy and toxicity. RCTs were simulated with 1:1 allocation at defined sample sizes and hazard ratios. We studied the simplest case of 3 levels each of toxicity and efficacy and the general case of the advanced cancer trial, with efficacy graded by five RECIST 1.1 health statuses and toxicity by the six-point CTCAE scale (6 x 5 matrix). The latter model was applied to a real-world dose escalation phase I trial in advanced cancer. Simulations in both the 3 x 3 and the 6 x 5 advanced cancer matrix confirmed that drugs with both superior efficacy and toxicity profiles synergize for greater statistical power with CWTA-RBA. The CWTA-RBA 6 x 5 matrix reduced sample size requirements over CWTA efficacy-only analysis. Application to the dose finding phase I clinical trial provided objective, statistically significant validation for the selected dose. CWTA-RBA, by incorporating both drug efficacy and toxicity, provides a single test statistic and plot that analyzes and effectively communicates therapeutic risks and benefits. CWTA-RBA requires fewer patients than CWTA efficacy-only analysis when the experimental drug is both more effective and less toxic. CWTA-RBA facilitates the objective and efficient assessment of new therapies throughout the drug development pathway. Furthermore, several advantages over competing tests in communicating risk-benefit will assist regulatory review, clinical adoption, and understanding of therapeutic risks and benefits by clinicians and patients alike.

As Kaplan-Meier (KM) analysis is limited to single unidirectional endpoints, most advanced cancer randomized clinical trials (RCTs) are powered for either progression free survival (PFS) or overall survival (OS). This discards efficacy information carried by partial responses, complete responses, and stable disease that frequently precede progressive disease and death. Chauhan Weighted Trajectory Analysis (CWTA) is a generalization of KM that simultaneously assesses multiple rank-ordered endpoints. We hypothesized that CWTA could use this efficacy information to reduce sample size requirements and expedite efficacy signals in advanced cancer trials. We performed 100-fold and 1000-fold simulations of solid tumour systemic therapy RCTs with health statuses rank ordered from complete response (Stage 0) to death (Stage 4). At increments of sample size and hazard ratio, we compared KM PFS and OS with CWTA for (i) sample size requirements to achieve a power of 0.8 and (ii) time-to-first significant efficacy signal. CWTA consistently demonstrated greater power, and reduced sample size requirements by 18% to 35% compared to KM PFS and 14% to 20% compared to KM OS. CWTA also expedited time-to-efficacy signals 2- to 6-fold. CWTA, by incorporating all efficacy signals in the cancer treatment trajectory, provides clinically relevant reduction in required sample size and meaningfully expedites the efficacy signals of cancer treatments compared to KM PFS and KM OS. Using CWTA rather than KM as the primary trial outcome has the potential to meaningfully reduce the numbers of patients, trial duration, and costs to evaluate therapies in advanced cancer.

The Kaplan-Meier estimator (KM) is widely used in medical research to estimate the survival function from lifetime data. KM is a powerful tool to evaluate clinical trials due to simple computational requirements, a logrank hypothesis test, and the ability to censor patients. However, KM has several constraints and fails to generalize to ordinal variables of interest such as toxicity and ECOG performance. We devised Weighted Trajectory Analysis (WTA) to combine the advantages of KM with the ability to compare treatment groups for ordinal variables and fluctuating outcomes. To assess statistical significance, we developed a new hypothesis test analogous to the logrank test. We demonstrate the functionality of WTA through 1000-fold clinical trial simulations of unique stochastic models of chemotherapy toxicity and schizophrenia progression. At several increments of sample size and hazard ratio, we compare the performance of WTA to both KM and Generalized Estimating Equations (GEE). WTA generally required half the sample size to achieve comparable power to KM; advantages over GEE include its robust non-parametric approach and summary plot. We also apply WTA to real clinical data: the toxicity outcomes of melanoma patients receiving immunotherapy and the disease progression of patients with metastatic breast cancer receiving ramucirumab. The application of WTA demonstrates that using traditional methods such as percent incidence and KM can lead to both Type I and II errors by failing to model illness trajectory. This article outlines a novel method for clinical outcome assessment that extends the advantages of Kaplan-Meier estimates to ordinal outcome variables., Comment: 29 pages, 3 tables, 10 figures

The field of histology relies heavily on antiquated tissue processing and staining techniques that limit the efficiency of pathologic diagnoses of cancer and other diseases. Current staining and advanced labeling methods are often destructive and mutually incompatible, requiring new tissue sections for each stain. This prolongs the diagnostic process and depletes valuable biopsy samples. In this study, we present an alternative label-free histology platform using the first transmission-mode Photon Absorption Remote Sensing microscope. Optimized for automated whole slide scanning of unstained tissue samples, the system provides slide images at magnifications up to 40x that are fully compatible with existing digital pathology tools. The scans capture high quality and high-resolution images with subcellular diagnostic detail. After imaging, samples remain suitable for histochemical, immunohistochemical, and other staining techniques. Scattering and absorption (radiative and non-radiative) contrasts are shown in whole slide images of malignant human breast and skin tissues samples. Clinically relevant features are highlighted, and close correspondence and analogous contrast is demonstrated with one-to-one gold standard H&E stained images. Our previously reported pix2pix virtual staining model is applied to an entire whole slide image, showcasing the potential of this approach in whole slide label-free H&E emulation. This work is a critical advance for integrating label-free optical methods into standard histopathology workflows, both enhancing diagnostic efficiency, and broadening the number of stains that can be applied while preserving valuable tissue samples., Comment: 10 pages, 10 figures

Histopathological visualizations are a pillar of modern medicine and biological research. Surgical oncology relies exclusively on post-operative histology to determine definitive surgical success and guide adjuvant treatments. The current histology workflow is based on bright-field microscopic assessment of histochemical stained tissues and has some major limitations. For example, the preparation of stained specimens for brightfield assessment requires lengthy sample processing, delaying interventions for days or even weeks. Hence, there is a pressing need for improved histopathology methods. In this paper, we present a deep-learning-based approach for virtual label-free histochemical staining of total-absorption photoacoustic remote sensing (TA-PARS) images of unstained tissue. TA-PARS provides an array of directly measured label-free contrasts such as scattering and total absorption (radiative and non-radiative), ideal for developing H&E colorizations without the need to infer arbitrary tissue structures. We use a Pix2Pix generative adversarial network (GAN) to develop visualizations analogous to H&E staining from label-free TA-PARS images. Thin sections of human skin tissue were first virtually stained with the TA-PARS, then were chemically stained with H&E producing a one-to-one comparison between the virtual and chemical staining. The one-to-one matched virtually- and chemically- stained images exhibit high concordance validating the digital colorization of the TA-PARS images against the gold standard H&E. TA-PARS images were reviewed by four dermatologic pathologists who confirmed they are of diagnostic quality, and that resolution, contrast, and color permitted interpretation as if they were H&E. The presented approach paves the way for the development of TA-PARS slide-free histology, which promises to dramatically reduce the time from specimen resection to histological imaging., Comment: 16 pages, 8 figures

In the past decades, absorption modalities have emerged as powerful tools for label-free functional and structural imaging of cells and tissues. Many biomolecules present unique absorption spectra providing chromophore-specific information on properties such as chemical bonding, and sample composition. As chromophores absorb photons the absorbed energy is emitted as photons (radiative relaxation) or converted to heat and under specific conditions pressure (non-radiative relaxation). Modalities like fluorescence microscopy may capture radiative relaxation to provide contrast, while modalities like photoacoustic microscopy may leverage non-radiative heat and pressures. Here we show an all-optical non-contact total-absorption photoacoustic remote sensing (TA-PARS) microscope, which can capture both radiative and non-radiative absorption effects in a single acquisition. The TA-PARS yields an absorption metric proposed as the quantum efficiency ratio (QER), which visualizes a biomolecules proportional radiative and non-radiative absorption response. The TA-PARS provides label-free visualization of a range of biomolecules enabling convincing analogues to traditional histochemical staining of tissues, effectively providing label-free Hematoxylin and Eosin (H&E)-like visualizations. These findings represent the establishment of an effective all-optical non-contact total-absorption microscope for label-free inspection of biological media., Comment: 22 pages, 6 figures in paper. 16 pages, 14 figures, 2 tables in supplemental Information

Malignant brain tumors are among the deadliest neoplasms with the lowest survival rates of any cancer type. In considering surgical tumor resection, suboptimal extent of resection is linked to poor clinical outcomes and lower overall survival rates. Currently available tools for intraoperative histopathological assessment require an average of 20 minutes processing and are of limited diagnostic quality for guiding surgeries. Consequently, there is an unaddressed need for a rapid imaging technique to guide maximal resection of brain tumors. Working towards this goal, presented here is an all optical non-contact label-free reflection mode photoacoustic remote sensing (PARS) microscope. By using a tunable excitation laser, PARS takes advantage of the endogenous optical absorption peaks of DNA and cytoplasm to achieve virtual contrast analogous to standard hematoxylin and eosin (H and E) staining. In conjunction, a fast 266 nm excitation is used to generate large grossing scans and rapidly assess small fields in real-time with hematoxylin-like contrast. Images obtained using this technique show comparable quality and contrast to the current standard for histopathological assessment of brain tissues. Using the proposed method, rapid, high-throughput, histological-like imaging was achieved in unstained brain tissues, indicating PARS utility for intraoperative guidance to improve extent of surgical resection., Comment: 17 pages, 3 figures

Histological visualizations are critical to clinical disease management and are fundamental to biological understanding. However, current approaches that rely on bright-field microscopy require extensive tissue preparation prior to imaging. These processes are labor intensive and contribute to delays in clinical feedback that can extend to two to three weeks for standard paraffin-embedded tissue preparation and interpretation. Here, we present a label-free reflection-mode imaging modality that reveals cellular-scale morphology by detecting intrinsic endogenous contrast. We accomplish this with the novel photoacoustic remote sensing (PARS) detection system that permits non-contact optical absorption contrast to be extracted from thick and opaque biological targets with optical resolution. PARS was examined both as a rapid assessment tool that is capable of managing large samples (>1 cm2) in under 10 minutes, and as a high contrast imaging modality capable of extracting specific biological contrast to simulate conventional histological stains such as hematoxylin and eosin (H&E). The capabilities of the proposed method are demonstrated in a variety of human tissue preparations including formalin-fixed paraffin-embedded tissue blocks and unstained slides sectioned from these blocks, including normal and neoplastic human brain, and breast epithelium involved with breast cancer. Similarly, PARS images of human skin prepared by frozen section clearly demonstrated basal cell carcinoma and normal human skin tissue. Finally, we imaged unprocessed murine kidney and achieved histologically relevant subcellular morphology in fresh tissue. This represents a vital step towards an effective real-time clinical microscope that overcomes the limitations of standard histopathologic tissue preparations and enables real-time pathology assessment., Comment: 32 pages, 11 figures, includes Supplementary Information section

Mohs micrographic surgery (MMS) is a precise oncological technique where layers of tissue are resected and examined with intraoperative histopathology to minimize the removal of normal tissue while completely excising the cancer. To achieve intraoperative pathology, the tissue is frozen, sectioned and stained over a 20- to 60-minute period, then analyzed by the MMS surgeon. Surgery is continued one layer at a time until no cancerous cells remain, meaning MMS can take several hours to complete. Ideally, it would be desirable to circumvent or augment frozen sectioning methods and directly visualize subcellular morphology on the unprocessed excised tissues. Employing photoacoustic remote sensing (PARS) microscopy, we present a non-contact label-free reflection-mode method of performing such visualizations in frozen sections of human skin. PARS leverages endogenous optical absorption contrast within cell nuclei to provide visualizations reminiscent of histochemical staining techniques. Presented here, is the first true one to one comparison between PARS microscopy and standard histopathological imaging in human tissues. We demonstrate the ability of PARS microscopy to provide large grossing scans (>1 cm2, sufficient to visualize entire MMS sections) and regional scans with subcellular lateral resolution (~300 nm)., Comment: 15 pages, 5 figures