WO2015157337A1 - Method and apparatus to diagnose the metastatic or progressive potential of cancer, fibrosis and other diseases - Google Patents

Abstract

A method and apparatus for determining the progressive potential of a disease is disclosed. The forward to backward, propagating second harmonic generation signal derived from a second harmonic generation instrument is used to assess the collagen microstructure of imaged body tissue by way of numerical values that are in turn used to determine the progressive or metastatic potential of the disease. The disease may, for example, be a cancer such as breast, cancer, lung fibrosis, colorectal adenocarcinoma, or the like. The apparatus may include in vivo instruments or laboratory diagnostic instruments with methods disclosed herein.

Description

METHOD AN D APPARATUS TO DIAG OSE THE METASTATIC OR PROGRESSIVE POTENTIAL OF CANCER, FIBROSIS AND OTHER DISEASES

CROSS REFERENCE TO RELATED PATENT APPLICATIONS

This application claims priority to United States Patent Application Serial No. 61/977,618 filed April 9, 2014 entitled "Method And Apparatus To .Diagnose Metastatic And Progressive Potential Of Cancer, Fibrosis, And Other Diseases" by Perry, Brown, Burke, Kottmann, Sime and Sharp, the entire disclosure of which is incorporated herein by reference as permissible by national or regional laws.

TECHNICAL FIELD

The present invention relates generally to the field of pathology, and, more particularly, to a method and apparatus to diagnose the metastatic or progressive potential of cancer, fibrosis and other diseases,

BACKGROUND ART

Determining the potential, for cancer io metastasize or other diseases such as lung fibrosis to progress and become fetal has profound implications in patient treatment as well as the development of new therapies and treatments. Predicting how a disease will progress given various or no treatment options is of tremendous value to the practitioner, the patient, and also to the medical research community. Unfortunately, the various diagnostic tools available today do not provide predictions with a sufficient degree of confidence, and as such, over treatment or non-targeted treatment is common. With treatments such as chemotherapy, this approach has profound, health implications for the patient, both physical and mental. What is needed is a .method and apparatus to diagnose the metastatic or progressive potential of various diseases such as cancer, fibrosis, and the like.

DISCLOSURE- OF THE INVENTION

in accordance with, the present invention, there is provided a method tor determining the progressive potential of a disease, the method comprising the steps of imaging body tissue using a second harmonic generation instrument; determining the ratio of the forward to backward propagating second harmonic generation signal, derived from the imaging of the body tissue with the second harmonic generation instrument; assessing the collagen microstructure of the i m aged body tissue using the ratio of the forward to backward propagating second harmo n ic generation signal; comparing the ratio of the forward to backward propagating second harmonic generaiiori signal to the ratio of the forward to backward propagating second harmonic generation signal of other tissue samples; and determining the progressive potential of the disease by numerical values derived from the ratio of the forward to backward propagating second harmonic generation signal of the imaged body tissue. The disease may, for example, be Sung fibrosis, a cancer such as breast cancer or colorectal adenocarcinoma, or the like.

The foregoing paragraph has been provided by way of introduction, and is not intended to limit the scope of the invention as described by this specification, claims and the attached drawings.

BRIEF DESCRIPTION OF TOE DRAWINGS

The invention will be described by reference to the following drawings, in which like numerals refer to like elements, and m which:

Figure 1 depicts methodology diagrams for the present invention;

Figure 2 is a graph depicting metastasis fee and overall, survival, as a function of F/B in ER+. LN breast cancer:

Figure 3 is a graph depicting progression free survival as a function of F/B in ER+ recurrent breast cancer patients treated with tamoxifen;

Figure 4 depicts overall survival of additional solid tumors (a; Stage I Colorectal Adenocarcinoma and b: Stage I Lung Adenocarcinoma) as a function, of F/B ratio;

Figure 5 is a graph depicting differences in F/B ratio ibr Healthy. COP and U3P lung tissue samples;

Figure 6 depicts healthy, COP, and UIP lung histopathoiogy compared to FSHG:

Figure 7 is a graph depicting differences in F/B ratio for lung tissue with preserved alveolar architecture adjacent to UIP ftbrotie lesions compared with Healthy and UIP tissue;

Figure 8 depicts increased Coll and Col3 deposition, and Coll :Col3 ratio differences, in UIP or COP versus healthv hsos; and

Figure 9 depicts Elastin and Elastm;Collagen ratio differences in UIP and COP versus healthy lung.

The present invention will be described in connection with a preferred embodiment, however, it will be ^"understood that there is no intent to limit the invention to the embodiment described. On the contrary, the intent is to cover all alternatives, modifications, and equivalents as may be included within, the spirit and scope of the invention as defined by this specification, claims, and drawings attached hereto. BEST MODE FOR CARRYING OUT THE INVENTION

A method and apparatus for determining the progressive potential of a disease is described. The method comprises the steps of imaging body tissue using a second harmonic generation instrument; deten»i»mg the rati of the forward to backward propagating second harmonic generation signal derived from the imaging of the body tissue with the second harmonic generation instrument; assessing the collagen microstmeture of the imaged body tissue using the ratio of the forward to backward propagating second harmonic generation signal; comparing the ratio of the forward to backward propagating second harmonic generation signal to the ratio of the forward to backward propagating second harmonic generation signal of other tissue samples; and determining the progressive potential of the disease by numerical values derived from the ratio of the forward to backward propagating second harmonic generation signal of the imaged body tissue. The disease may, for example, be lung fibrosis, a cancer such as breast cancer or colorectal ademx-arcinoraa. or the like.

An apparatus employing this method may be embodied in an endoscope arrangement, for example. Such an apparatus is further described herein.

By way of example, and not limitation, the speci fic use of the method and apparatus of the present invention is farther described using two examples- using second harmonic generation to predict patient outcome in solid tumors and the prediction of fata! lung fibrosis. After reading this specification, one can appreciate and understand the applicability of the present inventio to other diseases in addition to the examples provided herewith.

EXAMPLE 1- USI G SECOND HARMONIC GENERATION TO PREDICT PATIENT OUTCOME IN SOLID TUMORS

Introduction: Ove reatmenf. of estrogen receptor positive {ER+). lymph node-negative (LNN) breast cancer patients with chemotherapy is a pressing clinical problem that can be addressed by improving techniques to predict tumor metastatic potential. Here we demonstrate that second harmonic generation (SHG) analysis of primary tumor biopsies can provide prognostic information about the .metastatic outcome of ER+, LNN breast cancer, as well as stage 1 colorectal adenocarcinoma.

Methods: SHG is an optical signal produced by fibrillar collagen. The ratio of the for ard- to-backward emitted SHG signals (F B) is sensitive io changes in structure of individual col lagen fibers. F/B from excised primary tumor tissue was measured in. a retrospective stud of LNN breast cancer patients who had received no adjuvant systemic therapy and related to metastasis-free survival (MFS) and overall survival (OS) rates. In addition, F/B was studied for its association with the length of progression-tree survival (PFS) in a subgroup of ER+ patients who received tamoxifen as first-line treatment for recurrent disease, and for its relation with OS in stage 1 colorectal and stage I lung adenocarcinoma patients,

Results: In ER+, but not in ER-negative (E -), LN breast cancer patients an increased natural log of the P/B was significantly associated with a favorable MFS and OS. On the other hand, an increased natural log of the F/B was associated with shorter PFS in ER+ recurrent breast cancer patients treated with tamoxifen, in stage I colorectal, adenocarcinoma, an increased F/B was significantly related to poor OS, however this relationsiiip was not Statistically significant i stage 1 lung adenocarcinoma; and further testing is required.

Conclusion; Within ER.+, LNN breast cancer specimens F/B can. stratify patients based upon their potential for tumor aggressiveness. This offers a "matrix-focused^*' method to predict metastatic ^'outcome that is complementary to genomic "cell-focused^"' methods. This may contribute to improved metastatic prediction, and hence may help to reduce patient over- treatment. Introduction

Breast cancer is the leading cause of cancer related mortality in women (American Cancer Society. Cancer Facts & Figures 2012. Atlanta: American Cancer Society; 2012), predominantly due to metastasis (Fisher ER, Gregorio R , Fisher B, Redmond C, Y^'eiSios F, Sommers SC: The pathology of invasive breast cancer. A syllabus derived from findings of the ational Surgical Adjuvant Breast Project (protocol »o.4). Cancer 1 75, 36( 1 ): 1-85). After surgical resection of the primary tumor, the clinician must choose adjuvant therapy based upon the metastatic potential. Due to their aggressive biological behavior, liR-negative (ER-) tumors are treated with chemotherapy in the majority of patients. However, in ER+ patients whose cancer has not yet spread to the lymph nodes (LNN), the choice between hormonal therapy alone, or in combination with chemotherapy, is more uncertain. Following current standard of care, it is estimated that 40% of these patients will be "over-treated", receiving chemotherapy even though they would not go on to develop metastatic disease, causing many t endure the emotional distress and severe side effects accompanying chemotherapy (Wetge.lt B. Peterse JL, van't Veer tJ: Breast cancer metas asis; markers and m de s. Nature reviews Cancer 2005, 5(S);59i - 602), As such, there is a pressing clinical need to accurately predict which BR-h LNN patients have a lower metastatic potential and thus can be spared from over-treatment.

Metastatic potential and treatment response can be predicted to varying degrees of accurac using traditional histopathology, gene expression measurements (Paik S, Shale S, Tang G, Kim C, Baker J, Cronin M Baehner F.L, Walker MG, Watson D, Park T et ah A muittgene assay to predict recurrence of tanioxifen-treated, node-negative breast -cancer. The New England journal of medicine 2004. 35l(27):28 7-2826. Wang Y, Klijn JG, Zhang Y, Sleuwerts AM, Look: MP, Yang F, Talantov D, Timmermans M, Meijer-van Geider ME, Yu. J et oi: Gerte- expressioii profiles to predict distent metastasis of lymph-node-negative primary breast cancer. Lancet 2005, 365(946 ):67I -679. van 't Veer LJ, Dai H, van de Vijve MJ, He YD, Hart A A, Mao M. Peterse HL, van der Kooy , arton M,t, Witteveen AT et ah Gene expression profiling predicts clinical outcome of breast cancer. Nature 2002, 41.5(6871 );530~536. Parker JS, Mullins M, Cheang C, Leun S, Voduc D, Vickery T, Davies S, Fauron C, He X, lln Z et ah Supervised risk predictor of breast cancer based on int rinsic subtypes. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 2009, 27(8): 1 160- 11 7. Fi^'Hpits M, Rudas M, Jakesz R, Dubsky P, Fitxai F, Singer CF, Dietze O, Greil R, JeSen A, Sevelda P et at A new molecular predictor of distant recurrence in ER-positive, HER2- negative breast cancer adds independent information to conventional clinical risk factors. Clinical cancer research : an official journal of (he American Association for Cancer Research 201 L 17{ 18):60l2-6020), immunohistochtmiistry of gene related protein products (Ring BZ« ^•Seitz RS, Beck R. Shasteen WJ, Tarr SM, Cheang MC, Yoder BJ, Budd OT, Nielsen TO, Hicks DO et ah Novel prognostic hum unohistoeheitueal biomarker panel for estrogen receptor- positive breast cancer. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 2006, 2 <^" !9):3039-3047,

Pbilippar U, Roussos ET, Oser M, Yamaguehi H, Kim HD, Giampieri S Wang Y. Goswami S, WyekofF SB, Lauffenburger DA et oh A Mens invasion isoforin potentiates EGF-induced carcinoma cell invasion and metastasis. Developmental cell 2008, Ί5(6):813~828). mass-spectrometry based protein levels (Liu Q, Sting! C, Look MP, Smid M, Braakman R8, Dc arehi T, Sieuwerts AM, Span PN. Sweep FC„ Linderholm BK et at: Comparative proteome analysis revealing an 11-protein signature for aggressive triple-negative fereasi cancer. Journal of the National Cancer Institute 2014. !06(2);dji376). Image analysis of cell -stromal interactions within the tumor (Robinson BD, Ska GL, Liu YF, Rohan TE, Gertler FB, Condeeiis JS, Jones .1(3: Tumor microenvironmeat of metastasis in human breast carcinoma; a potential prognostic marker linked to hematogenous dissemination. Clinical ca cer research : an official Journal of the American Association for Cancer Research 2009, i5(7):2433-244i)_f and various other techniques. These techniques provide insights into neoplastic cell function, however, implicit in Steven Paget' s "Seed and Soil" hypothesis is the idea that -metastasis involves interactions between tumor cells and their microenvironinent (Paget S: The Distribution of Secondary Growths in Cancer of the Breast, T e lancet 1889, l33(342 i);S? !-573). Therefore, we have explored the possibility that the tumor extracellular matrix, specifically collagen structure quantified with second harmonic generation, microscopy, may provide additional information on tumor metastatic ability.

SHG is an intrinsic optical signal in which two incomin photons scatter off of material, producing one emission photon of half the incoming wavelength (Figure I), in tumors, SHG is generated by fibrillar collagen and. is sensitive to the microscopic structure of the scattering material. Hence SHG emission directionality is sensitive to the diameter of the fibrils that are bundled into collagen fibers, as well as their spacing withi the fiber, and the disorder in their packing (Han X, Burke M, Zettel ML, Tang !\ Brown EB: Second harmonic propertie of tumor collagen: determining the structural relationship between reactive stroma and healthy stroma. Optics express 2008, 16( 3 ) ; .184 - 1859. Lacomb R, Nadiani k O, Townsend SS, Campagnola Pi: Phase Matching considerations in Second Harmonic Generation from tissues: Effects on emission directionality, conversion efficiency and obse ved morphology. Optics commimicatiom 2008, 281(7): 1823- 1832. Williams RM, Zipfel WR, Webb WW; Interpreting second-harmonic generation images of collagen Ϊ fibrils. Biophysical journal 2005, 88(2): 1377-1386). The ratio of the forward-emitted to backward- emitted SHG (where ' oward" is the direction of the incident excitation: laser) is known as the F/B ratio and is sensitive to these structural properties of collagen fibers (Figure 1 ). We have shown that the average F/B of patient biopsy samples can differentiate healthy and breast tumor tissue, and changes with tumor grade and stage (Perry SW, Schueekler JM, Burke K, Arcuri GL, Brown EB; Stromal matrix nictalloprotease~13 knockout alters Collagen 1 structure at the tumor-host interface and increases lung metastasis of C57BL/6 syngeneic ΕΘ771 mammary tumor cells. BMC cancer 2013, 13:41 1). Since SHG is an intrinsic optical signature, measurements of F/B can. be performed on typical pathology slides without additional contrast reagents. Furthermore, determination of the average F/B in a sample involves only a straightforward, automated application of pixel intensity analysis thai does not require a trained observer. Therefore F/B analysis is an attractive candidate to apply to the prediction of tumor aggressiveness. Here we show that the natural log of the F/B can predict MPS in ER+, LN breast cancer patients. In a small subset of breast cancer patients treated with tamoxifen in a recurrent setting, the .natural log of the F/B is also found to be associated with shorter PFS. We further show that F/B was related to OS in stage 1 colorectal adenocarcinoma, pointing to the possibility that collagen, structure, as reported on by F/B, and tumor metastatic capacity are linked in both tumor types.

Methods

Patient S mpt s

Three-hundred, and forty-four human breast tumor samples were used from a collection at the Erasmus Medical Center (Rotterdam, Netherlands), which, were primarily from, one breast cancer genetic expression study (Wang Y, Klyii iG, Zhang Y, Sieowerts AM, Look MP, Yang F, Talantov D, Timmermans M, eijer-van Ge!der ME, Yu J ei at: Gene-expression profiles to predict distant metastasis of !yraph-node-negative primary breast cancer. Lancet 2005, 365( 460);67I- 7 } and later supplemented by 58 additional E - samples (Yu FX, Sieuwerts AM, Zhang Y, Martens JW, Smid M, lijn JG, Wang Y, Foekens JA: Pathway analysis of gene signatures predicting metastasis of node-negative primary breast cancer. BMC cancer 2007. 7:182). These fresh-frozen tissues were initially processed for microarray analysis, and were at a later stage processed for inclusion, on a tissae-jmkroarray (TMA) in cases where formal in-fixed paraffin embedded tissues were available as well Initial sample acquisition wa approved by the medical ethics committee (number 02*953) and conducted in accordance with the code of conduct of Federation of Medical Scientific Societies in the Netherlands (www.faiwv.nl). All patients were LNN and had. not been treated with, neoadjuvant nor adjuvant therapy. This allowed far the study of the natural course of the disease and pure tumor aggressiveness, without potentially bein confounded by systemic therapy. Some patients received radiation therapy, which has been shown not to affect distant metastases (Effects of Radiotherapy and Surgery in Early Breast Cancer— An Ov rview of the Randomized Trials, New Bnghmi Journal of Medici 1995, 333(22); 1444-1456), our main focus of this study. The median patient age was 52 years. Follow- up data was recorded every 3 months for 2 years, every 6 months for years 3-5_f and every 12 months afterwards. All samples were collected in triplicate as 5 μπι thick, 0.5 mm diameter core tissue samples and mounted as TMA slides, in which the uniform tumor presence was verified by hematox lin and eosin (H&E) staining. Note that the presence of H&E staining does not affect the reported F B (Lacornb R, Nadiaraykh O. Townsend SS, Campagnola PJ: Phase Matching considerations n Second Harmonic Generation from tissues: Effects on emission directionality, conversion efficiency and observed morphology. Optics communications 2008, 281(7): 1823- 3.832) , Patients were tested for BR and progesterone receptor (Pg ) status using inu uwhistocbemisiry, where the cutoff for receptor positivky was 1 % positive tumor cells. Bloom and Richardson grade and HER2 status data were assessed as described (Liu :NQ. De March! T, TMwneramos AM, Beekhaf R, Trapman-J^'ansen AM, Foekens R, Look MP, van Deurzen CH, Span FN, Sweep PC ef at Ferritin heavy c ain in triple negative breast cancer: a favorable prognostic marker that relates to a cluster of differentiation 8 positive (CD8+) effector T-cell response. Molecular ά cellular p reomics : MCP 2014, 13(7): 1814-182?) and were available as well for the tissues included in the TMA. In total, 221 TMA-cases were eligible for analysis of F/B ratio, of which 125 were ER÷ and 96 were E -,

Stage I colorectal adenocarcinoma samples were purchased from the Yale tissue pathology (YTMA-8, New Haven Connecticut). Samples were processed, as TMA with one 5 p.m thick, 0.5 mm diameter sample per patient, unstained, from within the primary tumor. Samples were collected from 1970-1 82 with up to 31. years of follow-up data, resulting in. a total, of 69 stage Ϊ primary colorectal tumors. Lung adenocarcinoma samples were acquired at the University of Michigan, providing a total of 55 stage 1 lung adenocarcinoma cases (Beer DG, Kardia SLR, Huang C-C, Giordano Ti, Levin AM, Misek DE, Lin L, Chen G, Gharib TG» Thomas DG et at Gene- expression profiles predict survival of patieats with lung adenocarcinoma. Nature medicine 2002, 8(8 );816-824), Tissue was collected between 1 94-2000 with the consent of the patient and approval from the local Institutional Review Board, All patients underwent the same treatment, surgical resection, with tntra-thoracic nodal sampling, The lung adenocarcinoma samples were provided as a 5 urn thick section through the foil diameter of the tissue. Analysis of H&E stained samples by a trained clinical pathologist was used to ensure images were taken within the tumor proper,

.Imaging

The method for determining F/B of a thin tissue sample has bee previously described |17j, Briefly, SHG imaging was performed on an Olympus B.X61WI upright microscope. A Spectra Physics atTai Ti: Sapphi e laser (circularly polarized. 810 nm, 100 fs pulses at 80 MHz) was directed through an Olympu Ruoview FV300 scanner. This was focused through an. Olympus UMPLFL20X water-immersion lens (20^x, 0.95 NA), which subsequently captured backward propagating SHG signal. This SHG signal was separated from the excitation beam using a 670 nm dichroic mirror, filtered using a. 405 am filter (HQ405/30m-2P, Chroma, Rockingham, ^'Vermont), and collected by a photomultlplier tube (Hamamatsu HC 125-02). The forward scattered SHG was collected through an Olympus 0.9 NA condenser, reflected by a 565 am dichroic mirror (565 DCSX, Chroma, Rockingham, Vermont) to remove excitation light, filtered by a 405 nm filter (HQ405/30m-2I\ Chroma, Rockingham, VT) and captured by photomuitiplier tube (Hamamatsu 1:1025-02). During acquisition of the daily calibration sample, a dilute fluorescein isothiocyanate (f lTC) solution, a 535/40 filter (535/40m-2P, Chroma, Rockingham, VT) replaced the 405 nm filters. Forward- and backward-sea tiered SHG images were simultaneously collected as a stack of .1 1 images spaced 3 urn. apart, with a 660 um field of view, imaging conducted on TMA slides of H&E stained, 0.5 mm diameter breast cancer and colon cancer samples permitted one image stack at the center of each sample. For the larger (approximately 3 em wide) lung cancer samples, 3 locations were chosen randomly in each sample and the 3 resultant F/B values (see below) were averaged. Image Analysis

Image analysis was conducted with image.! (Schneider CA. Rasband WS, Elieesri .W: NIB Image to linage,!; 25 years of ima e analysis. Nature methods 2012, 9(?):671-675), Tissue sections were 5 μηι thick, comparable to the axial resolution of the SHG images, hence there was effectively a single layer of collagen in each sample, "auto-focused*" with a maximum intensity projection of both the forward, and backward image stacks. This produced a single image pair (forward scattered SHG "F", and backwards scattered SHG "B") for each imaged location. A maximum intensity projection of an 1 1. image scan taken with a closed microscope shutter was used to determine the background noise of the Imaging system, which was then subtracted from each image. A common threshold (40 out of a maximum possible pixel count of 4095 a.u.) was initially determined by a blinded observer viewing -30 image pairs and choosing the threshold thai best distinguished pixels within fibers from those in the background. This single threshold was applied to each image to identity pixels within fibers by creating a pair of masks (one for F, one for B), in which all. of the pixels above threshold were set to h and all of the pixels below threshold were set to zero. These masks were multiplied to create one "forward x backward mask" whose pixels were equal to i only when they were equal to 1 in both the forward and ^'backward masks. The background subtracted F and B images were divided to produce an F/B image of the sample, which was multiplied by the "forward x backward mask", and the average value of all nonzero pixels yielded the sample^'s average F/^'B (Figure 1 ).

Day-to-day variations in optical alignments were normalized by imaging a standard solution of Fi^'fC daily and applying a normalization factor for each detector pathway that rendered the signal from the standard FITC sample constant over time.

Statistic*

STATA, release 13 (ScataCorp, Texas, USA) and Prism 5 software (GraphPad, La Jo!la, CA) was used for statistical analysis, MPS was defined as the date of confimvation of a distant metastasis after symptoms reported by the patient, detection of clinical signs, or at regular fol^'low-up. OS was defined as time until death, any cause, while patients who died without evidence of disease were censored at their last follow-up time. PFS was defined as the time from start of tamoxifen treatment until a second line of treatment was needed, or until death. The relationship between the natural log. of the F/B and survival rate was assessed using the Kaplan-Meier .method and evaluated using the log-rank test for trend. Multivariate Cox proportional hazard analysis was applied to evaluate the prognostic value of the natural log of^" the F B, age. menopausal status, tumor size, tumor grade. ER, Pg and HER2 status. Differences were considered statistically significant when the 2-sided p-value was below 0.05.

Results

F/B and Relationship with Patient and Tumor Characteristics

The median natural log of the F/B of and interquartile range in ail tumors was 2.228 (0.416) (Table .!.}. The natural log of the F/B was not significantly associated wit the age or menopausal status of the patient, There were also no significant correlations with tumor size, tumor grade, and HER2 status, in contrast, compared with steroid hormone-positive tumors, F/B was higher in BR- (p < 0.001 ) and PgR -negative tumors (p - 0.003), respectively (Table I K

Metastasis-Free. Survival in Breast Cancer Patterns

Univariate analysis of the natural log of the primary tumor F/B showed no statistically significant relationship between F/B and the length of MPS (Hazard. Ratio, FIR - 0.706; 95% confidence interval, CI 0.351 - 1 ,422; p ^~ 0.330) within the combined (ER+ and ER-) sample set. Because mechanisms of breast tumor progression varies based on ER status, and because ERi- and ER- tumors are biologically very different tumors (Gruvberger S, Ringnef M, Chen Y, Panava!iy S, Saal LR. Borg A, Ferno M, Peterson C, Meter PS: Estrogen receptor status in breast cancer is associated with remarkably distinct ge»e expression patterns. Cancer research 2001, 61(16}:5979-5984. Anderson WF, Chu KC, Chatterjee N, Brawley O, Brinton LA: Tumor variants by hormone receptor expression in white patients with node-negative breast cancer from the surveillance, epidemiology, and end results database. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 2001 , .19( 1): 18-27), we then analysed the prognostic value of F/B status n ER subgroups separately. Within the ER-t subgroup, in Cox regression analysis using F/B as a continuous variable there was a statistically signi ficant relationship bet ween the na tural log of the primary tumor F/B and MPS (HR ^« 0.23; 95% CI 0.08 - 0.65: p = 0.005) (Table 2), but within the BR- population the relationship was not statistically significant (HR === 2.72; 95% CI 0.8104 - 9,173; p =- 0.1.05). The ER+, LNN patient samples were then divided into four equal quarters consisting of a high natural log of the F/B (above 2.354: Q4), a low natural log of the F/B (below 1.954; Ql), and 2 mid-range F/B categories (range 1. 54-2.168: Q2, and 2..168-2354: Q3), and plotted in a Kaplan Meier curve (Figure 2$), Patients with tumors with low F/B (Ql) showed the worst MFS, while those with high F/B (Q4) .show d the best MFS. The 2-mid range categories iQ2 and Q3) showed an intermediate MFS (lograrik trend p -~ 0.004). In Cox multivariate regression analysis for MFS in ER+- patients, corrected for the traditional prognostic factors age, menopausal status of the patient tumor size, tumor grade, PgR, and HER2 status, an increasing natural log of the F/B was significantly associated with. longer MFS (HR ^{~ '}0.16; 95% G 0.05 - 0.55; - 0.004) (Table 2).

Overall Survival in Breast Cancer Patients

Next we tested whether F/B of the primary tumor was also significantly related, to OS in the ER+, LNN group of patients. Univariate Cox regression analysis showed that the natural log of primary tumor F B was borderline statistically significantly related to OS (HR.™^: 0.34; 95% CI 0.11 - 1,03; p - 0.057). A logrank test for trend analysis of Kaplan Meier curves with F/B divided into Q1 -Q4 shows a significant .relationship between increasing natural log of the primary tumor F/B and longer OS (Figure 2b, p - 0,03). A multivariate Cox analysis of this data showed that the natural log of the F/B ratio, when corrected for traditional prognostic factors, was borderline significantly related to OS (HR = 0.28; 95% CI 0.07 -·^■ 1.1 ; p = 0.068) (Table 3).

Tamoxifen treatment

The previous studies were conducted in untreated patients in order to analyze the relationship between F/B of the primary tumor and. tumor aggressiveness and. pure prognosis. A subset of these patients did metastasize to a distant site and were then treated with tamoxifen as first-line monotherapy. Therefore we evaluated this subset of ER÷ breast cancer patients to determine whether F/B of the primary tumor was significantly related to PFS after start of therapy for recurrent disease. The hazard ratio of the natural, lo of the natural log of the primary tumor F B was 3.39 (95% C! 1.22 - 9.37: p-0.01 ) and the logrank test for trend analysis of Kaplan Meier curves in equal quarters showed a significant relationship (p ^:~ 0.02} between primary tumor F/B and PFS (Figure 3). Interestingly, the trend in PFS (i.e. lower primary tumor F/B was associated with slower disease progression) was found to be the opposite of that observed in MPS and OS in the untreated ER+ patients (i.e. lower primary tumor F/B was associated with shorter MPS and OS times).

(Jvem!! survival as β function of F/B in other solid tumor types

Based on the significant relationships revealed .in the breast cancer samples, we investigated colorectal and lung adenocarcinoma, other solid tumor types in which tumor cell/matrix interactions may significantly affect metastasis. Similar to ER+, LNN breast cancer patients, stage 1 colorectal and lung adenocarcinoma are a subset of patients where there is a clinical need to assist the physician in deciding the appropriate level of treatment for the patient, in stage Ϊ colorectal adenocarcinoma there was a significant relationship between the F B of the primary tumor and patient. OS (Figure 4a), Notably, the observed trend (he. a lower F/B was associated with longer OS) was the opposite of the trend observed in the untreated ER-h LNN breast cancer samples, suggesting a different mechanistic relationship between metastasis and collagen fiber microstracture. In contrast, stage Ϊ lung adenocarcinoma showed no significant relationship between the F/B of the primary tumor and OS (Figure 4b). This suggests that not all solid tumors undergoing metastasis elicit identical collagen restructuring or utilize identical mechanisms relating metastatic ability and collagen microstructure. identification of these mechanisms through experimentation is important in order to apply the methods of the present invention to other solid tumors.

Currently the ER+, LNN breast cancer population suffers from over-treatment as many patients receive chemotherapy even though metastatic disease never would have arisen. As such, there is a pressing need to improve clinicians' ability to predict which tumors are likely to metastasize in this population. Current, methods to predic metastasis are "cell focused", using quantification of gene and protein expression levels, or cellular morphology and cell-cell interactions (Parker JS. Mul!in M, Cheang MC, Leung S, Vodac 0, Vickery T, Davies S.. Fauron C, He » I iu Z et at Supervised risk predictor of breast cancer based on intrinsic subtypes. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 2009, 27(8): 1 160-1 167. Filipits Vl udas , Jakesz R_f Dubsky P, Fitzal F, Singer CP, DIetze O, Greil R, Mm A, Sevekia P et at A. new raoleeniar predictor of distant recurrence in ER-positive, MER2~negative breast cancer adds independent information to conventional clinical risk factors. Clinical cancer research : cm official journal of she American Asso iati n far Cancer Research 201 !. , 17{.18);6^"012-6020, Ring BZ, Seitz RS, Beck R, Shasteen WJ, Tarr SM, Cheang_. MG, Yoder BJ, Biidd GT, Nielsen TO, Flick DG et at Novel prognostic tonjunohistoefaetnieal hionmrker panel for estrogen receptor-positive breast cancer. Journal, of clinical oncology : official journal of the American Society of Clinical Oncology 2006, 24f^'1 ):3039-3047. Liu NQ, Sting! C, Look MP, Smid » Braakman RB, De Marchi T, Steuwerts AM, Span PN, Sweep FC, Lmderhol B et ah Comparative r te se analysis revealing an i i-protein signature for aggressive triple-negative breast cancer. Journal of the National Cancer institute 2014, IM(2):dji..T76) . However, the process of metastasis is a complex interplay between tumor cells and their microenvironmem_* including the extracellular .matrix. (He.llem.an L Jamen MP. Rujgrok-Riisiier K. van Staveren IL, Look MP, Meijer-van Gelder ME, Sieuwerts AM, Klija JG„ Sleijfer S, Foekens JA et ah Association of an extracellular matrix gene cluster with breast cancer prognosis and endocrine therapy response. Clinical cancer research ; n official joitrnat of the American Association or Cancer Research 2008, 14{ Γ7):5555-5564. Joyce J A, Pollard. JW: Microenvironmental regulation of metastasis. Nature reviews Cancer 2009. 9(4):239-252). Therefore we explored the prognostic abilit of a "matrix focused" measurement, the SHG F/B of the primary iumor.

Siiidies demonstrating that SBG imaging can differentiate healthy and tumor tissue in ovarian (Nadiarnykh O, LaComb RB. Brewer MA, Campagooia Rf: Alterations of the extracellular matrix in ovarian cancer studied by Second Harmonic Generation imaging microscopy. BMC cancer 2010, 10:94), basal cell )Lin SJ, lee SH, Kuo CI. Wu K Lin WC, Chen JS, Liao YI L Hsu CJ, Tsai I F, Chen YF et at Dfecrimiiiation of basal ceil carcinoma from normal dermal stroma by quantitative mnitipfeoton imaging. Optics letters 2006, 31(18):275£-275&), and pulmonary cancers (Wang CC, Li FC, Wu RJ, Hovhan isyan VA, Lin WC, Lin S.L So PT, Dong CY: Differentiation of normal and cancerous lung tissues by muitiphoton imaging. Journal of biomedical optics 2009, i4(4);044034), establish two useful aspects of SHG: it is an intrinsic signal that does not require additional processing of tissue, and, if used to quantify intensity but not morphology, the analysis is automatable and does not require a trained technician. We recently applied this methodology in breast cancer, demonstrating that the simple intensity-based SHG F/B is significantly different amongst different breast tumor types (Perry SW, Schueckler JM, Burke , Arcuri GL. Brown E : Siroiisal matrix inetaJiftpw>tease~l3 knockout alters Collagen I structure at the tumor-host interface and increases lung metastasis of C57BL/6 syngeneic E0771 mammary tumor cells. BMC cancer 2013, 13:41 1 ), in the current work, we demonstrate that F/B analysis of the primary tumor is a prognostic indicator in the BR+, LNN population. Unlike the BR- or ER+- node-positive patients, in which adjuvant chemotherapy is universally applied, the choice of whether or not to prescribe adjuvant chemotherapy for ER+, LNN patients is not easily apparent Hence this is a population with a significant over-treatment problem requiring improved prognostic indicators. Our results suggest that. SHG F B from the primary tumor specimen may offer insight into eventual metastatic outcome of the patient and thus may help reduce over- treatment. Currently, predicting ike time to metastasis in this population is primarily facilitated by histopathology and by genetic .screens. These genetic screens quantify gene expression in cells within the tumor, including both the tumor and strom l ceils. The SMG-based method demonstrated here may be highly complementary to those genetic screens, as it derives its information from the structure of the extracellular matrix in the primary tumor, rather than from the tumor cells themselves. SHG imaging has been used previously to predict breast cancer survival times, however these studie focused on analysis of morphological information .from collagen images, requiring trained pathologists to score the orientation of collagen fibers in images (Conklin MW, Ekkhoff JC, iching KM, Pehlke CA, Elieerrl KW, Provenzano PP, Fried! A, Kee!y Pi: Aligned collagen fa a prognostic signature for survival in human breast carcinoma. The American journal of pathology 20! 1 , 178(3 ): 1221 -1232), Furthermore, the majority of that sample population was lymph node positive, while our study focuses on the LNN population, in which the key decision on adjuvant chemotherapy mast be made and .tor whom the risk of over-treatment is high.

Based on the important role that tamoxifen plays as a treatment in almost all ER+ breast cancer patients, after identifying the significant relationship between the natural log of the F/B and patient outcome in untreated patients, we were interested in exploring the prognostic capability of the F/B to determine the effects of tamoxifen on patients with recurrent tumors. Our results revealed thai the F/B as measured on the primary tumor was prognostic of PFS after patients who developed a metastasis at a distant site were treated with tamoxifen. Interestingly, the actual relationship between F B and outcome displayed a trend that was opposite to that in the MPS and OS findings from untreated. ER+ patients: in tamoxifen treated recurrent ER+ patients a high F/B was associated with a fester rate of progression, whereas in untreated F.R - ^•patients a high F/B was associated with improved MFS and OS. Tamoxifen is an ER antagonist, indicating this contrast between tamoxifen treated ER.+ tumors and untreated ER+- tumors could be due to the roles of ER in tumor progression. To explain this pattern of relationships between recurrence and F/B in ER-*- tamoxifen treated tumors, as opposed to untreated ER+- tumors, we therefore hypothesise that differences in primary tumor collagen microstructure may indicate differences in the mechanism by which tumor cells spread, which has the effect of altering susceptibility to later treatment, in an ER+ primary tumor with a low F B, cells spread into vasculature and to secondary locations, and upon tamoxifen administration these secondary tumors are effectively treated. In an ER+ primary tumor with a high F/B ratio, tumor cells metastasize via different mechanisms which decrease the tumor eel! sensitivity to tamoxifen treatment.

The results demonstrating another significant relationship between. F/B of the primary tumor and OS, in stage I colorectal adenocarcinoma, indicate that the mechanisms relating metastasis to collagen microstructure may be similar between breast cancer and other solid tumors. Analyzing collagen structure in colorectal adenocarcinomas may thus aid in predicting the OS rates in patients, consequently helping t determine which patients may be able to avoid over-treatment with chemotherapy in that tumor type as well. The fact thai the primary tumor F/B was not predictive of metastasis in stage I lung adenocarcinoma provides support for the idea that multiple mechanisms of tumor metastasis may exist, involving differential .interplay between tumor cells and matrix microstructure. These alternative mechanisms could be the result of different levels of fibrous tissue in the tissues of origin, (e.g. collagen density is high in breast and colon but not in lung tissue), in the future it may therefore be beneficial to investigate the relationship between primary tumor F/B and metastatic outcome in other solid tumors thai are typically characterized as more fibrous, such as pancreatic cancer.

In summary, we have identified the F/B, a simple and. easily automated, intensity-based measurement as an independent prognostic indicator of metastatic outcome in E + LNN breast cancer patients. Furthermore, escaped tumor cells with a low F/B at the primary site show a better responsiveness to tamoxifen treatment of the recurrence, indicating a possible mechanism by which collage structure at the primary site affects sensitivity to treatment. The primary tumor F/B is also prognostic in stage Ϊ colon adenocarcinoma, suggesting this assay may be useful in multiple types of solid tumors, By imaging the tumor "soil" this method, provides information complementary to that offered by current cdl-focrtsed techniques, and therefore in combination with those methods may improve prediction of recurrence and hence reduce over- treatment.

Table I; Log F/IJ ratio and association with breast cancer patient and tumor characteristics

C'hsrscterisifcs ediae levels (iHters srMisrasHfie

AS paifeats 221 ooo%) 2..2S(0.4i«)

A«s vea rs) 0.773"

≤40 B(H.9%)

41 · SS 9 {42.5%) 2.215(0.410)

SS - 78 7001.7¼) 2.29Ϊ (0.4561

?δ 24(10.9%) 2.198(0.32?)

0,4S7^r

Premeaopaasa! 113(51.1%) 2.200(0.44?)

oas3.i*it«l¾5.ti»sl t S(4S.9%) 2.250(0.379)

Tuawr size o.isr ρΎί (≤2 cm) 10§(4§.3 ) 2.239 (O.SSis)

pT2a - 5 cm) 105 (47.3%) 2..237 (0.505⁾

Sl¾«MC>5 em) 7 (3.2%) 1..8.30(0 4\

I S7(!6.?%) 2.20? (0.2.88)

E V 04,§¾) 2.233 φ.366)

ill 102 (45.7%) 2.264(0.491) ι:,·> : 6.^<>%· 2.I6S, (0.407)

96(45,4%) 2 JU (8.452)

0.003'

104(47.3%) 2.i59 (0.392⁾

! I? (52.9%) 2.302(0,425)

HEE2 states 0A2V

Positive- 26(!i.8%) 2..2S C0.3SS)

195 (SS .2%) 2.215(0.432)

* jruskaJ-WaHis test f Two-sample Wilcoxon rank-sum (Mann- Whitney) test ScarfT-Bioom-Richardson grade (6 missing values) Fable 2: Cox univariate and multivariate regression analysis for MFS in .125 E * patients t^'g)Y»riafe asafe-jtis ;vi»¾¾^~a-ria i¾ a aaiy sis*

Vatiatte HK 95% Ci 9 HK 95% CI

4S · 33 s40 «3fs o.ss .3? - £. 2 «70S o.so 0.500

53 - 70 30 yssrs 050 0.23- = 25 G..SS 0.53- ϊ.^'ί^ «340

V? S 45 -.,:ti 0.4S « :.^■ ;.:^:o * ··.<; 0.08-1.27 0.105:

MftROjsa usa 5 sia t*s

v -«s ess-!.?;? 0.33S 2.40 8.58?

ΪΒΒΚ»!· sts*

2 - 5 VS 2 i.n O.SS-S.K C.OJS o,s5 - 1.70 0.550

?i VS¾2 £t!3 l i 0.36 -6.SS 0.579 0.30 «30-2.32 0.3 se

TuBKis- gyaite

3.SS ¾-7,0$ 0.0 ί 27?ί x.i0-5.¾ 0,050

HtrsS 4,3S i.SS - 55.43 8.003 i ; Γ · ^:; 02 ο.δ 7

Ϊ¾.Κ. status

07i. C - ! ίί 0.207 o.<u δ. SO - i .3:4 0' 530

ΪΕΕΪίϊ status

4,55 L?S - ϋ,6 3.07 i 1 ο·?2 «,00* trig off fB ratio 0,23 0, ¾-0.65 0.00s o 56 0.00-0.35 0.304 multivariate model included 123 patients due to 2 missing values for tumor grade.

Table 3: Cox univariate and multivariate regression analysis for OS in 125 ER+ patients

CffivaHaf* aaaivsis MHSi ai-isS* satstysis*

49vsars 04? 0.25- !,;? O.iOS Ο.ίίί 3.23· ϊ.ίΐ: 0.2s? »<5-?Svs<S >'«srs S.S7 024-5.33 0.2 4 0,3? «.«?^■- 0.5¾ 0,035 > 20 vs 3 vests «33 5.:3* 3J 0.20 3.03 -e.S; 0.043

IM 0.3; ^,3 ·: 2333 2.5¾> 5.00 - 34v Ο.0Ϊ2

S 7,3 - m~2.il 0,49 0.SS 3.25-5.255 0. S3" 5.65 0 9 --l.il 0,45)2 3.5s.¾:72 0-720

2.53 5,02 0,044 2. SO ^■isi.: >:^■ 0-5. Π 502 5.S3 0.005 4.33 !.ft4-54¾-S 0.004

0,26 - S.01 0,033 0,48 «,23- 5,03 0.053

3.5? 5. Π - 3,50 O.S35 3. S3 5.5.3- 52.2.0 0.025

0.54 0.Π-Ϊ.0} 0,003 0.20 0.0?. ϊ.ΗΪ 0.0<SO

*The multivariate model included 123 patients due to 2 missing values for tumor grade. Figure 1 depicts methodology diagrams, where A. is a depiction of the forward- and backward- propagating SHG signal. Red excitation light is focused into the sample by objective lens 1» then SHG is emitted in the backwards direction (towards lens 1) or the forward direction

(towards lens 2), B. A flowchart of the methodology used to analyze SHG images and calculate the F B ratio. C. An F/B image of one patient, sample. Scale bar is 50 microns.

Figure 2 depicts Metastasis-free (a) and overall survival (b) as a function of F/B in ER r. LNN breast cancer. The patients are divided in four equal quarters (Q1-Q4) based on their F B tumor level. Patients at risk at various lime points are indicated.

Figure 3 depicts progression-free survival as a function of F/B in ER+ recurrent breast cancer patients treated with tamoxifen. The patients are divided in four equal quarters (Q1-Q ) based on their F/B tumor level. Patients at risk, at various time points are indicated.

Figure 4 depicts overall survival of additional solid tumors as a function of F/B ratio. Overall, survival in stage I colorectal adenocarcinoma (a) is significantly related to F/B of the primary tumor (p^∞ 0,03). F/B of Stage I lung adenocarcinoma, is not significantly related to OS (p^∞

0.53). The blue line is Group 1 has the lowest F/B and the brown line is Group 4 has the highest F/B ratio. Patients at. risk at various time points are indicated.

EXAMPLE 2- PREDICTION OF FATAL LUNG FIBROSIS

Rationale: it is not understood why some pulmonary fibroses such as cryptogenic organising pneumonia (COP) respond well to treatment, while others like usual interstitial pneumonia (UIP) (which may also be referred to as idiopathic Pulmonary Fibrosis (IPS ) are essentially equivalent to each other) do not, UIP and IPF being essentially equivalent for the purposes of the disclosure provided herein. Increased understanding of the structure and function of the matrix in this area is critical to improving our understanding of the biology of these diseases and developing novel therapies. The ability to differentiate between lung fibroses that respond well to therapies and others that are intractable using the methods of the present invention as described herein has profound implications for clinical approaches to trcatirient and patient care.

Objectives: Provide new insights into the underlying collagen- and matrix-related biological mechanisms driving COP versus UIP,

Methods: Two-photon second harmonic generation (SHG) and excitation fluorescence microscopies were used to interrogate and quantify differences between intrinsic fibrillar collagen and elasiin matrix signals in healthy, COP, and UIP long.

Measurements and Main Results: Collagen rmcrostructure was different in UIP versus healthy lung, but not in COP versus healthy, as indicated by the ratio of forward-to-backward propagating SHG signal (PSHO BSHO). This collagen microstruciure as assessed by FSHG BSHO was also different in areas with preserved alveolar architecture adjacent to LHP fihrotic lesions versus healthy lung. Fibrosis was. evidenced by increased coll and col3 content, .in COP and UIP versus healthy, with highest coll ;col3 ratio in UIP. Evidence of elasiin breakdown (i.e. reduced mature elastin fiber content), and increased coHagen:mature elasiin ratios, were seen in COP and UIP versus healthy.

Conclusions: Fibrillar coliaget s suhresolution structure (i.e. "mier structure") is altered in UIP versus COP and healthy lung, which may provide novel insights into the biological reasons why unlike COP, UIP is resistant to therapies, and demonstrates the ability o SHG microscopy to potentially distinguish treatable versus intractable pulmonary fibroses. INTRODUCTION

Pulmonary fibrosis is characterized by accumulation of extracellular matrix (ECM) proteins in lung tissue. The mechanisms leading to pathologic (or non pathologic) accumulation and organization of matrix proteins remain poorly understood. Although we have some insight into the composition., structure and/or organization of the matrix, many properties of the matrix remain uninvestigated. Numerous matrix proteins likely contribute to organ dysfunction in pulmonary .fibrosis, however, we are only beginning to understand how homeostasis and organization of these proteins impact cellular 'function.

Collagen, produced and organized mainly by fibroblasts and sear-forming myofibroblasts, is one of the most abundantly studied matrix proteins, At least twenty-eight different collagen subtypes have been described to date. All collagen species contain three alpha peptide sequences forming a triple helix. Collagen, type is determined by the type(s) of alpha peptides and post translations! modifications, hydroxy lation, and/or glycosylation. Further modification of collagen, structure occurs after release into the extracellular space. Here, crosslinking and joining of the helices occur to form collagen fibrils and larger collagen fibers, and fibrosis (aberrant excess deposition of collagen) may occur. The fibril-forming collagens include collagen types 1 -3, 5. 1 1, 24, and 2? ί Shoulders MD, Raines RT. Collage structure and stability. A m Rev Biochem 2009;78:929-958,), and at least several, of these fibrillar collagens (PCs) such as types 1, ill, and V are key players in lung fibroses including usual, interstitial pneumonia (UIP) and. cryptogenic organizing pneumonia (COP) ( Cordier JF, Cryptogenic organising pneumonia. The European respiratory journal : official journal of ihe European Society jbr Clinical Respiratory Physiology 2006;28:422-446. Cottin. V, Cordier JF. Cryptogenic organizing pneumonia. Seminar^'s in respiratory and critical care medicine 2012;33:462-475. Parra E , Teodoro W , Veiosa AP, de Oliveira CC Yoshinari Nil,. Capelozzi YL-. interstitial and vascular type v collagen morphologic disorganization in usual interstitial pneumonia. The journal of histochemistry and cytochemistry : official journal of the Histochemistry Soeiety 2006;54:1315-1325. Parra ER. Kairalla RA, de Carvalho CR, Capeloszi VL, Abnormal deposition of eollagen elastic vascular fibres and prognostic significance in idiopathic interstitial pneumonias. Thorax 2007;62:428-437.). These FCs are also uniquely detectable by Second Harmonic Generation (SHG) Microscopy (SHGM) (details below). Pulmonary fibrosis results from accumulation of fibroblasts, scar-forming myofibroblasts, and extracellular matrix proteins including collagen, and leads to irreversible loss of lung function. It. can be caused by various factors including toxins, radiation exposure, autoimmune disorders, and infection. Idiopathic Pulmonary Fibrosis (IPF) is a severe form, of fibrotie long disease that can progress to respiratory failure and has a prognosis worse than lung cancer, ί h re are currently few effective therapies, U.I.P is the histopathology underlying IPF and is characterized by heterogeneity of disease and accumulation of fibroblast foci and collagen with an emphasis on collagen type I (coll) over type 131 (co!3) (Cordier JF. Cryptogenic organising pneumonia. The European respiratory journal ; official journal of the European Society for Clinical Respiratory* Physiology 2006;28:422-446. Parra ER, Teodoro W , Velosa AP„ de Oliveira CC,. Yoshinari NH, Capelozzi VL. Interstitial and vascular type v collagen morphologic disorganization in usual interstitial pneumonia. The journal ofhistochemtetry and cytochemistry : official journal of the Histochemistry Society 2006;54:1315-1325.) ,and abnormalities in. other matrix molecules including elastin (Parra ER, airalla RA, de Carvalho CR. Capelom VL. Abnormal deposition of collagen/elastic vascular fibres and prognostic significance in idiopathic interstitial pneumonias. Thorax 2007;62:428-437).

IPF is one of many diseases associated with significant collagen and other matrix protein accumulation. It is die most common of the idiopathic interstitial pneumonias, is increasing in prevalence, and it is a progressive disease that causes significant morbidity and mortality, The median duration of survival from the time of diagnosis is only 2.9 years ( Nadrous HF, Ryu JH, Douglas WW, Decker PA, Olson EJ. Impact of angiotensin-eonvertmg enzyme inhibitors and statins on survival in idiopathic pulmonary fibrosis. Chest 2004;! 26:438- 446.). There are currently few effective FDA approved treatments for IPF, making research into IFF pathogenesis critical.

COP is another of the more common types of fibrotie lung diseases. It is also characterized by accumulation of matrix component resulting in organized areas of granulation tissue in the lung. Components of this pathologic matrix accumulation in COP include coil and col3 (with an emphasis on eoI3 over col l, in contrast to DIP), fibroneciin, and proteoglycan (Cordier JF. Cryptogenic organising pneumonia. The European respiratory journal : official journal of the European Society for Clinical Respiratory Physiology 2006:28 :422-446. Cottin V'.. Cordier JF. Cryptogenic organizing pneumonia. Seminars in respiratory and critical care medicine 2012;33:462-475), in stark contrast to U1P, COP is a treatable disease with most cases responding to corticosteroids. Although the mains components of U.IP and COP have some similarities, it is unknown why the excess matrix in COP cart be reabsorbed or cleared after treatment with corticosteroids while the matrix in UIP is resistant to treatment and resolution (Cordier JF. Cryptogenic organising pneumonia. The European respiratory journal : official journal of the European Society for Clinical Respiratory Physiology 2006;28:422-446).

A growing body of literature supports the roles of matri organization and structure as important effectors of fihrotic lung disease. EC-MI components have important mechanobiological properties including the abilities to activate pro-fibro ic cytokines; regulate cell trafficking; and modulate cell activation, proliferation, survival and differentiation (Tschufnperlin DJ, Boudreault F, Liu P. Recent advances and new opportunities in lung, mechanobioiogy. J Biomech 2010;43:99-107. Tsc-humperlin DJ, Liu F, Tager AM. Biomechanical regulation of mesenchymal cell function. Current opinion i rheumatology 2013;25:92-100) The organization and structure of the ECM, including collagen, also helps regulate availability of and interactions with a large variety of cell-matrix binding sites critical for controlling lung function. These findings further reinforce the notion that in biology, structure is a key determinant of function. Indeed, other data suggests that ECM stiffness regulates key cellular activities thai may contribute to IPF (Marinkovic A, Liu F, Tschumperlin DJ. Matrices of physiologic stiffness potently inactivate idiopathic pulmonary fibrosis fibroblasts. Am J Respir Cell Mol Biol 2013;48:422-430), as well as endogenous lung function (Suki B, Stamenovic LX Hubmayr R. Lung parenchymal mechanics. Comprehensive Physiology 2011 ;1 :1317-1351). Hence, there is heightened interest in the content and structure of the matrix, and how abnormal content and structure may impact lung pathophysiology, f r these reasons, we hypothesized that differences in ECM structure, and collagen microstructure in particular, underlie the different natural histories, prognoses, and responses to treatment of UIP and COS⁵.

To explore this question, we used SHGM to compare the matrix of UIP and COP to that of healthy lung tissue, SHGM is a variant of 2 photon (2P) microscopy that delects the PCs without exogenous labels, and can be used to interrogate changes in collagen's maerostruetural properties (e.g. collagen, fiber density, arrangement, and organization), as well as collagen' s subresolution raierostructurai properties (e.g. the diameter, order versus disorder, and/or packing density of collagen fibrils within larger collagen fibers) (reference Perr SW, Burke RM, Brown EB. Two-photon and second harmonic microscopy in clinical and translationai cfiiicer research. Annals of Biomedical Engineering 20.12:40:277-291 , Perry SW, Han X, Brown EB. Second harmonic generation in tumors: Scattering and polarization, in: Pavone FS, Cam ag.no la PJ_> editors. Second harmonic generation imaging, London, Ό : Taylor and Francis; 2012. Perry SW, Sehiteckfer J ₅ Burke , Arcuri GL, Brown EB. Stromal matrix metaHoproiease-13 knockout alters collagen i structure at the tumor-host interface and increases lung metastasis of c57bl/6 syngeneic e077J mammary tumor cells. BMC Cancer 2013;13:4.l 1 , Han X, Burke RM, Zette! ML, Tang P, Brown EB. Second harmonic properties of rumor collagen: [ erminmg the structural relationship between reactive stroma and healthy stroma. Opt Express 2008; 16: 1.846-1859. Laeorab R, Nadia ykh O, Townsend SS. Campagnola Pi. Phase matching considerations in second harmonic generation from tissues: Effects on emission directionality, conversion efficiency and observed morphology. Opt Commm 2008:281 : 1823- 1832.. Mem J, Moreaux L. Second harmonic generation by focused excitation of inhomogeneously distributed scatterers. Optics Communications 2001 ;196:325-330).

These rakrostractural features of individual, collagen, fibers, as they can influence SHO directionality from that fiber (i.e. ½ί.;Λ½-κ^ defined below), are herein collectively referred to as collagen "mi^'crosuwture ' In this aspect, SHGM is unique- in its ability to interrogate suhresohakm structure of FCs (e.g. coil and col 3) in intact and potentially live samples without exogenous labels, abilities which also tnake SHGM an attractive potential clinical and investigational diagnostic tool. Thus this technique can utilize intrinsic properties of matrix components to characterize the content and organization of the ECM. in these fibrotic lung diseases.

Using SHGM, herein we describe important differences in matrix content and organization in UiP/lPF and COP compared to healthy Sung tissue. Specifically, we found differences in collagcn's subresolutkm. structural properties in U1P compared to COP and healthy lung as assessed b SHGM. importantly, even adjacent normal USP tissue exhibited these diilerences in collagen microfracture compared to healthy lung, thus introducing the compelling possibilities that altered collagen mierostrocture might lead to or correlate with fibrosis in the relatively intractable disease 10 P, but not in the more treatable COP. We also report different coll :col3 ratios in UIP versu COP and healthy lung tissue, and other evidence suggests that altered cot1 :col3 ratios can drive (or perhaps be driven by) changes in FC microstructure such as fibril diameter. Finally, we show both UIP and COP have differences in mature elastm fiber content, and elast rxollagen ratio, suggesting thai both fibrotic disease have identifying physiological differences in matrix structure suggestive of lung disease, but only the less tractable disease, OTP, exhibits differences in underlying collagen microstracture. These results are important, because they provide new insights into the potential biological and biostriiciural underpinnings of refractory versus "treatable" lun fibroses, with an emphasis on subresohftion collagen microsiructure, and demonstrate the potential of the various methods described herein as a powerful new too! for aiding in the diagnosis and treatment of !ung fibrosis,

METHODS

Histology and Tmmunohistoeheinistry: Paraffin embedded human lung tissue sections were obtained from the Department of Pathology using an RSRB approved protocol after pathological confirmation of either UIP or organizing pneumonia. Healthy lung tissue specimens were obtained from non-smoker subjects who had a lung biopsy for a lesion that was confirmed either benign or not primary lung cancer, from regions adjacent to the lesions that did not contain any portion of the. lesion. Hematoxylin-eosin (H&E) staining and immunohistochemistry for coll and coB were performed as previously described (Perry SW, Sehueckler JM, Burke K, Arcuri GL, Brown EB. Stromal matrix metalloprotease-1.3 knockout alters collagen i structure at the tumor-host interface and increases lung metastasis of c57bl/6 syngeneic e0771 mammary tumor cells. BMC Cancer 2013;.l 3:4.1 1),

SHG Microscopy: Paraftln embedded human lung tissue sections for healthy, UIP and COP were prepared as described above, sectioned, then imaged for forward (F_SH_G) and backward (Bs_Hfj) SHG signals; coll and col3 knffiunofluorescenee (IF); and eiasti aiiiofiuoreseenee (AF), using a custom-built multi-photon microscope as previously described (Perry SW, Sehueckler JM, Burke , Arcuri GL, Brown. EB. Stromal matrix metailoproiease-13 knockout alters collagen i structure at the tumor-host interface and increases lung metastasis of c57bl 6 syngeneic e07?l mammary tumor cells. BMC Cancer 2013;i3:4 I 1), Images were captured, then F B SHG .ratio

data, coll and col3 IF, or eiastin AF, analyzed and quantified with image! as previously described..

Statistical Analyses: All data are expressed as means +/- SEM. A one way A OVA was used to establish statistical significance using Graph Pad Prism software. Results were considered significant. if/7 < 0.05. RESULTS

Fibrillar collagen microstructure in th ECM is different in UIP, tut not COP, versus healthy limg,

SHG in general is sensitive to changes in collagen micr tmciw including .regularity or ordering of collagen fibrils within larger collagen fibers; fibril compaction; and fibril diameter, tilt angle, or pitch angle, SHG is emitted both forwards and backwards (i.e. epi-directed) from the SHG-generating scatterers in the focal volume, and the FSHG<'¾HG ratio In particular is primarily sensitive to the spatial extent of SHG-generating scatterers along the optical axis, i.e. the effective diameter or packing arrangement/densit /order versus disorder of collagen fibrils within the SHG focal volume. Therefore_* to determine if a relatively intractable lung fibrosis such as UIP has a. different underlying FC microstructure in the ECM versus a treatable lung fibrosis such as COP, or versus healthy lung, we used SHGM. to interrogate the mean. FsMc/BsHCi ratio in the ECM of UIP, COP, and healthy lung tissues, Intriguingly, we found this •FSHO/BSHO tatio was significantly decreased UIP versus healthy lung, but unchanged in COP versus health l ung (Figure 5),

Figure 6 shows representative H&E staining (2A~C) matched to the same fields of view for P_$tfo (2I5-P) for healthy, COP. and UIP respectively, and illustrates that die SHG signal (white pixels, 2D-F} quantified from these lung tissues arises as expected chiefly from small airways (yellow arrows) and parenchymal alveolar space in healthy lung (2A/.D), and from ^•fibrotic collagen deposition (blue arrows) in COP (2B/E) and UIP (2C F).

Together these results show that FC microstructure is altered in LHP but not in COP versus healthy lung.

Lung tissue with preserved alveolar architecture adjacent to UlPfihmtic lesions hits different fibrillar collagen microstructure versus healthy lung

Next, we wondered whether lung tissue adjacent to UIP fibrotic lesions with preserved alveolar architecture also had different FC microstructure versus healthy lung as measured by Fs_Hc BsHij.. which might suggest the possibility of underlying collagen structural deficits that could predict or predispose development of UIP. Indeed, both fibrotic lesions and surrounding normal appearing lung tissue showed, differences in F¾ BSHG ratio versus healthy lung tissue (Figure 7), These results provide an exciting, previously unreported "'first glance" into the biologic underpinnings of UIP as it relates to FC mieroStiructure, and suggest the possibility that pre-existing alterations in PC microstructure even in "normal" hmg tissue may foreshadow or precipitate d velapment of UIP.

C lli Col3, a i Cofl/CoB ratio differences in UIP versus COP and healthy lung

Coll and co are implicated in the pathology of UIP and COP, and as fibrotie diseases, col l and col3 levels in UIP and COP are anticipated to be higher compared to healthy lung. Moreover, previous reports have suggested thai coll is the primar collagen deposited in DI P, whereas coB assumes this role in COP (Cordier JF. Cryptogenic organising pneumonia. The European respiratory journal : official journal of the European Society for Clinical Respiratory Physiology 2006;28:422-446). Importantly^ relative coll and coB expression levels can interact to regulate aspects of collagen microstructure such as collagen fibril or fiber diameter (Fleischmajer K, Perlish JS. Burgeson RE, Shaikh-Bahai F. Timpl R, Type i and type ill collagen interactions during fibrillogenesis, Ann M Y Acad Set 1990;580:161-175, Romanic AM, Adachi E, adler E, Hojima Y, Prockop DJ. Copolymerization of pncollagen Mi and collagen i. Pncollagen iii decreases the rate of incorporation of collagen i into fibrils, the amount of collagen i incorporated, and the diameter of the fibrils formed. The Journal of biological chemistry 1 91 ;266: 12703- 12709. Cameron G.1, Alberts 1L, Laing I f Wess TJ. Structure of type i and type iii heterotypic collagen fibrils: An x-ray diffraction study. J Struct Biol 2002;137:15-22).

Conversely, by altering availability of fibroblast (o other effector cell type) binding sties on collagen fibrils, changes in collagen" s subreso!ution fibril .microstructure may regulate relative collagen expression levels. Therefore, we wished to determine how changes in FS_HG BS_HO ratio (Figure 5), indicati ve of altered collagen microstructure in lung ECM, correspond with changes in coll co!3 deposition in U I P. COP, and healthy lung.

We found higher coll levels in both UI and COP compared to healthy lung, with UIP showing the highest coll levels versus COP and health (Figure 8A). Both UIP and COP had similarly ekvated col3 Ievek versus healthy lung (Figure SB), Overall, this resulted in relative coil:col3 ratios thai were significantly elevated in UIP versus healthy controls, but not in COP versus healthy controls (Figure EC). Figure 8D-F illustrate higher Co!3 levels in COP (4E) and UIP (4F) versus health (4D), as is shown ia 4B. Together, these results demonstrate the expected evidence of fibrosis in both UIP and COP compared to healthy lung controls, and confirm previous observations of higher relative coU :col3 deposition in UIP. versus more abundant coB over col deposition in COP (Cornier JP. Cryptogenic organising pneumonia. The European respiratory journal : official journal of the European Society for Clinical Respiratory Physiology 2006;28:422-446). These new results are particularly intriguing because UIP shows both a difference in FSHG/BSHG (i.e. PC microfracture) (Figure. 5) and a difference in coll ;eol3 ratio (Figure 8C) versus healthy lung, whereas COP shows neither a difference in FSHO BSHG « .r coll ;eol3 ratio versus healthy lung, Together, these results suggest a possible relationship between PC mierostructure differences and altered coll :co!3 ratios in intractable UIP fibrosis, but not in the more treatment responsive COP fibrosis,

Elmtitt and Elastit Collagen ratios differ in UIP and COP versus health)' lung in parallel with SHGM imaging, intrinsic tissue autofluorescence representing principally mature lung elastin can be captured simultaneously with SOG (Zipfei WR» Williams RM, Christie , Nikitin AY, Hyman BT, Webb W W. Live tissue intrinsic emission microscopy using niultiphoton-exciled native fluorescence and second harmonic generation. F roc Nad Acad Set U S A 2003;100:7075-7080), to provide additional insights into how ECM structure and organization may differ in UIP versus COP. Elastin is another lung ECM component that interacts closely with collagen to regulate lung function (Abraham T, Hirota J A, Wadsworth S, Knight DA. Minimally invasive moliiphoton and harmonic generation imaging of extracellular matrix structures in lung airway and related diseases. Pulm Pharmacol Ther 201 1 ;24:487-4%, Faffe DS, Zin WA, Lung parenchymal -mechanics in. health and disease. Physiol Rev 2009;89:759-775, Mijat!ovich SM, Stamenovie IX Fredberg J J. Toward a kinetic theory of connective tissue microraechanics, J Appl Physiol .1993;74:665-681. Yuan H. ingenito EP_> Suki B. Dynamic properties of lung parenchyma: Mechanical contributions of fiber network and interstitial cells. / Appl Physio! 1 97;83:1420-1431; discussion 1418-1429), and Is frequently dysreguiated in fibrotic lung diseases (Blaauboer MB. Boeijen FR, Emson CL, Turner SM, Zandieh-Doulabi I Hanemaaijer R, Smit Tii Stoop R. Everts V. Extracellular matrix proteins: A. positive feedback loop in lung fibrosis? Matrix Biol 2013. Pierce RA, Mariani TJ, Senior RM. Elastin in lung development and disease. Ciba Foundation symposium 1 95:192:199-212; discussion 212-1.94). Elastin ^*s intrinsic autofluorescence captured by two- photon excitation fluorescence (TPEF) microscopy arises from the pyridoxine-based pyridolamine cross-links (Zipfei WR, Williams RM, Christie R, Nikitin AY, Hyman BT, Webb WW. Live tissue intrinsic emission microscopy using raultiphoton-excited native fluorescence a»d second harmonic generation. Prac Natl Acad Scl U A 2003:100:7075-7080, Deyl Z, Macek K, Adam M, Vaneikova O. Studies on the chemical nature of elastin fluorescence; Bi chim Biophys Acta 1980;625:248-254) found only in mature elastin fibers Luisetti , Ma S, iadarola P, Stone PJ_} Viglio S, Casado B, Lin YY. Snider GL, Turino GM. Desmosine as a biomarkcr of elastin degradation in copd: Current status and future directions, Eur Respir J 2008;32:1146-1 157), thus making TPEF of elastin a useful indicator for the mature elastin fiber content of lung tissue. Therefore, we captured this signal for the same healthy, UIP, and COP specimens, then quantified and expressed it both by itself and relative to the total FC signal tie. total SHG signal, or FSHG + SHGJ, to see whether there were other underlying differences in ECM structure or organization that we could identify and quantify by SHGM and two-photon excitation fluorescence (TPEF) microscopy. Using this methodology, total mature elastin signal was similarly decreased in both UIP and COP compared to healthy lung tissue (Figure 9/Y), and FCimature elastin ratios (Fi ure B) were similarly increased in UIP and COP compared to healthy. However, in neither of these parameters was UIP different from COP. Panels 5C-D illustrate the lower FCrmature elastin ratio seen .in healthy versus UIP respectively.

These data demonstrate that compared to healthy lung, both fibrotic l ng diseases (UI P and COP) are characterized, by significant gross physiologic disruptions in ECM structure and organization that can be quantified with non-invasive and non-tissue destructive combined SHG and TPEF microscopy. Yet only the more intractable UIP fibrosis shows evidence of disrupted FC microstructure as interrogated by F_SH«/BS_HG. thus highlighting the compelling possibility that logetker them techniques may help make clinical distinctions between intractable and treatable hmg fibroses.

We use SHGM imaging to identify key differences in the ECM of UIP compared to COP and healthy control lung tissue. UIP and COP were chosen because they are both characterized by increases in matrix proteins, particularly PCs, yet they have contrasting natural histories, responses to corticosteroids, and prognoses. The reasons why UIP is progressive and difficult to treat are not clear. One possible explanation is that there may be a fundamental difference in c llagen's content, structure, and/or organization in the UIP ECM that renders collagen more structurally more resistant to degradation in UIP versus COP. We tested this hypothesis using SHGM, a microscopy approach that is sensitive to the intrinsic FC organization and microslruoture within the matrix, to co.ai1.rm whether FC in UIP has diffcrent krostruetural properties versus COP or healthy lung.

Using this approach, we have demonstrated for the first time that FC micmstructure in the ECM of DIP i significantly different from FC microfracture in either CHIP or healthy control lung tissue, as evidenced by the F$H»/8.S.HG ratio. Changes in this Ρ¾ΗΟ.Λ½50 ratio suggest that there is a significant difference in the density, structure, and/or organization of FC in UIP compared to COP and healthy king tissue, particularly with regard to the effective diameter or packing arrangement/density of collagen fibrils in the ECM. Further defining the exact nature of these "collagen microstructure" differences vvi.ll be an important goal of further studies, but these results are compelling by themselves because while previous studies have shown expression changes in several collagen subtypes in Sbroiic lung diseases, to our knowledge this is the first report of abnormalities of ECM microstructure - and FC microstructore in particular - in. U!P. Still more compelling is the fact that only the intractable fibrosis (UIP) demonstrated significant differences in FC -microstructure versus healthy lung, whereas the -treatable fibrosis (COP) did not, thus- providing we believe the first evidence that alterations in coilagerfs fundamental underlying structure may contribute to whether or not pul.mo.nary fibroses are treatment responsive. These results provide previously unavailable insights into the biological underpinnings of treatment-resistant pulmonary fibrosis, and also highlight the potential of SHGM as a novel clinical diagnostic and investigational tool for distinguishing between intractable and treatable lung fibroses.

We also found that lung tissue with preserved alveolar architecture adjacent to UIP fihrotie lesions has different FC microstructore than healthy lung, suggesting the possibility that- pre-existing alterations in FC structur even in ''normal" lung tissue may foreshadow or precipitate (or at minimum, associate with) development of UIP. As expected, both coll and col3 were elevated in UIP and COP versus healthy lung, with coll deposition being predominant to col3 in UIP, and vice-versa in COP, as has been previously reported (Cordier JF, Cryptogenic organising pneumonia. The European respiratory journal : official journal of the European Society for Clinical Respiratory Physiology 2006;28:422-446), These results are significant in the context of our other results reported herein because it is known that changes in FC ratios, particularly coll:eol3 ratios, plays a. significant, role in regulating collagen fibril diameter (one component of collagen microstructure) (FSeisclunajer R. Perlish JS, Burgeson RE, Shai i-Bahai F, Timpl R. ^'Type i and type Hi collagen interactions during fibril.logers.esis. Ann N Y Acad Sci 990:580:161-175. Romanic AM, Adacbi E, Kadler KE, Hojima. Y, Prockop DJ, Copolymerization of pncollagen iii and collagen i. Pncollagen iii decreases the rate of .incorporation of collagen i into fibrils, the amount of collagen i incorporated, and the diameter of the fibrils formed. The Journal of biological chemistry 1 91 ;266:12703-12709. Cameron Gl Alberts IL, Laing M, Wess TJ. Structure of type i and type iii heterotypic collagen fibrils: An x-ray diffraction study. J Struct .Biol 2002;137:15-22). Similarly, by regulating the availability of fibroblast (or other effector cell type) binding sites on collagen fibrils, changes in collagen's subresoltit on fibril mierostructure could in turn control relative levels of FC expression, In other words, different colEeoB ratios may in turn drive or be driven by altered collagen microstrueture in U1P. Together with the earlier data, these results demonstrate that, the ECM of UIP not only contains more collagen (particularly more coll ) than the ECM of COP and/or healthy lung tissue, but also that there are significant .differences in. the subresoltition microstrueture (i.e. diameter, density, and/or organization) of these collagen fibrils in UIP versus COP and healthy, independent of the absolute amount of collagen deposition in each disease.

Finally, we demonstrated that mature elastin content in both UIP and COP is reduced compared to healthy controls. Elastin's intrinsic autofluorescence originates from pyridoxi Debased pyrklolamine cross-links (Zipfei WR, Williams RM, Christie R, Nikitin AY, Hyman BT, Webb WW. Live tissue intrinsic emission microscopy using imut.tphoion~exeit.ed native fluorescence and second harmonic generation. Proc Nail Acad Sci U S A. 2003;! 00:7075-7080. Deyl Z Macek K, Adam M, Vancikova O. Studies on the chemical nature of elastin .fluorescence. Biochim Mophys Acta 1980:625:248-254) found primarily in. mature elastin fibers (Luisetti M, Ma S, ladarola P, Stone PJ, Viglio S, Casado B. Lin YY, Snider GL, Turino GM. Desmosine as a biomarker of elastin degradation, in copd: Current status and future directions. Eur Respir J 2008;32:1146-11.57) therefore TPEF of endogenous lung elastin preferentially identifies mature elastin fibers in lun tissue. These results are consistent with the concept that breakdown of mature elastin fibers in the lung, and their ''replacement^'' with often excess deposition of immature elastin fibers and elastin precursors, is belie ved to contribute to reduced lung function in a variety of pulmonary diseases (Souxa ABd, Santos FBd, Negri EM, Zin WA, Rocco REM. Lung tissue remodeling in the acute respiratory distress syndrome. Jomal de Pmumo ogia 2003;29:235-245). These results are also consistent with numerous reports of apparently increased elastin production, for example increased elastin gene expression and protein expression (Floff CR, Perkins DR. Davidson iM, Elastin gene expression is upregalated during pulmonary fibrosis. Connective tissue research 1999:40: 145-153). as well as increased enzymatic breakdown of mature .elastin in. COPD and IFF (Skjot-Arkil. H, Clausen RE, Nguyen OIL Wang Y, Zheng Q, Martinez FJ, Hogahoam CM, Han M, KKckstem LB, Larsen MR. Nawrocki A. Leemlng DJ, Kafsdal MA. Measurement ofmmp-9 and -12 degraded elastin (elm) provides unique informatio on lung tissue degradation, BMC' ulmonary medicine 2012; 12:34) in these and other (Stone PJ, Konstan MW, Berger M_* Dorkin HL, Franxblau C_> Snider GL. Elastin and collagen degradation products in urine of patients with cystic fibrosis. Am J Respir Qit Can Med 1995; 152:157-162} pulmonary fibroses. In other words, increased elastosis (i.e. breakdown of mature elastin fibers), as has been demonstrated for DIP and COP (Skjot-Arkil H, Clausen RE, Nguyen QH, Wang Y, Zheng Q_t Martinez FJ, Hogaboam CM, Han M, lickstein LB, Larsen MR, Nawrocki A, Leeming DJ, arsdal MA. Measurement of mmp-9 and -12 degraded elastin (elm) provides unique information on lung tissue degradation, BMC pulmonary medicine 2012; !.2:34), most likely leads to a compensatory increase in elastin production in an (ultimately unsuccessful) effort to restore the mature elastin fibers which have been lost. Hence our .results here together with these previous results all support the concept of increased elastin turnover (i.e. synthesis and "deposition" of "immature" elastin components) consequent to loss of mature elastin fibers in DIP and CO P, with resultant deficits in pulmonary function. Taken together with our findings on different FC raicrostruetore in UIP but not COP versus healthy lung, these observations on elastin content are especially compelling because they demonstrate that compared to healthy lung, both fibroses (ill and COP) have significant identifying physiologic disruptions in ECM structure and organization that are quantifiable with non-invasive and non-tissue destructive combined SHG and TPEF microscopy. Yet only the more intractable UIP fibrosis has disrupted FC microstructure identifiable by FSHO/B»«_¾ and thus -together these techniques may .represent novel clinical diagnostic tools for distinguishing between intractable and treatable lung fibroses, in summary, using SHG and TPEF microscopy, herein we identify several previously unreported key differences between UIP, COP and healthy lung tissue. The collagen microstructure differences we observed in UIP ECM provide novel insights as to why this pathology may be resistant to many therapies. For example, an ECM and/or collagen fibrils that are more densely packed, more ordered or disordered, and/or more Cross-linked may be more resistant to homeostatic turnover and exhibit, differences in matrix stiffness that are key to modifying cellular activity of resident cells and activation of pro-fibrogenl cytokines such as transforming growth factor beta (TGF-Π. identifying all the microstructural changes present in UIP and/or the mechanisms that regulate them will be a critical part of our future research. These ongoing studies will seek to determine more specifically exactly what features of collagen's microstruciure (e.g. fibril diameter, fibril density, and/or hetero- or homo-typic fibril composition or organization) are different in UIP versus COP and healthy lung, and identify .molecular targets that may effect these changes in collagen's underlying, microsiructure. Although further studies are required to ascertain whether or not the altered FC microsttucture as we demonstrate here is an underlying came of {^'rather than just associated with) differences in natural history, treatment responsiveness, and/or prognosis between UIP and COP, at a minimum these results introduce the intriguing possibility of using SHG microscopy in accordance with the present invention as a novel clinical hiomarker that may help predict treatment responsiveness of idiopathic iibrotie lung disease.

FIGURE LEGENDS

Figure 5: Fibrillar collagen mfcrostriicture is different in UIP, bat not COP, versus healthy lung. SHG imaging was performed on healthy, COP, and UIP formalin fixed paraffin embedded human lung tissue, and the FSHO/BXHG ratio was calculated to assess relative .differences i FC mkrostructure. Plot represents mean FSHG¾HG pixel intensity ± . SE^'M averaged, over - 12 fields of view (FOV) per patient (4-6 FOV/secrion from 2-3 sections/patient), from n^:::5, 3, and 10 patients per grou respectively (subject to tissue availability). Z-stacks from each FOV were average intensity projected and background subtracted, FSHG and SHG collagen signals masked to the same XY pixel areas, then divided to calculate the mean F_SH B_SHG value ± SBM as previously described. Compared to healthy lung tissue, FSHO BSHG was significantly decreased only in UIP (**p Q03) but not COP. Statistics were performed by one way ANOVA with Holm-Sida^'k post-hoc test and correction for multiple comparisons against healthy control. AH values are in relative arbitrary fluorescent units. Figure 6: Healthy, COP, and UIP lung hlstopatholugy compared to FSIKJ. Representative hematoxylin and eosin (H&E) FOVs showing healthy (A) versus fibrotie COP (B) and UIP (C) pathology were field-matched to the Fs_f i images (D, Έ, F) for the same FOVs, respectively. Note the eosin stained areas of concentrated collagen deposition (light pink color, indicated by bine arrows in B and C) that match areas of high PC FSHG signal intensity (white pixel regions, indicated by blue arrows in E and F} in COP and UIP respectively, hi contrast, the F_$HG (collagen) signal in healthy tissue (D) ari es primarily from al eolar parenchyma and small airway wails (examples of small airway walls are indicated by yellow arrows, in all images). Thus SH TM detects and allows quantification of altered microstructure (e.g. Figures 5, 7, 9} from both intrinsic normal and pathologic collagen content in. lung tissue. Levels (screen stretch) are linear and set the same for all images D-F,

Figure 7; Lung tissue with preserved alveolar architecture adjacent to UIP fibrotie lesi ns has different fibrillar collagen microstructure versus healthy lung, SHG imaging of the FsHo/BssK) mtio was performed on healthy, U I P. and lung tissue with preserved alveolar architecture adjacent to UIP .fibrotie lesions (UIP Adj), details otherwise as described i Figure 5. The Fs_fi« BsHG ratio was significantly decreased in both UiP Adj (*p<.04) and UIP (**p .G03) versus healthy lung tissue, suggesting that even "normal appearing^** lung tissue in UIP patients has altered. PC microstructure. All values are in relative arbitrary fluorescent units.

Figure 8: Increased Coll and C»I3 deposition, and CoiltCoB ratio differences, in UIP or COP versus healthy lung. The same patient sets or subsets as described in Figure S were immunotluorescently (IF) labeled for Coll and Co!3, and mean IF pixel intensity ± SEM quantified for Coll. (A), Col 3 (B), or Coll :Col3 ratio (C). Z-siaeks from each FOV were average intensity projected and background subtracted, and fluorescent intensities from the resultant images were quantified with Imaged and then expressed as mean anti-Coil anti-Col 3 IF SEM, as previously described. Co! UCoD ratio was quantified i the same fashion, then dividing Col.F oB signals, Compared to healthy. Col l deposition was increased in COP (**p<.G04>. and more so in UIP (***p .G003). Col.3 deposition was about equally increased in both COP (**p<.0O5) and UIP (**p<.005) versus healthy. Overall, this led to the Col.l :Col3 ratio being effectively equivalent in COP versus healthy, but significantly increased in UIP versus healthy (*ρ 01?). These findings are significant in the context of the F_$HO BSHG changes that were seen in only UIP (but not COP) versus healthy in Figures 5 and 6, because altered Coll :Coi3 ratios are known to regulate collagen, fibril diameter and/or structure (i.e. FC microstri!cture). Statistics were performed by one way ANOV A with Hoim-Sidak post-hoc test and correction for multiple comparisons against healthy control. All values are in relative arbitrary fluorescent units. For illustrative purposes, the originally grayscale Col 3 immunofluorescence is shown with. "Red" LUT applied in ima e!, with levels (screen stretch) linear and set the same for all images.

Figure 9: Elastin and Elastin: Collagen ratios differ in HIP and COP versus healthy lung. The same patient sets or subsets as described in Figure 5 were imaged and quantified for total

FC content (i.e. total FSHO ⁺ ¾H signals) and intrinsic auto.tluorescen.ee from mature lung elastin (captured at 515-555 nm), with the methods as described Figures 5, 7, 8 and in Results. Mature elastin fiber content (A) was similarly decreased in both COP (*ρ<„ί)Γ) and DIP (*p<.Ql) versus healthy lung tissue, and the total FC:mature elastin ratio (B) was similarly increased in both COP (**p 003) and UIP (**p<.003) compared to healthy. Plots represent, mean pixel intensity ± SEM for these elastin autofluorescence and F _HO + B_SHG signals, and statistics pe.rtbn.ned by one way ANOV A with Bolm-Sidafc post-hoc test and correction for multiple- comparisons against healthy control. All values are in relative arbitrary fluorescent units. Representative merged images (C) and (D) illustrate this Sower total FC SHG (biue):niature elastin (green) ratio seen in healthy compared to UIP (for each image, compare total, amount and intensity of the blue summed FSHO BSHG collagen SHG signal, relative to the green mature elastin signal, in healthy (C) versus UIP (D) panels respectively). For illustrative- purposes, the originally grayscale SHG and elastin fluorescence signals are shown with "Blue" and "Green" LUTs applied .in ImageJ respectively, with levels (screen stretch) linear and set the same for all channels in all images. Image scale is the same as in Figures 6 and 8 above.

Potential uses for the present invention, as it relates to lung fibrosis, include abilities to:

1. Predict much earlier (i.e. potentially many years earlier) what patients may develop fatal lung fibroses, which may in. turn allow: 1. earlier treatment intervention, and/or 2. development of more successful treatments for fatal lung fibroses. Currently there is o way to make such predictions, until diagnosis of fatal lung fibrosis is actually made, e,g. median 2.9 years before death from this disease, at which point it is too late to sto progression of this rapidly fatal disease.

2. Diagnose, with greater certainty, what patients have fatal lung fibrosis (either with bronchoscopy as described in s-1 below, or on biopsies as described in #2 below),

3. Provide new insights into etiology and new therapies/treatments for fatal (or other) long fibroses such as !PF/lff P.

Second Harmonic Generation (SHG) Instrument

The present invention includes an SHG instrument such as a bronchoscope or an SHG instrument in combination with, or in the form of, an endoscope. There are existing commercial confocal bronchoscopes (e.g. CellVizlo. Olympus), which could be developed into an. SHG bronchoscope. Notably, such an SHG endoscope would have applications for multiple diseases for which collagen SHG and F/.B SHG and related readout may provide clinical diagnostic value. For example, colon and gastrointestinal and gyneeologic l/urologica! diseases, as well as skin, cancer, etc - are disease areas that could all potentially benefit from this method, and for which endoscopy is typically used in diagnostics. A system and Method that enables the .measu ment of a second harmonic generation forward/backward ratio from an object by performing only a single image scan that may be used with the present invention is disclosed in commonly owned United States Piiblished Patent Application US2013/0057873 Al entitled "System And Method For Measuring The Ratio of Forward-Propagating to Back-Propagating Second Harmonic -Generation Signal, And Applications Thereof to Edward Brown III and Xiaoxing Han, the entire disclosure of which is incorporated herein by reference.

Diagnostics

While there is currently "some" ability to "diagnose" UIP/IPF (based on distinguishing features on lung biopsy and/or with High Resolution Computed Tomography (HRCT), family history, etc), currently such diagnoses of "fatal" fibrosis are typically only available and/or made somewhat late in the course of the disease (median survival is 2.9 years after diagnosis, for U!P/IFF). Moreover, there is some uncertainty with the current diagnostic methods, which as .noted above include radiologic imaging (HRCT)., and/or lung biopsy. In this context such a SHG bronchoscope or endoscope, as disclosed herein, would provide a less invasive and mare certain and/or confirming diagnosis compared to existing (biopsy) methods.

Secondly, our F/B SHG approach could also be applied to clinical diagnostics of lung biopsies for fibroses. This could provide a diagnostic approach with greater certainty than the existing methods (to predict fatal versus n.on fatal lung fibroses).

Second Harmonic Generation (SIIG) and muhiphoton microscopies predict and identify wuireatable lung fibrosis

Not all lung fibroses are fetal or unresponsive to therapies, but those that are, such as usual interstitial pneumonia (UIP), typicaliy progress rapidly with a median survival time of 2,9 years after diagnosis for UIP, ft is unknown why some lung fibroses respond well to therapies, yet others like UIP remain intractable and rapidly fatal Being able to predict, identify, or diagnose earlier which cases may progress to fatal long fibrosis, before advanced disease onset, may facilitate development or administration of therapies to help "stem the tide" of progressive and fatal lung fibroses. fibrosis of die lungs and other tissues is caused by aberrant and excess deposition of collagen, particularly of the fibrillar collagen subtypes. Second. Harmonic Generation microscopy (SHGM) and related techniques are a. variant of 2 photon (2P) microscopy that can detect (i.e. visualize and assess) these fibrillar eoi!agens in lung (and other) tissues without exogenous labels, As such, SHGM can be used to interrogate changes in collagen^* s macrostruetural properties (e.g. collagen fiber density, arrangement, and organization), as well as changes In collagen's subresolution microstructoral properties (e.g. the diameter, order versus disorder, and/or packing density of collagen fibrils within larger collagen fibers). In this aspect, SHGM is unique in its ability to inierrogate subresolution structure of fibrillar collagens (e.g. coll and eol.1) in intact and potentially live samples without exogenous labels, abilities which also make SHGM an attractive potential clinical and investigational diagnostic too!. One particular SHG measure, known as the fonvard-to-baekward SHG ratio, F/8 SHG. and/or FSHG BSHG_> n particular is primarily sensitive to the spatial extent of SHG- generaiing scatterers along the optical, axis, i.e. the effective diameter or packing arrangement/density/order versus disorder of collagen fibrils within the SHG focal volume. We use the term collagen "microstructure" to refer to these subresohrtion structural, properties of collagen fibrils or fibers, that can influence SHG directionality from that fibril or fiber, to alter the calculated F B ratio parameter. in the present invention, we determined that this measurable F/B SHG parameter was ^■different in: 1. Lung tissue with preserved alveolar architecture adjacent to UIP .fibrotic lesions- (i.e, "adjacent normal" lung tissue) compared to healthy lung (and fibrotic UIP .lung tissue had similar F/B to this adjacent normal" tissue from UIP long), and 2. Lung tissue from UIP fibrotic lung compared to healthy lung and compared to a non-fatal and "treatable" lung fibrosis, cryptic organizing pneumonia (COP) (which had similar F/8 to healthy Sung).

These results show for the first time that collagen ^*imicrostructure" (i.e. as interrogated by F B SHG) is different in each of these comparative scenarios, and teach at least two things about the F/B SHG parameter as measured in fibrotic versus norma! lung tissues: I, The F/B SHG parameter may "predict" which lung tissues are likely to become fatally fibrotic (since even normal-appearing, non-fibrotic lung tissue adjacent to fibrotic UIP lung tissue had the same F/B SHG "signature" as the fibrotic UIP lung tissue), and 2, The F/B- SHG parameter may "distinguish" fatal lung fibroses from non-fatal lung fibrosis and from healthy lung (since both, healthy lung and a non-fatal and treatable lung fibrosis. COP, shared a similar F B SHG signature, which was different from the F/B SHG signature found in Mai UI P lung tissue).

There are other parameters or "signatures"^' we can measure in lung tissue with SHG and multiph ton microscopies, such as but not limited to the mature eiastin content and the fibrillar colUtge elmtin mthh Both of these parameters were similarly changed in COP and

UIP compared to healthy lung tissue, but in neither of these parameters was UIP different from COP.

All together, these data and methods demonstrate that compared to healthy lung, both fibrotic lung diseases studied (i.e. the -rapidly fatal and untreatable-UIP, and the treatable and non-fatal. COP) are characterized, b significant gross physiologic disruptions in collagen and extracellular matrix (ECM) structure and organisation that can be quantified with non-invasive and non- tissue destructive combined SHG and mi ti photon microscopies, as interrogated by the mature elastin content and the fibrillar coUag : i stin ratio parameters. Yet only the more intractable UIP fibrosis shows evidence of disrupted fibril.! ar collagen microslrueture, as interrogated the FIE EG ratio.

Thus these new methods described above highlight that these techniques, methods and measures, particularly the F/B SHG parameter either b itself and/or in combination with the "mature elastin content" and/or the eolIagen:eJastm ratio .measures, may predict onset of, and/or make clinical distinctions between, intractable and treatable lung fibroses.

While the devices, systems and methods disclosed herein are described by way of example as being applied to various cancers and lung fibrosis, they are equally applicable to other diseases where disrupted fibrillar coliagen microstrueture is an indicator of progression or metastasis of the disease. li is, therefore, apparent, that there has been provided, in accordance with the various objects of the present invention, a method and apparatus for determining the progressive potential of a disease.

While the various objects of this invention have been described in conjunction with preferred embodiments thereof, it is evident that, many alternatives, modifications, and variations will be apparent to those skilled in the art, Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall, within, the spirit and broad scope of this .specification, claims and drawings appended herein.

Claims

What is claimed is:

1. A method for determining the progressive potential of a disease, the method comprising the steps of: imaging body tissue using a second harmonic generation instrument; determining the ratio of the forward to backward propagatin second harmonic generation signal derived from the imaging of the body tissue with the second harmonie generation instrument; assessing the collagen mierdstructure of the imaged body tissue using the ratio of the forward to backward propagating second harmonic generation signal; comparing the ratio of the forward to backward propagating second harmonic generation signal to the ratio of the forward to backward propagating second harmonic generation signal of other tissue samples; and determining the progressive potential of the disease by numerical values derived from the ratio of the forward to backward propagating second harmonic generation signal of the imaged body tissue,

2. The meihod of claim. 1, wherein the body tissue is obtained from a primary tumor biopsy.

3. The .method of claim 1, further comprising ^'the step of predicting the duration of metastasis free survival (MFS) from the numerical values derived from the rati of the forward to backward propagating second harmonic generation signal of the imaged body tissue.

4. The method of claim 1, further comprising the step of predicting the duration of progression free survival (PFS) from the numerical values derived from the ratio of the forward to backward propagating second harmonic generation signal of the imaged body tissue.

5. The method of claim. 1, further comprising the step of statistically analyzing the numerical values derived from the ratio of the forward to backward propagating second harmonic generation signal of the imaged body tissue to predict the duration of metastasis free survi val (MFS).

6. The method of claim 1, wherein the progressive potential of the disease is the metastatic potential for the disease,

7. The method of claim 1 , wherein the disease is estrogen receptor -positive (ER÷) breast cancer.

S. The method of claim t. wherein the disease is invasive ductal carcinoma.

9. The method of claim 8. further comprising the step of providing adjusted chemotherapy treatment levels to a -patient based on the determined progressive potential of their estrogen receptor positive (ER+) breas cancer.

10. The method of claim 1, wherein the disease is colorectal adenocarcinoma.

1 1. The method of claim L wherein the disease i s lung fibrosis.

12. The meihod of claim L wherein the method is at least -partially performed using a computer.

13. A method for determining the progressive potential of a disease, the method comprising the steps of: imaging in vivo body tissue using a second harmonic generation instrument in combination with an endoscope; determining the ratio of the forward to backward propagating second harmonic generation signal deri ved from the imaging of the in situ body tissue with the second harmonic generation instrument in combination with the endoscope; assessing the eolla&en mkrostructure of the imaaed in situ bodv tissue using the ratio of the forward to backward propagating second harmonic generation signal;

Comparing the ratio of the forward to backward propagating second harmonic generation signal to the ratio of the forward to backward propagating second harmonic generation signal of other tissue samples; and determining the progressive potential of the disease by numerical values derived from the ratio of the forward to backward propagating second harmonic generation signal of the imaged in situ body tissue,

14. The method of claim 13. iurther comprising the step of predicting the duration of metastasis free survival (MPS) from the ^'numerical values derived from the ratio of the forward to backward propagating second harmonic generation signal of the imaged in vivo body tissue,

1 5. The method of claim 13, further comprising the step of predicting the duration of progression free survival (PPS ) .from the numerical values derived from the ratio of the forward to backward propagating second harmonic generation signal of the imaged in vivo body tissue.

1.6. The method of claim 1.3, further comprising the step of statistically analyzing the numerical values derived from the ratio of the forward to backward propagating second harmonic generation signal of the .imaged body tissue to predict the duration of metastasis free survival (MPS).

17, The method of claim 13, wherein the progressive potential of the disease is the metastatic potential for the disease.

18. The method of claim 3.3, wherein the disease is lung fibrosis

1.9. The method of claim 13. wherein the disease is a cancer.

20. The method of claim 13, wherein the method is at least partially performed using a computer.