A method to assess presence of tumor tissue in a lymph node specimen using field cycling relaxometry, corresponding system and computer program product

Abstract

A method to assess the presence of tumour tissue in a lymph node specimen resected during surgery / biopsy using Field Cycling Relaxometry technique, corresponding system and computer program product.

Description

[0001] "Relaxometric method for ex vivo characterization of lymph nodes, corresponding system and computer program product" **** FIELD OF THE INVENTION The invention concerns a method for the assessment of positivity or negativity of a lymph node resected during surgery / biopsy using relaxometric measurements. The invention also concerns a system for the assessment of positivity or negativity of a lymph node (i.e., a relaxometer) and a corresponding computer program product for controlling the system. BACKGROUND OF THE INVENTION About ten million people die from cancer every year. It is the cause of every sixth deaths. It is one of the most relevant health problems in the world. It is estimated that metastasis is responsible for about 90% of cancer deaths. Metastasis is the general term used to describe the spread of cancer cells from the primary tumor to surrounding tissues and to distant organs and is the primary cause of cancer morbidity and mortality. Malignant tumors can give rise to metastasis following the lymphatic system in a sequential way. Accurate characterization of lymph nodes in patients with cancer is of major importance for correct prognosis and proper decision about the treatment planning. Size measurements are still the most frequently used method to discriminate malignant from non-malignant lymph nodes. However, the reliability of the method is highly questionable. To improve the diagnostic evaluation of lymph nodes, there is a need for modalities that go beyond anatomical observation to provide quantitative information on physiological and biochemical processes at the cellular level. Lymph node excision, followed by histopathological examination, is considered as the gold standard for the assessment of lymph nodes. Communication of a tumor with its sentinel lymph node (i.e., the lymph node that receives direct lymphatic drainage from a primary tumor site) takes place through preexisting afferent lymphatic vessels surrounding the tumor margin and / or through newly formed lymphatic vessels within the tumor. The rationale for sentinel lymph node mapping and biopsy is that the sentinel lymph node accurately reflects the status of the lymphatic basin draining a primary tumor. This assumption has been proven for several tumor types, e.g., malignant melanoma, early- stage breast cancer, and penile carcinoma. Sentinel lymph node (SLN) biopsy has gained popularity as a diagnostic procedure for several solid tumors such as melanoma, vulvar carcinoma, cervical cancer, penile cancer, colorectal cancer, neuroendocrine skin cancer, squamous cell cancer of the scrotum, head and neck cancer, and particularly breast cancer. It is considered a great advantage to the patient and surgeon if the regional lymph node dissection, when necessary, could be performed in the same surgery session of the excision of the primary tumor. If SLNs can be assessed during breast cancer surgery, secondary surgery could be avoided. This requires that an accurate and fast intraoperative assessment of the SLN status is available. In principle all the approaches proposed for the assessment of negative / positive tumor margins would work for SLN as well. The most commonly applied one is the frozen section analysis. This is usually done by a single Histopathological Evaluated (HE) frozen section. The sensitivity for detection of metastases by single HE frozen section analysis is (under optimal conditions) not higher than 80-85%, and lower in more difficult conditions as is often the case with frozen sections. The ability of the method to detect metastases is clearly higher in the presence of macrometastases rather than micrometastases. When doing frozen section analysis, it is unavoidable that some SLN tissue escapes the scrutiny, leading, in principle, to missing some SLN micrometastases. It has to be realized that for the average lymph node, only about 1% of the total nodal volume is actually observed under the microscope, even when making step sections and performing immunohistochemistry. The operative delay of 15 to 20 minutes caused by the frozen section investigation is, in principle, acceptable, since during this time the primary tumor can be excised. Another approach deals with the imprint cytology methodology but the sensitivity of this approach is markedly lower than that of frozen sections. Beyond imaging much attention is currently devoted to the detection of micrometastases via the amplification by reverse transcription polymerase chain reaction (RT-PCR) of mRNA, which is expressed in the cancer cells of interest but not by other lymph node cells. Thus, the method relies on the intraoperative analysis of the expression of some markers in sentinel lymph nodes as biomarkers for the presence of metastases. Overall, RT-PCR may be a valuable additional method although experimental drawbacks in its application have been reported. Further studies appear necessary to develop combinations of primers that are specific enough to establish the role of RT-PCR in detecting metastasis in SLNs and to assess the clinical value of RT-PCR. In summary, in oncological surgery at large, intraoperative single HE frozen section and imprint cytologic analysis of SLNs remain the techniques of choice. In general, they have been shown to be reasonably reliable for detection of breast cancer metastases, but a false negative rate of 30 to 40% will have to be accepted because of the possibility of micrometastases. More reliable detection of metastases in SLNs can be achieved with histopathologic investigations, including step HE-stained sections and immunohistochemistry. The main issue remains that all these techniques are time consuming, have limited detection abilities, and lack objectivity and reproducibility. They are strongly operator dependent and the attained accuracy may drop when lesser-experienced pathologists are involved. Importantly there is the need for methods able to make a total assessment of the resected SLNs. OBJECT AND SUMMARY OF THE INVENTION An object of the invention is to provide a method that allows the acquisition of highly reliable information on the presence of tumor tissue in a lymph node(s) resected during surgery / biopsy, using the Field Cycling Relaxometry. Another object of the invention is to provide a system for carrying out the method which enables such information to be obtained. Another object of the invention is to provide a computer program product to control such a system. According to the invention, the above objectives are achieved thanks to the subject matter recalled specifically in the ensuing claims, which are understood as forming an integral part of this disclosure. The present invention concerns a method to assess the presence of tumor tissue in a biological specimen resected during surgery / biopsy using the Field Cycling Relaxometry technique, wherein the specimen is one or more lymph nodes, the method comprising the following steps: a. obtaining a first sequence of at least m values of a measuring magnetic field strength Bri, wherein m ≥ 3, preferably m ≥ 5, a second sequence of at least n values of a time τ, a detection magnetic field strength Bd, a re-cycle delay time RD, a polarization magnetic field strength Bpand a polarization time tp; b. exposing the specimen to a polarization magnetic field having a polarization magnetic field strength Bpfor a time tp, thus allowing the magnetization build-up, if the current measuring magnetic field strength Briis ≤ 7 MHz; c. exposing the specimen to a measuring magnetic field having a measuring magnetic field strength Brifor a time τ, thus allowing the specimen magnetisation to relax towards a magnetisation equilibrium value determined by the actual Bri; d. acquiring a value of magnetisation MZof the specimen at time τ and at the measuring magnetic field strength Bri, the acquisition being carried out while exposing the specimen to a detection magnetic field having a detection magnetic field strength Bdafter a 90° pulse has been applied to MZ; e. switching off the magnetic field for the re- cycle delay time RD; f. repeating steps b. to e. a number of cycles n, wherein the value of time τ varies at each cycle; g. fitting the values of magnetization MZacquired after n repetitions of steps b. to e. (magnetization intensity vs. τ) according to a monoexponential Bloch equation to produce a respective magnetization recovery / decay curve and calculate the proton longitudinal relaxation rate R1Briat the respective magnetic field strength Bri; h. repeating steps b. to g. a number of times m, wherein the value of the measuring magnetic field strength Brivaries each time, thereby calculating at least three proton longitudinal relaxation rates R1Br1, R1Br2, R1Br3at the at least three measuring magnetic field strengths Bri; i. calculating: − a Ratio of two relaxation rates R1Bri; − a Sum of at least two relaxation rates R1Bri; − an extracellular water mole fraction p0by fitting the at least three magnetisation recovery / decay curves by means of the two- Site eXchange model; − an intracellular residence lifetime τiby fitting the at least three magnetisation recovery / decay curves by the two-Site eXchange model; j. processing the parameters calculated in step g. and i. for assessing the presence of tumor tissue in the specimen by: 1. calculating a product P = Ratio * p0, wherein tumor tissue is present in the specimen if P has a first positioning with respect to a cut- off and tumor tissue is absent in the specimen if P has a second positioning with respect to a cut-off; or 2. calculating a decision boundary S12(x) involving at least three parameters selected among the Ratio, the Sum, the extracellular water mole fraction p0, the intracellular residence lifetime τiand the relaxation rate R1Bri, wherein when the calculation of the decision boundary S12(x) involves three parameters at least one of the three parameters is the extracellular water mole fraction p0, or the intracellular residence lifetime τi, wherein tumor tissue is present in the specimen if S12(x) > 0, and tumor tissue is absent in the specimen if S12(x) < 0. The present invention also concerns a system for carrying out the method and a corresponding computer program product for controlling the system. BRIEF DESCRIPTION OF THE DRAWINGS The invention will now be described in detail, purely by way of illustrative and non-limiting example, with reference to the attached figures, wherein: FIGURE 1. The Fast Field Cycling (FFC) experiment. A) Not-Polarized (NP) sequence (for high field strengths, i.e., usually Br> 0.17 T): the nuclear spin magnetisation is built up during the evolution period (τ) at relaxation field Br, then the nuclear magnetic resonance (NMR) signal is detected at detection field Bd. The sequence is repeated, staggering τ each time. B) Pre-Polarized (PP) sequence (for measurements at low field strengths, i.e., usually at Br< 0.17 T): the nuclear spin polarization is built up during the pre- polarization phase, at polarization field Bp. Relaxation occurs during the evolution period (τ) at relaxation field Br, then the NMR signal is detected at detection field Bd. The sequence is repeated, staggering τ each time. FIGURE 2. An example of the magnetisation decay and recovery curves obtained by applying the pre-polarized (PP) and non-polarized (NP) sequences, respectively. FIGURE 3. A cartoon depicting the water compartmentalization in tissue. See text for symbol definitions. FIGURE 4. An example of application of the 2SX model. A) Set of magnetization decay data vs. ^ (acquired with the PP sequence in the range 0.01 – 1 MHz of magnetic field strengths) of healthy rat lymph nodes. The lines correspond to the fits of the data according to the 2SX model as described in the text. B) For the data acquired at 0.02 MHz, the contribution of the apparent a and b relaxing components to the overall relaxation rate constant are shown. FIGURE 5. The longitudinal relaxation rate constant values as a function of the applied magnetic field strengths for Matrigel (open circle) and the R1ivalues (filled square) calculated by means of the fitting procedure applied to the data set shown in figure 4. The bars represent the standard deviations. FIGURE 6. Block diagram of an FFC NMR relaxometer. FIGURE 7. Typical trend of the proton longitudinal relaxation rate R1of lymph node tissue as a function of the applied magnetic field: healthy (white dots) and metastatic lymph node (black dots, tumor > 40%). Error bars report the Standard Error. FIGURE 8. Schematic representation of two protocols L1 and L2 according to which the method to assess presence of tumor tissue in a lymph node herein disclosed is applied. FIGURE 9. Schematic representation of an embodiment of the two protocols L1 and L2 according to which the method to assess presence of tumor tissue in a lymph node herein disclosed is applied. FIGURE 10. Flow chart of the method object of the present disclosure. FIGURE 11. Example of the application of the L1 protocol. The P value was calculated for a set of lymph node samples after obtaining the corresponding relaxometric data. The samples were classified as healthy or metastatic according to the cut-off value with two misassignments: one false positive (FP) and one false negative (FN). FIGURE 12. Example of the application of the L2 protocol. The x-axis shows the classification of the samples according to the histopathological evaluation. On the y-axis, the samples were classified as healthy or metastatic by applying the discriminant rule with one misassignment: the false positive (FP) specimen is indicated in the plot with a star symbol. FIGURE 13: Performance of the relaxometric classifiers on respect to the reference method (histopathological analysis) for metastatic and healthy lymph nodes. On the right, the expression of the relaxometric classifier is shown, together with the related sensitivity (Sens.) and specificity (Spec.) values for each A-C case, wherein case A: P parameter, case B: Ratio parameter and case C: 2R1parameter. DETAILED DESCRIPTION OF THE INVENTION In the following description, numerous specific details are given to provide a thorough understanding of embodiments. The embodiments can be practiced without one or more of the specific details, or with other methods, components, materials, etc. In other instances, well-known structures, materials, or operations are not shown or described in detail to avoid obscuring aspects of the embodiments. Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. The headings provided herein are for convenience only and do not interpret the scope or meaning of the embodiments. The Field Cycling Relaxometry belongs to the field of the Nuclear Magnetic Resonance Techniques, as the more known Magnetic Resonance Imaging. The Field Cycling Relaxometry allows access to magnetisation decay / recovery curves by acquiring the Free Induction Decay (FID) signal (corresponding to the magnetisation intensity) from the sample after exposure to a given magnetic field (measuring magnetic field Br, also indicated as relaxation magnetic field) for different intervals of time τ (see Figs. 1 and 2). By changing the intensity of the measuring magnetic field Br, proton longitudinal relaxation rates R1can be determined at all frequencies permitted by the instrument and correlated to the chemical composition and dynamics of the proton containing species, mainly water, in the investigated tissue specimen. Figure 1 shows the typical acquisition sequences: Not Polarized (NP) and Pre-Polarized (PP) sequences, for measurements at high (usually > 0.17 T) and low (usually < 0.17 T) field strengths, respectively. For the sake of clarity, 0.17 T corresponds to a proton Larmor frequency (= ^^B0, where ^ is the proton gyromagnetic ratio constant and B0the magnetic field strength in Tesla) of 7 MHz. The PP sequence differs from the NP sequence because of the application of a high magnetic field (polarization magnetic field Bp) before the application of the measuring field Br, in order to have a good signal intensity, otherwise not present at low fields. Therefore, in this type of experiment the magnetic field cycles between the polarization field (Bp) (if applied), several fields (Br) at which the proton relaxation time has to be measured, and the detection field (Bd). The detailed description of the experiment is reported in many papers [i.e., Ferrante G. and Sykora S.: Technical aspects of Fast Field Cycling, Advances in Inorganic Chemistry, 57, 405-470]. Briefly, the basic field cycling experiment with the NP sequence consists of the following steps (see fig. 1 – panel A): 1. The magnetic field is switched to the value of a measuring magnetic field Brfor a time τ during which the magnetisation relaxes towards an equilibrium value, determined by the current Brvalue. 2. The magnetic field is switched to the value of a fixed detection magnetic field Bdand the equilibrium magnetisation established at Bris measured with a 90° pulse followed by acquisition of the magnetization recovery curve at Br. 3. The magnetic field is switched off for a certain time RD (Re-cycle Delay) that is the time between repetition of the cycles, equal to 0.5 - 1.5 s. The FID acquired at Bd(step 2) is proportional to the magnetisation at time τ at the given Br. The complete cycle should then be repeated a number n of cycles varying the value of τ (typically the number n of cycles of time τ is equal to 16 or 32, time τ ranging from 0.01 to 5 s in order to recover the entire magnetization recovery curve). Briefly, the basic field cycling experiment with the PP sequence consists of the following steps (see fig. 1 – panel B): 1. The sample is exposed to a high polarization field Bpfor a polarization time tpwith consequent building-up of the magnetisation of the sample. tpis selected in order to achieve a good signal intensity, i.e., with a signal to noise ratio (S / N) ≥ 3, preferably (S / N) ≥ 10. 2. The magnetic field is switched to the value of a measuring magnetic field Brfor a time τ during which the magnetisation relaxes towards an equilibrium value, determined by the value of Br. 3. The magnetic field is switched to the value of a fixed detection magnetic field Bdand the equilibrium magnetisation established at Bris measured with a 90° pulse followed by the acquisition of the magnetization decay curve (FID) at Br. 4. The magnetic field is switched off for a certain Re-cycle Delay time RD that is equal to 0.5 - 1.5 s. The FID acquired at Bd(step 3) is proportional to the magnetisation at time τ at the given Br. The complete cycle should then be repeated a number n of cycles varying the value of τ (typically the number n of cycles of time τ is equal to 16 or 32 values, time τ ranging from 0.01 to 5 s in order to recover a large number of points covering the entire magnetisation decay curve). A graphical representation of a magnetisation decay / recovery curve (Intensity vs. τ) is provided in Figure 2. Each dot is obtained applying the steps previously described, which are repeated n = 32 times to obtain a decay / recovery curve. More precisely, the PP sequence was applied at a measuring magnetic field Br= 0.01 MHz with 32 values of τ logarithmically distributed in the range 0.0035-2.8 s; the NP sequence was applied at a measuring magnetic field Br= 10 MHz with 32 values of τ logarithmically distributed in the range 0.01-4 s. The fit of a magnetisation decay / recovery curve to an exponential function (Bloch equation) provides the proton longitudinal relaxation rate R1(the reciprocal of the proton longitudinal relaxation time T1) at the field Br. In general, the Bloch equations are a set of macroscopic equations that are used to calculate the nuclear spin magnetisation M = (Mx, My, Mz) as a function of time τ when relaxation times T1and T2are present. These are phenomenological equations that were introduced by Felix Bloch in 1946 as disclosed, i.e., in F. Bloch, "Nuclear Induction", Physical Review 70, 4604– 73 (1946). In the case of NP and PP sequences, the magnetisation recovery / decay curve refers to the z component of the magnetisation, Mz, towards its thermal equilibrium value (M0) at field Br,associated to the constant T1(=1 / R1), i.e., the longitudinal (or spin- lattice) relaxation time, and, according to the Bloch equation, is defined by the following equations: −^^ ^^ ^^ ^^^^^^ ^^⁄ ^^^^^^^^ ^^^^^^^^for the NP sequence and −^^ ^^ ^^ ^^^^^^ ^^⁄ ^^^^^^^^^^^^^^^^for the PP sequence wherein ^^^^0^^^^and ^^^^0^^^^are parameters estimated from a fit procedure, where they are free to change as a function of the magnetic field strength Briapplied. ^^^^0^^^^corresponds to the highest measured intensity of the magnetization Mzat each selected Bri. For the magnetization recovery curve acquired by the NP sequence, ^^^^0^^^^is the difference between zero and the Mz value at τ = 0; for the magnetization decay curve acquired by the PP sequence, ^^^^0^^^^is the difference between zero and the Mzvalue at τ → ∞ (see Fig. 2). The magnetisation decay / recovery curves may also be subjected to other mathematical treatment, such as applying a biexponential model when two spin populations are present (for example hydrogen protons associated with free water and macromolecules) or the Two-Site eXchange (2SX) model when the relaxation process is modulated by the water exchange across the cell membrane [Landis, C. S. et al. Magn. Reson. Med. 1999, 42, 467– 47; Li, X, et al. Magn Reson Med. 2019, 82, 411–424; Hazlewood, C.; Chang, D.; Nichols, B.; Woessner, D. Nuclear Magnetic Resonance Transverse Relaxation Times of Water Protons in Skeletal Muscle. Biophys J. 1974, 14 (8), 583–606. https: / / doi.org / 10.1016 / S0006- 3495(74)85937-0]. The 2SX model considers that the major water compartmentalization in tissue is intracellular and extracellular (“inside” / “outside”), being the vascular space small and negligible (see Fig. 3). In the 2SX model the above reported Bloch equations [1, 2] for the analysis of the experimental recovery / decay of magnetization Mz(τ) are modified to describe this two-compartments, substituting the term −^^ ⁄^^^^^^^^^^ ^^= ^^−^^^^ ^^^^1with the following equation: −^^ ∙ ^^^^^^ ∙ ′′^^^^^^^^ + ^^^^ ∙ ^^^^^^ −^^ ∙ wherein aaand R’1,aare the fraction and rate constant for the apparent a relaxing component, aband R’1,bare the fraction and rate constant for the apparent b relaxing component, and τ is the running time for recovery by relaxation, as before. Because aaand abare related (aa+ ab= 1), there are only three independent parameters: R’1,a, R’1,b, and ab(or aa), expressed as: 1^^^^^^ ^^^^^^^^^^1^^^^^^ ^^^^^^^^2 ′^^ − − ^^ ^^ ^^−1− where R1oBriis the intrinsic longitudinal relaxation rate constant of the extracellular water protons at the magnetisation field strength Bri, R1iBriis the intrinsic longitudinal relaxation rate constant of the intracellular water protons at the magnetisation field strength Bri, τois the extracellular water lifetime and pois the tissue extracellular water mole fraction. poand τovalues are related to the intracellular water lifetime τiand the tissue intracellular water mole fraction piby the equilibrium mass balance: ^o = ^i (po / pi ) [7] This model can be applied when the condition │^i-1+ ^0-1│ ~ │R1i- R1o│ [8] is met, as happens at low magnetic field strengths (comprised between 10 MHz and 0.01 MHz), where the extracellular compartment is characterized by lower R1values with respect to the intracellular ones. In the fitting procedure (see Table 1, figure 4 and 5), the R1oBriare fixed to the relaxation rate measured for Matrigel (see figure 5), which mimics the extracellular compartment [Hughes, C.; Postovit, L.; Lajoie, G. Matrigel: A Complex Protein Mixture Required for Optimal Growth of Cell Culture. Proteomics 2010, 10 (9), 1886–1890. https: / / doi.org / 10.1002 / pmic.200900758; Ruggiero, M. R.; Baroni, S.; Pezzana, S.; Ferrante, G.; Geninatti Crich, S.; Aime, S. Evidence for the Role of Intracellular Water Lifetime as a Tumour Biomarker Obtained by In Vivo Field-Cycling Relaxometry. Angewandte Chemie - International Edition 2018, 57 (25), 7468–7472. https: / / doi.org / 10.1002 / anie.201713318]. Matrigel is the trade name for a gelatinous protein mixture secreted by Engelbreth-Holm-Swarm mouse sarcoma cells. It is considered a good model of the extracellular environment present in many tissues and used by cell biologists as a substrate (basement membrane matrix) for growing cells. The R1iBri, τiand p0parameters were obtained from the 2SX model fitting, applying a simultaneous fitting of at least three magnetization decay / recovery curves obtained over an extended range of magnetic fields comprised between 10 MHz and 0.01 MHz. The p0is allowed to vary within a feasible range, in accordance with results already reported in the literature (0.09-0.5) [C. S. Landis, X. Lin, F. W. Telaqng, P. E. Molina, I. Palyka, G. Vetek, C. S. Springer Jr, Magn. Reson. Med. 1999, 42, 467–478; X. Li, W. D. Rooney, C. S. Springer Jr, PNAS 2008, 105, 17937-17942; S. L. Barnes, A. G. Sorace, M. E. Loveless, J. G. Whisenant, T. E. Yankeelov, NMR Biomed. 2015, 28, 1345–1356; E. Panagiotaki, S. Walker-Samuel, B. Siow, S. P. Johnson, V. Rajkumar, R. B. Pedley, M. F. Lythgoe, D. C. Alexander, Cancer Res. 2014, 74, 1902-1912]. The fitting procedure imposes that the values of τiand p0parameters (which are independent of the magnetic field and intrinsic characteristics of the investigated specimen) are shared, i.e. they are the same for the three curves. Table 1 Independent variables of the 2SX model Magnetic As example, the fitting results for the data acquired on healthy rat lymph nodes (figure 4A) are reported in the following table 2, where the standard deviation of values is reported in bracket:

[0002] Table 2 Variable values determined by applying the 2SX model to the data acquired on healthy rat lymph nodes (in figure 4A) Br M0* y0 R1o R1i τ0 *** **From Matrigel; ***constrained value range from literature: 0.09-0.5 Discriminant Analysis (DA) may be applied for lymph node classification. DA developed historically along with multivariate statistics and can be traced back to R. A. Fisher (1890-1962) in the 1930s [Fisher, R. A. 1936. The use of multiple measurements in taxonomic problems. Annals of Eugenics 7: 179–188]. For a theoretical discussion of the subject, please refer to McLachlan, G. J. (1992) Discriminant analysis and statistical pattern recognition, New York, Wiley, doi: 10.1002 / 0471725293; CJ Huberty, S Olejnik (2005) Applied MANOVA and Discriminant Analysis. Wiley Series in Probability and Statistics, John Wiley & Sons, Inc., ISBN:9780471468158; WK Härdle, L Simar (2015) Applied Multivariate Statistical Analysis. Springer Berlin, Heidelberg, eBook ISBN 978-3-662-45171-7; MJ Adams (2005) Chemometrics and Statistics. Multivariate Classification Techniques. Editor(s): Paul Worsfold, Alan Townshend, Colin Poole, Encyclopedia of Analytical Science ISBN 9780123693976. DA is well-suited for classifying many aspects of medical diagnostics and is widely accessible and easy to perform using statistical packages such as OriginPro (OriginLab Corporation, Northampton, MA, USA); Stata Statistical Software (StataCorp LLC, College Station, TX); SAS software (SAS Institute Inc., Cary, NC, USA), IBM SPSS Statistics (IBM Corp., Armonk, N.Y., USA), MATLAB (The MathWorks Inc., Natick, Massachusetts, USA) and the free R Statistical Software (R Core Team, R Foundation for Statistical Computing, Vienna, Austria). DA allocates specimens to the positive or negative lymph node groups using information from specimens whose group memberships are known (i.e., training data). More precisely, the objectives of a DA are: i) Discrimination: to construct a classification model based on the parameters estimates of the training data. ii) Classification: to distribute unlabelled samples into labelled groups with the classification model. DA can be described as a four-step process: 1) Preliminary phase / operations. Identify from all available input features the useful feature variables for the formulation of the discriminant function(s), i.e., identify the training data set. With regard to the training data in this context, the variables / features considered belong to the MR measurements of the decay / recovery curve of magnetization, such as the derived R1values (and their combinations such as ratio of two R1values or sum of two or more R1values acquired at different Br, for example) and the extrapolated physical-chemical parameters (water intra- / extra-cellular residence times and mole fractions) acquired on representative specimens of the two groups under study (i.e. healthy and metastatic lymph nodes, as verified histologically). The data are analyzed to identify at which magnetic field strength the greatest differences in R1are observed and which variables best discriminate between the two groups. Thus, among all the possible ones, the variables to be considered for DA are those with significantly different mean values for the two groups, with a small variance within the group and with a low correlation. The correspondence between the training data set and the assumptions (multivariate normality, equality or not of variance-covariance within group and low multicollinearity of the variables) of the discriminant model are verified with the suitable statistic test. 2) Determination of the discriminant model. Discriminant analysis is designed to weigh and combine the variables of the training data set (discriminator variables) into discriminant functions, in such a manner as to force groups to be as mathematically distinct as possible. Several statistical criteria can be used. In practice, linear and quadratic discriminant models are frequently used, assuming an underlying multivariate normal data distribution. In this regard, please refer to A. Tharwat, “Linear vs. quadratic discriminant analysis classifier: a tutorial”, Int. J. Applied Pattern Recognition, Vol. 3, No. 2, 2016. Since the case of lymph nodes is a problem of classification between two groups (group 1 = metastatic lymph nodes; group 2 = healthy lymph nodes), two discriminant functions f1and f2(one for each group, as indicated by the subscript numbers) are used to determine the decision boundary (in the form of a linear or quadratic function) between the two groups and the region of each group in the data space. In particular, the first discriminant function f1will have the maximum value compared to the second function f2when the sample belongs to the first group, while the second discriminant function f2will have the maximum value compared to the first discriminant function f1when the sample belongs to the second group. Hence, the decision boundary between group 1 and group 2 (S12(x) = f1(x)- f2(x)) is calculated as follows: 1: for S12(x) > 0 sgn(S12(x))= sgn (f1(x)-f2(x))= Undefined: for S12(x)=0 2: for S12(x) < 0 [9] where sgn indicates the sign of the function. It means that each sample is assigned to the group that gives the highest value of the discriminant function. 3) Evaluation of the discriminant model. The efficacy of the discriminant model can be measured by the proportion of correct assignments. In practice, the model is applied on the training data set: the group labels of the training data set are predicted using the discriminant model and compared to the actual (determined by HE analysis) group labels. 4) Classification of an unknown sample. When the discriminant rule has been learned from the training data, it can then be used to predict the class label of new data points. In practice, in accordance with the previous scheme [9], the values of the variable vector of the unknown sample are substituted in the discriminant function f1(x) and f2(x) and the sample is assigned to the group at which corresponds the maximum value of the discriminant function. The current commercial Fast Field Cycling relaxometers operate with a solenoid magnet which may achieve magnetic fields from few kHz to a maximum of 42 MHz in terms of proton Larmor frequency (i.e., to about 1 T). The block diagram of a typical system (i.e., an NMR relaxometer) is shown in Fig. 6. The NMR system includes a resistive solenoid (indicated as “Magnet” in Fig. 6) connected to a suitable magnet power supply unit (controlled via a dedicated magnet interface) and a cooling system, specifically designed for Field Cycling purposes. The NMR system further includes an electronic control unit that in turn includes an interface to the magnet power supply unit, a Variable Temperature Controller (VTC), a RadioFrequency (RF) unit (transmitter and receiver, including a preamplifier) and an acquisition unit. The electronic modulation of the current flowing through the coil of the electromagnet permits fast variations of the magnetic field induction and the acquisition of very short T1, at present, down to fractions of a millisecond. The probe, the RadioFrequency (RF) unit (transmitter and receiver, including a preamplifier), the sample Variable Temperature Controller (VTC) and the Acquisition Unit are conventional systems for NMR instruments, and a corresponding detailed description will not be provided herein for the sake of brevity. In particular, the acquisition unit detects the NMR signal from the sample and digitizes that signal for processing by a host computer system (or, more generally, a processing unit), e.g., a workstation or a computer system embedded in the relaxometer apparatus. A computer program (e.g., software package) running on the host computer system supports all experimental procedures (e.g., data acquisition, visualization and evaluation). The present invention relies on the observation of the present inventors that a tailored comparative procedure between magnetisation recovery / decay curves acquired at given Brvalues allows to assess the presence of tumor tissue in resected lymph node(s). The relaxation rate R1of biological tissues is affected by the field strength at which the measurement is carried out. In particular, R1shows a dramatic increase on going from high to low fields. At the low fields the intracellular R1is high (very short T1), whereas R1of the extracellular compartment maintains a relatively low value. As water protons are the main source of the detected signal, the observed relaxation rates R1are weighted to values that reflect either the ratio between the volumes of the two compartments and the exchange rate between them. They contain relevant information on the cell types present in the investigated tissue specimen and on the physio-pathological transformations occurring inside the cells and at the level of membrane water permeability. The exchange of water molecules across the tumor cellular membrane reflects either an enhanced metabolism (the higher production of metabolites implies an increase of the osmotic pressure) and the over-expression / up / down- regulation of transporters at the cellular membrane. In Fig. 7 the R1values obtained from healthy lymph nodes (white dots) and metastatic lymph nodes (black dots), in the field range corresponding to Larmor Frequencies (= ^^B0, where ^ is the proton gyromagnetic constant) of 0.01 to 1 MHz, are reported. R1of the healthy lymph nodes results higher than the ones containing the tumor tissue. This behavior is due to the fact that, in the tumor specimen, there is an increase of the extracellular space and, at the same time, an increase of the water exchange rate between the intra- and extra-cellular compartments. In one embodiment, the present invention concerns a method to assess the presence of tumor tissue in a biological specimen resected during surgery / biopsy using the Field Cycling Relaxometry technique, wherein the specimen is one or more lymph nodes (the flow diagram of the method being shown in Figure 10), the method comprising the following steps: a. obtaining a first sequence of at least m values of a measuring magnetic field strength Bri, wherein m ≥ 3, preferably m ≥ 5, a second sequence of at least n values of a time τ, a detection magnetic field strength Bd, a re-cycle delay time RD, a polarization magnetic field strength Bpand a polarization time tp; b. exposing the specimen to a polarization magnetic field having a polarization magnetic field strength Bpfor a time tp, thus allowing the magnetization build-up, if the current measuring magnetic field strength Briis ≤ 7 MHz; c. exposing the specimen to a measuring magnetic field having a measuring magnetic field strength Brifor a time τ, thus allowing the magnetisation of the specimen to relax towards a magnetisation equilibrium value determined by the actual Bri; d. acquiring a value of magnetisation MZof the specimen at time τ and at the measuring magnetic field strength Bri, the acquisition being carried out while exposing the specimen to a detection magnetic field having a detection magnetic field strength Bdafter the application of a 90° pulse; e. switching off the magnetic field for the re- cycle delay time RD; f. repeating steps b. to e. a number of cycles n, wherein the value of time τ varies at each cycle; g. fitting the values of magnetization MZacquired after n repetitions of steps b. to e. (magnetization intensity vs. τ) according to a monoexponential Bloch equation to produce a respective magnetization recovery / decay curve and calculate the proton longitudinal relaxation rate R1Briat the respective magnetic field strength Bri; h. repeating steps b. to g. a number of times m, wherein the value of the measuring magnetic field strength Brivaries each time, thereby calculating at least three proton longitudinal relaxation rates R1Br1, R1Br2, R1Br3at the at least three measuring magnetic field strengths Bri; i. calculating: − a Ratio of two relaxation rates R1Bri; − a Sum of at least two relaxation rates R1Bri; − an extracellular water mole fraction p0by fitting the at least three magnetisation recovery curves by the two-Site eXchange model; − an intracellular residence lifetime τiby fitting the at least three magnetisation recovery / decay curves by the two-Site eXchange model; j. processing the parameters calculated in step g. and i. for assessing the presence of tumor tissue in the specimen by: 1. calculating a product P = Ratio * p0, wherein tumor tissue is present in the specimen if P has a first positioning with respect to a cut-off and tumor tissue is absent in the specimen if P has a second positioning with respect to a cut-off; or 2. calculating a decision boundary S12(x) involving at least three parameters selected among the Ratio, the Sum, the extracellular water mole fraction p0, the intracellular residence lifetime τiand the relaxation rate R1Briobtained for a measuring magnetic field strength Bri(preferably equal to 1 MHz), wherein when the calculation of the decision boundary S12(x) involves three parameters at least one of the three parameters is the extracellular water mole fraction p0, or the intracellular residence lifetime τi, wherein tumor tissue is present in the specimen if S12(x) > 0, and tumor tissue is absent in the specimen if S12(x) < 0. The method for determining the positivity / negativity of the lymph node(s) herein disclosed can be carried out either during or after the surgery. In one embodiment, the present invention concerns an NMR relaxometer system that includes: - a solenoid connected to a cooling system and a power supply unit, where the solenoid defines a space for hosting the probe in which the sample is settled; - a radio-frequency unit; - a magnet interface controlling the magnet power supply and thus the magnetic field generated by the solenoid; - a sample temperature control unit; - an acquisition module configured to acquire magnetisation data from the biological specimen; - a computer host equipped with a computer program (e.g., software package) for controlling the acquisition unit and controlling various steps of the experimental procedure (e.g., data acquisition, visualization and evaluation). In one embodiment, the present invention concerns a computer program product comprising instructions to operate an NMR relaxometer system according to the invention to execute the steps of the method according to the invention. The assessment of the optimal conditions of the Fast Field Cycling relaxometer will be acquired with an auto- test carried out before the start of the measurement session using a properly designed phantom endowed with relaxation properties in the range of those expected for the investigated specimen. The phantom is made by a cross-linked protein in a buffered solution with a preservative agent and it is sealed under inert gas atmosphere. For example, the phantom consists of a solution of Bovine Serum Albumin (BSA) 9% in phosphate buffer pH 7.4 plus sodium azide 0.3 %, cross-linked by heat (20’ at 80°C) or by glutaraldehyde. The skilled man, with his common general knowledge, knows how to produce a phantom endowed with the relaxation properties in the range of those expected for the investigated biological tissue under analysis. The check of relaxometer performance consists of the acquisition of the R1values at two magnetisation field strengths twice with the defined protocol. Both the signal intensity and the obtained R1values are evaluated. The limits of acceptability for signal intensity, average and standard deviation values of the repeated R1measurements have to be within the 5% of the values certified by the phantom manufacturer. The main parameters to be set for carrying out the method of the present invention are: - the number of times m and the values of the measuring magnetic field strengths Briused for the acquisition of the magnetisation recovery / decay curves; - the value of the polarization magnetic field strength Bpand the polarization time tp; - the number of cycles n and the values of the time intervals τ for the acquisition of the magnetisation relaxation curves at each Brivalue; - the waiting time before the starting of the new acquisition cycle (recycle delay, RD). In case the magnetization Mzmeasured by the relaxometer is lower than a threshold value fixed by the instrument manufacturer, the method steps b. to h. are repeated at least one more time and the magnetisations Mzdetermined in the first scan are summed to the magnetisations Mzdetermined in the second scan. For determining the positivity / negativity of the lymph node(s), the following parameters are calculated in steps g. and i.: - the relaxation rate R1Briat a given Bri, obtained by the fit of the magnetisation recovery / decay curves acquired in step c. (Intensity vs. τ) according to the monoexponential Bloch equation; - the Ratio that is the ratio of two relaxation rates R1Br1 / R1Br2measured at two different measuring magnetic field strengths Bri, wherein Br1is in a range of 0.01 - 0.15 MHz, and Br2is in a range of 0.15 - 10 MHz; - the Sum that is the sum of two relaxation rates R1Br2+R1Br3(2R1) or more (n) (nR1) relaxation rates measured at two or more (n) different Bri, wherein each Briis in the range of 0.15 – 10 MHz and wherein one of the Briis the highest Briapplied, for example the 2R1= R10.39 MHz+ R11 MHz; residence lifetime τiobtained by the simultaneous fit of the at least three magnetisation recovery curves (Intensity vs. τ) according to the 2SX model; - the tissue extracellular water mole fraction p0obtained by the simultaneous fit of at least three magnetisation recovery curves (Intensity vs. τ) according to the 2SX model. The above parameters are then subjected to a further elaboration: - calculation of a parameter P = Ratio * p0, and - calculating a decision boundary S12(x) involving at least three parameters selected from the Ratio, the Sum, the extracellular water mole fraction p0, the intracellular residence lifetime τiand the relaxation rate R1Bri. The specimen is classified as follows: - presence of tumor tissue in the specimen if the parameter P has a first positioning with respect to a cut-off and absence of tumor tissue in the specimen if the parameter P has a second positioning with respect to a cut-off; or - presence of tumor tissue in the specimen if S12(x) > 0, absence of tumor tissue in the specimen if S12(x) < 0, and undefined specimen if S12(x) = 0. The cut-off is determined in a training phase during which several resected tumor-affected lymph nodes are subjected to the method disclosed above and then to the histological analysis provided by a surgical pathologist. The histological analysis (comprising tissue formalin-fixation, processing, sectioning, and staining with Hematoxylin & Eosin) is the reference method for tumor assessment and allows the correct distribution of the analyzed specimen in the positive or negative classes, i.e., the True positive (TP) and True Negative (TN) groups, respectively. The cut-off value to assign the sample to the correct class (i.e., containing or not tumor tissue) is evaluated according to the Receiver Operating Characteristic (ROC) curve. The ROC curve is constructed by plotting the true positive rate (TPR) against the false positive rate (FPR) for the different possible cut-off values. The true positive rate is the proportion of samples that are correctly predicted to be positive out of all positive samples (TP / (TP + FN)), where FN are the False Negative samples. Similarly, the false positive rate is the proportion of samples that are incorrectly predicted to be positive out of all negative samples (FP / (TN + FP), where FP are the False Positive samples. The TPR and FPR correspond to the sensitivity and the (1 - specificity) of the method, respectively. Each point on the ROC curve represents a sensitivity / specificity pair. The cut-off value is the point which classifies most of the samples correctly, i.e., it has the best sensitivity / specificity pair. The decision boundary S12(x) is calculated from the difference of two discriminant functions which involve at least three parameters selected from the Ratio, the Sum, the extracellular water mole fraction p0, the intracellular residence lifetime τiand the relaxation rate R1Bri, wherein the discriminant functions are determined by performing a discriminant analysis of a training data set involving at least three of the parameters set forth above. In one embodiment, the acquisition is carried out at a detection magnetic field strength Bdcomprised between 5 MHz and 20 MHz expressed in terms of proton Larmor frequency. In one embodiment, the measuring magnetic field strength Briis comprised in the range 0.01 to 10 MHz in terms of proton Larmor frequency. In one embodiment, the relaxation rates R1Bri(=1 / T1Bri) are determined by applying the following Bloch equations: −^^ ^^ ^^^^^^ ^^⁄ ^^^^^^^^ ^^^^^^^^ and −^^ ^^ ^^^^^^ ^^⁄ ^^^^^^^^^^^^^^^^ wherein ^^^^0^^^^is the highest measured magnetisation Mzat Bri; ^^^^0^^^^is the magnetisation Mzat τ = 0 if Bri> 7 MHz (i.e., the NP sequence) or the magnetisation Mzat τ → ∞ if Bri≤ 7 MHz (i.e., the PP sequence); and equation [1] is applied for Bri> 7 MHz (i.e., the NP sequence), equation [2] is applied for Bri≤ 7 MHz (i.e., the PP sequence). In one embodiment, at least two values of Briare comprised between 0.01 and 0.15 MHz, and at least one value of Briis comprised between 0.15 and 10 MHz. In one embodiment, at least three values of Briare comprised between 0.01 and 0.15 MHz, and at least two values of Briare comprised between 0.15 and 10 MHz. In one embodiment, at least four values of Briare comprised between 0.01 and 0.15 MHz, and at least three values of Briare comprised between 0.15 and 10 MHz. In one embodiment, the Ratio is calculated by dividing two relaxation rates R1Briat two different measuring magnetic field strengths Bri, wherein the relaxation rate R1Briin the numerator is the relaxation rate R1Bridetermined to a value of the measuring magnetic field strength Bricomprised between 0.01 and 0.15 MHz, and the relaxation rate R1Briin the denominator is the relaxation rate R1Bridetermined to a value of the measuring magnetic field strength Bricomprised between 0.15 and 10 MHz. In one embodiment, the Sum is calculated by summing two relaxation rates R1Briat two different measuring magnetic field strengths Bri, wherein the two relaxation rates R1Briare determined at two values of the measuring magnetic field strength Bricomprised between 0.01 and 10 MHz. In one embodiment, the intracellular residence lifetime τi, and the tissue extracellular water mole fraction p0are calculated by the two-Site eXchange model by means of the following equation(s):^^^^ ^^ = ^^^^^^ − − ∙ ^^^^^^ ∙ ^^ ′ + ^^^^ ∙ ^^^^^^ ∙′^^^^^^ +^^ ^^^^^^ ^^^^ and wherein R1oBriis the longitudinal relaxation rate constant of the extracellular water protons of Matrigel at the measuring magnetic field strength Bri; R1iBriis the longitudinal relaxation rate constant of the intracellular water protons of the specimen at the measuring magnetic field strength Bri; ^^^^0^^^^and ^^^^0^^^^have the meaning set forth above; τo= τi(po / pi) [7], and equation [3a] is resolved for Bri> 7 MHz (i.e., the NP sequence), equation [3b] is resolved for Bri≤ 7 MHz (i.e., the PP sequence). In one embodiment, the specimen is a resected lymph node(s) having a weight comprised between 15 mg and 115 mg, preferably between 30 mg and 60 mg, as obtained by the surgeon in the surgery room. In one embodiment, the specimen is placed in a glass tube (having generally an outer diameter of 5 mm but other sizes are possible), the glass tube being introduced in the Field Cycling Relaxometer. In one embodiment, the steps d. to f. are carried out at a temperature ≤ 25 °C, preferably at about 10 °C. In one embodiment, when the steps d. to f. are carried out at a temperature lower than the room temperature (i.e., lower than 25 °C), a test tube containing the tissue specimen is brought to the selected temperature through the passage in a sample cooling device, wherein the sample cooling device is designed to host the test tube to allow the specimen to reach the desired temperature, preferably in less than one minute. In one embodiment, the polarization magnetic field strength Bpof the polarization magnetic field is comprised between 8 and 25 MHz. In one embodiment, the polarization step b. is carried out for a polarization time tp, wherein tpis comprised in the range 0.5 to 1.5 s. In one embodiment, the switching off of the magnetic field in step e. allows the specimen magnetisation to completely decay. In one embodiment, the switching off of the magnetic field in step e. lasts for at least 0.5 sec. In one embodiment, the number of cycles n ranges from 10 to 32. In one embodiment, the time value τ ranges from 0.01 to 4 sec. In one embodiment, in each cycle n, in which steps b. to e. are repeated, the time value τ is increased, preferably on a logarithmic basis. In one embodiment, the process steps b. to h. are carried out once employing a Matrigel specimen for determining the relaxation rate constants R1Briat the magnetic field strengths Briapplied according to the monoexponential Bloch equation, wherein the obtained R1Briare used as the longitudinal relaxation rate constants of the extracellular water protons R1oBriat the measuring magnetic field strength Briin the application of the two-Site eXchange model. In the following some embodiments of the present invention are disclosed. Assessment of positive / negative Lymph nodes In order to assess whether the lymph node is healthy (negative) or it has been invaded (positive) by tumor cells, two protocols L1 and L2 have been developed as shown in Figure 8. The two protocols imply to apply the following steps as reported in the Figure 8: − Acquisition step: acquisition of the magnetisation recovery / decay curves at a temperature ≤ 25°C at: 2-4 values of Larmor frequencies in the range 0.01-0.15 MHz using the protocol AP3 or AP4; 1-3 values of Larmor frequencies in the range 0.15-10 MHz using the protocol AP1 or AP2. Protocols AP1 to AP4 are disclosed below. In the case of protocol L1 (first panel of Figure 8): − Data treatment: the fitting of the magnetisation recovery / decay curves obtained in the acquisition step (Intensity vs. τ) is performed according to the monoexponential Bloch equation [1-2] to calculate the R1Brivalues at the given Bri. The obtained R1data are analyzed in terms of the above reported Ratio. The same data are also analyzed in terms of the 2SX model resolving equation [3a-b] to extract the extracellular water mole fraction p0. In the case of protocol L2 (second panel of Figure 8): - Data treatment: the fitting of the magnetisation recovery / decay curves obtained in the acquisition step (Intensity vs. τ) is performed according to the monoexponential Bloch equation [1-2] to calculate the R1Brivalues at the given Bri. The obtained R1Bridata are analyzed in terms of the above reported Ratio and 2R1methodology. The same data are also analyzed in terms of the 2SX model resolving equation [3a- b] to extract the intracellular residence lifetime τiand the extracellular water mole fraction p0. At least three of the previous parameter values (Ratio, 2R1, τi, p0and the R1value at 1 MHz) are used to calculate the decision boundary S12(x), i.e. the value of the discriminant functions f1and f2and to apply the sample classification scheme according to equation [9]. Examples of settings for the R1acquisitions at Brbetween 0.15 and 10 MHz are: − [AP1]: Bp= 9.5 MHz, tp= RD = 0.9 s, n = 14 τ distributed on a logarithmic basis in the interval 0.01-1.8 s; − [AP2]: Bp= 9.5 MHz, tp= RD = 0.9 s, n = 32 τ distributed on a logarithmic basis in the interval 0.01-4.0 s. Examples of settings for the R1acquisitions at Brbetween 0.01 and 0.15 MHz are: − [AP3]: Bp= 9.5 MHz, tp= RD = 0.9 s, n =14 τ distributed on a logarithmic basis in the interval 0.0035-1.0 s; − [AP4]: Bp= 9.5 MHz, tp= RD = 0.9 s, n = 32 τ distributed on a logarithmic basis in the interval 0.035-2.8 s. Application of the L protocols to assess the invasion of Lymph nodes by tumor cells 18 human lymph nodes were investigated (14 from breast surgery and 4 from prostate surgery). 2 lymph nodes from rats were also investigated. The results from the anatomopathological laboratory yielded 12 healthy lymph nodes (negative) and 8 invaded lymph nodes (positive). For data acquisition, the protocols reported in Figure 9, a specification of Figure 8, were followed. In particular, the acquisitions for both protocols were carried out at 10 °C and at the following Larmor frequencies: 0.01, 0.02, 0.037 and 0.07 MHz using the acquisition protocol AP2; 0.15, 0.39 and 1 MHz using the protocol AP1 (overall required time: ca. 20’). For both protocols, the Ratio was calculated using the data acquired at 0.02 and 1 MHz. The Mzdecay curves acquired at the previous 7 listed magnetic field strengths were subjected to the 2SX model treatment, obtaining the ^iand p0values. In the protocol L1, the P parameter is calculated as follows: ^^0.02 ^^^^^^ P = Ratio *1^^^^^^* p0In this case, it was the parameter P is less than or equal to 0.83 (the cut-off), the sample is classified in the negative lymph node group. When P is greater than 0.83 the specimen is likely to contain tumor tissue and is classified in the positive lymph node group (see Figure 11). By comparing the classification obtained with protocol L1 and the anatomopathologist analysis, one FN and one FP were found, thus yielding a sensitivity of 88%, specificity of 92% and accuracy (in terms of AUC, i.e., area under the curve) of 90%. The protocol L2 applies the discriminant rule learned from the training data set, developed by using the OriginPro software (Version 2023, OriginLab Corporation, Northampton, MA, USA) according to the QDA model and equal prior probabilities. The variables involved in the determined discriminant functions are Ratio, p0and R11MHz, f1(Ratio, p0and R11MHz) and f2(Ratio, p0and R11MHz) where 1 indicates the metastatic lymph node group and 2 the healthy lymph node group. The values of Ratio, p0and R11MHzof each sample are substituted in the discriminant function f1(x) and f2(x) and the sample is assigned to the class which corresponds the maximum score according to the classification scheme [9]. By comparing the classification obtained with protocol L2 and the anatomopathologist analysis (Figure 12), one FP was found, thus yielding a sensitivity of 100%, specificity of 92% and accuracy (in terms of AUC, i.e., area under the curve) of 96%. The histological evaluation The histological analysis is the gold-standard reference and the results from the relaxometric determinations have been referred to the histological evaluation for all the investigated specimens. The typical procedure is as follows: Hematoxylin & Eosin (H&E) staining was conducted using standard methods on formalin-fixed, paraffin-embedded tissues. Samples were fixed in 10% neutral buffered formalin and embedded in paraffin. For histopathological analysis, samples were serially sectioned (5 μm) and every section stained with H&E. Histopathological scoring of tumor was performed by an expert anatomopathologist. Comparative example The article by Bitonto V. et al. discloses a method to assess the presence of tumour tissue in a biological specimen resected during surgery / biopsy using the Field Cycling Relaxometry technique, according to the Ratio and the 2R1parameters, defined as follow: Ratio = R10.02 MHz / R11 MHz2R1 = R10.39 MHz+ R11 MHz The findings below demonstrate that the method object of the present invention produces superior classification results for lymph nodes compared to those achieved with the parameters specified in Bitonto V. et al. The relaxometric data set (related to 20 freshly excised lymph nodes) of the previous example was analysed using the protocol reported in Bitonto V. et al. based on the Ratio and 2R1parameters. Figure 13 shows the comparison of the results obtained using the parameter P of the present invention (case A) with those obtained using the Ratio (case B) or the 2R1(case C) parameters. As was the case for the P parameter, the ROC analysis was employed to identify the most suitable cut-offs, which are displayed in the figure as dotted lines (3.4 for the Ratio parameter and 14.5 s-1for the 2R1parameter, respectively). The two parameters of Bitonto V. et al. are not able to discriminate between positive and negative lymph nodes as the P parameter object of the present invention does (sensitivity of 88%, specificity of 92%). In fact, the calculated sensitivity and specificity are 87.5% and 83.3% for the Ratio parameter, 66.7 % and 62.5 % for the 2R1parameter, respectively. The L2 protocol previously outlined, which calculated the decision boundary S12(x) to discriminate between positive and negative samples employing the Ratio, p0, R11MHzparameters (Figure 9) was replicated for all possible triads of the feature variables (Ratio, 2R1, p0, ^i, R11MHz) in order to provide an illustrative example of the effects of parameter selection. The outcomes are enumerated (case 1-10) in the subsequent table 3: Table 3n Parameters FN,FP Sensitivity SpecificityAccuracy Case 11 serves as an illustration of the utilisation of four parameters. The increment of the number of variables, without including p0or ^iimproves the performance. It is noteworthy that case 5 corresponds to the aforementioned example. It is one of the cases related to the maximum attainable accuracy of 95%. Case 3, which exhibits the lowest level of accuracy, does not utilise the p0, tiparameters that are the result of data processing according to the 2SX model. In conclusion, the method object of the present invention is based on two protocols that can be applied to fresh tissue samples (intraoperative analysis allowed) using low-cost instrumentation. This approach demonstrates enhanced classification performance with respect to the results currently available according to the prior art.

Claims

CLAIMS 1. A method to assess presence of tumor tissue in a biological specimen resected during surgery / biopsy using the Field Cycling Relaxometry technique, wherein the specimen is one or more lymph nodes, the method comprising the following steps: a. obtaining a first sequence of at least m values of a measuring magnetic field strength Bri, wherein m ≥ 3, preferably m ≥ 5, a second sequence of at least n values of a time τ, a detection magnetic field strength Bd, a re-cycle delay time RD, a polarization magnetic field strength Bpand a polarization time tp; b. exposing the specimen to a polarization magnetic field having a polarization magnetic field strength Bpfor a time tp, thus allowing the magnetization build-up, if the current measuring magnetic field strength Briis ≤ 7 MHz; c. exposing the specimen to a measuring magnetic field having a measuring magnetic field strength Brifor a time τ, thus allowing the specimen magnetisation to relax versus a magnetisation equilibrium value determined by the actual Bri; d. acquiring a value of magnetisation MZof the specimen at time τ and at the measuring magnetic field strength Bri, the acquisition being carried out while exposing the specimen to a detection magnetic field having a detection magnetic field strength Bdafter a 90° pulse; e. switching off the magnetic field for the re- cycle delay time RD;f. repeating steps b. to e. a number of cycles n, wherein the value of time τ varies at each cycle; g. fitting the values of magnetization MZacquired after n repetitions of steps b. to e. (magnetization intensity vs. τ) according to a monoexponential Bloch equation to produce a respective magnetization recovery / decay curve and calculate the proton longitudinal relaxation rate R1Briat the respective magnetic field strength Bri; h. repeating steps b. to g. a number of times m, wherein the value of the measuring magnetic field strength Brivaries each time, thereby calculating at least three proton longitudinal relaxation rates R1Br1, R1Br2, R1Br3at the at least three measuring magnetic field strengths Bri; i. calculating: − a Ratio of two relaxation rates R1Bri; − a Sum of at least two relaxation rates R1Bri; − an extracellular water mole fraction p0by fitting the at least three magnetisation recovery curves by the two-Site eXchange model; − an intracellular residence lifetime τiby fitting the at least three magnetisation recovery / decay curves by the two-Site eXchange model; j. processing the parameters calculated in step g. and i. for assessing the presence of tumor tissue in the specimen by:

1. calculating a product P = Ratio * p0, wherein tumor tissue is present in thespecimen if P has a first positioning with respect to a cut-off and tumor tissue is absent in the specimen if P has a second positioning with respect to a cut-off; or 2. calculating a decision boundary S12(x) involving at least three parameters selected among the Ratio, the Sum, the extracellular water mole fraction p0, the intracellular residence lifetime τiand the relaxation rate R1Briobtained for a measuring magnetic field strength Bri, wherein when the calculation of the decision boundary S12(x) involves three parameters at least one of the three parameters is the extracellular water mole fraction p0, or the intracellular residence lifetime τi, wherein tumor tissue is present in the specimen if S12(x) > 0, and tumor tissue is absent in the specimen if S12(x) < 0.

2. The method according to claim 1, wherein the acquisition is carried out at a detection magnetic field strength Bdcomprised between 5 MHz and 20 MHz expressed in terms of proton Larmor frequency.

3. The method according to any one of the preceding claims, wherein the measuring magnetic field strength Bri is comprised in the range 0.01 to 20 MHz in terms of proton Larmor frequency.

4. The method according to any one of the preceding claims, wherein the relaxation rates R1Bri(=1 / T1Bri) are determined by applying the following Bloch equations: −^^ ^^ ^^ ^^^^^^ ^^⁄ ^^^^^^^^^^^^^^^^and −^^ ^^ ^^ ^^ ^^^^^^ ^^⁄ ^^^^^^^^^^^^wherein ^^^^0^^^^is the highest measured magnetisation Mzat Bri; ^^^^0^^^^is the magnetisation Mzat τ = 0 if Bri> 7 MHz or the magnetisation Mzat τ → ∞ if Bri≤ 7 MHz; and equation [1] is resolved for Bri> 7 MHz, equation [2] is resolved for Bri≤ 7 MHz.

5. The method according to any one of the preceding claims, wherein the Ratio is calculated by ratioing two relaxation rates R1Briat two different measuring magnetic field strengths Bri, wherein the relaxation rate R1Briin the numerator is the relaxation rate R1Bridetermined to a value of the measuring magnetic field strength Bricomprised between 0.01 and 0.15 MHz, and the relaxation rate R1Briin the denominator is the relaxation rate R1Bridetermined to a value of the measuring magnetic field strength Bricomprised between 0.15 and 10 MHz.

6. The method according to any one of the preceding claims, wherein the Sum is calculated by summing two relaxation rates R1Briat two different measuring magnetic field strengths Bri, wherein the two relaxation rates 5 R1Briare determined at two values of the measuring magnetic field strength Bricomprised between 0.15 and 10 MHz.

7. The method according to any one of the preceding 10 claims, wherein the intracellular residence lifetime τiand the tissue extracellular water mole fraction p0are calculated by means of the following equation(s): ^^^^ = ^^ ^^^^^^ − − ^^^^ ∙ ^^^^^^ ∙ ^^ ′ + ^^^^ ∙ ^^^^^^ ∙ ^^′^^^^^^ + ^^^^15 20 251 ^^^^^^ ^^ ^^^^^1^^ − ^^ ^^ ^^^^ −^^2 ′^^ ^^ ^^^^ ^−1^^^^ ^^^^−1and wherein R1oBriis the longitudinal relaxation rate constant of the extracellular water protons of Matrigel at the measuring magnetic field strength Bri; R1iBriis the longitudinal relaxation rate constant of the intracellular water protons of the specimen at the measuring magnetic field strength Bri; ^^^^0^^^^is the highest measured magnetisation Mzat Bri; ^^^^0^^^^is the magnetisation Mz at τ = 0 if Bri > 7 MHz or the magnetisation Mzat τ → ∞ if Bri≤ 7 MHz; τo= τi(po / pi) [7]; and equation [3a] is resolved for Bri> 7 MHz, equation [3b] is resolved for Bri≤ 7 MHz.

8. The method according to any one of the preceding claims, wherein the specimen is a resected lymph node(s) having a weight comprised between 15 mg and 115 mg as obtained by the surgeon in the operative room.

9. The method according to any one of the preceding claims, wherein the steps d. to f. are carried out at a temperature ≤ 25 °C, preferably about 10 °C.

10. The method according to any one of the preceding claims, wherein the polarization magnetic field strength Bpof the polarization magnetic field is comprised between 8 and 25 MHz.

11. The method according to any one of the preceding claims, wherein the polarization step b. is carried out for a polarization time tp, wherein tpis comprised in the range 0.5 to 1.5 s.

12. The method according to any one of the preceding claims, wherein the switching off of the magnetic field in step e. lasts for at least 0.5 sec.

13. The method according to any one of the preceding claims, wherein the number of cycles n ranges from 10 to 32.

14. The method according to any one of the preceding claims, wherein the time value τ ranges from 0.01 to 4 sec.

15. The method according to any one of the preceding claims, wherein the process steps b. to h. are carried out once employing a Matrigel specimen for determining the relaxation rate constants R1Briat the magnetic fieldstrengths Briapplied according to the monoexponential Bloch equation and the obtained R1Briare used as the longitudinal relaxation rate constants of the extracellular water protons R1oBriin the application of the two-Site eXchange model.

16. An NMR relaxometer system comprising: - a solenoid connected to a magnet power supply unit and to a cooling system, wherein the solenoid defines a space for hosting a probe in which a biological sample is insertable; - an electronic control unit that includes an interface coupled to the magnet power supply unit for controlling the power supply unit, a temperature controller coupled to the probe for controlling the temperature of the probe, a radiofrequency unit, and a data acquisition unit configured to acquire magnetisation data from the biological sample; and - a processing unit configured to control the data acquisition unit to execute the steps of the method according to any one of claims 1 to 15.

17. A computer program product loadable in the memory of a processing unit of an NMR relaxometer system according to claim 13 and comprising instructions to cause the NMR relaxometer system to execute the steps of the method according to any one of claims 1 to 15.

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