In vitro method for detecting thyroid cancer

Abstract

The present application discloses a novel and low-cost in vitro method for detecting thyroid cancer. The method comprises determining whether the lactate level of a malignant thyroid nodule is different to that of blood lactate, normal thyroid gland and benign thyroid nodule and applying this knowledge to clinical decision making of an indeterminate fine-needle aspiration (FNA) biopsy. The invention further relates to kits for detecting thyroid cancer comprising needles for obtaining samples, test strips to detect lactate levels and use indications.

Description

IN VITRO METHOD FOR THE DETECTION OF THYROID CANCER Field of invention

[0001] The present invention relates to a novel and low-cost in vitro method for determining the presence of thyroid cancer in a patient suspected of having thyroid cancer, as well as an in vitro method for the detection of thyroid cancer, comprising the use of specific lactate levels to determine whether a thyroid nodule is malignant and to know the diagnostic value of said lactate level in the cancerous thyroid nodule and to apply this knowledge in the “indeterminate” FNA. Background of the Invention

[0002] In the field, the following methods for diagnosing thyroid nodules are known, all of which are related to the evaluation of treatment for subjects diagnosed with thyroid cancer.

[0003] Some state-of-the-art solutions for the diagnosis of thyroid nodules and / or thyroid cancer are disclosed in the following documents.

[0004] WO 2012068483, Synta Pharmaceuticals Corp., 2011-11-18, PRESELECTION OF SUBJECTS FOR THERAPEUTIC TREATMENT BASED ON HYPOXIC STATUS, provides methods for preselecting a subject for therapeutic treatment with an agent based on modulated levels of hypoxia in the subject's cancer cells. In one modality, the document provides methods for preselecting a subject for therapeutic treatment with an agent based on modulated levels of lactate dehydrogenase (LDH) in a cell, for example, a cancer cell.

[0005] WO 2014194293, AMPLIMMUNE, INC, 2014-05-30, IMPROVED METHODS FOR THE SELECTION OF PATIENTS FOR PD-1 OR B7-H4 TARGETED THERAPIES, AND COMBINATION THERAPIES THEREOF, refers to improved methods for selecting and treating patients who would be suitable for PD-1 and B7-H4 pathway targeted and combination therapies. Specifically, the disclosure refers to improved PD-1 targeted and combination therapies for treating patients who have failed treatment with BRAF / MEK inhibitors or other RAS-RAF-MEK-ERK pathway inhibitors.

[0006] On the other hand, US 20230073725, ARIZONA BOARD OF REGENTS ON BEHALF OF THE UNIVERSITY OF ARIZONA, 2021-01-14, METABOLITE BIOMARKER PROFILE AND METHOD OF USE TO DIAGNOSE PULMONARY ARTERIAL HYPERTENSION (PAH), presents a method comprising a panel of metabolomic biomarkers that represents a metabolic profile / fingerprint and methods for applying the profile to diagnose, monitor, and guide treatment for pulmonary arterial hypertension (PAH). The profile comprises a unique panel of 36 metabolomic biomarkers / metabolites detected in plasma and / or urine obtained from the patient.

[0007] CN 116148472, Nanjing First Hospital, 2023-01-30, "A Kind of Metabolic Enzyme LDHA Succinylation Site and Its Specific Antibody and Application," describes a succinylation site of the metabolic enzyme LDHA, its specific antibody, and its application. The disclosure identifies the K155 amino acid site that affects LHDA protease activity and confirms that the K155R mutation significantly affects the level of succinylation modification of the LDHA protein.

[0008] WO 2006127537, JOHNSON & JOHNSON, 2006-05-18,THYROID FINE NEEDLE ASPIRATION MOLECULAR ASSAY, refers to methods, compositions and articles aimed at diagnosing thyroid carcinoma, differentiating between thyroid carcinoma and benign thyroid diseases, testing indeterminate thyroid fine needle aspiration samples from thyroid nodules and determining patient protocols and outcomes.

[0009] The thyroid nodule should be objectively evaluated by fine-needle aspiration biopsy (FNAB). This study is highly effective, with a sensitivity of 70 to 99% and a specificity of 50.7 to 70%. The FNAB result can be "non-diagnostic" or "indeterminate" if it falls within categories III, IV, or V of the Bethesda System. The reported probability of malignancy is 24.3 to 37.8% for Bethesda III, 26 to 73.5% for Bethesda IV, and 91.1 to 94.1% for Bethesda V. Bethesda category I can also be considered indeterminate, especially when the nodule occurs on two or more occasions and is accompanied by a Thyroid Imaging Reporting and Data System (TIRADS) ultrasound grade 4 or 5. up to 12.4%.

[0010] To resolve this uncertainty, a diagnostic surgery is traditionally performed, which consists of a thyroid lobectomy with intraoperative frozen section histopathological study.Currently, molecular testing is recommended to avoid unnecessary surgery. The most common tests are Thyroseq and the Afirma test, both with good diagnostic capabilities. In the most recent meta-analysis evaluating the diagnostic capacity of these tests, the ThyroSeq test (version 3) reported a sensitivity of 99% and a specificity of 64% with a negative predictive value of 96%, while the Afirma Genomic Sequencing Classifier (GSC) reported a sensitivity of 95%, a specificity of 51%, and a negative predictive value of 91%. These tests aim to avoid diagnostic surgery; however, their usefulness is limited by accessibility, as they are not available worldwide. Furthermore, their high cost—approximately $3,200 for Thyroseq V3 and approximately $4,600 for Afirma-GSC—significantly limits their application in social health programs.

[0011] Despite the solutions provided in the prior art, there remains a need for effective inventions that can determine whether the lactate level of a malignant thyroid nodule differs from that of capillary lactate, normal thyroid gland, and benign thyroid nodules. Knowing the diagnostic value of lactate in thyroid cancer allows for its application to determining this level in indeterminate fine-needle aspiration biopsy (FNAB). The present invention addresses this problem by providing a novel, low-cost in vitro method for determining the presence of thyroid cancer in a patient suspected of having the disease, as well as a method for thyroid cancer detection that utilizes specific lactate levels to determine whether a thyroid nodule is malignant. Brief Description of the Invention

[0012] The present invention has two objectives: first, to determine if the lactate level in the malignant thyroid nodule is different from capillary lactate, from that of a normal thyroid gland and a benign thyroid nodule; and second, to determine the diagnostic value of lactate measurement in the thyroid nodule with thyroid cancer and to apply this knowledge to indeterminate FNAs, which will help in making a clinical decision with lower cost, greater accessibility, and without the need for diagnostic surgery. Description of the Figures

[0013] The particular features and advantages of the invention, as well as other objects thereof, will be apparent from the following description, taken in connection with the accompanying figures, which should not be considered as limiting the invention:

[0014] Figure 1 shows an ROC curve for a cutoff point of 3 mmol / lt of lactate in a thyroid nodule.

[0015] Figure 2A shows decision thresholds and what to do in each of the three situations.

[0016] Figure 2B shows decisions regarding negative and positive tests in a complete sample.

[0017] Figure 3A shows decision thresholds and what to do in each of the three situations.

[0018] Figure 3B shows decisions for negative and positive tests in a sample of "indeterminate" nodes in the two situations studied. PPPN = Probability after negative test PPPP = Probability after positive test.

[0019] Figure 4A shows decision thresholds and what to do in each of the three situations.

[0020] Figure 4B shows a comparison between molecular and lactate tests. PPPN = Probability of a negative post-test; PPPP = Probability of a positive post-test. Detailed Description of the Invention

[0021] The following definitions are provided to allow a better understanding of the invention:

[0022] The use of the term “approximate / roughly” provides a certain additional range. The term is defined as follows. The additional range provided by the term is approximately ± 10%. For example, but not limited to, if one says “approximately 22 mmol / L,” the range is within ± 10% of the standard deviation, and so on for other measurements.

[0023] To achieve the aforementioned objectives, the present invention describes a method, preferably an in vitro method, for determining the presence of thyroid cancer in a patient suspected of having an indeterminate FNA, the in vitro method comprising:

[0024] measure at least one lactate level in a first sample obtained from a malignant thyroid nodule of the patient, at least one lactate level in a second sample obtained from capillary blood of the patient, and at least one lactate level in a third sample obtained from a healthy thyroid lobe of the patient;

[0025] Compare at least one lactate level from the first, second, and third samples

[0026] Determine a cutoff point for the lactate value in the first sample selected from an interval of approximately 0.1 to approximately 22 mmol / lt to differentiate whether the first sample is a benign or malignant nodule, where a result greater than or equal to approximately 3 mmol / lt with an area under a ROC curve of approximately 0.84 indicates the presence of thyroid cancer in the patient.

[0027] In one embodiment of the present invention, a method for detecting thyroid cancer in a patient suspected of having thyroid cancer is described, comprising:

[0028] determine at least one lactate level in a first sample obtained from a malignant thyroid nodule of the patient, at least one lactate level in a second sample obtained from capillary blood of the patient, and at least one lactate level in a third sample obtained from a healthy thyroid lobe of the patient; and

[0029] Determine a cutoff point for the lactate value in the first sample selected from a range of approximately 0.1 to approximately 22 mmol / lt to differentiate whether the first sample is a benign or malignant nodule.

[0030] In another embodiment of the present invention, the first sample of the malignant thyroid nodule is obtained by means of a fine-needle aspiration biopsy (FNAB), the second sample of capillary blood from the patient is obtained by means of a needle aspiration, and the third sample of the healthy thyroid lobe of the patient is obtained by means of a fine-needle aspiration biopsy (FNAB).

[0031] For the purposes of the present invention, the first sample is selected from solid tissue of a thyroid nodule or a mixed nodule comprising a liquid component and a solid component, wherein the liquid component is aspirated from the mixed nodule to obtain only a sample of the solid component.

[0032] In another embodiment of the present invention, the lactate levels in the first, second, and third samples are determined as follows:

[0033] i) place one drop of each of the first, second and third samples on lactate test strips, optionally BM Lactate test strips; and

[0034] ii) perform the reading of the quantitative photometric measurement of reflectance on the lactate test strips.

[0035] The lactate measurement is taken approximately 30 seconds to 1.5 minutes after placing a drop of each of the first, second, and third samples on the test strips. A diagnostic value test of the lactate is then performed using Bayesian analysis and clinical decision threshold levels. The Bayesian analysis is performed using the formulas described in the "Formulas" section below to inform the clinical decision.

[0036] In a further embodiment of the present invention, a kit for the detection of thyroid cancer in a patient suspected of having thyroid cancer is included, characterized in that it comprises needles for obtaining samples; reagent strips for detecting lactate levels and instructions for use.

[0037] Materials and methods:

[0038] We studied 219 thyroid nodules from patients who had various surgical indications such as compression, cancer, "indeterminate" FNA (Bethesda III, IV and V) as well as those nodules with Bethesda I on two occasions and ultrasound with TIRADS 4 or 5.

[0039] Lactate was determined in the samples, using the Accutrend® Plus portable equipment (Roche Diagnostics) which performs a photometric measurement of reflectance with BM Lactate reagent strips (Roche Diagnostics) that quantitatively determine lactate in fresh capillary blood. The samples were taken approximately between 20 and 60 minutes before the surgery and once the patient was intubated, in such a way that the 8-hour fasting and preoperative rest time was controlled. Once the patient was intubated and with an oxygen saturation of approximately 99 to 100%, the capillary lactate sample was taken, according to the manufacturer's instructions, from the index finger of the hand opposite the venous cannulation;Subsequently, a lactate sample was taken from the healthy thyroid lobe and from the solid tissue of the thyroid nodule. These samples were obtained using a puncture technique similar to that used for ultrasound-guided fine-needle aspiration biopsy (Todsen T, Bennedbaek FN, Kiss K, Hegedüs L. Ultrasound-guided fine-needle aspiration biopsy of thyroid nodules. Head Neck. 2021;43:1009-1013). In the case of a mixed nodule (with liquid and solid components), the liquid content was aspirated and another sample of the solid component was obtained, and lactate was measured in both samples.

[0040] Both the capillary blood drop and the drops obtained by fine-needle aspiration biopsy (FNAB) were placed on BMlactate reagent strips, according to the manufacturer's specifications, and the reading was taken approximately 30 seconds to 1.5 minutes later. The BM lactate analyzer automatically processes lactate with a reading range of approximately 0.8 to 22 mmol / L.

[0041] The first analysis was performed by comparing capillary lactate levels with those obtained from the healthy thyroid lobe, as well as from benign and malignant thyroid nodules. This comparison was performed using a Student's t-test for two independent samples with a significance level of approximately p < 0.05.

[0042] Subsequently, a ROC curve was performed to determine the best cutoff point of the lactate value (approximately 0.8 to approximately 22 mmol / lt) in the thyroid nodule to detect thyroid cancer.

[0043] The second part was to perform a diagnostic value test study (sensitivity, specificity, predictive values, positive and negative likelihood ratio, area under the curve and diagnostic accuracy) of lactate in the entire studied population and subsequently the diagnostic capacity of lactate was evaluated when the FNA was “indeterminate” in two conditions: First in thyroid nodules with Bethesda FNA I, III, IV and V and the second condition in thyroid nodules with Bethesda FNA III, IV and V.

[0044] Threshold levels were constructed as follows (Pauker SG, Kassirer JP. The threshold approach to clinical decision-making. N Engl J Med. 1980;302:1109–1117): the overall diagnostic threshold consisted of the prevalence of thyroid nodule cancer, as reported in the 2015 American Thyroid Association (ATA) Guidelines (Haugen BR, Alexander EK, Bible KC, Doherty GM, Mandel SJ, Nikiforov YE, et al. 2015 American Thyroid Association Management Guidelines for Adult Patients with Thyroid Nodules and Differentiated Thyroid Cancer: The American Thyroid Association Guidelines Task Force on Thyroid Nodules and Differentiated Thyroid Cancer. Thyroid. 2016;26:1–133), and is estimated to be between approximately 7 and approximately 15%, so the average of these prevalences is approximately 11%. In the case of "indeterminate" FNA, this prevalence changes, so that if the average probability of malignancy reported in the Bethesda system is calculated, it results in approximately 23%.

[0045] Formulas

[0046] The following formula is used for the therapeutic threshold (UT):

[0047] UT = probability of a negative result in a patient without disease (Pneg / nd) x risk of treatment in patients without disease (Rrx) – risk of the test (Rt) / Pneg / nd x Rt + probability of a negative result in patients with disease (Pneg / nd) x benefit of treatment in patients with disease (Brx).

[0048] Thus, the Brx rate was estimated at approximately 20%, considering that the survival rate for papillary thyroid cancer is approximately 98% at early diagnosis and approximately 78% when distant metastases are present. The Rrx rate was estimated at approximately 4.2% when calculating an overall average morbidity in total thyroidectomy, considering permanent injury to the recurrent laryngeal nerve, injury to the external branch of the superior laryngeal nerve, hypoparathyroidism, and hematoma (Gunn A, Oyekunle T, Stang M, Kazaure H, Scheri R. Recurrent Laryngeal Nerve Injury After Thyroid Surgery: An Analysis of 11,370 Patients. J Surg Res. 2020;255:42-49; Mohammad R, Huh G, Cha W, Jeong WJ.RecurrentLaryngealNerveParalysisFollowingThyroidectomy:AnalysisofFactorsAffectingNerveRecovery. Laryngoscope.2022;132:1692-1696;UludagM,AygunN,KartalK,CitgezB,BeslerE,YetkinG, et al. Contribution of intraoperative neural monitoring to preservation of the external branch of the superlaryngeal nerve: arandomized prospective clinical trial.LangenbecksArchSurg.2017;402:965-976;Hurtado-LópezLM,Díaz-Hernández PI,Basurto-Kuba E,Zaldívar-RamírezFR,Pulido-CejudoA.EfficacyofIntraoperativeNeuro-Monitoring toLocalizetheExternalBranch of the SuperiorLaryngealNerve.Thyroid.2016;26:174-8;EismontasV,SlepaviciusA,JanusonisV,ZeromskasP,BeisaV,StrupasK, et al.Predictorsofpostoperativehypocalcemiaoccurringaftera totalthyroidectomy:resultsof prospectivemulticenterstudy. BMCSurg.2018;18:55 yCaulleyL, Johnson-ObasekiS, Luo L,JavidniaH. Riskfactorsforpostoperativecomplications in totalthyroidectomy:Aretrospective,risk-adjustedanalysisfromthe NationalSurgicalQualityImprovementProgram.Medicine(Baltimore)2017;96:e5752), Rt en aproximadamente 1.4% al riesgo identificado en realizar una BAAF de tiroides (Park JY, Choi W, Hong AR, Yoon JH, Kim HK, Kang HC. AComprehensiveAssessmentof the Harms of Fine-NeedleAspirationBiopsyforThyroidNodules:ASystematicReview.EndocrinolMetab(Seoul).2023;38:104-116); Pneg / nd of approximately 98 representing the true negatives of a FNA; Pneg / d of approximately 1.8 representing the false negatives of a FNA (LundgrenCI,ZedeniusJ,SkoogL. Fine-needle aspiration biopsy of benign thyroid nodules: an evidence-based review. World JSurg.2008;32:1247-52), therefore the therapeutic threshold remained at approximately 61.5%.

[0049] Once the sensitivity and specificity of lactate in the thyroid nodule are known, the positive and negative likelihood ratio is calculated, and with this, the Bayesian analysis is performed using the formula:

[0050] Prior odds x probability ratio = Posterior odds

[0051] This is transformed into a probability and compared with the diagnostic and therapeutic thresholds, with the understanding that a negative post-test probability (PPPN) lower than the diagnostic threshold indicates that no further diagnostic test should be performed and rules out cancer, a positive post-test probability (PPPP) higher than the therapeutic threshold implies that treatment should be performed directly, and finally, if these values ​​fall between the diagnostic and therapeutic thresholds, another diagnostic test should be performed before making a decision.

[0052] The study was approved by the internal research and bioethics committees of a hospital in Mexico City, and all patients selected for the study were informed about it and their consent to participate was obtained.

[0053] Examples and Results

[0054] According to the present invention, a prospective, observational, comparative, cross-sectional study was carried out on 219 thyroid nodules surgically operated from June 2021 to June 2023 in the following generalized manner in 206 patients (13 patients with bilateral thyroid nodule) of which 181 were women and 25 men with an overall average age of approximately 47.5 years (19-79 years) average age of men approximately 47.3 (26-72 years) and average age for women approximately 47.5 years (19-79 years).

[0055] The final histopathological report for benign nodules was: 24 follicular adenomas, 3 Hurthle cell adenomas, 16 adenomatous goiters, 73 nodular colloid goiters, and 3 lymphocytic thyroiditis. The malignant nodules were: 94 papillary thyroid carcinomas, 3 follicular carcinomas, 2 medullary carcinomas, and 1 undifferentiated carcinoma.

[0056] Capillary lactate levels obtained from the patient's index finger had a mean of approximately 2.08 mmol / lt with a standard deviation (SD) of approximately 0.67 mmol / lt.

[0057] Lactate levels obtained from the FNA of a healthy thyroid lobe were approximately 1.98 mmol / lt with a SD of approximately 1.10 mmol / lt.

[0058] The lactate levels obtained from the FNA of the benign thyroid nodule were approximately 2.24 mmol / lt with SD of approximately 0.98 mmol / lt and for malignant thyroid nodules approximately 4.74 mmol / lt with SD of approximately 2.06 mmol / lt.

[0059] The lactate levels obtained from the fine-needle aspiration biopsy (FNAB) of 10 mixed thyroid nodules showed a lactate level in the cystic component (colloid) of approximately 6.67 mmol / L with a standard deviation (SD) of approximately 1.64 mmol / L. The lactate level in the solid component of these same nodules was approximately 2.49 mmol / L with an SD of approximately 0.28 mmol / L. The comparison of these two measurements showed a statistically significant difference, as determined by a Student's t-test with a p-value < 0.0000001. Given that these 10 nodules corresponded to 10 benign lesions, it is considered that the lactate measurement in the colloid does not correspond to physiological lactate levels, and only the lactate obtained from solid tissue should be taken into account.

[0060] The comparison, using Student's t-test, between the capillary sample and the healthy thyroid lobe sample showed no statistical difference (approximately p=0.33), similarly the comparison between the capillary sample and the benign thyroid nodule sample showed no statistical difference (approximately p=0.04).

[0061] The comparison between the healthy thyroid lobe sample and the benign thyroid nodule sample also showed no statistical difference (approximately p=0.08).

[0062] The comparison between the lactate sample from a benign thyroid nodule and a malignant thyroid nodule showed a statistically significant difference of approximately p=0.0000001, similarly the comparison between the capillary sample and a malignant thyroid nodule had a statistically significant difference of approximately p=0.0000001.

[0063] The comparisons made confirm that lactate measurement in the malignant thyroid nodule is different from that performed in capillary blood, in the healthy thyroid lobe, and in the benign thyroid nodule; therefore, it has diagnostic capacity to differentiate between benign and malignant; consequently, a ROC curve was performed to determine the ideal cutoff point of the lactate level to discriminate between benign and malignant; this analysis obtained a result of approximately 3 mmol / lt with an area under the curve of approximately 0.84 (Figure 1); therefore, this lactate level will be taken as positive for the detection of cancer in the evaluation of diagnostic value.

[0064] Of the 119 benign nodules, 107 had lower lactate and 12 had lactate above approximately 3 mmol / lt, while of the 100 malignant thyroid nodules, 10 had lower lactate and 90 had lactate above approximately 3 mmol / lt.

[0065] The diagnostic value of lactate for cancer detection in the complete sample had a sensitivity of approximately 90% and a specificity of approximately 89.9% (Table 1). The diagnostic value in “indeterminate” nodules comprised of Bethesda I, III, IV, and V had a sensitivity of approximately 88.9% and a specificity of approximately 86.8% (Table 2). The diagnostic value in “indeterminate” nodules Bethesda III, IV, and V had a sensitivity of approximately 88.1% and a specificity of approximately 80.4% (Table 3).

[0066] Table 1: Diagnostic value and Bayes' rule calculation in thyroid nodules, complete sample including Bethesda I to VI results: TESTS WITH DICHOTOMOUS RESULTS CHARACTERISTIC EVALUATED PRESENT ABSENT (Reference test +) (Reference test -) DIAGNOSTIC TEST + 90 12 102 DIAGNOSTIC TEST - 10 107 117 100 119 219 95% CI Sensitivity 90.0% 82.6% to 94.5% Specificity 89.9% 83.2% to 94.1% Positive predictive value 88.2% 80.6% to 93.1% Negative predictive value 91.5% 85.0% to 95.3% False positive proportion 10.1% 5.9% to 16.8% False negative proportion Negative 10.0% 5.5% to 17.4% Accuracy 90.0% 85.3% to 93.3% Diagnostic odds ratio 80.25 33.13 to 19 4.41 Youden J index 0.8 CPP or LR(+) 8.9 35.20 to 15.32 Taylor Miettinen CPN or LR(-) 0.11 0.06 to 0.20 Taylor Miettinen Pre-test probability (Prevalence) 45.7% CALCULATION OF POST-TEST PROBABILITIES (Bayes' Theorem) Estimated pre-test probability 11.0% 95% CI Positive post-test probability (PPPP) 52.5% 42.8% to 61.9% 1 - PPPP 47.5% 38.1% to 57.2% 1 - PPPN98.6%94.5% to 99.7% Post-test negative probability (PPPN)1.4%0.3% to 5.5% .

[0067] Table 2: Diagnostic value and Bayes' rule calculation in "indeterminate" thyroid nodules including Bethesda I, III, IV, V: TESTS WITH DICHOTOMOUS RESULTS CHARACTERISTIC EVALUATED PRESENT ABSENT (Reference test +) (Reference test -) DIAGNOSTIC TEST + 40 10 50 DIAGNOSTIC TEST - 56 67 1 45 76 12 1 95% CI Sensitivity 88.9% 76.5% to 95.2% Specificity 86.8% 77.4% to 92.7% Positive predictive value 80.0% 67.0% to 88.8% Negative predictive value 93.0% 84.6% to 97.0% False positive rate 13.2% 7.3% to 22.6% False negative rate Negative 11.1% 4.8% to 23.5% Accuracy 87.6% 80.6% to 92.3% Diagnostic odds ratio 52.80 16.83 to 165.61 Youden J index 0.8 CPP or LR(+) 6.76 3.76 to 12.15 Taylor Miettinen CPN or LR(-) 0.13 0.06 to 0.29 Taylor Miettinen Pre-test probability (Prevalence) 37.2% CALCULATION OF POST-TEST PROBABILITIES (Bayes' Theorem) Estimated pre-test probability 23.0% 95% CI Positive post-test probability (PPPP) 66.9% 53.0% to 78.3% 1 - PPPP 33.1% 21.7% to 47.0% 1 - PPPN96.3%89.1% to 98.8% Post-test negative probability (PPPN)3.7%1.2% to 10.9% .

[0068] Table 3: Diagnostic value and Bayes' rule calculation in "indeterminate" thyroid nodules including Bethesda III, IV, and V: TESTS WITH DICHOTOMOUS RESULTS CHARACTERISTIC EVALUATED PRESENT ABSENT (Reference test +) (Reference test -) DIAGNOSTIC TEST + 37 10 47 DIAGNOSTIC TEST - 54 14 6 42 51 93 95% CI Sensitivity 88.1% 75.0% to 94.8% Specificity 80.4% 67.5% to 89.0% Positive predictive value 78.7% 65.1% to 88.0% Negative predictive value 89.1% 77.0% to 95.3% False positive rate 19.6% 11.0% to 32.5% False negative rate 11.9% 5.2% 25.0% Accuracy 83.9% 75.1% to 90.0% Diagnostic odds ratio 30.34 9.49 to 96.97 Youden J index 0.7 CPP or LR(+) 4.49 2.55 to 7.92 Taylor Miettinen CPN or LR(-) 0.15 0.06 to 0.34 Taylor Miettinen Pre-test probability (Prevalence) 45.2% CALCULATION OF POST-TEST PROBABILITIES (Bayes' Theorem) Estimated pre-test probability 23.0% 95% CI Positive post-test probability (PPPP) 57.3% 43.1% to 70.4% 1 - PPPP 42.7% 29.6% to 56.9% 1 - PPPN95.8%85.6% to 98.8% Post-test negative probability (PPPN)4.2%1.2% to 14.4% .

[0069] The analysis with the Bayes test for the lactate level in the thyroid nodule, total sample had a PPPP of approximately 52% and PPPN of approximately 1.4%.

[0070] This same analysis for lactate levels in Bethesda I, III, IV, and V "indeterminate" thyroid nodules had a positive positive predictive value (PPPP) of approximately 66.9% and a negative negative predictive value (PPPN) of approximately 3.7%. When performed on Bethesda III, IV, and V "indeterminate" thyroid nodules, a PPPP of approximately 57.3% and a PPPN of approximately 4.2% were found.

[0071] The analysis of thresholds and clinical decision-making when the test is positive and negative, based on the diagnostic value in the total sample, is shown in Figure 2A-2B, and for the case of non-diagnostic FNA it is shown in Figure 3A-3B.

[0072] Discussion

[0073] For easy understanding of the results obtained, it should be noted that this was a study of 219 consecutive thyroid nodules, therefore, it is a representative sample in number and diagnostic opportunity, thus avoiding any selection bias.

[0074] On the other hand, various ideal diagnostic test conditions have been modeled to evaluate an “indeterminate” thyroid nodule, the appropriate scenario being a cancer prevalence of approximately 20 to 40%, with a sensitivity of approximately 90% and a specificity greater than approximately 80%39. These parameters are associated with both optimal clinical accuracy and clinical efficacy, and the results of the present invention fall within the same parameters.

[0075] Another important point for the proper use of lactate determination as a diagnostic method in the "indeterminate" thyroid nodule is that only the solid tissue sample should be taken, since that is where cells are found and these are the ones that show the change in lactate levels in relation to the presence or absence of the Warburg effect. If the sample is obtained from the colloid component in the case of a mixed nodule, the lactate level in the colloid is notably high and is not a product of the Warburg effect.

[0076] The results conclusively address both objectives. The first objective was to determine if capillary lactate levels in a healthy thyroid lobe and a benign thyroid nodule differ from those in a malignant thyroid nodule. The answer is conclusive, as capillary lactate levels are the same in both healthy thyroid lobes and benign thyroid nodules, but significantly different from those in malignant thyroid nodules. Consequently, if this level is equal to or greater than approximately 3 mmol / L, thyroid cancer should be considered. The test has an excellent capacity to differentiate between benign and malignant conditions, with an area under the curve of approximately 0.84.

[0077] The second objective of this invention relates to knowing the diagnostic value of lactate in the thyroid nodule, in general and in the condition of an "indeterminate" FNA, and the results indicate that the level of lactate in the thyroid nodule, in the complete sample, has an excellent diagnostic capacity with a sensitivity of approximately 90% and a specificity of approximately 89.9%, with an area under the curve of approximately 0.8.

[0078] In the case of an "indeterminate" fine-needle aspiration biopsy (FNAB), encompassing categories I, III, IV, and V of the Bethesda System, the diagnostic capacity is similar; however, the most important aspect is that, through the analysis of thresholds for decision-making, the lactate level allows cancer to be ruled out when it is below approximately 3 mmol / L. This lowers the a priori probability of cancer from approximately 23% to approximately 3.7%, therefore no further diagnostic testing is recommended, the patient is reclassified as benign, and observation is maintained. On the other hand, when the lactate level is equal to or greater than approximately 3 mmol / L, the a posteriori probability of cancer is approximately 66.9%, exceeding the therapeutic threshold of approximately 61.5%. Therefore, at this lactate level, no further testing is necessary, and treatment is performed, i.e., the patient undergoes surgery.This situation also occurs when only "indeterminate" FNA results are integrated into Bethesda III, IV, and V, ruling out cancer by decreasing the probability of this disease from approximately 23% to approximately 4.2% when lactate levels are below approximately 3 mmol / L in the patient under observation. Conversely, having a lactate level above approximately 3 mmol / L makes the possibility of cancer approximately 57.3%, a level very close to the therapeutic threshold (even within the 95% confidence interval). Therefore, the clinical decision in light of this result will be to perform diagnostic surgery with intraoperative study.

[0079] In order to compare the diagnostic capacity of lactate in thyroid nodules with "indeterminate" FNA results, the same Bayesian analysis was performed using the results published in meta-analyses. The diagnostic value for molecular tests was compared with the results of lactate in "indeterminate" thyroid nodules (Figure 4A-4B), yielding a positive predictive value (PPPN) of approximately 0.46% and a positive predictive value (PPPP) of approximately 45% for ThyroSeq V3. The same analysis for Afirma-GSC indicates a PPPN of approximately 2.8% and a PPPP of approximately 36.5%.If compared with the lactate results obtained for the "indeterminate" thyroid nodule, it can be seen that they have practically the same diagnostic value and, if we are more strict, they have a greater capacity to exceed the therapeutic threshold (Figure 4A-4B). Therefore, we can emphatically state that it resolves the diagnostic doubt when we have a fine-needle aspiration biopsy of a thyroid nodule with an "indeterminate" result, with the difference that it can be performed at the time of the aspiration biopsy without requiring complex logistics.

[0080] Another point to highlight is that the cost is notably different. Although this study was not designed for cost evaluation, these costs were mentioned in the introduction and are approximately $3,200 to $4,600 for molecular tests, while determining lactate requires portable equipment costing approximately $250 to $280, which is used multiple times, and each test strip costs approximately $4. Therefore, the economic advantage in favor of lactate is clear.

[0081] In fact, if the decision had been made to operate or not on patients with "indeterminate" FNA (I, III, IV, V) and those with a lactate below approximately 3 mmol / lt had not been operated on, 67 surgeries would have been avoided out of 77 patients operated on for benign disease, and this same situation for "indeterminate" FNA (III, IV, V) of 52 patients operated on for benign condition, 42 surgeries would have been avoided.

[0082] With these results we can conclude that the diagnostic value of lactate has a high diagnostic capacity and provides reliable information for clinical decision-making in the management of thyroid nodules with "indeterminate" FNA results.

[0083] Alterations to the invention described herein may be foreseen by those skilled in the art. However, it should be understood that this description relates to preferred embodiments of the invention, is for illustrative purposes only, and should not be construed as a limitation of the invention. All obvious modifications to the spirit of the invention, such as changes in the shape, material, and dimensions of the elements comprising the invention, shall be considered within the scope of the appended claims.

[0084] The invention has been described in an illustrative manner and it should be understood that the terminology used herein is intended to correspond to the nature of the words in the description rather than to any limitation.

[0085] Obviously, many modifications and variations of the present invention are possible in light of the foregoing teachings. Therefore, it should be understood that, within the scope of the described invention, the invention may be practiced in ways other than those specifically described.

Claims

An in vitro method for determining the presence of thyroid cancer comprising: measuring at least one lactate level in a first sample from a malignant thyroid nodule, at least one lactate level in a second capillary blood sample, and at least one lactate level in a third sample from a healthy thyroid lobe; comparing the at least one lactate level of the first, second, and third samples to determine a cutoff point of the lactate value in the first sample selected from a range of approximately 0.8 to approximately 22 mmol / L to differentiate whether the first sample is a benign or malignant nodule, wherein a result greater than or equal to approximately 3 mmol / L with an area under a ROC curve of approximately 0.84 indicates the presence of thyroid cancer. The in vitro method of claim 1, wherein the first sample of the malignant thyroid nodule is obtained by means of a fine-needle aspiration biopsy (FNAB); the second capillary blood sample is obtained by means of a FNAB puncture; and the third sample of the healthy thyroid lobe is obtained by means of a FNAB puncture biopsy. The in vitro method according to claim 2, wherein the first sample is selected from solid tissue of a thyroid nodule or a mixed nodule comprising a liquid component and a solid component. The in vitro method according to claim 3, wherein the liquid component is aspirated from the mixed nodule to obtain only a sample of the solid component. The in vitro method according to claim 1, wherein the at least one lactate level in the first, second, and third samples is determined as follows: placing at least one drop of each of the first, second, and third samples on at least one lactate test strip, optionally on at least one BM Lactate test strip; and reading the quantitative photometric measurement of the reflectance on the lactate test strips. The in vitro method according to claim 5, wherein the reading is taken approximately at an interval of 30 seconds to 1.5 minutes after placing at least one drop on the test strip. The in vitro method according to claim 1, further comprising testing a diagnostic value of lactate through a Bayesian analysis and clinical decision threshold levels. The in vitro method according to claim 7, wherein the Bayesian analysis is performed using the following formula: prior odds X likelihood ratio = posterior odds, wherein a negative post-test probability (PPPN) less than the diagnostic threshold indicates that no further diagnostic testing should be performed and rules out cancer; wherein a positive post-test probability (PPPP) greater than the therapeutic threshold implies that treatment should be performed directly. The in vitro method according to claim 7, wherein the clinical decision threshold levels are realized using the following formula: UT = probability of a negative result in a disease-free patient (Pneg / nd) x risk of treatment in disease-free patients (Rrx) – risk of the test (Rt) / Pneg / nd x Rt + probability of a negative result in disease-bearing patients (Pneg / nd) x benefit of treatment in disease-bearing patients (Brx). The in vitro method according to claim 1, wherein the comparison of at least one lactate level of the first, second and third samples is carried out by means of a Student's t-test. A method for treating thyroid cancer in a patient in need comprising measuring at least one lactate level in a first sample from a malignant thyroid nodule, at least one lactate level in a second capillary blood sample, and at least one lactate level in a third sample from a healthy thyroid lobe, wherein a result greater than or equal to approximately 3 mmol / L of lactate level compared among the three samples with an area under a ROC curve of approximately 0.84 indicates the presence of thyroid cancer, wherein the treatment of said cancer comprises surgery of the patient. A kit for use in the detection of thyroid cancer in a patient suspected of having thyroid cancer, characterized in that it comprises: needles for obtaining samples; reagent strips for detecting lactate levels; and instructions for use. A method for detecting thyroid cancer in a patient suspected of having thyroid cancer comprising: determining at least one lactate level in a first sample from a malignant thyroid nodule of the patient, at least one lactate level in a second sample obtained from capillary blood of the patient, and at least one lactate level in a third sample obtained from a healthy thyroid lobe of the patient. The method of claim 13, wherein the first sample of the malignant thyroid nodule is obtained by means of a fine-needle aspiration biopsy (FNAB); the second capillary blood sample is obtained by means of a FNAB puncture; and the third sample of the healthy thyroid lobe is obtained by means of a FNAB puncture biopsy. The method according to claim 14, wherein the first sample is selected from solid tissue of a thyroid nodule or a mixed nodule comprising a liquid component and a solid component. The method according to claim 15, wherein the liquid component is aspirated from the mixed nodule to obtain only a sample of the solid component. The method according to claim 13, wherein the at least one lactate level in the first, second, and third samples is determined by placing at least one drop of each of the first, second, and third samples on at least one lactate test strip, optionally on at least one BM Lactate test strip; and reading the quantitative photometric measurement of the reflectance on the lactate test strips. The method according to claim 17, wherein the reading is taken approximately at an interval of 30 seconds to 1.5 minutes after placing at least one drop on the test strip. The method according to claim 13, further comprising testing a diagnostic value of lactate through a Bayesian analysis and clinical decision threshold levels. The method according to claim 19, wherein the Bayesian analysis is performed using the following formula: prior odds X likelihood ratio = posterior odds, wherein a negative post-test probability (PPPN) less than the diagnostic threshold indicates that no further diagnostic test should be performed and rules out cancer; wherein a positive post-test probability (PPPP) greater than the therapeutic threshold implies that treatment should be performed directly. The method according to claim 19, wherein the clinical decision threshold levels are realized using the following formula: UT = probability of a negative result in a disease-free patient (Pneg / nd) x risk of treatment in disease-free patients (Rrx) – risk of the test (Rt) / Pneg / nd x Rt + probability of a negative result in disease-bearing patients (Pneg / nd) x benefit of treatment in disease-bearing patients (Brx). The method according to claim 13, further comprising comparing at least one lactate level of the first, second and third samples by means of a Student's t-test. A therapeutically effective amount of lactate for use in the diagnosis of thyroid cancer in a patient suspected of having such thyroid cancer. The therapeutically effective amount of lactate for use in accordance with claim 22, wherein said amount is greater than or equal to approximately 3 mmol / lt. The therapeutically effective amount of lactate for use according to claim 22 or 23, wherein said amount is determined by measuring at least one lactate level in a first sample from a malignant thyroid nodule, at least one lactate level in a second capillary blood sample, and at least one lactate level in a third sample from a healthy thyroid lobe.

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