A Rare Case of Autoimmune Hemolytic Anemia Caused by Autoantibody with Mimicking Anti-D and Anti-C Specificity.

BACKGROUND: Most of the autoantibodies that cause autoimmune hemolytic anemia (AIHA) are non-specific. Autoantibodies expressing alloantibody specificity are rare. METHODS: We present the case of a 4-year-old boy with no history of blood transfusion or underlying medical conditions who developed AIHA caused by autoantibody with mimicking anti-D and anti-C specificity. RESULTS: Following treatment with methylprednisolone sodium succinate and transfusion of red blood cells with negative antigens for D and C, along with administration of human immunoglobulin, the patient's condition gradually improved. He was ultimately discharged with a good prognosis. CONCLUSIONS: This report highlights a rare case of AIHA characterized by autoantibody with mimicking anti-D and anti-C specificity. Treatments of these patients could be antigen-negative red blood cells, glucocorticoid and immunoglobulin.

A national survey of current immunohematologic testing practices for the diagnosis of autoimmune hemolytic anemia in India.

Autoimmune hemolytic anemia (AIHA) is a common term for several disorders that differ from one another in terms of etiology, pathogenesis, clinical features, and treatment. Management of patients with AIHA has become increasingly evidence-based in recent years. While this development has resulted in therapeutic improvements, it also carries increased requirements for optimal diagnosis using more advanced laboratory tests. Unfortunately, limited data are available from developing countries regarding the testing and transfusion management of patients with AIHA. The main objective of this survey was to explore the current immunohematologic testing practices for the diagnosis of AIHA in India. This online survey consisted of 30 questions, covering the place of work, the number of AIHA cases encountered in the 3 preceding years, testing method(s), transfusion management, and so forth. Individuals representing 89 laboratories completed the survey; only 78 of which responded that AIHA testing was performed in their facility's laboratory. The majority of respondents agreed that the most commonly affected age-group comprised individuals of older than 20 years, with a female preponderance. Regarding transfusion management, respondents indicated that transfusion with "best-match" red blood cell units remains the most common practice. Column-agglutination technology is used by 92 percent of respondents as the primary testing method. Although a monospecific direct antiglobulin test is available at 73 percent of the sites, most of them have limited access to other resources that could diagnose cold or mixed AIHA. Merely 49 percent of responding laboratories have the resources to perform adsorption studies for the detection of alloantibodies. Furthermore, three-cell antibody screening reagents are unavailable at 32 percent of laboratories. In 72 percent of centers, clinical hematologists would prefer to consult a transfusion medicine specialist before administering treatment to AIHA patients. There is unanimous agreement regarding the need for a national registry. The survey data indicate wide variability in testing practices for patients with AIHA in India. Future studies are needed to focus on the feasibility and cost-effectiveness of different testing strategies for developing countries.

The Variation of RhD Blood Group in Different Periods in a Patient with Systemic Lupus Erythematosus.

BACKGROUND: Autoimmune hemolytic anemia disease often produces a large number of various autoantibodies, and some autoantibodies may be related to Rh blood group. In rare cases, autoantibodies can specifically target Rh antigen, thus interfering with the identification of Rh blood group. METHODS: A case of systemic lupus erythematosus (SLE) with inconsistent RhD blood group identification results in different periods was reported and the reasons were analyzed. RESULTS: Some autoantibodies can completely block D antigen on red blood cells, resulting in no redundant D sites on red blood cells binding to reagent anti D. In addition, the immunity of the body is extremely low, and the expression of red blood cell blood group antigens in part of the body is inhibited, which will cause the weakening of the expression of Rh antigen in red blood cells. Therefore, when testing the RhD blood type of the patient, the reagent anti D does not agglutinate with the patient's red blood cells, and a false negative result of the initial screening appears. Through the RhD negative confirmation test, the patient's blood type is a serologically weak D phenotype. CONCLUSIONS: If the result of serological preliminary screening test is RhD negative or RhD variant, the recipient should be treated as RhD negative, and RhD negative red blood cells should be transfused during blood transfusion. Conditional laboratories can implement RHD genotyping, which is conducive to improving the precise blood transfusion management level of RhD negative blood recipients, saving rare blood resources and improving the treatment efficiency of patients.

Case report: Decreased hemoglobin and multiple organ failure caused by ceftizoxime-induced immune hemolytic anemia in a Chinese patient with malignant rectal cancer.

Background: Drug-induced immune hemolytic anemia (DIIHA) is a rare but serious condition, with an estimated incidence of one in 100,000 cases, associated with various antibiotics. This study reports on a case of ceftizoxime-induced hemolysis observed in a patient in China. Case description: A Chinese patient diagnosed with malignant rectal cancer underwent antimicrobial therapy after laparoscopic partial recto-sigmoid resection (L-Dixon). After receiving four doses of ceftizoxime, the patient developed symptoms including rash, itchy skin, and chest distress, followed by a rapid decline in hemoglobin levels, the presence of hemoglobin in the urine (hemoglobinuria), renal failure, and disseminated intravascular coagulation. Laboratory analysis revealed high-titer antibodies against ceftizoxime and red blood cells (RBCs) in the patient's serum, including immunoglobulin M (IgM) (1:128) antibodies and immunoglobulin G (IgG) (1:8) antibodies, with noted crossreactivity to ceftriaxone. Significant improvement in the patient's hemolytic symptoms was observed following immediate discontinuation of the drug, two plasma exchanges, and extensive RBC transfusion. Conclusion: This case, together with previous reports, underscores the importance of considering DIIHA in patients who exhibit unexplained decreases in hemoglobin levels following antibiotic therapy. A thorough examination of the patient's medical history can provide crucial insights for diagnosing DIIHA. The effective management of DIIHA includes immediate cessation of the implicated drug, plasma exchange, and transfusion support based on the identification of specific drug-dependent antibodies through serological testing.

Autoimmune Hemolytic Anemias: Classifications, Pathophysiology, Diagnoses and Management.

Autoimmune hemolytic anemias (AIHAs) are conditions involving the production of antibodies against one's own red blood cells (RBCs). These can be primary with unknown cause or secondary (by association with diseases or infections). There are several different categories of AIHAs recognized according to their features in the direct antiglobulin test (DAT). (1) Warm-antibody AIHA (wAIHA) exhibits a pan-reactive IgG autoantibody recognizing a portion of band 3 (wherein the DAT may be positive with IgG, C3d or both). Treatment involves glucocorticoids and steroid-sparing agents and may consider IVIG or monoclonal antibodies to CD20, CD38 or C1q. (2) Cold-antibody AIHA due to IgMs range from cold agglutinin syndrome (CAS) to cold agglutin disease (CAD). These are typically specific to the Ii blood group system, with the former (CAS) being polyclonal and the latter (CAD) being a more severe and monoclonal entity. The DAT in either case is positive only with C3d. Foundationally, the patient is kept warm, though treatment for significant complement-related outcomes may, therefore, capitalize on monoclonal options against C1q or C5. (3) Mixed AIHA, also called combined cold and warm AIHA, has a DAT positive for both IgG and C3d, with treatment approaches inclusive of those appropriate for wAIHA and cold AIHA. (4) Paroxysmal cold hemoglobinuria (PCH), also termed Donath-Landsteiner test-positive AIHA, has a DAT positive only for C3d, driven upstream by a biphasic cold-reactive IgG antibody recruiting complement. Although usually self-remitting, management may consider monoclonal antibodies to C1q or C5. (5) Direct antiglobulin test-negative AIHA (DAT-neg AIHA), due to IgG antibody below detection thresholds in the DAT, or by non-detected IgM or IgA antibodies, is managed as wAIHA. (6) Drug-induced immune hemolytic anemia (DIIHA) appears as wAIHA with DAT IgG and/or C3d. Some cases may resolve after ceasing the instigating drug. (7) Passenger lymphocyte syndrome, found after transplantation, is caused by B-cells transferred from an antigen-negative donor whose antibodies react with a recipient who produces antigen-positive RBCs. This comprehensive review will discuss in detail each of these AIHAs and provide information on diagnosis, pathophysiology and treatment modalities.

Clinical profile and serological correlation with haemolysis in DAT-positive autoimmune haemolytic anaemia patients in Bangladesh.

Discerning the type of autoimmune haemolytic anaemia (AIHA) is crucial for transfusion support and initiation of treatment. This study aimed to establish the clinical profile and serological character of red cell autoantibodies and to investigate the relationship with haemolysis in AIHA patients who were direct antiglobulin test (DAT)-positive. A total of 59 DAT-positive AIHA patients were included in this study. Clinical, laboratory and serological findings were evaluated to find the gradation of haemolysis and to investigate its correlation with age, sex, type of autoantibody and level of autoantibody. Study findings revealed that most patients (89.8%) had haemolysis, wherein moderate haemolysis (67.8%) was predominant. Weakness, palpitations, fever, pallor, tachycardia and splenomegaly were common among patients with severe and moderate haemolysis. The majority (66.1%) had an associated disorder. Warm autoantibody was the most common, followed by cold and mixed cases. The severity of haemolysis correlated strongly with the strength of the DAT reaction (Cramer V 0.636, p<0.001). These findings may be useful to clinicians while determining a treatment plan. The direct relationship between severity of haemolysis and strength of DAT needs further exploration in a large population to establish whether it can be used as a tool to formulate a treatment plan when assessing AIHA patients in low resourced countries.

TLR8/TLR7 dysregulation due to a novel TLR8 mutation causes severe autoimmune hemolytic anemia and autoinflammation in identical twins.

Our study presents a novel germline c.1715G>T (p.G572V) mutation in the gene encoding Toll-like receptor 8 (TLR8) causing an autoimmune and autoinflammatory disorder in a family with monozygotic male twins, who suffer from severe autoimmune hemolytic anemia worsening with infections, and autoinflammation presenting as fevers, enteritis, arthritis, and CNS vasculitis. The pathogenicity of the mutation was confirmed by in vitro assays on transfected cell lines and primary cells. The p.G572V mutation causes impaired stability of the TLR8 protein, cross-reactivity to TLR7 ligands and reduced ability of TLR8 to attenuate TLR7 signaling. This imbalance toward TLR7-dependent signaling leads to increased pro-inflammatory responses, such as nuclear factor-κB (NF-κB) activation and production of pro-inflammatory cytokines IL-1ß, IL-6, and TNFα. This unique TLR8 mutation with partial TLR8 protein loss and hyperinflammatory phenotype mediated by TLR7 ligands represents a novel inborn error of immunity with childhood-onset and a good response to TLR7 inhibition.

Immunoglobulin subtyping and quantification in direct antiglobulin test: positive haemolysis in an HIV-prevalent setting.

AIMS: Positive direct antiglobulin tests (DATs) are valuable in identifying the aetiology of autoimmune haemolysis and in guiding therapeutic intervention. However, in HIV-positive individuals with background polyclonal gammopathy, a positive DAT in the absence of haemolysis is common. In this setting, IgG quantification and subtyping may be of value, as this is possible with the recently introduced gel cards. There is paucity of literature evaluating the diagnostic usefulness of IgG subtyping and quantification in HIV-positive individuals who are investigated for autoimmune haemolytic anaemia (AIHA). This study evaluated the usefulness of IgG quantification and subtyping in the diagnostic work-up of AIHA in patients with a positive DAT, with and without HIV infection. METHODS: This retrospective, cross-sectional study included patients investigated for AIHA in a quaternary care hospital. Those with a positive DAT had their IgG subtyped and quantified using the ID-Card DAT IgG1/IgG3 and IgG-dilution cards (Bio-Rad, Cressier, Switzerland). RESULTS: Ninety patients admitted from December 2019 to March 2020 were investigated for AIHA. Forty-four (49%) patients had a positive DAT of whom 26 (59%) had evidence of haemolysis, and 16 (36%) were HIV positive. Concurrent HIV and haemolysis were present in eight patients, two of whom had IgG1 although none had an IgG antibody titre >1:30. None of the HIV-positive patients without features of haemolysis had IgG1/IgG3 or IgG antibody titres >1:30. CONCLUSION: In our clinical setting, IgG quantification and subtyping were found to be of limited value in the diagnostic characterisation of AIHA in HIV-positive patients with false-positive DAT.

T-follicular helper cell expansion and chronic T-cell activation are characteristic immune anomalies in Evans syndrome.

Pediatric Evans syndrome (pES) is increasingly identified as the presenting manifestation of several inborn errors of immunity. Despite an improved understanding of genetic defects in pES, the underlying immunobiology of pES is poorly defined, and characteristic diagnostic immune parameters are lacking. We describe the immune characteristics of 24 patients with pES and compared them with 22 patients with chronic immune thrombocytopenia (cITP) and 24 healthy controls (HCs). Compared with patients with cITP and HC, patients with pES had increased circulating T-follicular helper cells (cTfh), increased T-cell activation, and decreased naïve CD4+ T cells for age. Despite normal or high immunoglobulin G (IgG) in most pES at presentation, class-switched memory B cells were decreased. Within the cTfh subset, we noted features of postactivation exhaustion with upregulation of several canonical checkpoint inhibitors. T-cell receptor ß chain (TCR-ß) repertoire analysis of cTfh cells revealed increased oligoclonality in patients with pES compared with HCs. Among patients with pES, those without a known gene defect had a similar characteristic immune abnormality as patients with defined genetic defects. Similarly, patients with pES with normal IgG had similar T-cell abnormalities as patients with low IgG. Because genetic defects have been identified in less than half of patients with pES, our findings of similar immune abnormalities across all patients with pES help establish a common characteristic immunopathology in pES, irrespective of the underlying genetic etiology.

In vitro Immunomodulatory Effects of Human Umbilical Cord-Derived Mesenchymal Stem Cells on Peripheral Blood Cells from Warm Autoimmune Hemolytic Anemia Patients.

INTRODUCTION: Autoimmune hemolytic anemia is a potentially lethal disease characterized by autoimmune hemolysis. Although human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) have been reported as a promising therapy, there is limited evidence regarding warm autoimmune hemolytic anemia (wAIHA) patients. This study aimed to investigate the potential therapeutic effects of hUC-MSCs via immune regulation in wAIHA patients. METHODS: Peripheral blood mononuclear cells (PBMCs) from 10 wAIHA patients and 8 healthy controls were isolated from peripheral blood and cultured for 3 days with or without the presence of hUC-MSCs; PBMCs were co-cultured with hUC-MSCs using Transwell assays. The supernatant cytokine levels were measured after culture through AimPlex Multiple Immunoassays for Flow, including IL-2, IL-4, IL-10, IFN-γ, TNF-α, and IL-17A. The percentages of regulatory T cells, regulatory B cells, and Th1/Th2 in PBMCs were also assessed before and after culturing. RESULTS: In the wAIHA group, hUC-MSCs could upregulate the Treg and Breg proportions after culturing for 3 days, and the Treg and Breg percentages increased after co-culturing with hUC-MSCs in the wAIHA group compared with PBMC cultured alone for 3 days (8.29 ± 8.59 vs. 6.82 ± 1.32, 3.82 ± 1.87 vs. 1.75 ± 1.20, respectively). Compared with the PBMC wAIHA group, the levels of TNF-α (2.13 ± 2.07 vs. 16.20 ± 21.13 pg/mL, p = 0.019) and IL-10 (10.51 ± 18.42 vs. 37.78 ± 44.20 pg/mL, p = 0.012) were significantly elevated in the PBMC + hUC-MSCs wAIHA group. CONCLUSION: The hUC-MSCs contributed to the increasing proportion of regulatory cell populations in PBMCs of wAIHA patients, thereby potentially regulating autoimmune response; thus, hUC-MSCs may be a promising approach for wAIHA treatment.

Complement Mediated Hemolytic Anemias in the COVID-19 Era: Case Series and Review of the Literature.

The complex pathophysiologic interplay between SARS-CoV-2 infection and complement activation is the subject of active investigation. It is clinically mirrored by the occurrence of exacerbations of complement mediated diseases during COVID-19 infection. These include complement-mediated hemolytic anemias such as paroxysmal nocturnal hemoglobinuria (PNH), autoimmune hemolytic anemia (AIHA), particularly cold agglutinin disease (CAD), and hemolytic uremic syndrome (HUS). All these conditions may benefit from complement inhibitors that are also under study for COVID-19 disease. Hemolytic exacerbations in these conditions may occur upon several triggers including infections and vaccines and may require transfusions, treatment with complement inhibitors and/or immunosuppressors (i.e., steroids and rituximab for AIHA), and result in thrombotic complications. In this manuscript we describe four patients (2 with PNH and 2 with CAD) who experienced hemolytic flares after either COVID-19 infection or SARS-Cov2 vaccine and provide a review of the most recent literature. We report that most episodes occurred within the first 10 days after COVID-19 infection/vaccination and suggest laboratory monitoring (Hb and LDH levels) in that period. Moreover, in our experience and in the literature, hemolytic exacerbations occurring during COVID-19 infection were more severe, required greater therapeutic intervention, and carried more complications including fatalities, as compared to those developing after SARS-CoV-2 vaccine, suggesting the importance of vaccinating this patient population. Patient education remains pivotal to promptly recognize signs/symptoms of hemolytic flares and to refer to medical attention. Treatment choice should be based on the severity of the hemolytic exacerbation as well as of that of COVID-19 infection. Therapies include transfusions, complement inhibitor initiation/additional dose in the case of PNH, steroids/rituximab in patients with CAD and warm type AIHA, plasma exchange, hemodialysis and complement inhibitor in the case of atypical HUS. Finally, anti-thrombotic prophylaxis should be always considered in these settings, provided safe platelet counts.

Circulating bioactive bacterial DNA is associated with immune activation and complications in common variable immunodeficiency.

Common variable immunodeficiency (CVID) is characterized by profound primary antibody defects and frequent infections, yet autoimmune/inflammatory complications of unclear origin occur in 50% of individuals and lead to increased mortality. Here, we show that circulating bacterial 16S rDNA belonging to gut commensals was significantly increased in CVID serum (P < 0.0001), especially in patients with inflammatory manifestations (P = 0.0007). Levels of serum bacterial DNA were associated with parameters of systemic immune activation, increased serum IFN-γ, and the lowest numbers of isotype-switched memory B cells. Bacterial DNA was bioactive in vitro and induced robust host IFN-γ responses, especially among patients with CVID with inflammatory manifestations. Patients with X-linked agammaglobulinemia (Bruton tyrosine kinase [BTK] deficiency) also had increased circulating bacterial 16S rDNA but did not exhibit prominent immune activation, suggesting that BTK may be a host modifier, dampening immune responses to microbial translocation. These data reveal a mechanism for chronic immune activation in CVID and potential therapeutic strategies to modify the clinical outcomes of this disease.

Involvement of Virus-Induced Interferon Production in IgG Autoantibody-Mediated Anemia.

Infection with viruses, such as the lactate dehydrogenase-elevating virus (LDV), is known to trigger the onset of autoimmune anemia through the enhancement of the phagocytosis of autoantibody-opsonized erythrocytes by activated macrophages. Type I interferon receptor-deficient mice show enhanced anemia, which suggests a protective effect of these cytokines, partly through the control of type II interferon production. The development of anemia requires the expression of Fcγ receptors (FcγR) I, III, and IV. Whereas LDV infection decreases FcγR III expression, it enhances FcγR I and IV expression in wild-type animals. The LDV-associated increase in the expression of FcγR I and IV is largely reduced in type I interferon receptor-deficient mice, through both type II interferon-dependent and -independent mechanisms. Thus, the regulation of the expression of FcγR I and IV, but not III, by interferons may partly explain the exacerbating effect of LDV infection on anemia that results from the enhanced phagocytosis of IgG autoantibody-opsonized erythrocytes.

Cold agglutinins revealed by abnormalities to the cell blood count: a case report.

Cold agglutinin are erythrocyte antibodies which possess the property of agglutinating red blood cells at temperatures of below 37°C, this phenomenon is reversible after heating. This is usually immunoglobulin M (IgM) class. Their pathogenicity is much more related to their temperature range of activity than their title. As we report in this observation, cold hemagglutination makes it difficult to interpret certain immunological tests such as ABO Rh blood grouping or searching for irregular antibodies (SAI). The discovery of cold agglutinins can be fortuitous revealing itself by disturbances and aberrations in the results of blood count or as part of a suggestive clinical or laboratory table cold hemagglutinin disease. The search for a lymphoid hematological at their diagnosis should be systematic.

Analysis of multiple organ damage and clinical immunological characteristics in systemic lupus erythematosus patients with hematologic involvement.

Objective: To investigate clinical immunological characteristics and imaging findings of multiple organ damage of systemic lupus erythematosus (SLE) patients with hematologic involvement. Methods: SLE patients diagnosed in the Second Affiliated Hospital of Nanchang University from June 2015 to March 2019 were selected, including 93 SLE patients with hematologic involvement and 68 SLE patients without hematologic involvement. Immunological indicators such as autoantibodies, immunoglobulin G (IgG), complement 4 (C4) and imaging data of several organs were measured respectively. The results were statistically analyzed. Results: SLE patients with hematologic involvement were more likely to have autoimmune hemolytic anemia (AIHA) (20.43%, P<0.05). The erythrocyte sedimentation rate (ESR) of SLE patients with hematologic involvement was 75.82 (±35.33) mm/h, IgG was 28.84 (±6.00) g/L and C4 was 0.073 (±0.031) g/L (P< 0.05). The area under the curve (AUC) of IgG was the highest among the above indicators (P<0.01). The positive anti-RO-52 antibody (OR=15.926, P<0.05) was an independent risk factor for pulmonary inflammatory lesions in SLE patients with hematologic involvement. Conclusion: Compared with the control group, abnormal immunological indicators and multiple organs damage are more obvious. Positive anti-RO-52 antibody may play an important role in the pathogenesis of pulmonary inflammation in SLE patients.

Immunoadsorption in a dog with severe immune mediated hemolytic anemia.

Immune mediated hemolytic anemia (IMHA) is a life-threatening disease with severe, acute hemolysis as a result of an autoimmune response directed against erythrocyte surface antigens. In veterinary medicine, IMHA is usually treated with immunosuppressants and often multiple blood transfusions. In human medicine, immunoadsorption (IA) is an established therapy for antibody removal in immune-mediated diseases. A female, spayed, five-year-old, 28 kg Entlebucher Mountain dog was presented with regenerative anemia and positive autoagglutination diagnosed as immune-mediated hemolytic anemia to the veterinary emergency service. Conventional treatment consisting immunosuppression with prednisolone and mycophenolate failed to improve hemolysis. As hematocrit dropped daily, multiple blood transfusions of blood group DEA 1 negative were required. IA was initiated at day 3 with COM.TEC and ADAsorb platforms and a LIGASORBstaphylococcus antitoxin A column. IA with citrate anticoagulation was performed over the treatment time of 77 minutes with a blood flow of 50 mL/min. Total plasma volume of 1.6 L was processed. Complications consisted of vomitus and lid swelling, shivering, excessive clotting in the tubing after a calcium bolus and hypotension. After IA, hemolysis stopped immediately, plasma concentrations of immunoglobulin G, immunoglobulin M and bilirubin decreased, and hematocrit remained stable. The dog was discharged without further hemolysis 4 days after immunoadsorption with immunosuppressive therapy. IA is a promising adjunctive therapy in severe cases of canine IMHA, but it cannot be concluded to which degree IA or concurrent immunosuppression contributed to cessation of hemolysis in the present case.

Primary Immunodeficiency in Children With Autoimmune Cytopenias: Retrospective 154-Patient Cohort.

Background: Primary immunodeficiency is common among patients with autoimmune cytopenia. Objective: The purpose of this study is to retrospectively identify key clinical features and biomarkers of primary immunodeficiency (PID) in pediatric patients with autoimmune cytopenias (AIC) so as to facilitate early diagnosis and targeted therapy. Methods: Electronic medical records at a pediatric tertiary care center were reviewed. We selected 154 patients with both AIC and PID (n=17), or AIC alone (n=137) for inclusion in two cohorts. Immunoglobulin levels, vaccine titers, lymphocyte subsets (T, B and NK cells), autoantibodies, clinical characteristics, and response to treatment were recorded. Results: Clinical features associated with AIC-PID included splenomegaly, short stature, and recurrent or chronic infections. PID patients were more likely to have autoimmune hemolytic anemia (AIHA) or Evans syndrome than AIC-only patients. The AIC-PID group was also distinguished by low T cells (CD3 and CD8), low immunoglobulins (IgG and IgA), and higher prevalence of autoantibodies to red blood cells, platelets or neutrophils. AIC diagnosis preceded PID diagnosis by 3 years on average, except among those with partial DiGeorge syndrome. AIC-PID patients were more likely to fail first-line treatment. Conclusions: AIC patients, especially those with Evans syndrome or AIHA, should be evaluated for PID. Lymphocyte subsets and immune globulins serve as a rapid screen for underlying PID. Early detection of patients with comorbid PID and AIC may improve treatment outcomes. Prospective studies are needed to confirm the diagnostic clues identified and to guide targeted therapy.