St Thomas’ Hospital, York Road Education Centre, London Validation study of CG cut-off and a retrospective study – the Italian experience Antonio Pecoraro PhD Student in Clinical and Experimental Medicine Division of Allergy and Clinical Immunology Department of Translational Medical Science University of Naples Federico II Center for Basic and Clinical Immunology Research (CISI) St Thomas’ Hospital, York Road Education Centre, London Friday 7 September 2018

Antibody deficiency represents an heterogenous group of diseases characterized by an impaired antibody production in response to pathogens. This is a screenshot of the web page of the European Society for Immunodeficiencies (ESID), that report the last accessible update on the epidemiology of primary immunodeficiency. We can see as antibody deficiencies represent the major part of the registered cases and within antibody deficiency, CVID is the mosto common and severe disease. So it has to be considered as a public health problem, relevant not only to clinical immunologists.

Diagnostic delay in antibody deficiency: results from the European Society for Immunodeficiencies (ESID) 2014 1746 patients with common variable immunodeficiency (CVID) Mean diagnostic delay: 4.5 years Antibody deficiencies, and CVID in particular, are characterized by a significant delay between the onset of symptoms and the establishment of the diagnosis. Delay in the diagnosis of CVID ranges widely in the various geographic areas and is approximately of 4 to 5 years in this large European study of 2014 from the European Society for Immunodeficiencies that did not include Italian patients. This is the largest cohort of CVID patients ever described, counting 1746 European patients with available data. The mean diagnostic delay di 4.5 years. Interestingly, there is no delay reduction in patients diagnosed after the year 2000 compared to those diagnosed before, suggesting there has been no improvement in the diagnostic tools for early detection of PIDs. This is an important issue, since undiagnosed and untreated immunodeficiencies may result in organ damage such as bronchiectasis. Gathmann et al. JACI. 2014

Diagnostic delay in antibody deficiency: results from an Italian cohort 224 patients with common variable immunodeficiency Mean diagnostic delay: 8.9 years Similarly, in a previous study reporting the clinical features of an Italian cohort of 224 CVID patients, the mean diagnostic delay was 8.9 years. This is an heterogenous cohort, as we can see by the distribution of the number of patients for the age at diagnosis (in panel A). Although the major part of the subjects had a diagnostic delat of two to five years (as we can see in panel B), a minor part presented a much greater delay, also greater than 20 years, strongly affecting the long-term outcome. Age at diagnosis Years of diagnostic delay Quinti et al. J Clin Immunol. 2007

Causes of diagnostic delay in antibody deficiency Heterogeneity of the clinical picture Poor education and awareness in both primary and secondary care As consequence, one of the most important challenges for the researchers and the clinicians who deal with antibody deficiency is to achieve the shortening of diagnostic delay. In order to accomplish this mission is necessary to pinpoint the factors contributing to the establishment of this diagnostic delay. These are: - The heterogeneity of the clinical picture. Indeed, in contrast to many other forms of primary immunodeficiency, antibody deficiencies may be diagnosed at almost any age. Moreover, non-infectious diseases, such as inflammatory, autoimmune and neoplastic complications represent, sometimes, the clinical onset of the immunodeficiency making diagnosis even more challenging. The poor education and awareness in both primary and secondary care about both the clinical course and the epidemiological size of this group of diseases. For example, there is the misconception that immunodeficiency always presents in infancy. - The lack of a definitive and univocal screening test, contrary to what happens to severe combined immunodeficiencies, where successful neonatal screening programmes have been developed in a number of countries. Lack of screening tests

Jolles et al. Clin Exp Immunol. 2014 Calculated globulin as screening test for antibody deficiency: the Wales experience In this regard, as we have listened before, a screening test based on the use of calculated globulin, which is the difference between total protein and albumin, has been validated and employed across the clinical biochemistry laboratories of Wales. This study was designed and conducted at University of Cardiff by Stephen Jolles. First, they conducted a pilot study to assess the correlation between values of IgG and calculated globulin in order to assess a calculated globulin cutoff to detect hypogammaglobulinemia. Then, they employed this CG cutoff to conduct a one-year perspective study in 9 hospitals across Wales: 826 samples with low CG were collected and the 89% of samples had an IgG lower than 6 g/l, thus demonstrating the validity of the CG cutoff. Pilot study to assess the correlation between values of IgG and CG Perspective study: 826 samples with low CG detected in 9 hospitals in Wales in 1 year Jolles et al. Clin Exp Immunol. 2014

Graziano et al. Clin Immunol. 2017 Delay in diagnosis affects the clinical outcome in CVID patients with marked IgA deficiency 75 CVID patients enrolled at the University of Naples Federico II Based on the interesting results of the Welsh experience, we decided to «import» this approach in the University of Naples Federico II and to validate this screening test in the Italian setting. In fact, in a recent study conducted by our research group in a cohort of 75 CVID patients enrolled at the University of Naples Federico II, we demonstrated that an higher age at the diagnosis of CVID, due to diagnostic delay, was significantly associated with a reduction of patients survival in the subset of CVID patients with a marked IgA deficiency, that are represented by the red light in this Kaplan-Meier analysis. An higher age at the diagnosis of CVID due to diagnostic delay was significantly associated with a reduction of patients survival if stratified per median of IgA (less than 8.00 mg/dl) Graziano et al. Clin Immunol. 2017

Deficiency in an Italian University Hospital Validation of Calculated Globulin (CG) as a Screening Test for Antibody Deficiency in an Italian University Hospital Study design: Selection of 200 anonymized serum samples (25 samples for each value of CG between 15 and 22 g/l) Biochemistry Laboratory Dosage of IgG serum levels for each sample Clinical Pathology Laboratory Statistical analysis (correlation IgG vs CG, ROC curve analysis) Division of Allergy and Clinical Immunology Therefore, in order to shortens the diagnostic delay of antibody deficiency and to improve the long-term outcome of immunodeficency patients in our Centre, we decided to conduct a pilot study to validate a cutoff of calculated globulin in our Clinical Biochemistry laboratory. For this purpose, we adopted a study design similar to this employed in the University of Cardiff: In collaboration with the Biochemistry laboratory of our University Hospital, we selected 200 anonymized adult serum samples during a six-month period. Serum samples were selected through a software that allows to see on a table the total protein and albumin results of each samples. In fact, Italian Biochemistry laboratories generally do not calculate or report the value of calculated globulin which has never been used in our country. Each selected sample was identified through a numerical code that guaranteed the anonymity of the samples. In particular, 25 anonymized samples were collected for each value of calculated globublin between the values of 15 and 22 g/l. This because the laboratory employed only the colorimetric bromocresol green (BCG) method for albumin and the Architect Biuret method for total protein All the samples selected were moved to the Clinical Pathology laboratory, where, in the same day, was performed the dosage of IgG. After having performed the IgG dosage for each of the 25 samples for each value of calculated globulin, we conducted the statistical analysis to assess the correlation between calculated globulin and IgG values to chose the adequate calculated globulin cut-off. Finally, to further verify the utility of CG, we retrospectively evaluated the relationship between CG values and IgG levels in 38 patients diagnosed with CVID at our Centre, finding that 37/38 patients would have been right dignosed with CVID using the CG cutoff established with our validation study . The protocol of the study was examinated and approved by the Ethic Commettee of the University of Naples Federico II Retrospective analysis of the correlation between CG and IgG values in 38 CVID patients Division of Allergy and Clinical Immunology

Results: Correlation of CG values with IgG serum levels As mentioned above, Twenty-five samples obtained anonymously for each level of CG at 15, 16, 17, 18, 19, 20, 21 and 22 g/l were tested for immunoglobulin levels. The correlation with IgG, performed through Spearman’s test, is shown (r = 0.61; p <0.0001). A CG level of 19 g/l corresponded to a mean IgG of 531 mg/dl (±204)

Results: ROC curve analysis of CG as independent discriminative value to detect hypogammaglobulinemia IgG 600 mg/dl IgG 500 mg/dl Then, based on the previous results, Four receiver operating characteristic curve (ROC) analysis were performed to verify if CG was an independent discriminative value to detect subjects with IgG serum levels lower than 600, 500, 400 and 300 mg/dl, respectively IgG 400 mg/dl IgG 300 mg/dl

Results: Comparison of sensitivity and specificity of different calculated globulin (CG) cutoffs for IgG serum levels below 600, 500, 400 and 300 mg/dl. Sensitivity and specificity of each CG value for each IgG level were calculated through the analysis of the ROC curves as shown in this Table. An IgG value of 600 mg/dl was considered as cutoff to define hypogammaglobulinemic subjects. The comparison between 119 samples with IgG<600 mg/dl and 81 samples with IgG>600 mg/dl demonstrated that a cut-off value of CG of 19 g/l corresponded to a sensitivity of 70% and a specificity of 75%.

Results: Retrospective analysis of the correlation between CG values and IgG serum levels at diagnosis in 38 CVID patients To further address the potential utility of CG as a screening test for hypogammaglobulinemia, we performed a retrospective analysis of laboratory and clinical information in patients diagnosed with CVID at our Division of Allergy and Clinical Immunology. Thirty-eight patients had retrospective information available from medical charts and/or from the data storage system. Interestingly, 37/38 patients (97.3%) had CG values lower than or equal to 19 g/l at the time of CVID diagnosis. The CG cut-off of 19 g/l was chosen based on the results of our study and detected antibody deficiency in 97.3% of the analyzed CVID patients. Similarly, to the analysis on anonymized samples, a linear correlation between CG and IgG levels was observed (r=0.52, p=0.0009) as we can see in this figure, further confirming the effectiveness of CG to predict IgG serum levels.

Deficiency in an Italian University Hospital: Summary of the findings Validation of Calculated Globulin (CG) as a Screening Test for Antibody Deficiency in an Italian University Hospital: Summary of the findings CG cut-off of 19 g/l detected patients with IgG ˂than 600 mg/dl with a sensitivity of 70% and a specificity of 75% Results are consistent with the findings of the study on CG by Jolles et al., where a cut-off of 18 g/l, with a sensitivity of 66% and a specificity of 78% for samples with IgG<500 mg/dl So, To summarize the findings of this study, we can say that: A CG cut-off of 19 g/l detected patients with IgG ˂than 600 mg/dl with a sensitivity of 70% and a specificity of 75% The results are consistent with the findings of the study on CG by Jolles et al., where a cut-off of 18 g/l, with a sensitivity of 66% and a specificity of 78% for samples with IgG<500 mg/dl And, in the retrospective analysis, our CG cut-off of 19 g/l detected antibody deficiency in 97.3% (37/38) of the subjects of the cohort of patients diagnosed with CVID at our Centre However, we do not have any retrospective data to evaluate how many years of disease we would have prevented and how many resources we would have saved for CVID patients. In the retrospective analysis, our CG cut-off of 19 g/l detected antibody deficiency in 97.3% (37/38) of the subjects of the cohort of patients diagnosed with CVID at our Centre

Next step….Perspective study Study design: Phase 1: Identification and collection of the serum samples with a CG level lower than or equal to 19 g/l from adults (age > 18 years) during a period of 1 year Biochemistry Laboratory Phase 2: For each sample selected: Dosage of IgG, IgA, IgM Serum electrophoresis Serum immunofixation (when appropriate) Clinical Pathology Laboratory The next step to do to employ calculated globulin as screening test for antibody deficiency in the Italian setting, is to perform a perspective study to evaluate the real effectiveness of this screening approach. This is the design of the study that will involve three division of our Department. In first phase, serum samples with a CG value lower than or equal to 19 g/l will be identificated and collected at Biochemistry Laboratory, during a period of one year. Simoultanously, in the second phase, all the serum samples collected will be transferred at the Clinical Pathology Laboratory, where they will perform the dosage of immunoglobulins, the serum electrophoresis and (when appropriate) the serum immunofixation. Then all laboratory results and the clinical data regarding the samples (age, gender, clinical speciality of source, and reason for the test) will be stored and analyzed at our Division of Allergy and Clinical Immunology Phase 3: Collection of laboratory results and clinical data (age, gender, clinical speciality of source, and reason for the test) and Statistical Analysis Division of Allergy and Clinical Immunology

Perspective study: Timeline This is the Time-line of the study: the collection of the samples, the laboratory analysis and the record of the information will take place in a time of 12 months. While, the processying of the results will last about three months.

Primary Objectives of the Perspective study To assess the sensitivity, the specificity and the positive predictive value of the CG cut-off to detect hypogammaglobulinemia To assess the ratio between the new diagnosed immunodeficiency cases (using CG screening) and the total population analyzed Finally, these are the primary objectives of the perspective study: To assess the sensitivity, the specificity and the positive predictive value of the CG cut-off to detect hypogammaglobulinemia To assess the ratio between the new diagnosed immunodeficiency cases (using CG screening) and the total population analyzed To determine the source (in terms of clinical specialties) of the hypogammaglobulinemic patients To determine the source (in terms of clinical specialties) of the hypogammaglobulinemic patients

Calculated Globulin (CG) as a Screening Test for Antibody Deficiency: Open Questions Sensitivity and Specificity of CG are potentially limited by the fact that IgG accounts for only about 30% of total serum globulin fraction Acute inflammation may cause an increase of alpha and beta globulins and potentially mask low gamma-globulin levels There are some open question or possible limations for the use of Calculated Globulin (CG) as a Screening Test for Antibody Deficiency. For example.. Sensitivity and Specificity of CG are potentially limited by the fact that IgG accounts for only about 30% of total serum globulin fraction Acute inflammation may cause an increase of alpha and beta globulins and potentially mask low gamma-globulin levels There has been any validation for children, whose the normal IgG values vary according to age And finally we have to consider the costs of performing reflex tests in virtually healthy subjects. Actually, the costs of missing antibody deficiency diagnoses largely outweigh the costs required for this screening program No validation for children, whose the normal IgG values vary according to age Costs ?

Costs of Diagnosed vs Undiagnosed Immunodeficiency In fact, in this regard, in a study on the economic impact of primary immunodeficiency conducted by the Jeffrey Modell foundation, the initiation of immunoglobulin replacement therapy led to an annual saving per patient of $ 78,166 despite the costs of immunoglobulin that amount to 30000 dollars per patients. In fact, as we can see in the last row of this table, the cost of an undiagnosed patients (that include the costs for the treatment and the hospitalization related to infection and the school or work days missed) are of 138000 dollars vs 30000 dollars for diagnosed patients. As consequence the economic benefits for the health care system as well as the possibility of early detection of disease before the onset of end-organ damage outweigh the economic disadvantages of the proposed screening test. Modell et al. Immunol Res. 2011

Conclusions The results of this study confirm the potential utility of a screening test based on CG as a tool to: Reduce diagnostic delay of antibody deficiency Improve long-term prognosis of patients living with antibody deficiency In conclusion, The results of this study confirm the potential utility of a screening test based on CG as a tool to: Reduce diagnostic delay of antibody deficiency, thus preventing the development of irreversible organ damage, as bronchiectasis or COPD Improve long-term prognosis of patients living with antibody deficiency Reduce the healthcare costs of antibody deficiency Reduce the healthcare costs of antibody deficiency.

Aknowledgements Immmunodeficiency Centre for Wales Stephen Jolles Mark Ponsford Department of Biochemistry Soha Zouwail Division of Allergy and Clinical Immunology Giuseppe Spadaro Arturo Genovese Ludovica Crescenzi Biochemistry Laboratory Marcella Savoia Antonio Del Rio Clinical Pathology Laboratory Margaret Ricciardone Thank you very much for your attention, I would like to thank all the people who contributed to the development of this study From the University of Cardiff…. Etc etc Fromt the University of Naples….