Clinical and laboratory characteristics of patients with symptomatic secondary immunodeficiency following the treatment of haematological malignancies

Abstract Secondary immunodeficiency (SID), manifesting as increased susceptibility to infection, is an emergent clinical problem in haematoncology. Management of SID includes vaccination, prophylactic antibiotics (pAbx) and immunoglobulin replacement therapy (IgRT). We report clinical and laboratory parameters of 75 individuals, treated for haematological malignancy, who were referred for immunological assessment due to recurrent infections. Forty‐five were managed with pAbx while thirty required IgRT after failing to improve on pAbx. Individuals requiring IgRT had significantly more bacterial, viral and fungal infections resulting in hospitalization at least 5 years after their original haemato‐oncological diagnosis. Following immunological assessment and intervention, a 4.39‐fold reduction in the frequency of hospital admissions to treat infection was observed in the IgRT cohort and a 2.30‐fold reduction in the pAbx cohort. Significant reductions in outpatient antibiotic use were also observed in both cohorts following immunology input. Patients requiring IgRT were more hypogammaglobulinaemic and had lower titres of pathogen‐specific antibodies and smaller memory B cell populations than those requiring pAbx. Test vaccination with pneumococcal conjugate vaccine discriminated poorly between the two groups. Patients requiring IgRT could be distinguished by combining wider pathogen‐specific serology with a frequency of hospital admissions for infection. If validated in larger cohorts, this approach may circumvent the need for test vaccination and enhance patient selection for IgRT.


INTRODUCTION
Symptomatic secondary immunodeficiency (SID) may be defined as an increased susceptibility to bacterial, viral or fungal infections arising from environmental factors (e.g. nutritional state) or other disease processes (e.g infection, inflammation, malignancy) and their treatments (e.g. cytotoxic or biologic chemotherapy). Symptomatic SID is estimated to be 30-fold more common than primary immunodeficiencies, but the epidemiology, risk factors and immunopathogenesis of symptomatic SID remain poorly understood [1].
The UK has observed a sustained increase in demand for immunoglobulin replacement therapy (IgRT) to manage patients with recurrent infections due to SID [2,3], mainly from individuals previously treated for haematological malignancies. A growing armamentarium of biological and small molecule therapeutics is now employed to treat haematological malignancies leading to well-documented improvements in overall survival [4,5]. However, improvements in overall survival may lead to the emergence of clinically significant, long-term immunocompromise in cancer survivors [1]. responses should be assessed [7]. Furthermore, the evidence supporting the use of IgRT in haematological malignancies derives from studies undertaken before the introduction of modern therapeutic strategies, most notably anti-CD20 B cell depleting agents and CAR-T cell therapy [8][9][10][11], and prior to the institution of widespread childhood vaccination programmes that have impacted the incidence of invasive pneumococcal disease in adults [12].
The purpose of this study was to describe the clinical and immunological characteristics of a heterogeneous cohort of patients referred to Clinical Immunology for the assessment of recurrent infections following treatment for haematological malignancy. In describing this cohort of patients, we aimed to identify biomarkers that might differentiate individuals who might benefit from IgRT at the point of referral without subjecting them to unnecessary pAbx trial periods.

METHODS
A comprehensive, retrospective note review was conducted on all patients referred to the Immunodeficiency service at the Queen Eliza- and those with B cell counts below the lower limit of reliable determination were excluded from flow cytometric analysis. Pathogen-specific antibodies were determined using an in-house Luminex immunoassay [14]. Seroprotective levels of IgG were defined as 0. 10

RESULTS
Seventy-five consecutive patients referred to Clinical Immunology for the investigation of recurrent infections between 2014 and 2019 are included in this study (Table 1). A flow diagram describing the assessment and management of these patients is provided in Figure 1A (Table 2). Infections characteristic of classical antibody deficiency (e.g. lower respiratory tract infections and sinusitis) occurred more frequently in patients requiring IgRT, but a significantly higher incidence of fungal pneumonia and non-neutropenic sepsis/bacteraemia was also observed, suggesting broader, combined immune defects in these individuals, compared to those on pAbx. These patients also suffered significantly more late infections-defined as those occurring at least 5 years after their initial diagnosis of haematological malignancy-requiring hospital admission ( Figure 1C).
A broad range of bacteria, viruses and fungi were identified from microbiological samples from this cohort (Table 3). Parainfluenza and respiratory syncytial virus were more likely to be detected in the group requiring IgRT; however, there was no difference in influenza detection. Non-typable Haemophilus influenzae was isolated more frequently 57.3% of patients referred to Clinical Immunology were already taking at least one pAbx, which they had been receiving for a median of 39 months, the commonest being penicillin V, a narrow spectrum antibiotic commonly prescribed for presumed functional asplenia following transplant. The average number of pAbx employed prior to referral was not significantly different between those who required long-term IgRT and those who did not (1.50 vs. 1.13, p = 0.264). Following referral to Clinical Immunology, the most commonly used pAbx was azithromycin and a much wider spectrum of pAbx prophylaxis was utilised to ensure infections could not be controlled with antibiotic prophylaxis alone ( Figure S1). Haematological parameters including total neutrophil and lymphocyte counts did not discriminate between patients who could be maintained on pAbx and those requiring IgRT ( Table 1). Enumeration of the total peripheral blood CD3+ T cell population and the CD4+ or CD8+ T cell subpopulations also did not discriminate between treatment groups (Figure 2A). 8% of the total cohort were found to have a CD4+ T cell count < 0.2 × 10 9 /L, a known risk factor for Pneumocystis jiroveci injection. A small, but statistically significant difference  (CD19+ IgD-CD27+) were all significantly lower in patients requiring long-term treatment with IgRT ( Figure 2B).

Following assessment and intervention by
As a cohort, patients requiring IgRT had lower total immunoglobulin G, A and M levels compared with those on pAbx ( Figure 3A), however, overlap exists between the groups with no discriminatory level. 44% of patients in the pAbx group had an IgG level below the lower limit of the normal reference range (6 g/L) and 13% had an IgG level < 4 g/L, a level that would potentially make them eligible for immunoglobulin replacement for secondary antibody deficiency under the 2019 NHS England guidelines [6]. Equally, a small number of patients in the IgRT group had baseline IgG > 5 g/L but still had recurrent infections that had improved following the initiation of IgRT. There was no linear relationship between total IgG levels and the overall number of hospital admissions to treat infections prior to referral to Clinical Immunology was significantly lower in those requiring long-term IgRT ( Figure 3C).
Response to test vaccination with pneumococcal conjugate vaccine 13 (PCV13) was also poor in both treatment groups; no patients in the IgRT and only 35% of patients in the antibiotic cohort mounted a satisfactory response to vaccination with PCV13, defined by an increment in protective serotypes to over 70% of those measured ( Figure 3D). The incidence of microbiological isolates in each group is compared statistically using Poisson's incidence rate ratio (i.e. the total number of positive microbiological isolates occurring in each group corrected for the total time between the first infection and referral to clinical immunology in each group). Only pathogens where four or more individual isolates across the total cohort are shown). The cohort was divided into two subgroups; individuals who had undergone bone marrow transplantation and individuals who had not (Table S1). Mirroring the overall cohort, patients requiring long-term  (Table S1). The timing of anti-CD20 depletion may be important as we found patients requiring long-term IgRT had significantly greater exposure to B cell depletion after transplantation ( Figure 5) for indications including Epstein Barr virus reactivation and autoimmune cytopenias.

DISCUSSION
Increased susceptibility to infection following the diagnosis and treatment of haematological malignancy is predictable and well-described. Serum IgG concentrations and response to pneumococcal vaccination are currently used to stratify the severity of immunodeficiency and manage the demand for immunoglobulin products [6]. However, we show the significant limitations of these approaches. Although individuals requiring IgRT were significantly more hypogammaglobulineamic than those maintained using antibiotic prophylaxis, total IgG concentrations showed no correlation with the overall number of serious infections or late-occurring serious infections requiring hospital admission. This is unsurprising as effective humoral immunity depends on the generation of high-affinity, somatically hypermutated, class-switched antibodies. Our data suggest that baseline pathogen-specific antibodies may provide greater insight into this axis and the need for IgRT than total IgG or pneumococcal vaccine responsiveness.
We identify that absolute titres of IgG directed against pneumococcal serotype 14 and HiB are potential biomarkers of future IgRT requirements. Pneumococcal serotype 14 was, in the prepneumococcal vaccine era, the most common cause of invasive pneumococcal infection worldwide [17]. Pneumococcal serotype 19A subsequently emerged as the most common cause of invasive pneumococcal disease following the introduction of the seven valent pneumococcal vaccines [18]; of note, the absence of a protective IgG titre to Pn19A was also associated with the need for IgRT in our cohort. Due to the age of our cohort, none would have benefited from childhood vaccination against HiB or pneumococcal disease; humoral immunity would have developed following infection or colonisation and be maintained by long-lived plasma cells within the bone marrow [19][20][21]. The loss of humoral immunity to Pn14 and HiB may be a surrogate marker for the disruption of the reservoir of high-quality humoral immunity and therefore provide valuable insight into the severity of the immune deficiency. CoV-2 vaccination schedule, rising to 76.0% after three doses [23][24][25].
This raises the possibility of using alternative test vaccination strategies to interrogate immune competence and, that with better vaccine design and protocols, immune responses to existing non-SARS-CoV-2 vaccines may be improved in immunocompromised cohorts. Furthermore, as pneumococcal polysaccharide vaccination becomes widely adopted in haemato-oncology management guidelines [26], immunologists must be cognizant of the intersection between therapeutic vaccination and test vaccination, in particular, the potential for vaccine hyporesponsiveness through repeat exposure to pneumococcal vaccinations [27,28].
The severity of secondary immunodeficiency is dynamic; we have previously demonstrated that kinetics of B cell reconstitution following anti-CD20 therapy and its association with vaccine responses to SARS-CoV-2 vaccines differs between haematology and rheumatology cohorts as a factor of their underlying disease and other therapeutic immunosuppression [29]. In this cohort, we provide further evidence of the heterogeneity of the effects of B cell depletion on humoral immune competence. In the non-bone marrow transplantation cohort of patients, total exposure to rituximab was not a significant risk factor determining the future requirements for IgRT, in keeping with rapid B cell reconstitution and restitution of vaccine responsiveness observed in most lymphoma patients following treatment [29]. However, in the bone marrow transplantation cohort, exposure to rituximab after transplantation was associated with a future need for immunoglobulin. Although rituximab use in this context is to treat Epstein Barr virus reactivation, viral reactivation may be a surrogate for a combination of poor immune reconstitution and/or increased immune suppression to treat graft versus host disease (GvHD). Aberrant B cell function is observed in chronic GvHD [30] and chronic GvHD has previously been identified as a risk factor contributing to late fatal infections in patients undergoing bone marrow transplantation alongside increasing age, male sex, and mismatched unrelated grafts [31].