Clinical and Immunologic Characteristics of Non-Hematologic Cancers in Patients with Inborn Errors of Immunity

Simple Summary Comprehensive studies on non-hematologic tumors in patients with inborn errors of immunity are scarce. Using a well-established registry of patients with long-term follow-up, molecular defects associated with these cancers were reported for the first time among these patients. Using the retrospective data available of this national registry of patients with primary immune defects, we clarified that almost all cancer hallmarks are involved in the development of non-hematologic cancers in patients presenting with non-hematologic cancers. Abstract Inborn errors of immunity (IEI) are a heterogeneous group of inherited disorders, and almost 500 genes associated with these disorders have been identified. Defects in IEI genes lead to diverse clinical manifestations including increased susceptibility to recurrent or prolonged infections, immune dysregulation phenotypes (such as severe atopy, allergy, autoimmunity, and uncontrolled inflammation, lymphoproliferation), as well as predisposition to malignancies. Although the majority of IEI patients present hematologic cancers, the characteristics of other types of cancers are not well described in these groups of patients. By investigating 5384 IEI patients registered in the Iranian national registry the clinical and immunologic phenotypes of patients with non-hematologic cancers were compared with other malignant and non-malignant patients. Solid tumors were reported <20% of malignant IEI patients (n = 27/144 patients) and appeared to be very heterogeneous by type and localization as well as molecular defects (mainly due to DNA repair defect resulted from ATM deficiency). The correlation between the type of malignancy and survival status was remarkable as patients with non-hematologic cancers survive higher than IEI patients with hematologic cancers. Our findings showed that different types of malignancy could be associated with specific entities of IEI. Therefore, the education of physicians about the risk of malignancies in IEI is required for personalized treatment and appropriate management of patients.


Introduction
Inborn errors of immunity (IEI), previously referred to as primary immunodeficiency disorders constitute heterogeneous genetic diseases, commonly presented with infections or other immune-related manifestations including malignancies [1,2]. Thanks to the improvements in molecular diagnosis methodologies, approximately 500 genes associated with IEI have been described affecting both innate and adaptive immunity [3]. These genetic alterations result in developmental arrest and/or the functional defects of different immune system components [4]. Among all reported IEI patients in the world, <15% of patients received a final molecular diagnosis demonstrating the essential need for clarification of pathogenic mechanisms in the remaining cases [5].

Patients
A total of 5384 patients with IEI were registered in the Iranian national IEI database between the years 1999 and 2020 [19,20]. To have a more comprehensive investigation of patients with malignancy, this retrospective longitudinal study was conducted between August 2022 and October 2022 among registered patients to analyze the clinical, immunologic, and molecular data. After establishing a research design, the study was approved by the ethics committee of Tehran University of Medical Science, Tehran, Iran. Patients with incomplete data or those who did not meet the criteria were excluded. The diagnosis of IEI was carried out based on the Middle East and North Africa (MENA) and European Society for Immunodeficiency (ESID) diagnostic criteria [21,22]. The patients were categorized into IEI subcategories using the International Union of Immunological Societies (IUIS) classification [3]. Written informed consent was obtained from all subjects involved in the study or their parents.

Clinical Evaluation in IEI Patients
We gathered all IEI patients' medical relevant data using a comprehensive questionnaire that included demographic data, clinical complications, immunological and paraclinical tests covering the diagnostic basic lab tests at the time of the first presentation until specific diagnostic evaluation for confirmation of cancer, and during and after cancer treatment. For each patient, malignant presentations before and/or after IEI diagnosis were documented. The accurate diagnosis of cancer was based on international criteria considering medical, imaging, biochemical and histopathological documents as described previously [10,14,15]. The evaluation for cancer diagnosis was re-assessed for all IEI cases by an oncologist and a subspecialist related to the involved tissue/organ and was matched with topography and morphology codes obtained from the International Classification of Diseases, 10th revision (ICD-10). Due to the aim of the study, if these patients had concomitant non-hematologic and hematologic cancers, it was classified in the group of non-hematologic cancers but also reported in detail separately.

Genetic Investigation and Diagnoses in IEI Patients
Targeted sequencing was conducted on extracted genomic DNA from a selected group of IEI cases with a classical clinical presentation suggestive of a specific entity and agreed to genetic testing as described previously [3,[23][24][25][26][27]. Whole-exome sequencing was performed to detect single nucleotide variants insertion/deletions and large deletions for patients in whom targeted sequencing failed or had medical features resembling several genetic defects. Candidate genetic alterations were weighed based on the American College of Medical Genetics and Genomics criteria including considering the allele frequency in the population, computational data, immunological data and clinical phenotyping. Only variants with pathologic and likely pathogenic scores and correct Mendelian inheritance pattern were included as the final molecular diagnosis [28][29][30][31].
After confirmation of their clinical and genetic diagnosis patients were classified according to the International Union of Immunological Societies (IUIS) updated classification. This classification includes nine categories of immunodeficiencies affecting cellular and humoral immunity (non-syndromic combined immunodeficiency or CID), combined immunodeficiencies with associated or syndromic features (syndromic CID and bone marrow failure), predominantly antibody deficiencies (PAD), diseases of immune dysregulation, congenital defects of phagocyte number or function (phagocytic disorders), autoinflammatory disorders, defects in intrinsic and innate immunity, complement deficiencies, bone marrow failure and phenocopies of inborn errors of immunity [3].

Statistical Analysis
Data were analyzed using the SPSS statistical software package version 25.0 (IBM corporation, Chicago, IL, USA) and R statistical systems (version 3.4.1., R Foundation for Statistical Computing, Vienna, Austria). The Shapiro-Wilk test was used to validate the assumption of normality for a variable, and the nonparametric or parametric tests were carried out according to the normality assumption. A p-value < 0.05 was considered statistically significant.

Results
Altogether 144 patients (2.6% of the total 5384 patients in IEI registry) with a definite diagnosis of malignancy were recruited for this study, from which 41.6% were female (Table 1). At the time of this study, the median age of cancer patients was 23.2 (interquartile range, IQR 11.0-28.5) years which was significantly higher than the median age of the IEI patients without cancer (9.5 (1.5-37.0) years, p < 0.001). Among these IEI patients with cancer presentation, hematologic malignancies were the most common type of malignancy expectedly. However, non-hematologic malignancies constituted 18.7% of patients (n = 27). Of note, the main non-hematologic malignancies included gastrointestinal cancers (n = 10, 8 gastric adenocarcinomas, 2 colorectal cancer), head/neck cancers (n = 8, 4 squamous cell carcinoma of tongue, 2 mandibular squamous cell carcinoma, 2 brain tumor), skin cancers (n = 3, 2 squamous cell carcinoma and 1 melanoma), breast cancers (n = 3, 2 invasive ductal carcinomas, 1 in situ ductal carcinoma), ovarian cancers (n = 2, both ovarian cystadenoma), and thyroid cancer (n = 1, papillary thyroid cancer). The correlation between the type of malignancy and IEI clinical features was remarkable as patients with non-hematologic cancers present a higher rate of chronic lung complications including bronchiectasis than IEI patients with hematologic cancers (40.7% vs. 19.6%, p = 0.02). Of note, chronic enteropathy was also among major-specific gastrointestinal manifestations observed significantly more frequently in patients with non-hematologic cancers (40.7% vs. 11.9%, p < 0.001). This association with chronic enteropathies were even higher in patients with gastrointestinal cancers compared to other non-hematologic cancers (9/10 vs. 1/17, p = 005). In general, patients with non-hematologic cancers were predisposed less than hematologic cancers to severe infection, autoimmunity and lymphoproliferative disorders, but these differences were not statistically significant (Table 1). Expectedly, the age of diagnosis of malignancy were correlated with the current age of patients at the time of study (r = 0.65. p = 0.03). Patients with a clinical diagnosis of CID and immune dysregulation presented exclusively with hematologic malignancies. However, non-hematologic malignancies were recorded in patients with PAD (n = 14), syndromic CID (n = 8) and defects in intrinsic and innate immunity (n = 5). No tumor was documented in IEI cases with phagocyte disorders and autoinflammatory disease and complement deficiency. Among PAD patients, the most prevalent type associated with non-hematologic malignancy was clinically diagnosed as common variable immunodeficiency (10 patients), selective IgA deficiency (3 patients) and hyper IgM syndrome (1 patient). Regarding patients with the syndromic CID group, Ataxia-Telangiectasia and hyper IgE syndrome patients had solid tumors in 7 cases and 1 cases, respectively. In patients with inborn errors of innate immunity cases of Chronic mucocutaneous candidiasis (n = 3) and Mendelian susceptibility to mycobacterial diseases (n = 2) were reported with non-hematologic malignancies. General immunologic profiles of patients in both groups are presented in Table 2.

Discussion
In the current retrospective study, we evaluated for the first time the overall incidence of non-hematologic malignancy in IEI patients enrolled in the national registry in detail. We observed almost 20% of malignant patients with documented non-hematologic cancers, consisting of a prevalence of 500 per 100,000 (mainly due to gastrointestinal cancers, head/neck cancers and breast cancers) at the median age of 27.4 years. The prevalence of cancers in the normal population (age-standardized rate 300 per 100,000, mainly due to breast cancers, gastrointestinal cancer and lung cancers) indicates almost twice the higher risk of developing non-hematologic malignancies in our cohort [32,33]. Of note, this risk still is far lower than the predisposition of IEI patients to hematologic malignancy, presenting almost 20-fold higher risk mainly to non-Hodgkin's lymphoma. In contrast in the normal population hematologic malignancies are mainly due to leukemia with a prevalence of 10 per 100,000. The age-standardized mortality rate of cancer in the normal Iranian population was also 80 per 100,000 which showed a similar rate in comparison with IEI patients with non-hematologic cancers, but remarkably lower than the mortality in IEI patients with hematologic malignancies (800 per 100,000, 10-fold higher mortality rate compared to cancers in immunocompetent population) [32,33]. Consistently our observation within the current cohort also showed a significantly better survival of cases with non-hematologic malignancy than hematologic cancers.
The promise of individualized management of cancer patients relies on the discovery of actionable genetic alterations and the evaluation of novel targeted modalities. Despite this important practical usage, identification of a pattern of gene expression and epigenetics may help for elucidation of underlying pathogenesis and in-directly suggest potential targeted therapies. The most prevalent genes with somatic mutations identified in population-based cancer databases (mainly with non-hematologic cancers detected in immunocompetent individuals) were TP53, KRAS and APC. Of note, almost all of these top hints are immune-related genes and their germline mutation can be associated with IEI [3,34]. The current cohort of IEI patients with non-hematologic cancers and solved molecular diagnosis showed defects in DNA repair pathway (ATM deficiency), T cell development (AIRE deficiencies and STAT1 gain-of-function), B cell development (IKZF1 and PI3KR1 deficiencies), and innate immune pathway (IL12RB1 deficiency). Especially our finding highlighted mutations in ATM genes were associated with a high level of mortality and presentation of both hematologic and non-hematologic cancers in children and young-

Discussion
In the current retrospective study, we evaluated for the first time the overall incidence of non-hematologic malignancy in IEI patients enrolled in the national registry in detail. We observed almost 20% of malignant patients with documented non-hematologic cancers, consisting of a prevalence of 500 per 100,000 (mainly due to gastrointestinal cancers, head/neck cancers and breast cancers) at the median age of 27.4 years. The prevalence of cancers in the normal population (age-standardized rate 300 per 100,000, mainly due to breast cancers, gastrointestinal cancer and lung cancers) indicates almost twice the higher risk of developing non-hematologic malignancies in our cohort [32,33]. Of note, this risk still is far lower than the predisposition of IEI patients to hematologic malignancy, presenting almost 20-fold higher risk mainly to non-Hodgkin's lymphoma. In contrast in the normal population hematologic malignancies are mainly due to leukemia with a prevalence of 10 per 100,000. The age-standardized mortality rate of cancer in the normal Iranian population was also 80 per 100,000 which showed a similar rate in comparison with IEI patients with non-hematologic cancers, but remarkably lower than the mortality in IEI patients with hematologic malignancies (800 per 100,000, 10-fold higher mortality rate compared to cancers in immunocompetent population) [32,33]. Consistently our observation within the current cohort also showed a significantly better survival of cases with non-hematologic malignancy than hematologic cancers.
The promise of individualized management of cancer patients relies on the discovery of actionable genetic alterations and the evaluation of novel targeted modalities. Despite this important practical usage, identification of a pattern of gene expression and epigenetics may help for elucidation of underlying pathogenesis and in-directly suggest potential targeted therapies. The most prevalent genes with somatic mutations identified in population-based cancer databases (mainly with non-hematologic cancers detected in immunocompetent individuals) were TP53, KRAS and APC. Of note, almost all of these top hints are immune-related genes and their germline mutation can be associated with IEI [3,34]. The current cohort of IEI patients with non-hematologic cancers and solved molecular diagnosis showed defects in DNA repair pathway (ATM deficiency), T cell development (AIRE deficiencies and STAT1 gain-of-function), B cell development (IKZF1 and PI3KR1 deficiencies), and innate immune pathway (IL12RB1 deficiency). Especially our finding highlighted mutations in ATM genes were associated with a high level of mortality and presentation of both hematologic and non-hematologic cancers in children and youngadult IEI patients. Within the four main categories of IEI associated with non-hematologic cancer in our study, several lines of evidence have been highlighted for the underlying mechanisms of tumorigenesis. Combined immunodeficient and antibody-deficient patients can develop several different cancer hallmarks including avoiding immune destruction, resisting cell death, inducing angiogenesis, deregulating cellular energies, activating invasion and metastasis, tumor-promoting inflammation, and enabling replicative immortality. Moreover, cases with syndromic CID due to DNA repair can increase genome instability and mutation and evade growth suppression (Figure 3) [9]. adult IEI patients. Within the four main categories of IEI associated with non-hematologic cancer in our study, several lines of evidence have been highlighted for the underlying mechanisms of tumorigenesis. Combined immunodeficient and antibody-deficient patients can develop several different cancer hallmarks including avoiding immune destruction, resisting cell death, inducing angiogenesis, deregulating cellular energies, activating invasion and metastasis, tumor-promoting inflammation, and enabling replicative immortality. Moreover, cases with syndromic CID due to DNA repair can increase genome instability and mutation and evade growth suppression (Figure 3) [9]. A wide range of ages at the last visit was recorded in our study, however, the median age observed was consistent with the previous report of the Immunodeficiency Cancer Registry (ICR) [35]. The male predominance in both hematologic and non-hematologic patients was in line with other studies [36]. The result can be justified by the fact that many types in the hematologic group were due to X-linked transmission disease including BTK deficiency. However, we could not observe any gene with X-linked inheritance in patients with non-hematologic cancers and the role of hormonal and other gender-specific environmental factors may play a role in this group of IEI patients. Moreover, the population with available genetic sequencing in this study were limited and prevents final conclusion over the entire population, indicating the need for investigation of genetic defects of patients with solid tumors in other IEI international registries in future studies.

Conclusions
Our data indicated that different types of genetic defects are underlying non-hematologic malignancies of IEI patients with higher prevalence compared to the normal population. Therefore, increased awareness of immunologists and oncologists about the risk of these types of malignancies in IEI patients, required regular monitoring and the performance of genetic evaluation for prognostic estimation and probable personalized treatment are essential. Although very few studies address these types of malignancies in the IEI cohorts, future studies and a better understanding of all molecular mechanisms of this phenotype ultimately improve management and, eventually, the survival of patients can be achieved. A wide range of ages at the last visit was recorded in our study, however, the median age observed was consistent with the previous report of the Immunodeficiency Cancer Registry (ICR) [35]. The male predominance in both hematologic and non-hematologic patients was in line with other studies [36]. The result can be justified by the fact that many types in the hematologic group were due to X-linked transmission disease including BTK deficiency. However, we could not observe any gene with X-linked inheritance in patients with non-hematologic cancers and the role of hormonal and other genderspecific environmental factors may play a role in this group of IEI patients. Moreover, the population with available genetic sequencing in this study were limited and prevents final conclusion over the entire population, indicating the need for investigation of genetic defects of patients with solid tumors in other IEI international registries in future studies.

Conclusions
Our data indicated that different types of genetic defects are underlying non-hematologic malignancies of IEI patients with higher prevalence compared to the normal population. Therefore, increased awareness of immunologists and oncologists about the risk of these types of malignancies in IEI patients, required regular monitoring and the performance of genetic evaluation for prognostic estimation and probable personalized treatment are essential. Although very few studies address these types of malignancies in the IEI cohorts, future studies and a better understanding of all molecular mechanisms of this phenotype ultimately improve management and, eventually, the survival of patients can be achieved.