2.1 COVID-19 vaccines and lymphadenopathy

Almost all COVID-19 vaccines may cause reactive lymphadenopathy; most cases are subclinical and detectable only instrumentally, whereas a minority of cases show overt clinical C19-LAP. Increased lymph node 18F-FDG uptake has been detected in up to 36% of vaccinated patients⁶⁹ up to 10 weeks after vaccination, with women and people aged over 65 years being most frequently affected.^{69, 70} Lymph node enlargement occurs less frequently; different studies have reported an incidence of ~1% among those vaccinated against COVID-19^{15, 16, 35, 58} depending on the specific vaccine; namely, it has been reported in 0.3% of Pfizer-BioNTech and 1.1% of Moderna vaccines, respectively⁵⁸ (Table 1). While most cases of SLDI do not show clinically evident enlargement of lymph nodes, an awareness of SLDI among doctors is of fundamental importance in a variety of clinical contexts, particularly with regard to cancer staging and follow-up to avoid the risk of overdiagnosis. In our research, surgical excision or CNB of C19-LAP- or SLDI-enlarged lymph nodes have been performed in 55 cases.^{20, 22, 23, 35, 39, 41-43, 47, 49-63} The histological diagnoses were as follows: negative for neoplasia, reactive hyperplasia, and florid reactive hyperplasia (39 cases),^{23, 39, 50-61} progressive transformation of germinal centres (1 case),⁵⁴ atypical follicular hyperplasia with light chain-restricted germinal centres (1 case)⁶² granulomatous reaction (2 cases),^{50, 63} metastases (4 cases),^{20, 22, 50} Kikuchi-Fujimoto disease (KFD; 6 cases),^{40-42, 49, 56, 64} Rosai-Dorfman-Destombes disease (RDD; 1 case),⁴⁸ and Langerhans cell histiocytosis (LCH; 1 case).⁵⁷ In cases of reactive hyperplasia, the histological features were described as a preserved lymph node structure with cortical follicular hyperplasia, enlarged germinal centres and interfollicular expansion by small lymphocytes. Prominent germinal centres and tingible-body macrophages were frequently reported. In expanded interfollicular regions, capillaries with focally prominent endothelial cells have been described.⁵⁵ Diffuse or partial capsule thickness was frequently reported. The immunohistochemical phenotype (IHC) was reported in eight cases.^{54, 58, 61, 62, 71} The results of flow cytometry were also reported in one case.⁴⁴ A case of atypical follicular hyperplasia with light chain-restricted germinal centres after a COVID-19 booster was described,⁶² but the specific vaccine was not reported. A case of C19-LAP with eosinophilic abscesses has also been reported.⁴³ The histological features showed eosinophil-rich inflammation with micro-abscesses.⁴³ COVID-19 cases associated with KFD and RDD have been described as exhibiting the typical histological features of the corresponding, non-vaccine-related entities.^{40-42, 48, 56, 64}

2.2 Fine needle aspiration cytology in cases of SLDI and C19-LAP

The present study is a pooled review based on 14 reports^{20-22, 35-38, 44-47, 52, 72} and one unpublished case observed by the authors. In total, 26 cases are reported, including 17 (65%) female patients, 8 (32%) male patients, and one patient whose gender was not reported,²¹ with an age range of 27-70 years. Nine cases (34%) had a previous or active history of malignancy, including breast cancer, papillary thyroid carcinoma, renal cell carcinoma, melanoma or lung carcinoma; one case had a positive family history of breast carcinoma. C19-LAP was reported after the first or second administrations of two types of vaccines, namely Pfizer-BioNTech (18 cases, 72%) and Moderna (5 cases, 20%); the vaccine type was reported as mRNA COVID-19 vaccine in 2 cases (8%). C19-LAP was observed in imaging examinations after the first and second administrations of the Pfizer-BioNTech vaccine, with a median delay of 10.5 days (range, 5-18 days) and 5 days (range, 1-7 days), respectively.

Lymphadenopathies were developed on the same side as the vaccine shot in 13 cases, and on the opposite side in 3 cases; the side on which the lymphadenopathy occurred was not reported in 10 cases. The specific lymph nodes reported were supraclavicular (10 cases), cervical (6 cases), axillary (7 cases), mediastinal (1 case) and submandibular (2 cases) (Table 2). All lymph nodes were evaluated using US, and were reported as enlarged and oval, with major diameters ranging from 5 to 50 mm (mean 23.7 mm) (Figure 1); spherical shape was reported in one case (Hagen et al.³⁸). Another reported US feature was diffuse or focal cortical thickening and preserved, visible hilum in almost all cases; effaced hilum was reported in one case (Hagen et al.³⁸). The 18F-FDG-PET/CT data were not reported for any of these cases.

2.3 C19-LAP Cytological Features

Our research found 14 detailed reports describing the FNAC features of SLDI and C19-LAP.^{20-22, 35-38, 44-47, 52, 59, 72} From these 14 reports, FNAC findings for 25 cases of C19-LAP were retrieved; one unpublished case observed at our institution was also added, for a total of 26 cases. Aalberg et al.²¹ reported a case in the axillary lymph node of a patient suffering from stage IV renal cell carcinoma; FNAC smears were negative for neoplasia and showed reactive lymphoid cells. Trikannad et al.⁵⁹ reported a case in the mediastinal lymph node of a patient suffering from melanoma. Cytological findings showed a reactive granulomatous process, negative for malignant cells. Gullotti et al.²⁰ reported a case of metastasis for melanoma. Heaven et al.⁴⁴ reported three cases; one showed polymorphous lymphoid cells suggestive of a reactive process; the second showed a reactive lymphoid population with scattered large, atypical cells of uncertain significance; in the third, they observed atypical lymphocytes suggestive of a lymphoproliferative disorder. In the last two cases a biopsy was performed, and in one case flow cytometry assessed a polyclonal reactive process. García-Molina et al.³⁶ reported two cases and described similar cytological features, with a polymorphous cellularity, where numerous large cells with ovoid nuclei stood out, occupying almost the entire cytoplasm. Nuclei were sometimes bilobed or hyperchromatic, all of which exhibited nuclear hyperchromatism or large nucleoli. Numerous histiocytes with dense cytoplasm, sometimes polylobulated with prominent nucleoli, and a few histiocytes with granular bodies in their cytoplasm were also reported. Occasional apoptotic cells were also observed. All of this occurred in a lymphocytic background with lymphocytes with some atypia and irregular morphology, with nuclear notches and few plasma cells. Occasional neutrophil polymorphonuclear leukocytes and very scarce eosinophils could also be seen. These cytological findings were considered representative of nonspecific chronic adenitis. Dirven et al.⁷² reported one case in a patient suffering from a multiple endocrine neoplasia syndrome type 1 (MEN1), in which the FNAC of an axillary adenopathy revealed a benign reactive pattern without metastatic disease. Tan et al.³⁷ described one case in which the smears showed a mixed lymphoid population with a range of small to large lymphocytes. An increased proportion of large, activated lymphocytes was also observed with a thin rim of bluish cytoplasm, and small, sometimes peripheral nucleoli. Germinal centre components, including lympho-histiocytic aggregates with follicular dendritic cells, tingible body macrophages and centroblasts, were also present. Tingible-body macrophages were particularly prominent, and many contained abundant karyorrhectic debris. Plasma cells and eosinophils were not prominent. No necrosis or granulomas were seen. The FNAC diagnosis was reactive process, suggestive of reactive follicular hyperplasia. Cardoso et al.⁴⁵ reported one case of a roundish, 13.7 × 13.5 mm lymph node in which FNAC showed atypical lymphoid cytology; neither Reed-Sternberg cells nor malignant epithelial neoplastic cells were identified. The LN was removed and histological examination showed reactive follicular hyperplasia, with a concordant immunohistochemical evaluation. Fernández-Prada et al.⁴⁶ reported five cases in which FNAC findings were described as merely showing reactive inflammatory signs, with lymphocytic infiltrate and active germinal centres. Kang & Kim²² reported one case in which FNAC of the left cervical lymph node revealed reactive LAP. Only small lymphoid cells were detected at the suspicious left lymph node. The authors of the present study (Caputo et al.) observed a case of cervical C19-LAP (unpublished). Smears showed polymorphous lymphoid cells ranging from small mature lymphocytes to large follicular centre cell populations (Figure 2), and macrophages with tingible bodies and capillary structures were present. Flow cytometry revealed balanced T and B cells; the latter were, in part, CD10-positive, with polyclonal light chain expression. Yoshimoto et al.⁴⁷ reported one case of C19-LAP in the axillary lymph node in a patient suffering from breast carcinoma. FNAC excluded metastasis but cytological features were not provided. Yu et al.³⁵ reported a case of C19-LAP in the axillary lymph node, for which cytological features were not detailed; however, clinical-cytological data favoured a suppurative adenitis, which was subsequently healed. Ganga et al.⁵² reported a large (50 mm) submandibular, hypoechoic lymph node with a cytological diagnosis of reactive inflammation, which shrank and disappeared after therapy. Hagen et al.³⁸ reported five cases in which smears showed a florid reactive lymphadenopathy pattern, characterised by a mixed lymphoid population with lymphocytes at different stages of maturation, including many centroblasts admixed with numerous tingible body macrophages. Immunohistochemistry in one case revealed a reactive pattern with predominant CD3- and CD5-positive T cells, admixed with CD20-positive B cells without co-expression of CyclinD1, CD5, CD10 or CD23. Flow cytometry was performed in three cases; for two cases, the findings revealed T-cell predominant populations, with 72% CD3-positive T-cells vs 23% CD19-positive B cells, and 79% CD3-positive T cells vs 19% CD19-positive B cells; the other case showed a predominance of B-cells, with 55% CD19-positive B-cells vs 37% CD3-positive T cells. IgH rearrangement polymerase chain reaction testing revealed a polyclonal B-cell population, consistent with follicular hyperplasia. LN-FNAC diagnoses were histologically controlled in three cases and by clinical follow-up in the remaining 23 cases.

In summary, LN-FNAC of SLDI and C19 LAP cases has been performed in 9 patients during follow-up for different neoplasms, in 2 patients with comorbidity and 15 cases with negative clinical history. LN-FNAC diagnosed reactive hyperplasia in 22 cases and atypical or suspicious features in 2 cases: granulomatous process in one and metastasis in the other. Flow cytometry was performed in three cases to assess the polyclonality of the corresponding processes. Histological controls and follow-up confirmed the FNAC diagnoses. The clinical and cytological features of the reported cases are summarised in Table 2.

3.1 Post-vaccine lymphadenopathy

Post-vaccine lymphadenopathy due to reactive changes is a well-known phenomenon that may occur as a side effect of different vaccines, including bacillus Calmette-Guerin (BCG), hepatitis B, human papillomavirus, and tetanus vaccines,^{4-9, 12, 14} sometimes simulating a lymphoma,^{24, 57, 62} either clinically or pathologically. SARS-CoV-2 vaccines are the first mRNA vaccines to have been used on a large scale. Their mechanism of action differs from that of other vaccines, which are often based on attenuated or dead microorganisms, toxins, or bacterial surface proteins. SARS-CoV-2 vaccines are based on mRNA delivered into host cells, where it is translated into a protein, which is the target of the immune response.^{2, 15, 73, 74} Clinical trials have shown that the mRNA COVID-19 vaccines are highly immunogenic, and this quality might explain the higher rates of LAP reported as a side effect relative to other vaccines.^{26, 75, 76} The entity and the efficacy of the immune response to the same vaccine administration may depend on the constitutive and temporal differences in the reactivity between individuals. The actual incidence of C19-LAP is difficult to assess because of the heterogeneity of the sampled vaccinated populations, the lack of systematic investigations and selection bias. An additional limit to the evaluation of the incidence of SLDI and C19-LAP is that patients were controlled for pre-existing morbidities and for staging or follow-up of neoplastic diseases by 18F-FDG PET-CT, in which SLDI is more likely to be detected.^{24, 25, 47, 55, 66, 76} The morphological features of post-vaccine lymphadenopathies depend largely on the nature of the vaccine. Calmette-Guerin LAP has been reported as caseating granulomatous inflammation not dissimilar from tubercular lymphadenitis.⁷⁷ Rubella LAP has been reported as lymphadenopathy with a prevalence of sinus histiocytosis.¹¹ A case of tetanus LAP was suspected to be a T- cell lymphoproliferative disorder and was then diagnosed as vaccine-related “pseudolymphomatous” CD4 – cells, florid proliferation.¹⁴ All other LAPs (associated with measles, rubella, smallpox, cholera, typhus, tetanus, diphtheria, pertussis, salk (polio), influenza, and HPV) have been reported as diffuse, follicular, or combined diffuse and follicular lymph node hyperplasia with increased numbers of lymphoblasts, vascular and sinusoidal changes, and a variable number of eosinophils, plasma cells, and mast cells.^{11, 12} In addition to granulomatous or follicular hyperplasia, non-Covid-vaccine-related cases of lymphadenopathy caused by KFD,¹⁰ RDD and Langerhans cell histiocytosis,⁶¹ as well as post-transplantation like lymphadenopathy, have been reported.⁷⁸

3.2 Pathology of COVID-19 post-vaccine lymphadenopathy

Lymph node excision and core-needle biopsy (CNB) were the most frequently performed procedures for the pathological evaluation of COVID-19 post-vaccine lymphadenopathy. We retrieved 48 cases of C19-LAP with histological control (excision or CNB)^{22, 23, 39, 43, 48, 49, 51, 53, 56, 57, 61, 62} and diagnosed as negative or reactive with or without follicular hyperplasia (37 cases). Two cases were reported as florid lymphoid hyperplasia^{54, 57}; KFD was reported in four cases^{37, 39, 49, 56} (16.7%). Larkin et al.⁵⁴ reported a focal increase in Epstein-Barr Virus (EBV)-positive cells, with other findings suggestive of prior infection. In one case, progressive transformation of germinal centres was also reported.⁵⁴ Tintle et al.⁵⁷ reported a case of Langerhans cell hyperplasia. Gogia et al.⁴⁸ reported a case of RDD after COVID-19 infection. The pooled analysis of 38 cases showed a mean age of 40.2 ± 15.6 years old, 50.0% (18/36) of whom were female. Half of these patients had no prior medical history, while three patients (3/18, 16.7%) had a prior non-neoplastic medical history, including asthma, eczema, and hypothyroidism in the study by Tintle et al.⁵⁷; steroid-dependent minimal-change renal disease was reported by Al Soub et al.⁵⁶ and diabetes mellitus and hypertension by Tan et al.³⁹ Cases reported by Ozutemiz et al.⁵⁵ had a family history of breast cancer. Most cases of lymphadenopathy occurred on the same side as the vaccination site, with contra-laterality reported in two cases (2/18, 11.1%).⁷⁹ The most common site of lymphadenopathy was the supraclavicular region (6/13, 46.2%), followed by the axillary (4/13, 30.8%) and cervical regions (3/13, 23.1%). The most commonly reported associated symptoms were fever and pain. The mean reported lymph node dimensions were 22.1 ± 14.7 mm. Ultrasound features were lymph node enlargement, cortical thickening, hypoechogenic areas, lost or partially detectable hilum and ill-defined borders.⁷¹

The cases of florid lymphoid hyperplasia and KFD had mean ages of 18.0 ± 5.0 years and 23.3 ± 7.5 years, respectively, which are significantly younger than those diagnosed with reactive changes or as negative for malignancy (44.2 ± 13.1 years old, p = 0.02). The mean length of the larger of the two lymph node dimensions did not differ significantly among these three diagnoses (reactive changes or negative for malignancy: 22.1 ± 18.2 mm, florid lymphoid hyperplasia: 15.5 ± 5.5 mm, KFD: 22.3 ± 7.1 mm). In the histopathological reports of C19-LAP, lymph nodes have mostly been described as florid follicular hyperplasia; a case of progressive transformation of germinal centres was also reported by Larkin et al.⁵⁴ and a case of Langerhans cell hyperplasia⁵⁷ was also reported. KFD has been reported in several studies.^{40-42, 64}

3.3 Lymph node fine needle aspiration cytology

LN-FNAC is a useful and effective procedure in the diagnosis of lymphadenopathy.^{27-30, 32-34, 80, 81} The COVID-19 pandemic led to a worldwide reduction in the number of FNAC procedures performed, in general and in lymph nodes specifically. Paradoxically, the pandemic has indirectly enhanced and highlighted the role of LN-FNAC, which was the procedure of choice when more invasive core-needle or surgical biopsies were not available, particularly during the lockdowns, when access to hospitals was extremely restricted, even for therapeutic procedures.⁸² The role of LN-FNAC in C19-LAP can be also deduced by the evaluation of cytological and histological reports of C19-LAP; in a comprehensive review⁶¹ 61 cases were collected from 25 different reports, including cases diagnosed by CNB, histology and LN-FNAC. In this review, 22 cases of C19-LAP were diagnosed by LN-FNAC and 39 by CNB or surgical excision, showing that LN-FNAC had a significant clinical role during the Covid pandemic. As reported above, in the cases analysed, in addition to reactive cases (22 cases) and malignant cases (1 case), “atypical lymphoid proliferation” was reported in 3 cases.^{44, 45} In one of these cases,⁴⁴ LN-FNAC was repeated, and the case was diagnosed as reactive and confirmed on follow-up. In the remaining two cases, lymph nodes were excised and diagnosed as reactive on histology. The authors of the present study believe these cases might match the case described by Patil et al.⁶² of atypical follicular hyperplasia with light chain-restricted germinal centres after a COVID-19 booster. It is conceivable that the immunological hyperstimulation or the hyperreactivity caused by RNA vaccines in certain patients might be responsible for the marked hyperplasia of germinal centres and pseudo-clonality, as is the case in some cases of autoimmune diseases.⁸³ It is worth noting that ancillary techniques were not performed on cytological material in any of these cases. It is not clear whether this was due to a shortage of diagnostic material or was merely the operators' choice; however, in these cases, ancillary techniques may be able to solve many of the diagnostic problems through proper phenotypisation and/or clonality assessment of the cell populations.^{28, 33, 80, 81, 84} Because LN-FNAC does not perform as well as the gold-standard histological evaluation in the identification of some specific lymphadenopathies and lymphomas,²⁸ it has been broadly criticised.^{85, 86} This is unfair, in the authors’ opinion, because LN-FNAC is extremely valuable in excluding and sometimes confirming malignant processes, and it is particularly useful when CNB or histological excisions are hampered or difficult to perform, as was the case during the Covid lockdowns. In fact, as reported above, of all of the reported cases of C19-LAP diagnosed by LN-FNAC, 3 cases were confirmed by histology and 19 by follow-up, thus avoiding more complex and invasive procedures. Regarding the clinical background of C19-LAP cases diagnosed by LN-FNAC, a history of neoplasm was present in 8/26 cases (30%), and in the metastatic case the clinical history was negative. These findings confirm the crucial role of LN-FNAC in the management of oncological patients for the quick evaluation of new lymphadenopathies. Notably, Hagen et al.,³⁸ who combined clinical data and cytological features and applied the proposed Sydney System,²⁸ included C19-LAP in their cytological report in addition to excluding malignancy, thus enhancing the value of LN-FNAC.