Opportunistic infections complicating immunotherapy for non-small cell lung cancer.

Immunotherapy has produced durable responses in numerous advanced and metastatic cancers, especially advanced non‐small cell lung carcinoma (NSCLC). However, opportunistic infection has become a major risk for patients who have received immune checkpoint inhibitors (ICIs). Early diagnosis of infection is difficult due to an acute disease course and heterogeneity in clinical manifestation. We retrospectively analyzed four cases with NSCLC who received ICIs and developed opportunistic infections. Two of our cases received antecedent glucocorticoids to treat immune‐related adverse events (irAEs), whereas immunosuppressive agents were not used beforehand in the other cases. We highlight that opportunistic infections complicating immunotherapy can be severe and even fatal. When patients deteriorate after initial remission from irAEs by glucocorticoids, infections should be thoroughly investigated and carefully distinguished from an irAE flare. Bronchoscopy and bronchoalveolar lavage (BAL) are essential. In patients where limited results from traditional microbiological tests have been obtained, next‐generation sequencing (NGS) of BAL fluid is beneficial in guiding a precise antimicrobial treatment. An antipneumocystis prophylaxis may also be considered in selected patients.


Introduction
Immune checkpoint inhibitors (ICIs) have produced durable responses in numerous advanced and metastatic cancers, especially advanced non-small cell lung carcinoma (NSCLC). 1 The mechanism of ICIs is to promote reactivation of antitumor immunity. Importantly, infections have become a major risk for patients who receive ICIs. 2 In particular, for patients who develop immune-related adverse events (irAE), concurrent use of systemic glucocorticoids further increase the susceptibility to severe infections leading to fatal outcomes. 3 In our study, we aim to share our experiences and thoughts from four cases with NSCLC who received ICI treatment and developed opportunistic infection.

Methods
Patients with NSCLC who developed pneumonitis combining opportunistic infections during immunotherapy were retrospectively recruited from Peking Union Medical College Hospital. Chest computed tomography (CT) scans and bronchoscopy were performed to diagnose pneumonitis. Cytological classification, pathogen culture and next generation screening of bronchoalveolar lavage were performed to confirm infections.

Results
Four male patients with a mean age of (60 AE 2.9) years were analyzed. Clinical and laboratory data of the patients are presented in Table 1.

Case 2
A 61-year-old male with advanced lung adenocarcinoma and diabetes mellitus was admitted to hospital due to acute onset of fever, dyspnea, and productive cough for two days. The patient was treated with paclitaxel, carboplatin and pembrolizumab (Keytruda; Merck Sharp & Dohme; 200 mg) for six cycles (Fig 2a). Three cycles of 200 mg pembrolizumab were then administered, and he received the last dose four days prior to admission. On presentation, chest CT showed extensive GGOs and multifocal consolidations (Fig 2b). Intravenous methylprednisolone was administered (1.0 mg/kg/day) empirically, and his symptoms improved slightly. Bronchoscopy with bronchoalveolar lavage (BAL) was performed. Percentage of lymphocytes in bronchoalveolar lavage fluid (BALF) was 1%. Pathogen culture of BALF identified Corynebacterium striatum and Candida albicans. NGS of BALF found Corynebacterium striatum. Vancomycin (1 g per 12 hours) and caspofungin (75 mg once!50 mg per 12 hours) were initiated accordingly. The patient clinically and radiographically improved (Fig 2c).

Case 3
A 62-year-old male with squamous lung carcinoma presented with fever, cough, and hemoptysis for four weeks. The patient had undergone a right lower lobectomy in 2015. A recurrence was found in the residual lung at the surgical site. He completed radiotherapy, then received two cycles of pembrolizumab (Keytruda; Merck Sharp & Dohme; 100 mg). Three weeks after the last dose of pembrolizumab, CT scans indicated multiple irregular opacities on the right lung (Fig 3a). Radiation pneumonia was suspected. The patient received intravenous methylprednisolone 80 mg/day for 10 days, and was clinically and radiographically improved (Fig 3b). His steroid dose was tapered gradually. Unfortunately, the patient then developed fever, cough, and hemoptysis. Chest CT showed bilateral ground-glass opacities (GGOs) (Fig 3c). Although empirical antibiotics and methylprednisolone (120 mg/day) were administered, the patient did not improve. On admission, PaO 2 was 58 mmHg. Serum (1-3)-ß-d-glucan test was 277.7 pg/ml, and serum CMV-DNA was 31000 copies/ mL. BALF NGS was positive for Pneumocystis jirovecii and CMV DNA. TMP-SMX (20 mg/kg/day), intravenous methylprednisolone (40 mg/day), and ganciclovir (5 mg/kg, per 12 hours) were given. Meanwhile, intravenous immunoglobulin (10 g/day for five days) was administered. However, the patient deteriorated progressively. Repeat CT scans indicated the presence of extensive GGOs and multifocal consolidations bilaterally (Fig 3d). Two weeks after admission, he was intubated and died of respiratory failure.

Case 4
A 62-year-old male with advanced squamous cell lung carcinoma presented with fever and productive cough for two days. He was treated with two cycles of etoposide and cisplatin, and concurrent radiotherapy was performed. The patient obtained partial remission, and was transitioned to two cycles of toripalimab (PD-1 inhibitor; 240 mg). He developed progressive dyspnea 10 days after the last dose of toripalimab.CT scans revealed multiple patchy infiltration on the right lung (Fig 4a). An IrAE was considered likely to have occurred, and he received intravenous methylprednisolone (40 mg/day) as well as empirical antibiotics (ceftazidime, 1 g per 12 hours; moxifloxacin, 0.4 g/day). His steroid dose was tapered within six weeks as his  symptoms improved (Fig 4b). However, the patient subsequently developed fever, dyspnea, and productive cough after ceasing steroid treatment. CT scan showed scattered GGOs in a pattern of reticular opacities (Fig 4c). BALF lymphocytes percentage were 6%. BALF pathogen culture was negative, while NGS revealed Pseudomonas aeruginosa, Pneumocystis jirovecii, and Candida albicans. Intravenous moxifloxacin (0.4 g/day), fluconazole (200 mg/day), oral SMZ-TMP (20 mg/kg/day), and intravenous methylprednisolone (40 mg per 12 hours) were administered. The patient's fever subsided, and the dyspnea was gradually relieved. Repeated CT scans showed the infiltration had resolved (Fig 4d).

Discussion
Opportunistic infections accompanying immunotherapy are severe and can often be fatal. For patients with NSCLC, infections should be carefully distinguished from checkpoint inhibitor pneumonitis (CIP), pseudoprogression, and hyperprogression. Fever is a strong indicator of infection. However, it often requires intensive investigation owing to heterogeneity on clinical presentation. Two of the above cases did not receive antecedent glucocorticoid therapy, yet developed immune-related, steroidfree infection. Similarly, previous reports have described tuberculosis in patients with no previous use of immunosuppressive agents. 4,5 Although random control trials of CTLA-4 and PD-1/PD-L1 blocking agents did not confirm an independent relationship of ICIs with infection, 6,7 we do see patients treated with ICIs who suffer from severe infections. Multiple predisposing factors for infection often coexist in patients who receive ICIs, including cytotoxic chemotherapy, radiotherapy, and associated immunosuppressive comorbidities. Noticeably, both of the above two cases had diabetes mellitus. In a previous cohort published by Fujita et al. 2 32 out of 167 (19.6%) patients with NSCLC who received nivolumab developed infectious diseases, and diabetes was identified as an independent risk factor for infection. We therefore recommend a careful evaluation of patients with diabetes. Further research is needed to better understand the mechanism of increased risk of infection accompanying immunotherapy.
Two of the four patients in our study developed immunerelated, steroid-induced pneumonia. Current guidelines recommend 1-2 mg/kg/day of prednisone/equivalent glucocorticoids for lower-grade (ie, grade 2) CIP, and 2-4 mg/kg/ day for higher-grade (ie, grade 3-4) CIP. 8 In a recent retrospective study involving 740 patients with melanoma treated by ICIs, the rate of serious infection was 13.5% in the subgroup of patients exposed to glucocorticoids or infliximab. 9 When suspected, opportunistic infections should be thoroughly screened, including Aspergillus fumigatus pneumonia, PCP, tuberculosis, and viral infections.
Bronchoscopy and bronchoalveolar lavage (BAL) are essential to confirm an infection. An increased lymphocytic infiltration supported CIP. 10 In a 2017 case series, 68% (24/35) of CIP cases had T lymphocytic alveolitis. 11 In our clinical cases, percentages of lymphocytes in BALF were low, suggesting infection rather than irAE. Given the unfavorable effect of steroids on controlling infections, it is important to carry out bronchoscopy BAL in a timely manner in patients with suspected CIP before significant supplemental oxygen requirements are needed.
NGS opens up revolutionary opportunities in diagnostics of infectious diseases. It is especially useful for cases that challenge the limits of traditional laboratory testing. 12 A 2017 prospective study involving 101 immunocompromised adults showed that NGS had a high negative predictive value, and detected more clinically relevant pathogens than conventional microbiological methods. 13 In our case reports, patients underwent NGS of BALF and obtained revealing results indicating mixed infection. Pathogens included bacteria (Cases 3 and 4), CMV (Case 1), Pneumocystis jirovecii (Case 4), and aspergillus fumigatus (Cases 1 and 3). To patients who obtained limited results from traditional microbiological culture, NGS of BALF is beneficial in guiding a precise antimicrobial treatment.
The treatment strategies of opportunistic infections complicating immunotherapy have not as yet been defined. Our clinical cases demonstrated rapid courses, and early diagnosis was pivotal. We also suggest that an antipneumocystis prophylaxis may be considered in selected patients. Prospective trials are required to investigate the optimal procedures and therapeutic management.

Disclosure
The authors report that there are no conflict of interests.