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Immunotherapy of solid tumors: how safely treat the patients.

Immunotherapy with immune checkpoint inhibitors (ICIs) was shown to improve survival of patients with solid tumors like: melanoma, renal carcinoma, non-small cell lung cancer, cutaneous carcinomas or head and neck carcinoma. However, a special type of ICIs toxicity is observed, namely non-infectious inflammation of different organs connected with autoimmunity known as immune related adverse events (irAEs). This non-infectious inflammation may affect different organs and systems as endocrine organs, the gastrointestinal tract, heart, skin and the nervous system. The lungs are also often involved and this condition is referred to as checkpoint inhibitor pneumonitis (CIP). ICIs toxicity is graded from 1 to 5 depending on the clinical course, the 5 grade being a fatal complication. Corticosteroids are the treatment of choice, generally with good efficacy. In some difficult cases, escalation of immunosuppression is required. The knowledge on irAEs should be promoted among clinicians of all specialties, nurses, patients and their families. The aim of this review is to present the wide spectrum of irAEs: clinical signs and symptoms, differential diagnosis, diagnostic procedures and treatment. The data are supported by our own clinical observations.

The frequency of diarrhea ranges from 19% to 54% [13]. Diarrhea usually appeared between 5 th and 10 th week of treatment initiation but it may occur at any time after the first dose of ICI or even after the four months after the end of therapy [1 ;14].
The reason of diarrhea associated with immunotherapy is not clear. Diarhea may be related to fungal, bacterial (Cl. difficile), viral (CMV) or parasitic infection and these reasons should be considered in differential diagnosis. The potential cause is colitis, which may be a life-threatening complication leading to perforation (0.7-1.5%) [9;15], megacolon toxicum and even death (0.6-1.0%) [10]. The risk of immune-related colitis (irC) is described in 5 to 22% [1;14] [16]. Recently, important risk factors for the occurrence of irC have been identified: the dose, concomitant intake of non-steroidal anti-inflammatory drugs, coexistence of inflammatory bowel diseases and intestinal microbiota disturbance [10;17;18]. IrC seems to be more frequent in patients treated for melanoma in comparison to nonsmall cell lung cancer (NSCLC) or renal cell cancer [19;20].
The occurrence of diarrhea is an indication for differential diagnosis. It should be carried out as soon as possible because early and correct diagnosis allows for effective treatment [21]. The infectious reasons must be excluded-the stool analyses for enteropathogens and Clostridium difficile toxin should be performed [1;2;10].
The immunotherapy as a reason must be taken into account when the infectious causes of diarrhea have been excluded [14]. Coexisting symptoms such mouth ulcers, perianal abnormalities, arthritis, skin lesions, liver damage or endocrinopathy suggest relation of diarrhea with side effect of immunotherapy. Blood tests may reveal anaemia, elevated Creactive protein, hypoalbuminemia, increase of faecal calprotectin [22].
A definitive diagnosis of irC is based on endoscopic and histopathological evaluation.
In the majority of patients the rectum or/and the left colon are involved therefore the flexible sigmoidoscopy is sufficient for diagnosis. However some patients need colonoscopy [22][23][24]. The endoscopic lesions include erythrema, luminal bleeding, erosions and ulcerations [22][23][24]. The presence of these changes but also a normal appearance of the mucosa are usually not sufficient. The final diagnosis can be made only after histological (immunohistological) evaluation of the biopsy. This examination allows for definitive exclusion the infectious disease and confirm inflammatory bowel disease (if no macroscopic changes and histopathological features of inflammation a microscopic colitis is diagnosed).
Regardless of the cause and severity of the symptoms, the first therapeutic step should be a diet to slow down intestinal peristalsis and reduce intestinal secretion [1;10]. In mild form of diarrhea (National Common Terminology Criteria for Adverse Events-CTAE grade 1) there is no necessity to discontinue immunotherapy. Constipating drugs such loperamide and atropine sulphate may be used only after the exclusion of infection [1 ;14].
If the above methods are ineffective and there is an ultimately diagnosis or strong suspicion of inflammatory bowel disease, the immunotherapy must be discontinued and corticosteroids should be introduced (Table 1) [14;25;26]. Corticotherapy is highly effective leading to resolution of symptoms in 87.5% [24]. However, in a small group of patients resistant to steroids infusion of single dose of infliximab (IFX) should be used.
Response to a single dose is very good, sometimes the dose is repeated [27;28].
Very rarely a severe damage to the large bowel occurs, a colectomy with ileostomy is indicated [22]. In severe colitis a definitive discontinuation of immunotherapy is recommended. In patients with moderate form of colitis it is debatable but immunotherapy should be discontinued at least temporarily. After resolving, a return to immunotherapy with low dose steroids may be considered, alternatively, if occurred after administration of anti-CTLA-4, this may be converted to anti-PD-1/PD-L1 inhibitors [29].
Immune-related hepatitis (irH) is the second frequent gastroenterological complication of immunotherapy. It usually appears between 6 th and 14 th weeks of treatment [9; 14;29] and affects 1 to 17% of patients [30].
The clinical signs of irH appear late and are usually associated with severe liver damage. Therefore, liver function tests have to be intensively monitored to recognize IrH early (activity of aminotransferases (AT), γ-glutamyltranspeptidase and alkaline phosphatase).
The results of laboratory indices of liver damage strongly supports its immunological reason. However, the exclusion of other possible causes of liver injury always applies: progression of the neoplasm or its complications (e.g. thromboembolism), which can be determined by imaging examinations: ultrasonography or computed tomography. It is also necessary to perform serological tests to exclude acute viral infection [31] and to perform an anamnesis to eliminate other possible causes such as alcohol, drugs, herbs. Liver biopsy is not necessary but may be useful individually in rare cases of doubt or fulminant course [9].
The initial finding of liver damage is an indication to repeat laboratory tests at least once a week [1;31]. Mild irH usually disappears after 4-6 weeks of appropriate treatment (table 1) and the return to immunotherapy is accepted [1]. It has recently been shown that azathioprine at 1-2 mg/kg dose may be effective in patients who have not completely respond to prednisone or who flare during tapering steroid [26; [31][32][33]. The administration of IFX for irH is contraindicated due to possible immune mediated hepatitis [31].

Endocrine immune-related adverse events
ICI-induced immune system activation often leads to the endocrine irAEs. Endocrinopathies are usually mild (grade ≤2), whereas severe or life-threatening (grade ≥3) are very rare.
Endocrinopathies generally do not require permanent discontinuation of ICIs (even in grade ≥3 CTCAE) and rarely require high-dose corticosteroids, although lifelong management may be required when persistent [34][35][36]. The most frequent endocrinopathies include thyroiditis and hypophysitis and the symptoms are rather non-specific. The centers using immunotherapy should have access to appropriate hormonal diagnostics and cooperate with endocrinologists.
ICI-induced hypophysitis (IH) has been described as the most frequent endocrine irAEs associated with anti-CTLA-4 administration with the incidence up to 17% [1], although in recent meta-analyzes it is estimated at 3. Hypogonadism can be corrected if the gonadal axis did not recover after 3 months and no contraindications are present [40].
Thyrotoxicosis may be asymptomatic or cause mild symptoms of hyperthyroidism. Extremely rarely, thyroiditis is the cause of life-threatening disorders (0.1%) [38]. The phase of hyperthyroidism resolves spontaneously within 4-6 weeks or in majority (80%) progresses to permanent hypothyroidism. The anti-thyroid antibodies (anti-TPO, anti-Tg) can be detected in some patients. ICI-induced Graves' disease is very rare [38]. The destructive thyroiditis and Graves' disease may also coexist in some patients.
The acute phase of thyroiditis with asymptomatic and transient thyrotoxicosis does not require treatment, only monitoring of TSH, fT4, fT3 at intervals of 2-3 weeks. In symptomatic patients (with tachycardia) beta-blockers are recommended . In severe hyperthyroidism, corticosteroids should be introduced (e.g., prednisone 1 mg / kg). Antithyroid drugs should be considered in case of Graves' hyperthyroidism. LT4 replacement for symptomatic hypothyroidism should be started with a dose of 25-50 μg in the morning and adjusted (after 4-6 weeks) to achieve normal TSH level. Immunotherapy may be continued in most cases of thyroid dysfunction. In our opinion TSH level should be also regularly monitored in patients treated for hypothyroidism before using ICIs, as TSH may increase or transient thyrotoxicosis may occur after starting immunotherapy.
Autoimmune diabetes mellitus (DM) is a rare complication (<1%) [44] [30]. The time to onset of hyperglycemia varies from the first weeks to 12 months after starting ICIs [45;46]. A characteristic feature is a very rapid increase in glycemia with complete lack of insulin secretion, confirmed by undetectable C-peptide levels at the time of diagnosis. Patients often present with signs and symptoms of hyperglycemia or ketoacidosis [44]. Glucose levels should be routinely checked during the course of immunotherapy. ICI-induced DM resembles the so-called fulminant DM, a form of type 1 DM first described in Japan [47].ICI-induced diabetes is a life-threatening condition, patients often require admission to intensive care units. ICI-related DM results in long-term need for insulin; restarting ICIs can be considered when adequate glucose control has been achieved.
Primary adrenal insufficiency is a very rare complication of immunotherapy [38;48]but can lead to life-threatening adrenal crisis. Laboratory tests show hyponatraemia with hyperkalaemia, and may include hypoglycaemia and hypercalcaemia. Elevated ACTH in the presence of low morning cortisol levels indicate primary adrenal insufficiency.

Skin toxicity
Skin toxicity is among the most prevalent irAEs reported with ICIs. It occurs in 30-40% of patients receiving PD-1 / PD-L1 inhibitors and in approximately 50% of patients treated with ipilimumab [2].
The mechanism of formation of dermatological irAEs is not fully understood. However, their close association with T-cell activation bound by checkpoint blockers is noteworthy. Dermal toxicity is the first to occur during treatment with ICIs and it appears to be independent of the dose of drug used [49]. In addition, some of the irAEs like maculopapular rash and vitiligo may be correlated with a better therapeutic response [50 -52]. The overall incidence of dermatological irAEs is higher with ipilimumab compared to anti-PD-1 or anti-PD-L1 agents and occur more frequently, appear earlier, last longer and are more severe when anti-CTLA-4 and anti-PD-1 antibodies are used in combination [49;53]. Most of irAEs are mild (grade 1-2) and their nature is very similar. The most common is nonspecific maculopapular rash and pruritus. A pruritic maculopapular rash is the most common irAEs ( Fig.1A-C) ( with incidence 24.3% for ipilimumab, 16.7% pembrolizumab and 14.3% for nivolumab [49;54;55]). Rash occurs mainly on the trunk, less frequently on the upper limbs, next it spreads peripherally to the extremities. Most often it appears after a couple of treatment cycles, and the severity of the changes may increase during subsequent cycles. The average time to onset of maculopapular rash is 3-4 weeks from start of anti-CTLA-4, 5 weeks-anti-PD-1, and 2 weekscombination of ipilimumab and nivolumab [49]. Changes may also appear many months after the introduce of treatment [53]. In assessing the severity of lesions (CTCAE), the extent of the lesions and the negative impact on health-related quality of life is taking into account. Grade 1 includes lesions occupying less than 10% of the skin's surface, grade 2 -10-30% and grade 3 more than 30% of the surface. Grade 4/5 is rare. In patients treated for advanced melanoma, changes ≥grade 3 were observed in ≤2% in monotherapy and 3-5% in combination [2 ;49]. The rash may also be the first clinical manifestation of a severe cutaneous drug reaction. During ICIs therapies, the occurrence of Stevens-Johnson syndrome, erythema multiforme, toxic epidermal necrolysis acute generalized exanthematous pustulosis or drug reaction with eosinophilia and systemic symptoms (DRESS) has been reported [49;56]. Itching is a common and troublesome symptom. It typically develops concomitantly with maculopapular rash, albeit it can also be associated with a normal-appearing skin.
The frequency in patients during treatment for advanced melanoma ranges between 14-47% and is the lowest in the case of anti-PD-1 therapies and the highest in the combination anti-CTLA-4 / anti-PD-1.
Pruritus with severity ≥grade 3 occurred in ≤2% of treated patients [2; 49;53]. Vitilligo-induced ICIs occurs during melanoma therapy (7.5-11%) ( Fig.1.D). The mechanism of its formation is probably related to a cross-reaction against antigens shared by melanoma cells and normal melanocytes [49]. PD-1 / PD-L1 blockers can activate pre-existing immune disorders or/also induce the development of de novo autoimmune skin diseases. The occurrence of bullous pemphigoid Also, the pre-existing autoimmune diseases seem not to predict complications [62;63]. The analysis of observations shows that lung fibrosis does not exclude immunotherapy [60]however the course of pulmonary complications may be accomplished by comorbidities.
Many patients suffer from chronic obstructive lung disease (COPD) and interstitial lung diseases. The recognition of CIP is complicated by the similarity of symptoms, on the other hand, the course is unfavourable, especially in the elderly [58-60;64]. Among pulmonary complications, tuberculosis and opportunistic infections need to be considered. Thus, the knowledge of patient's history is essential for a differential diagnosis. Furthermore, the signs in patient examination are also unspecific and these are mainly crackles in auscultation. The analysis of blood gases and pulmonary function tests with the diffusion capacity is necessary to assess the severity of restriction or hyperinflation as well as gas exchange disturbances The key method of CIP recognition is chest imaging with high resolution computed tomography (HRCT) as superior. Different patterns in HRCT could be visible and the most frequent ones are: ground glass opacities, consolidations, reticular opacities and micronodules [60;64]. The overlap of changes is frequently observed [58]. The interstitial inflammation process is dynamic and the HRCT pattern may show more or less active or more or less irreversible (fibrotic) changes. There are some attempts to incorporate the classification of interstitial lung diseases (ILD) to the CIP classification and the nonspecific interstitial pneumonia (NSIP) pattern seems to be leading followed by organising pneumonia (OP) -like a pattern [60;64]. Bilateral distribution and localisation away from lung tumor are often observed [58]. Pleural effusion and mediastinal lymph nodes involvement are rare.
The issue of CIP is non-infectious parenchymal inflammation and the COP, AIP or NSIP pattern was confirmed by histological examination, however the data are scanty [59].
Sarcoid granulomatosis is a special kind of possible toxicity, however rare, it may involve extrapulmonary organs though [60]. The differential diagnosis of new lung infiltrations in ICIs treated patients include tumor progression or pseudoprogression, pneumonia or pneumonitis. Thus, the full microbiological tests and histopathological examination of sputum/material from bronchoscopy is often needed and decisive. The course of CIP is often rapid and the decision of therapy is urgent, hence the application of bronchoalveolar lavage (BAL) fluid examination might be suggested [60;65] (evidently, acute pneumonitis may be a contraindication to bronchoscopy with BAL). BAL fluid analysis allows the recognition of infection (also opportunistic), the presence of malignant cells and, by total and differential immune cell count, the character of interstitial lung disorder [66]. The predominance of lymphocytes is suggestive for active non-infectious inflammation [65] . According to our experience the considerable BALf evaluation could be ensured by microscopic examination of slides stained with haematological and histological methods, combined with flow cytometry analysis for immune cell subtyping [66;67] .
The treatment and management of CIP include the modification of ICIs therapy with or not immunosuppression and depends on the clinical course [68]. The resume of current recommendations is shown in table 2 [1;2;6]. In general, in the stages 1 and 2 improvement is possible. However, a relapse could incidence and if corticosteroids are ineffective, another immunosuppressant is indicated: mycophenolate mofetil, infliximab, cyclophosphamide . The prognosis is worse in patients with symptomatic pneumonitis, coexisting other lung diseases, combined therapies, previous chemo-radiotherapy [69]. Interestingly the better efficacy of ICIs was reported in patients with associated irAEs [70;71].

Immune checkpoint inhibitorsassociated cardiotoxicity.
In real practice true incidence of ICIs cardiotoxicity is unknown and it seems that the problem is underestimated. This is particularly important as not only the range of ICIs sensitive cancers but also treatment strategies that include ICIs are increasing rapidly. Clinical observations show that immune involvement of the cardiovascular system, particularly of the heart, lead to the highest case fatality rate among the IrAEs [72].

The ICIs block inhibitory molecules expressed on T lymphocytes what cause
activation of systemic T-cell response as well as those in the cardiovascular system, especially myocardium. In the experimental autoimmune myocarditis T lymphocytes contribute not only to the induction but also myocardial damage. In experimental studies eg. CTLA-4/PD-1 deficient animals displayed increased inflammation and myocardial damage [73;74]. PD-L1 is also expressed on cardiomyocytes. It is postulated that PD-L1 exerts dual cardio-protective function by transmit direct cardioprotective signaling. This seems particularly important in patients with pre-existing cardiovascular dysfunction as the cardiac stress / ischemia / hypertrophy upregulated PD-L1 expression [75]. Thus, in cardiovascular system, ICIs can results not only in autoimmune T-cell related injury, but also PD-L1 inhibition which might accelerate pre-existing heart disease.

The cardiac antigens being tumor related factors stimulate tumor/cardiac T-cell clones
and are capable of increase ICIs toxicity. The concurrent ICIs related toxic effects, e.g. myositis is common in patients with myocarditis and may reflect a shared antigen profile between cardiac and skeletal muscle. Pre-existing cardiovascular diseases as well as autoimmune diseases are potential risk factor for irAEs.
The risk factors of cardiotoxicity are not the same among patients and due to diverse presentation the strategy for personalizing surveillance should be incorporated based on initial assessment [76;77].
The incidence of myocarditis is higher during ICIs combination therapy, and usually occurs early after exposure. Of concern, around 50% cases are fatal. Myocarditis may occur with acute manifestation as acute heart failure with cardiogenic shock, multiorgan failure, pulmonary oedema (mimicking pneumonitis), new event of left ventricle impairment, malignant ventricular arrhythmia or advanced conduction disease [78]. ICIs associated cardiotoxic effects can extend beyond myocarditis; however the first approach is to exclude myocarditis; if confirmed it is crucial to urgently follow myocarditis management protocol.
Cardiotoxicity is also manifested as arrhythmias included atrial fibrillation, atrioventricular conduction diseases, pericarditis with or without pericardial effusion [76;79]. should be selected depending on the kind of ICI related toxicity and its clinical manifestation [79]. (Figure 2, Table 3).
The first and most urgent step is to consider ICI discontinuation. The final decision should be taken jointly by oncologist and cardio-oncologist. The second approach is the implementation of typical conventional cardiac treatment to alleviate complications. In the most severe cases, intensive monitoring and adequate invasive management is recommended.
The third approach is the introduction of immunosuppression. In the most serious events eg. myocarditis, severe heart failure, effusion pericarditis, advanced conduction disturbance, serious arrhythmia with confirmed (or highly probable relationship) ICIs relation, highintensity immunosuppression followed by oral corticosteroids is recommended.
Finally the restart of ICI after the interruption is very difficult to make. It is crucial to define the clinical certainty that event is ICIs-related cardiac complication. The restart in myocarditis is not recommended because of the potential risk for fatal relapse. In cases of less severe cardiotoxic ICIs related complications (eg. subclinical myocardial dysfunction or pericarditis) it may be possible to restart immunotherapy with close surveillance for recurrence. The close cooperation between oncologist and cardiologist is crucial for appropriate decision [76][77][78][79].

Other rare immune-related toxicities
Rheumatologic (musculoskeletal), renal, neurologic, ophthalmologic and hematologic belong to relatively less frequent IrAEs. The most of these irAEs are rather mild in severity, but occasionally may be life-threatening. Nephrotoxicity is considered as rare adverse event. In large meta-analysis on 3695 patients ICIs-related acute kidney injury was estimated to occur in approximately 2.2% of patients [82], higher in other studies [83] or isolated cases of interstitial nephritis [2], granulomatous nephritis, thrombotic microangiopathy or lupus nephritis [82][83][84]. Renal involvement is usually asymptomatic. Serum sodium, potassium, creatinine and urea should be measured before every infusion of ICIs. When renal dysfunction is suspected it is necessary initially to stop nephrotoxic drugs, to perform differential diagnostics to rule out other reasons for renal insufficiency. ICIs therapy should be withheld the administration of systemic corticosteroids considered. Close monitoring of serum creatinine should accompany the treatment.
Nephrotoxicity typically resolves, and ICIs treatment can resume if grade 2-3 adverse events resolve promptly, but therapy should be permanently discontinued in case of persistent or recurrent grade 2-3 adverse events or grade 4 toxicity with a help of nephrologist [2].
Incidence of neurotoxicity related to ICI is low but probably underreported, on the other hand its incidence is related to the terminology and reporting common symptoms as headache or dysgeusia [1; 85;86]. The time for onset of neurologic irAEs is usually early within first two weeks, nevertheless there were some reports of neuro-related toxic events late after stopping ICIs. The most common are encephalitis and myopathies, however there is a diversity of reported events (Table 4 Table 1 The rules of treatment of gastrointestinal adverse events of immunotherapy depending on the severity of symptoms according to National Common Terminology Criteria for Adverse Events (CTAE) Table 2 Management and treatment of pulmonary adverse events of immunotherapy with check point inhibitors according to severity. Table 3 Immune checkpoint inhibitorsproposed management of the associated cardiotoxicity (modified and based on [76]) Table 4 The most common symptoms associated with immune related adverse events of rheumatic, kidney, neurologic, eye or hematologic origin /according to [7] Reference List  The rules of treatment of gastrointestinal adverse events of immunotherapy depending on the severity of symptoms according to National Common Terminology Criteria for Adverse Events (CTAE)

Immune related colitis (IrC)
Grade 1 (G1) increase of <4 stools per day over baseline -continue treatment -diet, antidiarrheals Grade 2 (G2) Increase 4-6 stools per day over baseline, abdominal pain, blood in stool, general symptomsstop immunotherapybudesonid 9mg/day 8 weeks, if symptoms aggravte-prednisone 0.5-1mg/kg 7 days Grade 3/4 (G3/G4) Increase of ≥7 stools per day over baseline, severe or persistent abdominal pain, fever, peritoneal signs, blood in stool, deficiencies in blood tests, severe lesions in colonstop treatmenti.v. metylpredniosolne 1-2mg/kg 5 days followed by oral steroids or IFX 5mg/kg one dose    Table 4 The most common symptoms associated with irAE of rheumatic, kidney, neurologic, eye or hematologic origin [according to Champiat S. et Figure 1 Clinical images of skin toxicity in a patients treated immune checkpoint inhibitors. A-C.
Author confirms that patients have agreed to use their skin lesion images in a scientific publication.

Figure 2
Management in the case of myocardial infarction related to immune check inhibitors (ICIs).