Pancytopenia And Limbic Encephalopathy Complicating Immunotherapy For Clear Cell Endometrial Cancer With Microsatellite Instability-High (MSI-H).

BACKGROUND: Clear cell carcinoma of the endometrium (CCE) has a tendency to occur in a mismatch repair protein deficient molecular background. Treatment with immunotherapy can predict a favorable response. CASE
PRESENTATION: We are presenting a 53-year-old female, diagnosed with CCE 17 years ago, who was treated initially with hysterectomy and left salpingo-oophorectomy, who relapsed a few months later, and was then treated with left pelvic mass excision and sigmoidectomy. Recently, the disease recurred as a retroperitoneal lymphadenopathy, which was resected but then relapsed locally, spread to the lungs, and progressed further after three lines of chemotherapy. On pathological review of the tumor, it was found to harbor loss of nuclear expression of MLH-1 and PMS-2. Based on a strong predictor of response to immunotherapy, pembrolizumab was tried. However, within a few days of the single dose of pembrolizumab, immune thrombocytopenia followed by pancytopenia, recurrent seizures, visual hallucination, and cerebellar signs consistent with limbic encephalitis developed, which were not responding to steroid and intravenous immunoglobulin.
CONCLUSION: We are presenting a case of a CCE with deficient mismatch repair that developed two autoimmune side effects, pancytopenia and limbic encephalitis, within a few days of a single injection of pembrolizumab.

Introduction

A frequent mismatch repair protein deficiency can be seen in mixed endometrial and clear cell carcinoma of the endometrium (CCE).1 Mismatch-repair status can predict clinical benefit from immune checkpoint blockade.2

Different immune checkpoint inhibitors had been investigated in advanced endometrial cancer including PD-1 inhibitors as pembrolizumab and PDL-1 inhibitors as atezolizumab and avelumab.3

Immune-related adverse events complicating immunotherapy can mimic autoimmune conditions, affecting the thyroid, lung, colon and liver.4 With the broad use of anti-PD1 in clinical practice, rarer side effects are emerging.

To date, hematological immune-related adverse events remain occasionally described;5 for instance, bi-cytopenia (severe anemia and thrombocytopenia) possibly induced after the sixth cycle of injection of Nivolumab (anti-PD-1 antibody), given to a patient with primary malignant melanoma of the esophagus with inefficiency of high-dose intravenous methylprednisolone,6 immune-mediated thrombocytopenia,7 immune-mediated agranulocytosis,8 immunotherapy-associated hemolytic anemia with pure red-cell aplasia,9 immune medicated pancytopenia,10 and even central immune cytopenia.11

Limbic encephalopathy due to checkpoint inhibitor has also been reported,12–18 and as with encephalitis from other causes, the most frequent signs and symptoms are fever, headache, confusion, memory impairment, gait ataxia, seizures, and hallucinations. The onset was typically acute to sub-acute over days to a few weeks.19

Case Report

A 53-year-old female patient, known to have diabetes mellitus, and hypothyroidism, and no family history of cancer, was diagnosed in 1999, with endometrial cancer and was treated with hysterectomy and left salpingo-oophorectomy, relapsed few months later, as left pelvic mass, excised with sigmoidectomy, without adjuvant chemotherapy.

She was well until May 2016, when she presented with few months’ history of abdominal pain and rising CA 125. MRI and PET CT scan showed retroperitoneal mass that invaded inferior vena cava with no distant metastasis (Figure 1A).

Figure 1

(A) Initial PET scan showing retroperitoneal mass invading inferior vena cava. (B) PET scan showing retroperitoneal mass progression with right hydronephrosis and lung metastasis post 3 lines of chemotherapy.

The mass was excised together with inferior vena cava angioplasty and the pathology showed lymph node metastasis with poorly differentiated carcinoma, forming cribriform/papillary growth pattern (Figure 2: image 1) and focal clear cell changes (Figure 2: image 2) in favor of endometrial primary. The excisional margin was positive. The tumor board decided either adjuvant chemotherapy or radiotherapy, which was declined by the patient.

Figure 2

H&E of the excised retroperitoneal lymph node showing poorly differentiated carcinoma, forming cribriform/papillary growth pattern [image 1] and focal clear cell changes [image 2]. Complete loss of nuclear expression of MLH-1 [image 3] and PMS-2 [image 4]. Intact expression of MSH-6 [image 5] and MSH-2 [image 6]. Low power section demonstrates invasive malignant tumor-infiltrating tissue by a solid sheet of tumor cells with obvious voluminous clear cytoplasm (hematoxylin and eosin stain, 4×, [image 7]. High power section demonstrates malignant tumor composed of large voluminous clear cytoplasm, distinct margins, enlarged angulated pleomorphic hyperchromatic bizarre nuclei with prominent nucleoli (hematoxylin and eosin stain, 40×, [image 8].

In September 2016, the tumor relapsed in the retroperitoneal lymph node between L3-4 and in the lungs. Since then until April 2017, the patient received three lines of chemotherapy: Carboplatin/Paclitaxel/Bevacizumab, Topotecan and then Liposomal Adriamycin that were poorly tolerated. The disease progressed further locally causing mass effect on the right ureter and spread to the lungs (Figure 1B).

In July 2017, immunohistochemical stains for DNA Mismatch Repair proteins of the resected retroperitoneal lymph node, demonstrated significant complete loss of nuclear expression of MLH-1 (Figure 2: image 3) and PMS-2 (Figure 2: image 4), with intact expression of MSH-6 (Figure 2: image 5) and MSH-2 (Figure 2: image 6).

The retroperitoneal mass and paracaval lymph node were sent to pathology for evaluation. The retroperitoneal mass consists of an irregular, lobulated firm mass surrounded by fibro-adipose tissue. Sections submitted revealed an invasive high-grade malignant tumor with mixed cribriform/papillary growth and infiltrative clear cell solid growth. The area of the cribriform/papillary growth pattern (Figure 2 images 1,2). Complete loss of nuclear expression of MLH-1 (Figure 2 image 3) and PMS-2 (Figure 2 image 4). Intact expression of MSH-6 (Figure 2 image 5) and MSH-2 (Figure 2 image 6). The area of the clear cell tumor demonstrated obvious voluminous clear cytoplasm (Figure 2 image 7). High power section demonstrates malignant tumor composed of large voluminous clear cytoplasm, distinct margins, enlarged angulated pleomorphic hyperchromatic bizarre nuclei with prominent nucleoli (Figure 2 Image 8).

Based on a strong predictor of response to treatment, i.e. MSI High status, combined with the overall favorable toxicity profile of Pembrolizumab,20 she was given Pembrolizumab on the 2nd of September 2017.

At this time, the patient ECOG status was 2, with neurological examinations. Biochemistry results found WBC at 4.7 ×109/L, Hb at 8.6 g/dL, platelets at 201 ×109/L, and MPV at 6.5 fl. Two days later, after receiving two units packed red blood cells, the WBC were at 5.6 ×109/L, Hb at 11.7 g/dL, but the platelets dropped to 72 ×109/L. Six days later, the patient developed seizure and upon regaining consciousness, she started to have visual hallucination, with frank cerebellar signs (horizontal nystagmus, dysmetria, with ataxic gait), WBC at 3.5 ×109/L, Hb 11.6 g/dL, platelets 21 ×109/L, HIT test was negative and fibrinogen level was normal.

Recurrent seizures with confusion were recurred, MRI head, on 11th of September 2017, did not show bleeding, brain metastasis or features of encephalopathy.

The patient’s performance status dropped further to become bed bound. The consulted neurologists suggested clinical recurrent confusion, lack of comprehension, and on and off lip smacking were in favor of non-convulsive status epilepticus; however, the existence of cerebellar symptoms, visual hallucination, episodes of on and off unresponsiveness was in favor of limbic encephalitis which were suggestive of paraneoplastic syndrome versus immunotherapy side effect. Therefore, an EEG was requested (Figure 3)

Figure 3

EEG background presented with diffuse delta slowing, but without hemispheric asymmetry nor epileptiform discharges. The impression picture was in favor of severe encephalopathy.

On the 14th of Sept 2017, WBC 2.9 ×109/L, Hb 7.6 g/dL, platelets reached 9 ×109/L and MPV were 12.2 fl; a picture in favor of peripheral consumption. Direct Coombs test was positive, and the peripheral smear showed pancytopenia.

Bone marrow was assessed (Figure 4 A, B & C), and the patient was kept on Methylprednisolone 1 mg/kg for seven days, was planned to be tapered by 10 mg weekly.

Figure 4

(A) Bone marrow aspirate (100×) showing enhanced megakaryopoiesis and anisocytosis with some small-hypolobated megakaryocytes and few forms showing hyperlobulation with widely separated nuclear lobes (arrow). (B) vWF immunostain highlights active megakaryopoiesis. (C) Bone marrow biopsy (H&E, 10×): Normocellular for age with trilineage hematopoiesis and active megakaryopoiesis. No evidence of BM infiltration by carcinoma cells.

IVIG was given as per neurologist request for five days to treat limbic encephalopathy. During the following days the WBC reached 1 × 109/L, platelets 10 × 109/L and Hb 7 g/dL (Figure 5). She developed septic shock, then passed away on the 24th of September 2017.

Figure 5

Blood counts from 01.08.2017 to 24.09.2017 showing the WBC, Hemoglobin and the Platelet curves during the period of follow-up.

Genetic Counseling And Testing

Due to the patient’s loss of MLH1 and PMS2 on immunohistochemistry, the patient was referred to the genetic counseling clinic to be evaluated for Lynch syndrome. Unfortunately, due to the patient’s poor health condition at that time, patient’s guardian did not agree to perform germline genetic testing and neither agreed for DNA banking.

Later, one of the patient’s sisters whose unaffected 51 years old (Figure 6. III-3) presented to the genetics clinic with an interest to be evaluated due to her sister’s history of endometrial cancer. Due to the patient’s III-4 MMR deficient young onset diagnosis of endometrial cancer, her sister was offered germline genetic testing for Lynch syndrome genes including MLH1, MSH2, MSH6, PMS2, and EPCAM. III-3 results revealed that she carries a heterozygous pathogenic mutation c.2805dupT (p.Asp936Ter) in the MSH6 gene.

Figure 6

Patient’s three-generation pedigree.

This duplication creates a nonsense variant, which changes an Aspartic Acid to a premature stop codon.

This variant is predicted to cause loss of normal protein function through either protein truncation or nonsense-mediated mRNA decay. This identified MSH6 mutation might explain the MMR deficient endometrial cancer in patient III-4 and there is a high probability that she could have been also a carrier for MSH6 mutation especially that MSH6 germline mutations has a strong correlation with endometrial cancer than other Lynch syndrome genes. Patient’s sister was later offered risk-reducing strategies including prophylactic total hysterectomy and bilateral salpingo-oophorectomy and colonoscopies.

Discussion

Checkpoint inhibitors (Anti PD-1 drugs) are approved in a variety of cancers, including solid tumors with a microsatellite instability phenotype.21 The side effects of anti-PD-1 are similar to autoimmune conditions.4

The hematological immune-related side effects were reported in few cases, such as bicytopenia,6 two cases of immune thrombocytopenias within two weeks and after 42 days of immunotherapy. Platelets recovered by given Prednisolon, Immunglobulin and Rituximab.7 Agranulocytosis inducing Staphylococcus infection in patient treated with Nivolumab, that only responded to high dose of Corticosteroid therapy.8 Immunotherapy-associated hemolytic anemia with pure red-cell aplasia occurred after the third dose of Pembrolizumab, with high lactic dehydrogenase, positive coomb’s test, but low retics count, while the bone marrow findings were consistent with pure red-cell aplasia.9 A summary of selected literatures about immune checkpoint inhibitors associated hematological adverse effects is shown in Table 1.

Table 1

Summary Of Selected Literatures About Immune Check Point Inhibitors (Icpis) Associated Hematological Adverse Effects

Reference	Therapeutic Agent	Diagnosis	Number Of Cases	Hematological Adverse Effect	Occurred Post How Many Cycles/Days After ICPIs	Intervention Or Management Of Hematological Adverse Effect	Outcome Of Management Of Hematological Adverse Effect
Inadomi et al6	Nivolumab	Melanoma	1	Severe anemia and thrombocytopenia	6th cycle	Blood transfusion and high-dose IV steroids	Ineffective
A. Le Roy et al7	Pembrolizumab	Melanoma	2Case ACase B	Immune thrombocytopenia	A: 1st cycleB: NA	A: steroids and intravenousinfusions of immunoglobulins (IVIG).B: A course of steroid	A: EffectiveB: Effective
T. Samer et al8	Nivolumab	NSCLC	1	Severe agranulocytosis	2nd cycle	IVIG plus oral and IV steroids	Effective
Nair R, et al9	Pembrolizumab	Melanoma	1	Warm-antibody autoimmune hemolytic anemia and pure red-cell aplasia	3rd cycle	High-dose steroids	Pure red-cell aplasia flared when prednisone tapered to 20 mg.Subsequent treatment with one dose of IVIG enabled tapering of the glucocorticoids
Atwal, Dinesh, et al10	Pembrolizumab	Melanoma	1	Pancytopenia	18th cycle	High-dose prednisolone and a 5-day course of IVIG	Resolved after IVIG course
J.-M. Michot et al11	Nivolumab	Stage IV adenocarcinoma of the lung	3Case ACase BCase C	Bone marrow failure as an immune-related aplastic anemia	NA	A: IGIV, Antibiotics,4 RBC units + 3 platelets unitsB: Steroids, GCSF, 4 RBC and9 platelets unitsC: Steroids, IGIV, GCSF,antibiotics, 20 RBCand 15 platelets units	A: ineffectiveB: Partial and transientresponse to steroids;persistent pancytopeniastill ongoing at 4 monthsC: ineffective.

Abbreviations: ICPIs, immune checkpoint inhibitors; IVIG, intravenous immunoglobulin; GCSF, granulocyte colony-stimulating factor; RBC, red blood cells; NA, not available; ITP, Idiopathic thrombocytopenic purpura.

Pure encephalitis occurred a median of 2.5 months following initiation of checkpoint inhibitor with a median of 3.5 doses administered. Focal abnormalities were reported on MRI of the brain in 11 of 15 patients (73%) with immune checkpoint blockade-mediated encephalitis. Electroencephalograms showed generalized slowing in four of five encephalitis patients examined (80%). Subclinical epileptiform activity was also identified in one patient.19 A summary of selected literatures about immune checkpoint inhibitors associated encephalopathy is shown in Table 2.

Table 2

Summary Of Selected Literatures About Immune Check Point Inhibitors (Icpis) Associated Encephalopathy

Reference	Therapeutic Agent	Diagnosis	Number Of Cases	Immune Related Adverse Effect	Occurred Post How Many Cycles/Days After ICPIs	Intervention Or Management Of Hematological Adverse Effect	Outcome Of Management Of Hematological Adverse Effect
Salam S, et al12	Pembrolizumab	Melanoma	1	Antibody-negative limbic encephalitis	After 12 months from c1	Course of steroids (iv and oral)	Ineffective
M.P. Brown et al13	Pembrolizumab	Melanoma	1	Autoimmune limbic encephalitis	Between cycles 7 to 10	Course of steroids (iv and oral)	Effective
S. Feng et al14	Pembrolizumab	NSCLC	1	Diffuse encephalopathy	2nd Cycle	Course of steroids (iv and oral)	Effective
M. Niki et al15	Pembrolizumab	NSCLC	1	Autoimmune limbic encephalitis	After 8 months from c1	Course of oral steroids	Effective
T. J. Williams et al16	Nivolumab and ipilimumab	Melanoma	2Case ACase B	Autoimmune Encephalitis	After cycle 1	A: IVIG, IV steroids and IV RituximabB: Oral steroids	A: EffectiveB: ineffective
S. Shah et al17	Nivolumab	NSCLC	2Case ACase B	Autoimmune Encephalitis	A: After 4 months from cycle 1B: After cycle 5	A: IVIG, IV steroids and IV Rituximab and tetrabenazineB: IVIG, IV steroids and IV Rituximab	A: IneffectiveB: Effective
S. Schneider et al18	Nivolumab	NSCLC	1	Autoimmune limbic encephalitis	After cycle 14	Course of oral steroids	Effective

Abbreviations: ICPIs, immune check point inhibitors, IVIG, intravenous immunoglobulin.

Our patient developed the above-mentioned neurological manifestations, with generalized slowing with delta brush on EEG and non-diagnostic MRI head associated with autoimmune pancytopenia within few days of Pembrolizumab administration, suggestive of autoimmune etiology.

Conclusion

We are presenting a case of a CCE with deficient mismatch repair that developed two autoimmune side effects, pancytopenia and limbic encephalitis, within few days of single injection of Pembrolizumab & to the best of our knowledge, this is the first report in the middle east of these two distinct immune-related side effects occurred for the same patient. We hope that reporting such rare side effects more and more will help in raising the awareness of the oncology community about them.