Acute graft-versus-host disease (aGvHD), together with infections, constitute the two main early complications of allogeneic hematopoietic cell transplantation (alloHCT).¹ The mainstay of first-line therapy in aGvHD is systemic administration of high-dose glucocorticoids, but only 40%–60% of patients respond to this treatment depending on the grade of severity of the disease.¹ At present, there is no established standard-of-care second-line therapy. The high mortality rate of steroid-refractory/dependent (sr/d) aGvHD, especially in patients with grade III-IV lower gastrointestinal (GI) tract involvement,² is a major drive for exploring novel therapeutic strategies.

One of them, ruxolitinib, a selective Janus kinase (JAK1 and JAK2) inhibitor, is associated with significant general improvement, and improved remission rates in patients with sr/d aGvHD.³

A second, emerging therapy is fecal microbiota transplantation (FMT) (being the most effective method of gut microbiota replacement), which we recently assessed in a prospective, multicenter study regarding antibiotic-resistant bacteria decolonization in which sr/d aGvHD patients reached 57% of overall response rate.⁴

Taken together, considering the possibly complementary mechanisms of ruxolitinib and FMT, we progressed on from positive initial clinical observations to offer combination therapy of ruxolitinib and FMT to our patients with sr/d GI-aGvHD. To our knowledge, this is the first report on such combination therapy.

CASE 1: A 66-year-old male, a recipient of alloHCT from a matched unrelated donor (MUD; see Table 1 for details), attended our clinic because of fatigue, dehydration, cachexia, and exacerbation of severe gastrointestinal symptoms. Despite thorough investigation, we could not identify other reasons for the patient's symptoms (i.e. > 2 L/day of loose stools with mucus and blood occasionally, with ileus and peritoneal signs). On the day +45 after alloHCT, the patient was diagnosed with possible (colonoscopy with biopsy) GI aGvHD stage IV MAGIC, IV Glucksberg, IBMTR D,⁵ and treatment was initiated with high-dose methylprednisolone (2 mg/kg). No clinical response was seen and mycophenolate mofetil was introduced with steroid tapering, but the patient's condition progressively worsened. We decided to treat the patient with ruxolitinib and FMT, but pancytopenia was the reason for postponing ruxolitinib. Because of GI tract colonization with antibiotic-resistant bacteria (ARB; Pseudomonas aeruginosa MBL), our patient qualified for intervention with an FMT procedure (as a part of a prospective clinical study assessing ARB decolonization rate by the use of FMT [NCT02461199]). The first FMT dose was associated with improvement of the patient's condition and diarrhea alleviation (Figure S1). The patient subsequently received a second and third FMT dose. We saw marked clinical improvement, including complete resolution of diarrhea and abdominal pain. Unfortunately, due to the patient noncompliance, diarrhea, ileus, and peritoneal signs recurred. As the last, fourth treatment line, we decided to add ruxolitinib. Following this, the patient's status stabilized, and he obtained clinical partial remission, although he still had diarrhea up to five stools a day (formally active disease, according to EBMT criteria⁵). He was lost to follow-up and died on day +264.

CASE 2: A 52-year-old female, post-alloHCT from matched related donor (MRD), began to have diarrhea and extensive skin rash on day +17. She was finally diagnosed as overall stage IV MAGIC, Glucksberg IV, IBMTR D⁵ aGvHD (skin IV biopsy proven and gut IV biopsy proven). She was given methylprednisolone (2 mg/kg), budesonide, parenteral nutrition, and—due to steroid resistance—mycophenolate mofetil; temporary improvement occurred. Because of the deterioration of diarrhea, ruxolitinib was added (initially 2 × 5 mg, then 2 × 7.5 mg per day). Because of ARB colonization, the patient received FMT twice. After FMT, gastrointestinal symptoms resolved. The obtained response was consolidated with another FMT and intravenous methotrexate (15 mg). Ruxolitinib was continued. After all treatments were administered, the patient's diarrhea resolved, and the patient entered complete remission of gut aGvHD. Furthermore, her overall performance status significantly improved, and she was discharged. During follow-up, there was no aGvHD exacerbation and no chronic GvHD occurred, but the patient ultimately died due to relapse on day +398 after alloHCT.

CASE 3: A 55-year-old female, post-alloHCT from MRD, started to have diarrhea with increasing intensity from day +11. Biopsies taken during colonoscopy revealed features of severe aGvHD. We diagnosed aGvHD in overall stage IV according to MAGIC criteria, Glucksberg IV and D according to IBMTR⁵ (skin II probable and gut IV proven). High-dose methylprednisolone (2 mg/kg) was introduced to the patient's treatment, after which all symptoms from the gastrointestinal tract alleviated. During the weaning of the steroid dose, the diarrhea gained in its intensity again (> 2 L of stool daily); additionally, the patient started to have severe, spastic abdominal pain with peritoneal signs. Again, high-dose steroids were administered, but after 10 days of therapy, there was no response (NR). Given this, we introduced second-line treatment with ruxolitinib; this was associated with a subsequent slow and gradual improvement in diarrhea severity, but without any progress in alleviation of abdominal pain. Even though the patient was treated for more than 14 days with ruxolitinib, we did not see any further improvement in the patient's symptoms. Suboptimal clinical improvement to the administered treatment (steroids and ruxolitinib) prompted us to combine ruxolitinib therapy with FMT, as we did in previous cases. After obtaining the Institutional Review Board (IRB) acceptance [KB/203/2020] for three FMTs every week, the first and second administration of FMT was performed. Following this, we observed rapid and significant improvements. A third FMT administration was performed, with further normalization of the patient's condition and stabilization of the frequency of patient's defecation to once a day. Finally, the CR of aGVHD was diagnosed. She is now in +351 day after alloHCT with aGvHD CR, without significant morbidity or further complications.

CASE 4: A 22-year-old female, post-alloHCT from MUD, started to have diarrhea on day +31, after which we suspected gut GvHD. Colonoscopy with histopathology showed GI-aGvHD and MAGIC grade IV overall, IV Glucksberg, and IBMTR D⁵ was diagnosed. Therapy with methylprednisolone (2 mg/kg) and budesonide was initiated. After 10 days of steroids, the patient was assessed as having partial clinical response, but formally NR according to MAGIC and active disease according to EBMT.⁵ In light of the unsatisfactory result of therapy with high-dose steroids, ruxolitinib treatment (2 × 5 mg) was started, and IRB application for FMT was made (with the same protocol as above [KB/202/2020]). Finally, the first FMT was performed and from the next day, the volume of stool gradually decreased. The abdominal pain almost completely resolved. Then, second and third FMTs were performed. We observed general improvement of the patient's condition. Despite this, she continued to have up to five bowel movements a day, less profuse, more concentrated but still diarrheal stool with episodic abdominal pain. Therefore, a further three doses of FMT were planned, and after additional IRB approval [KB/202/2020], she obtained the fourth, fifth, and sixth FMT; after this, she had one stool a day, formed, without any abdominal pain. CR was assessed, and she is now in perfect condition, on day +326 without any morbidity, cGvHD and aGvHD recurrence, with ruxolitinib stopped.

The main comment about the first case is that FMT and ruxolitinib were administered very late during the post-alloHCT course—our patient did not report diarrhea in post-transplant care, although it did occur. We independently observed early and satisfying improvement, but unfortunately, it lasted for only a short period, and ended rapidly because of our patient's noncompliance. Similar to the situation in case 1, case 2's performance status was advanced and severely complicated. We decided to perform FMT first, before ruxolitinib administration, with the intention of ARB decolonization and simultaneously, to initiate immunomodulatory effects by the gut microbiota, without possible cytopenias as a treatment complication. Our patient's general condition significantly improved and ruxolitinib could be safely added. She reached durable CR of gut aGvHD.

Encouraged by previous observations, case 3 was treated with FMT and ruxolitinib intentionally early after the diagnosis of steroid resistance. We initially planned FMTs sessions (3×) in combination with ruxolitinib. This patient showed a particularly marked improvement, and reached complete remission of aGvHD just after FMT introduction, on ruxolitinib taken for several weeks. A possible explanation for such an outstanding response is the relatively short previous time-span from the diagnosis of steroid dependency, with treatment prior to the development of severe gut wall damage facilitating microbiota engraftment.

The same strategy as in case 3 was applied in case 4, when ruxolitinib and FMT was administrated even earlier than in case 3. Undoubtedly, after ruxolitinib administration and the first three FMTs, the patient's condition improved. Eventually, despite visible improvements, complete remission was not reached. In order to deepen the response, we decided to continue our treatment with the next three FMTs combined with a higher dose of ruxolitinib that resulted in CR.

After this initial experience, we see great potential in this strategy, and suggest that randomized controlled trials are needed to find the answer, and explore how deeply FMT enhances outcomes of patients similar to those in the trial published by Zeiser et al.³ We see a real chance that the additive effect of FMT will show significantly better results. Furthermore, modifications of protocols for combined treatment with FMT and ruxolitinib merit consideration. First clinical trial, besides of this report, was registered (NCT04269850).⁶

急性移植物抗宿主病 (aGvHD) 与感染一起构成同种异体造血细胞移植 (alloHCT) 的两个主要早期并发症。¹ aGvHD 一线治疗的主要支柱是全身给予大剂量糖皮质激素，但根据疾病严重程度的不同，只有 40%–60% 的患者对这种治疗有反应。¹目前，没有建立标准的二线治疗。类固醇难治性/依赖性 (sr/d) aGvHD 的高死亡率，特别是在 III-IV 级下消化道 (GI) 受累的患者中，²是探索新治疗策略的主要动力。

其中之一，鲁索替尼，一种选择性 Janus 激酶（JAK1 和 JAK2）抑制剂，与显着的全面改善相关，并提高了 sr/d aGvHD 患者的缓解率。³

第二种新兴疗法是粪便微生物群移植 (FMT)（是最有效的肠道微生物群替代方法），我们最近在一项关于抗生素抗性细菌去定植的前瞻性多中心研究中对此进行了评估，其中 sr/d aGvHD 患者达到 57%的总体响应率。⁴

总之，考虑到 ruxolitinib 和 FMT 的可能互补机制，我们从积极的初步临床观察进展到为我们的 sr/d GI-aGvHD 患者提供 ruxolitinib 和 FMT 的联合治疗。据我们所知，这是关于此类联合治疗的第一份报告。

案例 1：一名 66 岁男性，接受来自匹配无关供体（MUD；详见表 1）的 alloHCT，因疲劳、脱水、恶病质和严重胃肠道症状加重就诊。尽管进行了彻底的调查，但我们无法确定导致患者症状的其他原因（即 > 2 L/天的稀便，偶尔伴有粘液和血液，伴有肠梗阻和腹膜症状）。在 alloHCT 后第 +45 天，患者被诊断为可能（结肠镜检查和活检）GI aGvHD IV 期 MAGIC、IV Glucksberg、IBMTR D、⁵治疗以高剂量甲泼尼龙 (2 mg/kg) 开始。未见临床反应，并在类固醇逐渐减量的情况下引入吗替麦考酚酯，但患者的病情逐渐恶化。我们决定用鲁索替尼和 FMT 治疗患者，但全血细胞减少是推迟鲁索替尼的原因。由于耐抗生素细菌（ARB；铜绿假单胞菌）在胃肠道定植MBL)，我们的患者有资格接受 FMT 手术干预（作为通过使用 FMT 评估 ARB 去定植率的前瞻性临床研究的一部分 [NCT02461199]）。第一次 FMT 剂量与患者病情改善和腹泻缓解有关（图 S1）。患者随后接受了第二次和第三次 FMT 剂量。我们看到了显着的临床改善，包括腹泻和腹痛完全消失。不幸的是，由于患者不依从，腹泻、肠梗阻和腹膜体征再次出现。作为最后的第四条治疗线，我们决定添加鲁索替尼。此后，患者的状态稳定下来，获得了临床部分缓解，尽管他仍然每天腹泻多达 5 次（正式活动性疾病，根据 EBMT 标准⁵）。他失访并于第 +264 天死亡。

案例 2：一名 52 岁女性，来自匹配相关供体 (MRD) 的 alloHCT 后，在第 +17 天开始出现腹泻和大面积皮疹。她最终被诊断为整体IV期MAGIC，Glucksberg IV，IBMTR D ⁵aGvHD（经皮肤 IV 活检和经肠 IV 活检证实）。她接受了甲基强的松龙 (2 mg/kg)、布地奈德、肠外营养和 - 由于类固醇抵抗 - 吗替麦考酚酯；出现了暂时的改善。由于腹泻恶化，加用鲁索替尼（最初2×5mg，然后每天2×7.5mg）。由于 ARB 定植，患者接受了两次 FMT。FMT 后，胃肠道症状消失。获得的反应用另一种 FMT 和静脉注射甲氨蝶呤（15 毫克）巩固。继续使用鲁索替尼。在进行所有治疗后，患者的腹泻得到缓解，患者进入肠道 aGvHD 的完全缓解期。此外，她的整体表现状态显着改善，她出院了。在随访期间，

案例 3：一名 55 岁女性，来自 MRD 的 alloHCT 后，从第 +11 天开始出现腹泻，腹泻的强度越来越大。在结肠镜检查期间进行的活组织检查显示了严重 aGvHD 的特征。我们根据 MA​​GIC 标准、Glucksberg IV 和 D 根据 IBMTR ⁵在总体 IV 期诊断出 aGvHD（皮肤 II 可能和肠道 IV 证明）。大剂量甲基强的松龙（2 mg/kg）被引入患者的治疗中，之后胃肠道的所有症状都得到了缓解。在停用类固醇剂量期间，腹泻的强度再次增加（每天 > 2 L 粪便）；此外，患者开始出现严重的痉挛性腹痛并伴有腹膜体征。再次给予高剂量类固醇，但在治疗 10 天后，没有反应 (NR)。鉴于此，我们引入了鲁索替尼的二线治疗；这与随后腹泻严重程度的缓慢逐渐改善有关，但在减轻腹痛方面没有任何进展。即使患者接受了鲁索替尼治疗超过 14 天，我们也没有看到患者有任何进一步的改善。” 症状。所给予治疗（类固醇和鲁索替尼）的次优临床改善促使我们将鲁索替尼治疗与 FMT 相结合，就像我们在以前的案例中所做的那样。在获得机构审查委员会 (IRB) 接受 [KB/203/2020] 每周三次 FMT 后，进行了第一次和第二次 FMT 管理。在此之后，我们观察到了快速且显着的改进。进行了第三次 FMT 给药，进一步使患者的病情正常化，并将患者排便的频率稳定到每天一次。最终诊断出aGVHD的CR。她现在在 alloHCT 后 351 天，aGvHD CR，没有明显的发病率或进一步的并发症。所给予治疗（类固醇和鲁索替尼）的次优临床改善促使我们将鲁索替尼治疗与 FMT 相结合，就像我们在以前的案例中所做的那样。在获得机构审查委员会 (IRB) 接受 [KB/203/2020] 每周三次 FMT 后，进行了第一次和第二次 FMT 管理。在此之后，我们观察到了快速且显着的改进。进行了第三次 FMT 给药，进一步使患者的病情正常化，并将患者排便的频率稳定到每天一次。最终诊断出aGVHD的CR。她现在在 alloHCT 后 351 天，aGvHD CR，没有明显的发病率或进一步的并发症。所给予治疗（类固醇和鲁索替尼）的次优临床改善促使我们将鲁索替尼治疗与 FMT 相结合，就像我们在以前的案例中所做的那样。在获得机构审查委员会 (IRB) 接受 [KB/203/2020] 每周三次 FMT 后，进行了第一次和第二次 FMT 管理。在此之后，我们观察到了快速且显着的改进。进行了第三次 FMT 给药，进一步使患者的病情正常化，并将患者排便的频率稳定到每天一次。最终诊断出aGVHD的CR。她现在在 alloHCT 后 351 天，aGvHD CR，没有明显的发病率或进一步的并发症。在获得机构审查委员会 (IRB) 接受 [KB/203/2020] 每周三次 FMT 后，进行了第一次和第二次 FMT 管理。在此之后，我们观察到了快速且显着的改进。进行了第三次 FMT 给药，进一步使患者的病情正常化，并将患者排便的频率稳定到每天一次。最终诊断出aGVHD的CR。她现在在 alloHCT 后 351 天，aGvHD CR，没有明显的发病率或进一步的并发症。在获得机构审查委员会 (IRB) 接受 [KB/203/2020] 每周三次 FMT 后，进行了第一次和第二次 FMT 管理。在此之后，我们观察到了快速且显着的改进。进行了第三次 FMT 给药，进一步使患者的病情正常化，并将患者排便的频率稳定到每天一次。最终诊断出aGVHD的CR。她现在在 alloHCT 后 351 天，aGvHD CR，没有明显的发病率或进一步的并发症。每天排便一次。最终诊断出aGVHD的CR。她现在在 alloHCT 后 351 天，aGvHD CR，没有明显的发病率或进一步的并发症。每天排便一次。最终诊断出aGVHD的CR。她现在在 alloHCT 后 351 天，aGvHD CR，没有明显的发病率或进一步的并发症。

案例 4：一名 22 岁女性，来自 MUD 的 alloHCT 后，在第 +31 天开始腹泻，之后我们怀疑是肠道 GvHD。组织病理学结肠镜检查显示 GI-aGvHD 和 MAGIC 总体 IV 级，IV Glucksberg，并且诊断出IBMTR D ⁵。开始使用甲基强的松龙 (2 mg/kg) 和布地奈德进行治疗。使用类固醇 10 天后，患者被评估为具有部分临床反应，但根据 MA​​GIC 的正式 NR 和根据 EBMT 的活动性疾病。⁵鉴于大剂量类固醇治疗效果不理想，开始使用ruxolitinib治疗（2×5mg），并申请IRB治疗FMT（方案同上[KB/202/2020]）。最后，进行了第一次 FMT，从第二天开始，粪便量逐渐减少。腹痛几乎完全缓解。然后，进行了第二次和第三次 FMT。我们观察到患者状况的总体改善。尽管如此，她仍然每天排便多达五次，排便次数减少，排便次数增多，但仍是腹泻性大便，并伴有阵发性腹痛。因此，计划再进行三剂 FMT，在获得额外 IRB 批准 [KB/202/2020] 后，她获得了第四、第五和第六次 FMT；此后，她每天排便一次，没有任何腹痛。CR被评估，

关于第一个病例的主要评论是 FMT 和鲁索替尼在 alloHCT 后的过程中很晚才使用——我们的患者在移植后护理中没有报告腹泻，尽管确实发生了腹泻。我们独立观察到早期和令人满意的改善，但不幸的是，它只持续了很短的时间，并且由于我们患者的不依从而迅速结束。与案例 1 的情况类似，案例 2 的性能状态是先进的并且非常复杂。我们决定在给予 ruxolitinib 之前首先进行 FMT，目的是 ARB 去定植，同时通过肠道微生物群启动免疫调节作用，而不会出现血细胞减少症作为治疗并发症。我们患者的一般状况显着改善，可以安全地添加鲁索替尼。

受先前观察结果的鼓舞，病例 3 在诊断出类固醇耐药后的早期有意使用​​ FMT 和鲁索替尼进行治疗。我们最初计划与鲁索替尼联合进行 FMTs 会议（3 次）。该患者在服用鲁索替尼数周后表现出特别显着的改善，并在 FMT 引入后就达到了 aGvHD 的完全缓解。对这种出色反应的一个可能解释是，从诊断出类固醇依赖的时间跨度相对较短，在发生严重肠壁损伤之前进行治疗促进了微生物群的植入。

病例4采用与病例3相同的策略，鲁索替尼和FMT的给药时间甚至比病例3还要早。毫无疑问，在给予鲁索替尼和前3次FMT后，患者的病情有所改善。最终，尽管有明显的改善，但并未达到完全缓解。为了加深反应，我们决定继续我们的治疗，接下来的三个 FMT 结合更高剂量的鲁索替尼，导致 CR。

在此初步经验之后，我们看到了该策略的巨大潜力，并建议需要随机对照试验来找到答案，并探索 FMT 在多大程度上增强了与 Zeiser 等人发表的试验中相似的患者的结果。³我们看到 FMT 的加性效应将显示出明显更好的结果的真正机会。此外，FMT 和鲁索替尼联合治疗方案的修改值得考虑。除本报告外，第一个临床试验已注册（NCT04269850）。⁶