These considerations raise another important point regarding the optimal chemotherapy backbone for immunotherapy. The chemotherapy and chemo-immunotherapy arms PFS and OS curves separated after 4–7 months in clinical trials, which might suggest the lack of a synergistic effect between platinum–etoposide and ICIs. In experimental models, this lack of synergy was explained by a mechanism involving calreticulin (CALR) in immunogenic cell death. CALR is a protein normally located in the endoplasmic reticulum; it translocates to the cell membrane in response to the endoplasmic reticulum (ER) stress and provides an "eat-me" signal to antigen-presenting cells. The anthracyclines, but not etoposide, are efficient immunogenic cell death inducers and strongly immunogenic in preclinical mouse models [23]. In the phase I CheckMate 012 study on patients with non-small-cell-lung cancer (NSCLC), nivolumab was evaluated in three different chemotherapy regimens, including gemcitabine– cisplatin (for the squamous histology) or pemetrexed–cisplatin (for the nonsquamous tumours) or paclitaxel–carboplatin (for all the histologies). Although it was for a limited number of patients, the combination of nivolumab 5 mg/kg with paclitaxel–carboplatin yielded a similar overall response rate and higher 24-month OS than pemetrexed–cisplatin [33]. Further studies are needed to confirm whether a different chemotherapy backbone and platinum-containing regimens than platinum–etoposide may produce more favourable outcomes in ED-SCLC by the addition of the immunotherapy. A combination of lurbinectedin and atezolizumab is currently ongoing in a phase I/II study, with atezolizumab at a fixed dose of 1200 mg followed by lurbinectedin at a starting dose of 3.2 mg/m2 as a 1-hour infusion on day 1, every three weeks, in patients with SCLC progressing on first-line platinum-based chemotherapy (NCT04253145).

Furthermore, there is a renewed interest in developing new platinum compounds by exploiting the recent knowledge on the sensitivity and resistance mechanisms of cancer cells, their epigenetic modifications, which translate into a platinum drug-tolerant cancer phenotype, and the immunomodulatory effects of platinum compounds to limit immune cell exhaustion [34].

#### **3. Oral Versus Intravenous (i.v.) Formulations of Chemotherapy**

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has been challenging the oncology services and how we currently administer systemic treatments to patients with cancers, particularly in the palliative setting [35]. Patients with lung cancer could represent a vulnerable population to this infection [36–38] with high related mortality in the range of 25–39% [36–42].

Oral alternatives to i.v. anticancer therapies have gained attention, given efforts to reduce visits to the hospitals and the associated risk of infective transmission [42,43]. There is also a benefit in terms of patient convenience and preference with PO (per os) anticancer drugs, provided that their efficacy is equivalent to their i.v. counterparts [44].

Oral formulations of either topotecan and etoposide, two of the most active and used drugs in SCLC, are available and have the following sufficient data to support their use.

#### *3.1. Oral Topotecan*

Oral topotecan (2.3 mg/m2/d, from day 1 to day 5, every 21 days) has demonstrated an absolute benefit in OS (of 12 weeks), slower quality of life deterioration, and greater symptom control, as compared to best supportive care (BSC) by a phase III randomised clinical trial in 141 patients with relapsed SCLC, regardless of their treatment-free interval (< or ≥60 days) [45].

As a second-line treatment, a phase III randomised trial comparing oral topotecan (2.3 mg/m2/d from day 1 to day 5) with i.v. topotecan (1.5 mg/m2/d from day 1 to day 5, every 21 days) demonstrated similar activity and tolerability between the two formulations in 309 patients with SCLC sensitive to initial chemotherapy (i.e., with a treatment-free interval of > or = 90 days). The absolute difference in response rates between oral and i.v. topotecan was −3.6% (18.3 vs. 21.9% for oral and i.v., respectively), whilst no difference in OS was observed. The toxicity profile was different with more thrombocytopenia and diarrhea for oral topotecan, but less neutropenia and anemia, as compared to i.v. topotecan [46]. Another phase III randomised trial compared oral topotecan (2.3 mg/m2 from day 1 to day 5) every 21 days for six cycles to carboplatin (area under the curve 5 mg/mL per min day 1) plus etoposide (100 mg/m2 from day 1 to day 3), as a second-line treatment for 164 patients with sensitive relapsed (at least 90 days after completion of firstline treatment) SCLC [22]. The median PFS was significantly longer in the combination chemotherapy group than in the oral topotecan group with an absolute benefit of 2.0 months (HR 0.57, 90% CI 0.41–0.73; *p* = 0.0041), but no OS difference was observed (HR 1.03, 95% CI 0.87–1.19; *p* = 0.94). The toxicity was comparable between the two groups, although two treatment-related deaths occurred in the oral topotecan group (both were febrile neutropenia with sepsis) as compared to none in the combination group.

As first-line treatment, a phase III study randomised 784 patients with untreated ED-SCLC to either oral topotecan (1.7 mg/m2/d from day 1 to day 5) with i.v. cisplatin (60 mg/m2 on day 5) (TC) or i.v. etoposide (100 mg/m2/d from day 1 to day 3) with i.v. cisplatin (80 mg/m2 on day 1) (PE) every 21 days. No difference in OS was observed between the two groups with the absolute difference of −0.03 (95% CI, −6.53 to 6.47), meeting the predefined criteria for non-inferiority of TC relative to PE. The regimens were similarly tolerable, with more frequent grade 3/4 neutropenia with PE (84% vs. 59%), grade 3/4 anemia and thrombocytopenia with TC (38 vs. 21% and 38 vs. 23%, respectively) [11].

#### *3.2. Oral Etoposide*

As an alternative to the i.v. formulation, the use of oral etoposide is supported by randomised data and a registry real-world population-based study of 2066 chemotherapy naïve patients with LD- (*n* = 762) and ED-SCLC (*n* = 1264) [47]. These studies showed, both in the LD- and in the ED-SCLC, no significant difference in OS between i.v. versus oral etoposide, although the oral group did require more dose reductions as compared to the i.v. group [47]. The oral etoposide was given on days 2 and 3 at the doubled dose of 200 mg/m2 than 100 mg/m2 of the i.v.

#### *3.3. Conclusions*

Based on these data, oral topotecan and etoposide could be considered as convenient alternatives to other i.v. therapies either in first-line, as a substitute for i.v. etoposide in platinum-combinations, and, for the oral topotecan, in subsequent treatment lines, as compared to other i.v. options, particularly for the platinum-resistant disease. Furthermore, the reduced incidence and severity of neutropenia observed with oral topotecan in two of the three above-mentioned phase III studies, either when compared to the i.v. etoposide and to its i.v. formulation, may represent a further advantage during the SARS-CoV-2 pandemic and speculatively for novel chemo-immunotherapy combinations.

#### **4. Lurbinectedin**

Lurbinectedin is an oncogenic transcription inhibitor. The drug is an analogue of trabectedin which covalently binds guanine residues in the minor groove of DNA, creating adducts that are able to induce double-strand breaks. As a consequence, a cascade of events occurs affecting the activity of DNA binding proteins, involving transcription factors and DNA repair mechanisms, leading to double-strand breaks and finally to cell death [48]. According to its mechanism of action, lurbinectedin showed antiproliferative and cytotoxic functions in several tumour cell lines and increased activity in cell lines bearing defects in the DNA mismatch repair. Moreover, lurbinectedin causes ICD (immunogenic cell death) and elicits anticancer immunity [49]. A single preclinical study demonstrated a direct effect of lurbinectedin on the tumour microenvironment, as it decreases the tumour-associated macrophages and circulating monocytes, and the angiogenic factor VEGF (vascular endothelial growth factor), with consequent reduced blood vessel density [50]. Lurbinectedin received accelerated approval by the FDA in 2020 for adult patients with metastatic SCLC progressing on or after platinum-based chemotherapy after demonstrating favourable ORR and DOR in an open-label, single-arm phase II trial [51] (Table 2).

**Table 2.** Lurbinectedin trials on small cell lung cancer (SCLC). Median progression-free survival (mPFS) and median overall survival (mOS) expressed in months, overall response rate (ORR) as a percentage, with 95% CI in graphs. Abbreviations: N/A = not available; mOS = median Overall Survival; mPFS = median Progression Free Survival; ORR = Overall Response Rate; ITT = Intent To Treat population.

#### **5. Clinical Development**

#### *5.1. Phase I Trials*

A phase I study indicated the dose of 4 mg/m2 or a flat dose of 7 mg i.v. every 21 days as safe dosing for lurbinectedin [57]. Based on preclinical data of synergy, another phase I study investigated the combination of lurbinectedin at 4 mg flat dose with doxorubicin 50 mg/m2 [53]. The study enrolled 27 relapsed SCLC patients. When administered as second-line, the combination showed relevant activity as 91.7% of patients with a platinumsensitive disease (defined as with platinum-free interval ≥ 90 days) and 33.3% of those

with a platinum-resistant disease (with platinum-free interval < 90 days) achieved a disease response, with a PFS of 5.8 and 3.5 months, respectively. As a third-line treatment, all patients had a platinum-resistant disease, and the median PFS was 1.2 months. The major toxicities were hematologic, with high rates of grade 3–4 neutropenia (95%), leukopenia (79%), anemia (47%) and thrombocytopenia (26%). Aiming at improving the safety of this combination, an expansion cohort with a reduced dose of both drugs (lurbinectedin 2 mg/m2 and doxorubicin 40 mg/m2) was implemented, with an observed ORR of 37% and 53% in patients with a resistant and sensitive disease, respectively. The median PFS was 3.4 months in all patients, 1.5 and 5.7 months in those with a resistant and sensitive disease, respectively. The median OS was 7.9 months in all patients, 4.9 and 11.5 months in those with a resistant and sensitive disease, respectively [58].

Another combination with lurbinectedin and irinotecan has been explored in a phase Ib/II trial for advanced solid tumours (NCT02611024). Patients with SCLC included in this study were 13. The study investigates an escalating dose of lurbinectedin starting from 1 mg/m2 on day one with a fixed dose of irinotecan 75 mg/m2 on days 1 and 8, every 21 days. The recommended dose of lurbinectedin was 2.0 mg/m2, and the maximum tolerated dose was 2.4 mg/m2 in combination with irinotecan 75 mg/m2 and prophylactic granulocytecolony stimulating factor (G-CSF). In the SCLC cohort, the ORR was 61.5% [54,56].

#### *5.2. Phase II Trial*

Recently, the results of a phase II, open-label, basket trial exploring the safety and efficacy of lurbinectedin as a single-agent in several tumour types were published, including 105 patients with progressive SCLC [52]. The eligibility criteria required previous platinumbased chemotherapy and the absence of central nervous system (CNS) involvement. Fortyfive and 60 patients had chemotherapy-resistant and -sensitive disease, respectively, with a chemotherapy-free interval of ≥90 days. The median follow-up was 17.1 months. The ORR was 35.2% (95% CI, 26.2–45.2), the median DOR and mOS were 5.3 and 10.8 months, respectively, in all SCLC patients. Patients with platinum-sensitive disease showed better outcomes as compared to platinum-resistant disease, with a median DOR of 6.2 months (95% CI, 3.5–7.3) and 4.7 months (95% CI, 2.6–5.6), respectively. The most frequent grade 3–4 AEs were hematological. Furthermore, a preplanned subset analysis of patients with chemotherapy-free interval ≥ 180 days was conducted on 20 patients. The ORR was 60% (95% CI, 36.1–86.9) in these patients, with a median DOR of 5.5. months (95% CI, 2.9–11.2) and a disease control rate (DCR) of 95% (95% CI, 75.1–99.9). The median PFS was 4.6 months (95% CI, 2.6–7.3), and the median OS 16.2 months (95% CI, 9.6-not reached). The most common grade 3–4 adverse events were hematological and increased liver function tests [59].

#### *5.3. Phase III Trial*

The phase III ATLANTIS study (NCT02566993) compared the combination of lurbinectedin and doxorubicin to topotecan or CAV at the physician's choice. Sixty-hundredthirteen patients with SCLC progressing to one prior platinum-based chemotherapy were randomised 1:1. Lurbinectedin was given at 2 mg/m2, lower than that of 3.2 mg/m2 approved by the FDA. Patients were stratified by their chemotherapy-free interval, ECOG PS of 0 or 1–2, CNS involvement, prior use of ICIs (anti-PD-1/PD-L1 agents), and investigator's preference of topotecan or CAV. The recent press release from Jazz Pharmaceuticals and PharmaMar announced that the experimental treatment with lurbinectedin and doxorubicin missed its prespecified OS endpoint [55]. The secondary outcome endpoints and subgroup analyses, however, favoured the investigational combination in the intent-to-treat patient population, including the analysis of OS differences between the subgroup of patients treated with lurbinectedin and doxorubicin versus CAV; the OS and PFS in patients with and without CNS metastases; the ORR and DOR as assessed by an independent review committee.

Patients with relapsed SCLC still represent an unmet medical need as they have limited treatment options, lack of druggable targets, and poor prognosis. Despite the innovative mechanism of action and promising phase I and II trial results, lurbinectedin in combination with doxorubicin did not meet its prespecified OS endpoint in the phase III trial. As above-mentioned, however, there is still rationale and room for exploring combinations of lurbinectedin with ICIs +/− other chemotherapy agents, based on its immunomodulatory effects. (Table 2).

#### **6. Novel Formulations of Traditional Chemotherapy Agents and "Promising" Drug Derivatives**

Few cytotoxic agents with modest benefit and poor tolerability are currently available for SCLC, especially in later treatment lines. Although potentially associated with prolonged survival, their toxicity profile often represents a barrier to their administration at the full recommended dose. In addition to new agents with a different mechanism of action, the development of newer formulations of traditional chemotherapy drugs might represent a promising strategy to improve either activity and safety (Table 3).

#### *6.1. Nanoparticle Albumin Bound-Paclitaxel (Nab-Paclitaxel)*

Paclitaxel, alone or in combination with carboplatin, has shown activity in the refractory relapsed SCLC; however, its use is limited by potentially severe infusion reactions and peripheral neurotoxicity [60–62]. A solvent-free formulation of paclitaxel, the nanoparticle albumin-bound (nab)-paclitaxel, has been developed to improve the tolerability of paclitaxel, confirming efficacy and a safer profile than paclitaxel in pancreatic, breast cancer and NSCLC [63]. The activity of single-agent weekly nab-paclitaxel was tested in phase II, single-arm, NABSTER trial on 68 patients with relapsed SCLC, divided into two cohorts based on platinum-sensitivity (with a cut-off of 60 days). The primary endpoint was investigator-assessed ORR; PFS and OS were secondary endpoints. The RR was 8% and 14% in the refractory and sensitive cohort, respectively. The median PFS was similar in both the cohorts (1.8 and 1.9 months), whilst the median OS was longer in the sensitive than in the refractory cohort (6.6 vs. 3.6 months, respectively). The treatment was well tolerated with grade 3–4 neutropenia in 10% of patients and anemia in 4%. The study did not meet the primary endpoint on ORR, and the authors concluded that further investigations and a head-to-head comparison with topotecan were not justified [64].

#### *6.2. Liposomal Irinotecan (Nal-IRI)*

Liposomal formulations of chemotherapy agents, such as irinotecan and topotecan, have been developed to improve their efficacy through a slow and controlled release of the drug expected to prolong the exposure of tumour cells to these agents [65,66]. Irinotecan is active in the first-line treatment of SCLC in combination with cisplatin and is commonly used in Japan. In the second-line setting, single-agent irinotecan showed similar outcomes to those from topotecan and is included in several guidelines as a possible treatment option [67–69].

The liposomal encapsulation of irinotecan has been designed to prolong its circulation levels, exploiting the tumour vascular permeability and accumulation in the tumour tissue, where the macrophages can activate the drug [65]. Compared to irinotecan, its liposomal formulation demonstrated a longer persistence of plasma levels of SN-38, the active metabolite (50 vs. 8 h in mice), resulting in sustained topoisomerase-1 inhibition, increased DNA damage and cell death [70].

In metastatic pancreatic cancer, liposomal irinotecan in combination with fluorouracil and folinic acid showed significant improvement in OS in phase III Napoli-1 trial and was approved by the FDA and EMA as second-line treatment after failure of gemcitabine-based chemotherapy [71].

**Table 3.** Novel formulations of traditional chemotherapy and drug derivatives clinical trials results. mPFS and mOS expressed in months, ORR as percentage, with 95% CI in graphs. Abbreviations: ITT = Intent To Treat population; N/A = not available; mOS = median Overall Survival; mPFS = median Progression Free Survival; ORR = Overall Response Rate.

SCLC is highly vascularised and enriched by tumour-associated-macrophages (TAM). These two characteristics suggest a possible high penetration of liposomal irinotecan (nal-IRI) in the tumour tissue and activation by local phagocytic through high CES (carboxylesterase) levels, the enzyme that converts irinotecan in its active metabolite SN-38, resulting in effective cytotoxic activity [72–74]. In preclinical models, nal-IRI showed superior antitumour activity than topotecan and irinotecan, either in cell-line derived models and patients-derived xenograft models built after progression to carboplatin and etoposide [75]. These preclinical data supported the clinical development of nal-IRI in patients with SCLC.

The RESILIENT study is an ongoing two-part phase II/III trial assessing the potential use of nal-IRI in patients with SCLC who progressed on or after platinum-based regimens. Part one of the trial involved dose-finding and dose-escalation analyses, whilst in the second part, patients were randomised to liposomal irinotecan or topotecan to compare efficacy in terms of PFS and OS. In the dose-finding phase, patients were divided into two cohorts to receive liposomal irinotecan every 2 weeks at 70 mg/m2 or 85 mg/m2. The cohort treated with 85 mg/m2 dose was closed early for dose-limiting toxicities. In the second cohort, 12 patients received the 70 mg/m2 dose, which was deemed by the investigators as tolerable. Among these patients, preliminary exploratory efficacy endpoints were promising, with a RR of 33.3%, the median time to response of 6 weeks and an overall DCR of 58.3% [76]. This led to a dose-expansion phase with additional 13 patients, whose safety data were presented at the 2020 World Conference on Lung Cancer (WCLC). Among the 25 patients who received the 70 mg/m2 dose, the grade ≥3 treatment-emergent AEs occurred in 40% (*n* = 10) of patients, mostly represented by diarrhea (*n* = 5), neutropenia (*n* = 4), anemia (*n* = 2) and thrombocytopenia (*n* = 2) [77]. The trial is still recruiting for the second part (www.ClinicalTrials.gov Identifier: NCT03088813; accessed on 20 January 2021).

#### *6.3. Belotecan*

Belotecan is a new camptothecin analogue and topoisomerase I inhibitor. Preclinical data on mice models suggested superior antitumour efficacy and wider therapeutic margins than topotecan [78]. In phase II clinical trials, belotecan monotherapy showed encouraging activity and good tolerability in patients with relapsed SCLC [79,80]. More recently, secondline belotecan has been compared to topotecan in a phase IIb trial on 164 SCLC patients progressing to platinum-based chemotherapy. Patients were randomised in a 1:1 ratio to receive five consecutive daily intravenous infusions of topotecan (1.5 mg/m2) or belotecan (0.5 mg/m2) every 3 weeks for six cycles. The study was powered to assess the noninferiority of belotecan to topotecan in ORR. Belotecan significantly improved ORR (33 vs. 21%, respectively, *p* = 0.09) and DCR (85 vs. 70%, *p* = 0.030) as compared to topotecan. Furthermore, the median OS was significantly longer with belotecan than topotecan (13.2 vs. 8.2 months, HR = 0.69, 95% CI 0.48–0.99), with a favourable safety profile. On the basis of these promising results, belotecan might be another treatment option for second-line treatment in SCLC, pending phase III trials to confirm its efficacy in this setting [81].

#### *6.4. Amrubicin*

Amrubicin is a third-generation synthetic anthracycline with potent inhibiting activity on the topoisomerase II. Among drugs belonging to the same class, amrubicin has fewer chronic cardiological effects (e.g., cardiomyopathy) and no cumulative-dose heart damage in animal models [82]. Amrubicin is approved in Japan as a single-agent for SCLC after the failure of platinum-based chemotherapy, whilst it is under evaluation in other countries. In this setting, it has been investigated by several studies on Asian patients, showing ORR ranging from 36 to 52% and median OS of 7–12 months [83–87]. Two phase II studies demonstrated clinical efficacy in the Caucasian population also, with higher ORR as compared to topotecan (44 vs. 15%, respectively) [88,89]. A phase III study compared amrubicin with topotecan in patients with SCLC progressing to platinum–etoposide

chemotherapy. A total of 637 patients were randomly assigned in a 2:1 ratio to receive amrubicin 40 mg/m2 on days 1–3 every three weeks or topotecan 1.5 mg/m2 on days 1–5 of 21 days cycles. Amrubicin did not improve the primary endpoint of OS as compared to topotecan (7.5 vs. 7.8 months, HR 0.88, *p* = 0.170), despite an improvement in the median PFS (4.1 vs. 3.5 months, HR, 0.80, *p* = 0.018) and ORR (31.1 vs. 16.9%, odds ratio [OR] 2.22, *p* < 0.001). A slight survival benefit of two weeks was observed in the subgroup of patients with refractory disease. The safety profile favoured amrubicin as far as hematologic events are concerned; however, higher rates of infections (16 vs. 10%, respectively) and febrile neutropenia (10 vs. 3%, respectively) were linked to amrubicin [90].

In the first-line setting of ED-SCLC, the combination of amrubicin plus cisplatin has been compared to cisplatin–etoposide in phase III non-inferiority study in Chinese patients. The amrubicin–cisplatin regimen was non-inferior to standard platinum–etoposide chemotherapy on OS (median OS of 11.8 vs. 10.3 months, *p* = 0.08), with a slight nonsignificant improvement of 1.5 months [91].

The potential interest of this drug derives from its possible synergy with other agents. Although the results of the lurbinectedin/doxorubicin combination showed by the AT-LANTIS trial press release were disappointing [55], amrubicin still remains a plausible alternative to the doxorubicin as a potential companion drug for lurbinectedin in future clinical trials. Furthermore, based on its immunomodulatory effect, the association of amrubicin and pembrolizumab is under evaluation in a phase II trial in patients with refractory SCLC [92]. Therefore, despite the modest activity showed in non-Asian patients as single-agent, amrubicin may still have a role as a new companion for combination strategies in clinical trials.

#### *6.5. Temozolomide*

Temozolomide is an oral alkylating agent that induces cytotoxic damage and apoptosis through single-strand DNA breaks. The rationale for its application in SCLC is strong, as temozolomide has a good penetration through the blood–brain barrier, which can be useful for brain metastasis, and SCLC has aberrantly methylated O6-methylguanine-DNA methyltransferase MGMT, the enzyme involved in the repair mechanism of the DNA damage induced by temozolomide [93].

Two phase II single-arm studies investigated the role of the single agent temozolomide in SCLC patients who progressed to one or two lines of chemotherapy, stratified on the basis of platinum sensitiveness. In the first by Pietanza et al., 64 patients were enrolled and received temozolomide 75 mg/m2/die for 21 days in a 28-day cycle. The primary endpoint was ORR in the platinum-sensitive and refractory cohort. The ORR was 22% (95% CI, 9–40%) and 19% (95% CI, 7–36%), respectively. One complete remission was observed in the platinum-sensitive cohort. The main limiting toxicities were grade 3 thrombocytopenia and neutropenia, which were observed in nine patients (14%) [94].

In the second study by Zauderer MG et al., a different schedule was used to improve tolerability on hematologic toxicities. Temozolomide was given 200 mg/m2/die for 5 days in a 28-day cycle. Among 25 SCLC patients, five patients have grade 3–4 events (mostly anemia, thrombocytopenia and neutropenia). The ORR was 12% (95% CI 3–31%), with two responses also observed in refractory patients, median PFS was 1.8 months (95% CI 0.9–3.5 months) and median OS 5.8 months (95% CI: 3.3–9.8 months) [95].

Data on brain metastasis response were conflicting among the two studies. In the first study, at the standard dose, 38% had a CR (complete response) or PR (partial response) (95% CI, 14–68%) [94], whilst no response was seen in the eight patients with target brain lesions of the second study (four had stable disease and four progression) [95].

MGMT promoter methylation is a well-known predictive factor of response to temozolomide in glioma [96]. For this reason, it was evaluated in both the aforementioned studies. In the first one, ORR improved in patients with methylated MGMT promoter with respect to patients without methylation (38 vs. 7%; *p* = 0.08) [94]. In the second one, 50% of

patients had methylation in MGMT promoter, but the number of patients was too small to derive statistical conclusions on response [95].

However, due to the scarce data on efficacy and hematologic toxicity, temozolamide single agent is not routinely used in clinical practice.

More recently, the association of temozolamide with PARP (poly ADP-ribose polymerase) inhibitors, veliparib and olaparib, has been investigated [97,98]. Alterations in the PARPdependent base excision repair pathway are an established resistance mechanism to temozolamide, and preclinical models validated the rationale for the clinical development of combinations with temozolamide and PARP-inhibitors [99]. The combination of temozolamide plus veliparib or olaparib improved ORR compared to temozolamide alone in phase II trials [97,98]. However, data on larger cohorts and phase III trials are ongoing to assess the possible application in clinical practice.

#### **7. Conclusions**

After many years of inactivity, the treatment of SCLC has been observing a renewed interest thanks to the introduction of immunotherapy in the first-line setting.

Cytotoxic agents remain so far the backbone treatment for immunotherapy in the first-line and the only current options in later therapeutic lines, although limited benefit and relevant toxicity, particularly in the platinum-resistant population.

Re-interpreting and exploiting the mechanisms of action of old cytotoxic agents, such as cisplatin and anthracyclines, with a view to their immunomodulatory effects, can unveil new therapeutic scenarios with combination strategies based on ICIs and different chemotherapeutic agents or their new formulations.

Despite the limitations highlighted by all the chemotherapy molecules currently investigated, the discovery of transcription factors and their overexpression will allow the segmentation of SCLC into different molecular subgroups, which could benefit from combinations of chemotherapy with other small molecules (such as PARP inhibitors, Aurora kinase inhibitors).

The chemotherapy chapter for SCLC is not yet closed, and new studies are needed to better understand its role in these future therapeutic scenarios.

**Funding:** This research received no external funding.

**Acknowledgments:** The authors thank Elisa Sala, Ph.D. medical writer, for her contribution in drafting and editing the text.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

## *Review* **History of Extensive Disease Small Cell Lung Cancer Treatment: Time to Raise the Bar? A Review of the Literature**

**Chiara Lazzari 1,†, Aurora Mirabile 1,†, Alessandra Bulotta 1, Maria Grazia Viganó 1, Francesca Rita Ogliari 1, Stefania Ippati 1, Italo Dell'Oca 2, Mariacarmela Santarpia 3, Vincenza Lorusso 1, Martin Reck 4 and Vanesa Gregorc 1,\***

**Simple Summary:** Small cell lung cancer (SCLC) remains the most aggressive form of neuroendocrine tumor of the lung, for which treatment options remain limited. The introduction of immune checkpoint inhibitors has modified for the first time the therapeutic strategies in patients with extensive disease after decades. New therapeutic approaches are required. Deeper knowledge of tumor biology is required to gain new insights into this complex disease.

**Abstract:** Several trials have tried for decades to improve the outcome of extensive disease small cell lung cancer (ED-SCLC) through attempts to modify the standard treatments. Nevertheless, platinum/etoposide combination and topotecan have remained respectively the first and the second line standard treatments for the last 40 years. With the advent of immunotherapy, this scenario has finally changed. Our review aims to provide an overview of the primary studies on the actual therapeutic strategies available for ED-SCLC patients, and to highlight emerging evidence supporting the use of immunotherapy in SCLC patients.

**Keywords:** small cell lung cancer; chemotherapy; Immunotherapy; extensive disease

#### **1. Introduction**

Small cell lung cancer (SCLC) is an aggressive tumor, with a high mitotic rate and early metastasis occurrence. It is observed in approximately 15% of new cases of lung cancer. Smoking represents the main risk factor for its development. For a long time, SCLC has been classified according to the Veteran's Administration Lung Cancer Study Group as limited stage (tumor located in the thorax and included in a single radiation field) or extensive stage (when not confined into a single radiation field, or in the presence of distant metastases). To better define patients' prognosis, in 2009, the International Association for the Study of Lung Cancer proposed the use of the tumor, node, and metastasis (TNM) staging system [1]. Following the introduction of the eighth TNM edition for the classification of non-small cell lung cancer (NSCLC), and the survival analysis of patients with SCLC, classified according to the seventh or the eighth TNM editions, the eighth TNM classification was adopted, and it is currently used [2]. Approximately 60–70% of patients are diagnosed with metastatic disease at the onset [3]. During the last four decades, platinum–etoposide has been the

**Citation:** Lazzari, C.; Mirabile, A.; Bulotta, A.; Viganó, M.G.; Ogliari, F.R.; Ippati, S.; Dell'Oca, I.; Santarpia, M.; Lorusso, V.; Reck, M.; et al. History of Extensive Disease Small Cell Lung Cancer Treatment: Time to Raise the Bar? A Review of the Literature. *Cancers* **2021**, *13*, 998. https://doi.org/10.3390/cancers 13050998

Academic Editor: Jeffrey A. Borgia

Received: 26 January 2021 Accepted: 19 February 2021 Published: 27 February 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

only recognized treatment in the first-line setting. Despite the response rate of 60–80%, responses are not durable, and patients develop resistance and unfortunately die within ten months. Less than 7% of patients are still alive at five years [4,5]. While aiming to improve patients' outcomes, several treatment strategies have been tested, but with poor results. At odds with NSCLC, where the deep understanding of tumor biology and the identification of actionable molecular alterations have been translated into efficient molecularly targeted therapies, no driver-targetable molecular alterations have been identified in SCLC, and its therapeutic portfolio has not been improved for several years. Recently, immune checkpoint inhibitors have significantly prolonged patient survival, thus resulting in a practice-changing strategy for the first time.

The current review provides an overview of the progress made in treating patients with extensive disease SCLC (ED-SCLC).

#### **2. First Line Chemotherapy in ED-SCLC**

Originally, cyclophosphamide, doxorubicin, and vincristine (CAV) represented the standard treatment used in untreated patients with ED-SCLC. For a long time, research has focused on identifying the most effective combinatorial chemotherapy regimen to prolong patients' survival with acceptable toxicity and good quality of life. Table 1 summarizes the main phase III trials conducted in naive patients with ED-SCLC.

Because of the high proliferative rate of SCLC, the high chemosensitivity, and the high percentage of relapses, in order to increase tumor-cell death, among the strategies evaluated, the use of alternating non-cross-resistant regimens was tested. A phase III study was designed to compare in 437 patients with ED-SCLC, stratified according to performance status (PS), cisplatin–etoposide (EP) with CAV for four cycles, or the alternation of CAV/EP for six cycles [6]. Overall survival (OS) was the primary end-point of the trial. No significant difference was observed in terms of objective response rate (ORR) or OS. The alternating regimen was associated with a significantly prolonged time to progression (TTP) in comparison to CAV, but with more hematologic toxicity. Moreover, CAV and EP resulted in comparable efficacies in terms of OS, TTP, and ORR.

Results from a meta-analysis, including data from 36 randomized phase II and III trials, demonstrated, for the first time, OS improvement with etoposide alone, or in combination with cisplatin [7], in patients with ED-SCLC. A further meta-analysis evaluated the differences between cisplatin and carboplatin [8] in terms of efficacy and toxicity. The results were comparable for OS and PFS, and were associated with a different spectrum of toxicity. The included a higher percentage of hematologically adverse events with carboplatin, and more non-hematological events with cisplatin, including nausea, vomiting, and renal failure. Based on these findings and the good efficacy/toxicity ratio of EP, this regimen has become the standard first-line treatment. The choice between cisplatin or carboplatin is secondary to the expected toxicity profile, the organ function, the PS, and the patients' co-morbidities [8].

Different phase III studies have explored the efficacy of adding one or two drugs to EP chemotherapy, the use of alternative compounds to etoposide, such as irinotecan, topotecan or amrubicine, or the use of a high dose of chemotherapy [9]. A phase III trial evaluated the benefit of ifosfamide combined with EP (VIP) in 171 patients with ED-SCLC [10]. Patients were stratified according to PS and randomized between EP or VIP. Patients with brain metastases were eligible and received concurrent whole-brain radiotherapy. The study was designed to demonstrate an OS increase of 50% in the VIP arm compared with the EP regimen. No significant difference was shown in terms of ORR, while prolonged median progression-free survival (PFS) and OS were observed in patients receiving VIP over EP. However, the benefit was less than expected, and VIP was associated with more hematologic and non-hematologic toxicity. Another phase III study tested the advantage of combining cyclophosphamide and epidoxorubicin with EP (PCDE) as a strategy to intensify and improve the therapeutic options in patients with ED-SCLC [11]. Two hundred twenty-six patients lacking previous treatment were randomized between EP or PCDE. Brain metastases represented an exclusion criterion to enter the study. The primary end-point was OS, and the trial was designed to demonstrate a 15% improvement at 1 year with PCDE over EP. At the end of chemotherapy, prophylactic cranial irradiation was recommended for patients with complete responses, while thoracic radiotherapy was suggested for those with partial responses in the case of a residual tumor. PDCE significantly increased ORR, and prolonged PFS and 1-year survival, over EP, with higher hematologic toxicity, and without affecting the quality of life. However, these results did not translate into a practice-changing strategy. In addition, the attempt to use a high dose of EP failed to demonstrate any significant advantage over standard EP in terms of ORR or OS, and was associated with increased toxicity [12]. Another strategy tested was the comparison of a combination therapy containing carboplatin, etoposide, and vincristine with paclitaxel, etoposide and carboplatin, in a phase III study enrolling 614 naive patients with SCLC of any stage [13]. The primary end-point was OS. The results showed a statistically significant increase in median and long-term survival, PFS, and a decrease in toxicities for patients with SCLC receiving a paclitaxel-containing regimen compared with standard chemotherapy.

The preliminary efficacy observed with the topoisomerase I inhibitor irinotecan [14], and the positive findings from a phase II study showing an ORR of 86%, with a median OS of 13.2 months in patients with ED-SCLC receiving cisplatin combined with irinotecan (IC) [15], led to the design of four phase III trials to compare the efficacy of IC to that of EP. All the studies included OS as the primary end-point. The Japanese JCOG 9511, designed to enroll 230 patients, was prematurely closed, following the inclusion of 154 patients [16]. The preliminary results, observed during the interim analysis and showing an OS difference in favor of IC combination, which was further confirmed at the second interim analysis, led to the early termination of patient accrual. The definitive results showed a significant improvement of ORR, PFS, and OS in the IC arm. Patients in the EP arm experienced higher grade 3–4 neutropenia and thrombocytopenia, but lower grade 3–4 diarrhea, than the IC group. However, these results were not confirmed by another phase III trial, designed to compare EP with IC in a larger number of patients from North America [17]. Three hundred thirty-one patients were enrolled. Conversely, from the Japanese trial, no significant difference in terms of ORR, PFS, or OS was observed between the two arms. Different reasons might explain the divergent findings of the two studies, including the differences in the characteristics of the patients enrolled (with a lower percentage of cases with more advanced disease in the JCOG study), the different dose and schedule of IC used, with an increased dose of irinotecan administered in the Japanese trial, and pharmacogenomic differences between the North American and Japanese populations. Similar findings were reported by the phase III SWOG S0124 study, enrolling 651 patients and designed using the same eligibility criteria and treatment regimens as the JCOG 9511 [18]. No difference was observed in ORR, PFS, or OS between the two arms, with less hematologic and greater gastrointestinal toxicity for IC over EP. The different results of the JCOG 9511 trial might be related to the smaller number of patients enrolled, and imbalances in the distribution of patients, including less patients with PS2, more women, and fewer cases with brain metastases in the Japanese study. Moreover, the differences in the ethnicities and the genetic backgrounds of the populations included might have influenced the final results. Conversely, when irinotecan in combination with carboplatin was compared with oral etoposide in combination with carboplatin in a phase III study, enrolling 220 patients with ED-SCLC, irinotecan prolonged OS without affecting the quality of life [19]. More recently, a meta-analysis, including data from six randomized trials [20] enrolling 1476 naive patients with ED-SCLC, compared the efficacy and toxicity of irinotecan/platinum with etoposide/platinum. Two trials were phase II studies, and in two trials carboplatin was used instead of cisplatin. Although the results showed that the combination of irinotecan/platinum improves ORR and prolongs OS over EP, the subgroup meta-analysis performed after excluding the two trials using carboplatin failed to confirm this advantage. The heterogeneity of the population, the different drugs, and the doses used has not allowed for a conclusion on the most efficient regimen, and EP has remained the reference standard treatment for western countries. Another topoisomerase I inhibitor, topotecan, has been explored in combination with cisplatin (TP) in untreated patients with ED-SCLC. Seven hundred and ninety-five patients were enrolled in a phase III trial, designed

to assess OS superiority or at least the non-inferiority of TP over EP [21]. Despite a higher ORR, a prolonged PFS, and a comparable OS observed in the TP arm, the higher percentage of hematological toxicities, including anemia and thrombocitopenia requiring transfusions, associated with a higher rate of treatment-related deaths, has not allowed the replacement of standard EP with TP. Similarly, the topoisomerase II inhibitor amrubicin, an anthracycline with the advantage of not determining cardiovascular toxicity, was tested in a phase III study, designed to evaluate its non-inferiority in terms of OS, and eventually its superiority over EP when combined with cisplatin in 300 naive Chinese patients with ED-SCLC [22]. The results showed significantly higher ORR in the AP arm, with comparable PFS and OS, but this was associated with a higher percentage of hematopoietic toxicities in the amrubicine group.

**Table 1.** Phase III trials exploring chemotherapy in first-line setting of patients wing extensive disease small cell lung cancer (ED-SCLC).

EP: cisplatin–etoposide. CAV: cyclophosphamide, doxorubicin and vincristine. VIP: ifosfamide, cisplatin, etoposide. PDCE: cyclophosphamide, epidoxorubicin, cisplatin, etoposide. CEV: carboplatin, etoposide, and vincristine. TEC: paclitaxel, etoposide, and carboplatin. IC: irinotecan, cisplatin. CBDCA: carboplatin. E: etoposide. I: irinotecan. \* oral. TP: topotecan, cisplatin. AP: amrubicin, cisplatin. OS: overall survival. PFS: progression-free survival. TTP: time to progression. m: months.

> In conclusion, due to the toxicity and the lack of substantial efficacy, all the alternative regimens tested in patients with untreated ED-SCLC were unsuitable for becoming a new standard, and EP has remained the first therapeutic choice.

#### **3. Immune Checkpoint Inhibitors in the Treatment of Patients with ED-SCLC**

Recently, in patients with ED-SCLC, the treatment landscape has evolved because of the advent of immune checkpoint inhibitors. The genomic instability of SCLC [23] favors the accumulation of DNA damages, which in turn favor the development of immunogenic clones recognized by the antigen-presenting cells and the dendritic cells. As a consequence, CD8+ T cells are simulated. The activation of the immune system contributes to the killing of tumor cells.

Due to the correlation between SCLC and smoking, many somatic mutations occur. A higher T cells ratio was found in long-term survivors with SCLC compared to patients with recurrent disease [24], thus suggesting the potential efficacy of immune checkpoint

inhibitors in SCLC. As such, immunotherapy has been tested in patients with ED-SCLC as a single agent, combined with chemotherapy or different immune checkpoint inhibitors, in the first-line setting, the maintenance, and the second line (Table 2).

Few data are available regarding the immune composition of SCLC tumors. Retrospective analyses indicate the expression of PD-L1 in a variable percentage of cases (0–71.6%). PD-L1 upregulation has been found in patients with high levels of CD3ε and CD68 mRNA, and in those with increased levels of CD8. CD8 T cells are detectable in 13% of patients [25]. However, a lower percentage of CD8 has been observed in SCLC compared with patients affected by NSCLC. These differences might partly explain the different outcomes obtained with immune checkpoint inhibitors in NSCLC in comparison with SCLC.

Recently, chemotherapeutic agents' immunologic effects on the tumor microenvironment's modulation have been established [26]. Preclinical evidence suggests that chemotherapy upregulates PD-L1 on tumor cells, increases the expression of nuclear factor kappa-light on B cells, favors the activation of antigen-presenting cells, and inhibits the infiltration of immunosuppressive cells in the tumor [27–30]. Based on these findings, clinical trials have been designed to evaluate platinum–etoposide's synergistic effect with immune checkpoint inhibitors in SCLC [31].

#### *3.1. Immune Checkpoint Inhibitors in First Line Setting-CTLA4 Blockade*

The anti-CTLA-4 monoclonal antibody ipilimumab was the first immune checkpoint inhibitor evaluated in combination with chemotherapy in untreated patients with ED-SCLC in a phase II study [32]. To define the best timing for chemotherapy and immunotherapy administration, two regimens were tested in 130 patients: ipilimumab concurrent with paclitaxel/carboplatin, or ipilimumab in combination with paclitaxel/carboplatin following two cycles of chemotherapy alone (phased regimen). The primary end-point of the study was immune-related progression-free survival (irPFS). Results showed improved irPFS in the group receiving the phased regimen over chemotherapy alone, while no difference was observed with the concurrent regimen, thus suggesting that induction chemotherapy might favor the release of tumor antigens, thus resulting in a more effective treatment strategy. Based on these promising findings, the phase III CA 184-156 study was designed to compare ipilimumab or placebo in combination with etoposide and platinum [33]. The phased regimen was selected. One thousand one hundred and thirty-two patients were enrolled to demonstrate an OS improvement in the experimental arm. Conversely, from what was expected, ipilimumab did not result in a statistically significantly improved OS over chemotherapy alone, and immunotherapy did not enter into the therapeutic armamentarium of patients with ED-SCLC.

#### *3.2. Immune Checkpoint Inhibitors in First-Line Setting-PD-1/PD-L1 Blockade*

Previous reports showed that PD-L1 is expressed in immune cells infiltrating the SCLC stroma, thus suggesting the potential of enhancing tumor-specific T-cell immunity by targeting the PD-1/PD-L1 pathway. Three phase III studies and one phase II study have been designed to evaluate the safety and efficacy of atezolizumab, durvalumab, pembrolizumab and nivolumab in combination with platinum–etoposide-based chemotherapy.

The efficacy of atezolizumab combined with carboplatin etoposide was evaluated in the phase III IMpower 133, designed to demonstrate the OS and PFS improvement of the combination over carboplatin–etoposide in 403 patients with naïve ED-SCLC. Patients were stratified according to sex, ECOG PS, and the presence of brain metastases [34]. In the presence of clinical benefit, patients were allowed to continue therapy beyond progression. Prophylactic cranial irradiation was allowed following the first four cycles, during the atezolizumab/placebo maintenance phase. The trial reached the primary end-points, showing a statistically significant reduction in the risk of death of 30%, and a progression of 23% in patients receiving atezolizumab. One-year OS rate was 13% higher in the atezolizumab group than in the placebo group (51.7 vs. 38.2%). The benefit was independent of the biomarkers analyzed, since neither PD-L1 expression nor blood

tumor molecular burden were predictive of the outcome, and this was observed in all the subgroups analyzed except for those patients with treated brain metastases. However, this was an exploratory, and not a pre-planned, analysis performed on a limited number of cases, and no definitive conclusion can be drawn. Based on these results, the combination of carboplatin, etoposide and atezolizumab has become the new referral standard treatment for naive patients with ED-SCLC.

The CASPIAN trial was a randomized, phase III study, designed to compare the efficacy of the PD-L1 inhibitor durvalumab, with or without the CTLA-4 inhibitor tremelimumab, in combination with platinum–etoposide in 805 patients with untreated ED-SCLC [35]. In the case of progression, continuation of treatment was allowed in the presence of clinical benefits. The co-primary end-points were OS for durvalumab platinum– etoposide versus chemotherapy, and for durvalumab tremelimumab platinum–etoposide versus chemotherapy. The adding of durvalumab tremelimumab did not significantly prolong OS over chemotherapy. Conversely, as already observed in the IMpower 133 trial, the combination of durvalumab with platinum–etoposide significantly improved OS. No significant difference was evidenced in terms of PFS between the treatment arms. A survival benefit was also found with durvalumab in the group of patients with untreated brain metastases. These results confirm the lack of any additional benefit with the CTLA-4 blockade in unselected patients with ED-SCLC. Based on these findings, durvalumab in combination with cisplatin or carboplatin etoposide has been approved by the Food and Drug Administration (FDA) and the European Medicine Agency (EMA) for use in untreated patients with ED-SCLC.

The phase III KEYNOTE 604 trial was designed to investigate the efficacy of the PD-1 inhibitor pembrolizumab, combined with platinum–etoposide, over chemotherapy alone in 453 naive patients with ED-SCLC [36]. The co-primary end-points were OS and PFS. The trial reached one of its primary end-points, showing a significant PFS improvement in chemotherapy and immunotherapy, which did not translate into prolonged OS, despite the numerical benefit in OS being comparable to those achieved by IMpower 133 and CASPIAN. However, double the percentage of patients in the experimental arm were alive at two years compared with chemotherapy alone, thus suggesting that a subgroup of patients obtain a durable benefit from the combination. Compared to the IMpower 133 and the CASPIAN trials, more patients with brain metastases (with ECOG PS of 1) with large tumor dimensions, elevated LDH, and more metastatic sites were enrolled in the KEYNOTE 604 trial. These differences in the populations might have influenced the final results.

Finally, the PD-1 inhibitor nivolumab was tested in combination with platinum– etoposide in the phase II EA5161 trial, enrolling 160 naive patients with ED-SCLC [37]. The study reached its primary end-point, showing a significant PFS improvement in the experimental arm.

In conclusion, the durable benefit observed with the addition of PD-1 or PD-L1 inhibitors to platinum–etoposide has modified the therapeutic strategies recommended in patients with ED-SCLC after several decades. This is particularly remarkable in such an aggressive disease, where, historically, it has been challenging to overcome the long-term survival benefit obtained with platinum–etoposide.

#### *3.3. Immune Checkpoint Inhibitors as a Maintenance Strategy*

Aiming to prolong survival, immune checkpoint inhibitors have been tested in the maintenance setting in those patients with stable or responsive ED-SCLC following four to six cycles of platinum–etoposide, but with poor results.

Pembrolizumab was evaluated in a phase II study, designed to demonstrate in 45 patients a PFS improvement of 50%, compared with historical data [38]. Pembrolizumab was administered within eight weeks from the end of chemotherapy. Despite no significant PFS or OS improvement being observed among the patients enrolled, the 1-year PFS and OS rates were 13% and 37%, respectively, thus suggesting that a subgroup of patients might receive durable benefits from this strategy.

Similar results were found in the phase III CA 451 study, designed to compare nivolumab with the combination of ipilimumab + nivolumab or a placebo in 834 patients with ED-SCLC, who did not progress following four cycles of platinum-based chemotherapy [39]. The study failed to demonstrate OS improvement in nivolumab + ipilimumab patients over placebo patients, and of nivolumab over placebo.

These findings suggest that the timing of checkpoint inhibitors administration is important in improving the survival benefit in patients with ED-SCLC, and a maintenance strategy with immune checkpoint inhibitors is not suggested.

#### *3.4. Immune Checkpoint Inhibitors in Recurrent Patients*

Controversial results have been observed in the studies evaluating the efficacy of pembrolizumab, nivolumab, durvalumab and atezolizumab in patients progressing after platinum–etoposide-based chemotherapy.

The activity of pembrolizumab as a monotherapy in the second/third-line setting was assessed in two trials, the phase Ib KEYNOTE 028 trial and the phase II KEYNOTE 058 trial. The KEYNOTE 028 trial [40] was a multi-cohort phase Ib study, designed to define the preliminary effects of pembrolizumab in different cohorts of patients with solid tumors. The presence of PD-L1 expression in at least 1% of tumors and inflammatory cells represented one of the inclusion criteria to enter the study, the primary end-point of which was to determine the percentage of objective responses. One of the cohorts included 24 patients with ED-SCLC progressing to at least one previous line of chemotherapy. Among these, approximately half of them had received the second line with topotecan or irinotecan. An ORR of 33.3% was identified, with a median duration of response of 19.4 months, thus suggesting a promising clinical activity of pembrolizumab in this setting. In order to confirm these findings in a larger population, the phase II KEYNOTE 058 trial enrolled patients progressing to standard treatment [41]. Similarly, to the KEYNOTE 028 trial, the primary end-point here was the percentage of objective response. Differently from KEYNOTE 028, here, PD-L1 positivity was not an inclusion criterion, even though to enter the trial the collection of evaluable tumor samples was mandatory, in order to centrally test PD-L1 expression. One hundred and seven patients with ED-SCLC were included, and an ORR of 18.7% was registered. The percentage of responses was six times higher in PD-L1-positive compared to PD-L1-negative tumors (35.7% vs. 6.0%), and longer OS was observed in PD-L1-positive patients (14.6 vs. 7.7 months). A pooled analysis of the two studies, including only those patients who received two lines of chemotherapy (83/131), showed that 88% of responder patients had PD-L1-positive tumors, thus suggesting the importance of selecting patients according to PD-L1 expression [42]. Nivolumab was tested in the CheckMate 032 and CheckMate 331 trials.

The CheckMate 032 was a phase I/II study investigating the activity and safety of nivolumab as a monotherapy, or in combination with ipilimumab, in metastatic patients with different solid tumor types [43]. Two hundred and sixteen patients with LD or ED-SCLC progressing after one or more previous regimens were enrolled into four cohorts in a sequential manner. In each cohort, a different regimen was administered, including nivolumab 1 mg/kg + ipilimumab 1 mg/kg, nivolumab 1 mg/kg + ipilimumab 3 mg/kg, or nivolumab 3 mg/kg + ipilimumab 1 mg/kg. The combination was continued for four cycles, followed by nivolumab until progression or unacceptable toxicity. The percentage of objective response was the primary end-point of the trial. The ORR was 10% in patients receiving nivolumab, 23% in those included in the nivolumab 1 mg/kg + ipilimumab 3 mg/kg cohort, 19% in those receiving nivolumab 3 mg/kg + ipilimumab 1 mg/kg, and 33% in the group treated with nivolumab 1 mg/kg + ipilimumab 1 mg/kg. Tissue was available for PD-L1 assessment in 69% of cases. Conversely from what was observed with pembrolizumab, tumor responses occurred independently of PD-L1 expression. Despite the limited number of patients enrolled, the preliminary analysis showed a similar efficacy between platinum-sensitive and -resistant patients, and between those previously treated with one, or two or more, lines of therapy [44]. To confirm these findings, the phase III CheckMate 331 trial was designed. However, the study did not reach the primary end-point of showing the improved OS of nivolumab over

chemotherapy (topotecan or amrubicin) in 569 patients with SCLC previously treated with one line of platinum–etoposide [45].

**Table 2.** Clinical trials exploring immune checkpoint inhibitors in patients with ED-SCLC.

I: ipilimumab. PC: paclitaxel, carboplatin. CE: etoposide, carboplatin. DPE: durvalumab, platinum\*, etoposide. DTPE: durvalumab, tremilimumab, platinum\*, etoposide. \* cisplatin or carboplatin was allowed according to investigators' choice. PFS: progression-free survival. ORR: objective response rate. (#): includes all the patients receiving nivolumab + ipilimumab irrespective of the schedule used.

> The efficacy of durvalumab was evaluated in a phase I/II study, enrolling 21 patients with pretreated ES-SCLC [46], whose primary objective was to determine the safety profile of durvalumab. An ORR of only 9.5% was registered.

> Finally, atezolizumab did not demonstrate any advantage over topotecan or reinduction of chemotherapy in 73 patients with pretreated ED-SCLC, enrolled in the phase II IFCT-1603 trial [47].

> In conclusion, immune checkpoint inhibitors have not shown a significant advantage in relapsing patients with ED-SCLC, despite the fact that a subgroup of them might experience a durable clinical benefit. Biomarkers able to select those patients who benefit more have not been identified yet, despite results from the KEYNOTE 028 and the KEYNOTE 058 studies suggesting that PD-L1 expression could be considered. However, these findings were not confirmed in the studies testing nivolumab, where the activity was observed irrespective of PD-L1 status. Finally, in the trials evaluating durvalumab and atezolizumab, patients were not selected according to PD-L1 expression, and no definitive conclusions could be drawn [48].

#### **4. Second Line and Beyond**

Those patients relapsing or progressing during first-line treatment are classified as refractory. In the case of progression within 3 months, they are considered platinumresistant. If relapse is observed after 3 months (the last cycle of first-line chemotherapy), they are considered platinum-sensitive.

According to ESMO guidelines, in sensitive patients, a rechallenge with drugs used in first-line therapy is allowed. However, the lack of PFS and OS benefit was observed in a retrospective analysis, including data from 65 patients with sensitive ED-SCLC [49]. On the other hand, a systematic analysis, including data from 1692 sensitive and refractory patients with SCLC, showed higher ORR and longer OS in sensitive patients [50].

Different compounds have been tested as therapeutic strategies in refractory patients with ED-SCLC (Table 3).

**Table 3.** Clinical trials exploring second-line therapies in patients with ED-SCLC.

PFS: progression-free survival. ORR: objective response rate.

Among these, topotecan failed to demonstrate improved efficacy in terms of ORR over CAV in a randomized trial enrolling 211 relapsing patients with ED-SCLC. No significant difference was observed in terms of ORR, PFS or OS between the two arms, although a greater proportion of patients in the topotecan group showed improved symptoms [51]. Thanks to these findings, topotecan was approved for the second-line treatment of sensitive SCLC patients.

The efficacy of topotecan was compared to amrubicin in a phase II trial enrolling 76 patients with ED-SCLC who were sensitive to previous first-line platinum-based chemotherapy [52]. ORR was the primary end-point of the study. The results showed significantly higher ORR with amrubicin, and longer, but non-statistically significant, OS. The limited number of patients enrolled in the phase II study and the lack of independent confirmation of responses might have influenced the final results. A phase III trial is currently ongoing to confirm (or not) these data.

The promising results from a phase II study with lurbinectedin in 105 refractory patients with SCLC, showing an ORR of 35% with a median response duration of 5.3 months [54]. secured lurbinectedin the accelerated approval of the FDA for patients with metastatic SCLC previously treated with platinum-based chemotherapy. However, the phase III ATLANTIS trial, comparing lurbinectedin in combination with doxorubicin versus the physician's choice of topotecan or CAV, failed to demonstrate an OS improvement in 613 patients with SCLC progressing to one previous line of platinum-based chemotherapy.

Alternative strategies to chemotherapy and immune check point inhibitors, including compounds targeting angiogenesis, have been tested. Among these, a single-arm phase II trial safely combined NGR-hTNF, a vascular-targeting agent (0.8 μg/m2), which increases intra-tumoral chemotherapy penetration and T-lymphocyte infiltration [55], with doxorubicin (75 mg/m2 every 3 weeks), showing manageable toxicity and promising activity in unselected platinum-resistant or platinum-sensitive patients with relapsed ED-SCLC. The primary endpoint was PFS. Safety, ORR and OS were the secondary end-points. The median PFS was longer in platinum-sensitive compared to platinum-resistant patients (4.1 vs. 2.7 months, respectively). Prolonged OS was observed in those patients with increased lymphocyte counts [53]. The preclinical data indicate that NGR-hTNF modifies the composition of the tumor microenvironment, including the infiltration of CD8+ T cells, and favors the secretion of cytokines and chemokines [55], thus suggesting that NGR-hTNF might enhance the efficacy

of immune checkpoint inhibitors [56]. NGR-hTNF combined with immunotherapy might represent a strategy to be evaluated in patients with relapsed SCLC.

All the agents tested have shown modest activity. To date, topotecan represents the only approved drug for the second-line treatment of patients with ED-SCLC.

#### **5. Discussion and Future Directions**

SCLC remains the most aggressive form of neuroendocrine tumor of the lung, for which treatment options remain limited. The characterization of the tumor biology of SCLC has been challenging, mainly due to the limited availability of tumor tissue, since surgery represents an option confined to a small number of cases. The high number of somatic mutations, generally including loss of function mutations or deletions in tumor suppressor genes, have further increased the difficulty of developing selected targeted therapies. Alterations in the genes involved in cell cycle regulation or DNA damage response, including RB transcriptional corepressor 1 (RB1) and tumor protein p53 (TP53), are commonly observed in SCLC [57]. The amplification of the chromosomal regions, including L-myc [58] or C-myc [59], the overexpression of the cyclin D1 [57], the alteration of PTEN [60] or of genes involved in transcriptional regulation and chromatin modification, the presence of inactivating mutations in NOTCH, and the overexpression of genes responsible for DNA damage response, represent some of the molecular aberrations characterizing the genomic landscape of SCLC [61]. To date, the majority of the trials investigating a molecular approach have provided disappointing results, mainly because the patients enrolled were not selected according to specific driver molecular alterations. The introduction of immune checkpoint inhibitors has improved systemic treatments after decades (Figure 1). However, patients' prognoses remain dismal, and new therapeutic approaches are required. Tissue collection is strongly advocated to gain new insights into the biology of this complex disease. The implementation of non-invasive methods, including the analysis of circulating tumor DNA and the use of circulating tumor cells (CTC), might become tools to overcome the inadequate amounts of tumor samples, in order to dissect the pathogenesis of SCLC and discover new targetable molecular alterations. Preclinical studies using CTC-derived explants (CDX) might be useful to identify the bypass signaling of acquired resistance, and design combinatorial strategies to overcome these pathways.

**Figure 1.** (**A**) Immunotherapy treatment strategies currently approved or tested in patients with ED-SCLC. (**B**) Approved chemotherapy treatments for patients with ED-SCLC. (**C**) Potentially targetable and identified molecular pathways in ED-SCLC.

**Author Contributions:** Conception: V.G.; collection and assembly of data: C.L. and A.M.; manuscript writing: all authors; (VII) final approval of manuscript: all authors. Image: S.I. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** N/A.

**Informed Consent Statement:** N/A.

**Data Availability Statement:** N/A.

**Acknowledgments:** Alliance Against Cancer.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

## *Review* **Pathology and Classification of SCLC**

**Maria Gabriela Raso \*, Neus Bota-Rabassedas and Ignacio I. Wistuba \***

Department of Translational Molecular Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA<EMAIL_ADDRESS>
**\*** Correspondence<EMAIL_ADDRESS>(M.G.R<EMAIL_ADDRESS>(I.I.W.);

Tel.: +1-713-834-6026 (M.G.R.); +1-713-563-9184 (I.I.W.)

**Simple Summary:** Small cell lung carcinoma (SCLC), is a high-grade neuroendocrine carcinoma defined by its aggressiveness, poor differentiation, and somber prognosis. This review highlights current pathological concepts including classification, immunohistochemistry features, and differential diagnosis. Additionally, we summarize the current knowledge of the immune tumor microenvironment, tumor heterogeneity, and genetic variations of SCLC. Recent comprehensive genomic research has improved our understanding of the diverse biological processes that occur in this tumor type, suggesting that a new era of molecular-driven treatment decisions is finally foreseeable for SCLC patients.

**Abstract:** Lung cancer is consistently the leading cause of cancer-related death worldwide, and it ranks as the second most frequent type of new cancer cases diagnosed in the United States, both in males and females. One subtype of lung cancer, small cell lung carcinoma (SCLC), is an aggressive, poorly differentiated, and high-grade neuroendocrine carcinoma that accounts for 13% of all lung carcinomas. SCLC is the most frequent neuroendocrine lung tumor, and it is commonly presented as an advanced stage disease in heavy smokers. Due to its clinical presentation, it is typically diagnosed in small biopsies or cytology specimens, with routine immunostaining only. However, immunohistochemistry markers are extremely valuable in demonstrating neuroendocrine features of SCLC and supporting its differential diagnosis. The 2015 WHO classification grouped all pulmonary neuroendocrine carcinomas in one category and maintained the SCLC combined variant that was previously recognized. In this review, we explore multiple aspects of the pathologic features of this entity, as well as clinically relevant immunohistochemistry markers expression and its molecular characteristics. In addition, we will focus on characteristics of the tumor microenvironment, and the latest pathogenesis findings to better understand the new therapeutic options in the current era of personalized therapy.

**Keywords:** pathology and classification of SCLC; biology of SCLC; immune-checkpoint inhibitors in SCLC

#### **1. Introduction**

Lung carcinoma has consistently remained the leading cause of cancer-related death worldwide [1,2]. Currently, in the United States, it ranks as the second most frequent type of new cancer case, both in males and females, accounting for more than 228,800 new cases in 2020 and 12.7% of all new cancer cases [3,4]. Small cell lung carcinoma (SCLC) is a very aggressive, poorly differentiated, and high-grade neuroendocrine carcinoma representing approximately 13% of all lung carcinomas. Most commonly seen in heavy smokers as an advanced stage disease, it clinically presents with early metastatic spread and good responsiveness to initial therapy, and in most patients, it is consistently followed by relapse with a chemo resistant disease [5].

Remarkably, no preneoplastic lesions have been identified in SCLC [6]. This is in stark contrast with the recognition of the diffuse idiopathic pulmonary neuroendocrine

**Citation:** Raso, M.G.; Bota-Rabassedas, N.; Wistuba, I.I. Pathology and Classification of SCLC. *Cancers* **2021**, *13*, 820. https://doi. org/10.3390/cancers13040820

Academic Editors: Alessandro Morabito and Christian Rolfo Received: 21 December 2020 Accepted: 10 February 2021 Published: 16 February 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

cell hyperplasia (DIPNECH) as the precursor lesion for neuroendocrine carcinoids of the lung [7,8], pulmonary neuroendocrine cells (PNECs) are considered putative precursors of small cell lung cancer [9], while the expression of ASCL1 (both in tumors and cell lines) confirms SCLC neuroendocrine lineage [10,11]. However, this concept was challenged with the identification of a distinctive tuft cell variant, showing high POU2F3 expression while lacking neuroendocrine markers, postulating a SCLC-P entity distinctive from the classical neuroendocrine variants, and strongly suggesting a distinct cell-of-origin for this subtype [12]. Alternatively, trans-differentiation towards a tuft cell expression profile from a cell of origin shared with other subtypes remains an alternative explanation. Data from genetically engineered mouse models and human tumors have revealed multiple levels of heterogeneity in SCLC. Additionally, using genetically defined mouse models of SCLC, investigators have uncovered distinct metastatic programs attributable to the cell-of-origin cell-type, concluding that intra-tumoral heterogeneity is influenced by the cell of origin and proposing that SCLC can arise from molecularly distinctive cells.

#### **2. Pathological Classification of SCLC**

SCLC was first described in 1879 by Harting and Hesse and classified as a lymphosarcoma [13]; later, in 1926, it was classified as small 'oat cell' carcinomas of the lung [14]. From that point forward, it has been historically known as oat cell carcinoma [15,16], and belongs to the neuroendocrine lung tumors group defined in the last WHO Classification of Lung Tumors [17]. In 2015, the World Health Organization (WHO) Classification of Tumors of the Lung, Pleura, Thymus, and Heart was published with numerous important changes from the previous 2004 publication. One of the most significant changes in this edition involves the inclusion of neuroendocrine tumors (NE) grouped together in one category, recognizing four major NE tumors: typical carcinoid tumor (TC), atypical carcinoid tumor (AC), SCLC, and large cell NE carcinoma (LCNEC) [17]. The current sub-classification recognizes two subtypes: pure SCLC and combined SCLC, the latter is determined by the presence of features of a different histological carcinoma, non-small cell lung carcinoma (NSCLC), variant or at least containing 10% of large cell carcinoma component. Up to 28% of surgically resected SCLC tumors are combined SCLC, and of these, 16% belong to the large cell carcinoma variant [18,19].

#### **3. Macroscopic Features of SCLC**

As a highly aggressive disease, SCLC is found disseminated in the initial clinical presentation in the majority of cases, and surgery is limited to a small subgroup of patients with confined disease [20]. Usually, these tumors undergo surgical resection only when diagnosis of SCLC has not been previously established, and it is from those infrequent surgical specimens that a macroscopic description of SCLC tumors is described. These tumors are grossly identifiable as fleshy, ranging from a white and grayish to a light tan color, soft and friable irregular masses, with necrotic cut surface areas. The majority of them are situated in hilar or perihilar areas, with less than 5% of the cases presenting in peripheral locations. Invasion into the peribronchial tissue and lymph node is often grossly identifiable, typically spreading in circumferentially along the submucosa of the bronchi.

#### **4. Microscopic Features of SCLC**

#### *4.1. Cytological Features*

Cytology is a very reliable method to establish a SCLC diagnosis and often a valuable complement to bronchial biopsies where crush artifacts may hamper a definitive diagnosis. The most common cytologic feature is highly cellular aspirates with presence of small blue cells with very scant or null cytoplasm, loosely arranged or in a syncytial pattern (Figure 1). Within tightly cohesive sheets, nuclear molding is well developed. Additionally, a background of single cells, doublets, triplets, and small cell cords with extensive necrosis are characteristic of this entity. The mitotic rate is usually very high (10 mitosis per 10 high power fields) and chromatin smearing is very frequent. Well-preserved cells show the

g

characteristic "salt and pepper" chromatin patter, while less well-preserved cells show dark blue chromatin. Cytoplasmic globules, also named paranuclear blue bodies, are considered distinctive of a SCLC diagnosis [21,22].

**Figure 1.** Small cell lung carcinoma (SCLC) cytological and histological characteristics. (**a**,**b**) High power field view of SCLC in cellular aspirates. Loosely arranged small blue cells with scant cytoplasm and fine chromatin features (arrow).

#### *4.2. Histopathological Features*

Hematoxylin and eosin (H&E) assessment of a SCLC tumor under a light microscope main features is defined by the presence of diffuse sheets of small round to fusiform cells, with scant cytoplasm, and inconspicuous or absent nucleoli with finely granular nuclear chromatin (Figure 2). Nuclear chromatin smearing, also called crushed artifact, is a common feature together with nuclear molding (Figures 2 and 3). Extensive intratumoral necrosis is often recognized, as well as the high mitotic index that defines the tumor (average of 40 mitosis/mm2 area) [18,23,24] (Figure 2). In addition to the common diffuse sheetlike growth pattern, SCLC tumor can demonstrate other growth features as peripheral palisading, streams, ribbons, organoid nesting, and rosettes [18,23]. Another characteristic feature is the frequent basophilic nuclear debris encrustation of the blood vessel wall, widely known as Azzopardi effect (Figure 4) [25,26]. Larger specimens may present a variety of cellular characteristics like larger cells, pleomorphic cells, giant tumor cells, and dispersed chromatin with prominent nucleoli, among others.

**Figure 2.** Small cell lung carcinoma (SCLC) cytological and histological characteristics. High power field view of a formalin fixed paraffin embedded tissue H&E stained slide showing in (**a**). sheets of small cells with scant cytoplasm and nuclear molding and (**b**). Sheets of tightly packed small cells and intratumoral necrosis.

**Figure 3.** Chromatin smearing "crush artifact". High magnification showing Small cell lung carcinoma (SCLC) with extensive chromatin smearing "crush artifact".

**Figure 4.** Azzopardi phenomenon. High magnification showing Small cell lung carcinoma (SCLC) with basophilic nuclear debris encrustation of the blood vessel wall (arrow).

SCLC can be observed as a pure SCLC histology or combined with NSCLC histology features. The latter refers to the admixture of NSCLC elements; where adenocarcinoma, squamous cell carcinoma (SCC), and large cell carcinoma can be seen, as well as, although at a lower frequency, giant cell carcinoma and spindle cell carcinoma [17]. To establish a diagnosis of combined SCLC and large cell carcinoma, large cells have to constitute at least 10% of the tumor; however, no assessment of the percentage of the secondary histology is necessary to render a combined SCLC diagnosis for other NSCLC histologies [19].

#### *4.3. Immunohistochemistry*

The histopathological diagnosis is based on light microscopy using routine H&E stained slides, as stated in the current 2015 WHO classification. Immunohistochemistry (IHC) assays are required when confirmation of equivocal features is necessary or when a differential diagnosis must be addressed, especially in small biopsies with crush artifacts (Table 1).

**Table 1.** Immunohistochemistry (IHC) markers and immunohistochemical characteristics of SCLC.

SCLC stains positively for low molecular weight keratins, showing a dot-like paranuclear or diffuse cytoplasmic staining, and is negative for high molecular weight keratins. SCLC shows a variable expression of neuroendocrine differentiation markers such as neural cell adhesion molecule (NCAM/CD56), chromogranin, synaptophysin, and insulinomaassociated protein 1 (INSM1), and usually presents lower protein levels than low-tointermediate grade neuroendocrine tumors. It is important to note that a minority of SCLCs are negative for all standard neuroendocrine markers. Importantly, immunohistochemistry has only a limited role in distinguishing LCNEC and SCLC. One potentially helpful, although with low-sensitivity, feature is focal and weak napsin A immunoreactivity, which can be seen in up to 15% of LCNEC [27] while SCLC is consistently negative for this marker. Thus, when present, weak napsin A labeling in a high-grade NE carcinoma may favor the diagnosis towards LCNEC. Thyroid transcription factor-1 (TTF-1) is a homeodomain-containing transcription factor selectively expressed in pulmonary adenocarcinomas, thyroid tumors, and small cell carcinomas. Interestingly, TTF-1 is expressed in close to 90% of SCLC, which can be useful for differentiating SCLC from neuroendocrine cancers not originating from the lung.

High Ki67 (MIB-1) labeling index (>50%, usually 70–100%) is a hallmark of SCLC, helping in distinguishing it from low- and intermediate-grade neuroendocrine cancers [28]. Other markers used to characterize SCLC are: B-cell lymphoma 2 (BCL2; expressed often), P16 nuclear staining (expressed in 95–100% of cases), tyrosine-protein kinase KIT (CD117/c-KIT; expressed in about 60% of cases, retinoblastoma protein (P-RB; always negative), and homeobox protein orthopedia (OTP, always negative) [29].

#### **5. Differential Diagnosis of SCLC**

Although SCLC has a very well-defined diagnostic criteria, sampling issues, fixation artifacts, and the morphologic variability of pure SCLC tumor cells, can all potentially make the diagnosis of SCLC challenging.

The current WHO classification of lung cancer recognizes four neuroendocrine tumors, namely typical carcinoid tumor (TC), atypical carcinoid tumor (AC), SCLC, and large cell NE carcinoma (LCNEC). Neuroendocrine tumors have a wide differential diagnosis based on their mitotic rate and necrosis extension. Other histologies, such as basaloid squamous cell carcinoma, small round cell sarcoma, metastatic breast carcinoma, and non-Hodgkin's lymphoma, may be considered in the differential diagnosis of SCLC. In these cases, it is important to perform IHC in order to confirm its neuroendocrine differentiation and epithelial nature, detect other differentiation markers, as well as to assess the site of origin

and proliferation rate. As an example, p63 and TTF-1 expression is useful in distinguishing SCLC from poorly differentiated nonkeratinizing SCC: p63 positive and TTF-1 negative expression consequently indicates a poorly differentiated nonkeratinizing SCC, while an opposite immunostaining pattern identifies a SCLC diagnosis.

#### **6. Landscape of the Immune Tumor Microenvironment in SCLC**

Current evidence shows that the immune system is capable of generating antitumor responses against various tumors, including lung cancer, suggesting that immunotherapy may be a viable therapeutic approach for patients with SCLC [30]. Increasing evidence shows that the immune system is involved in the pathophysiology of SCLC [31]. Moreover, confirmation that SCLC is immunogenic comes from the relationship between immune activity and prognosis. For instance, more infiltrating CD45+ T-cells in SCLC tumors were found to be a predictor of better OS, independently of stage and performance status. In addition, more effector T-cells were found in limited stage disease (LD) SCLC compared with extended stage disease (ED) SCLC cases, and higher effector-to-regulatory T-cell ratios were associated with longer survival [22,30,32].

As it is known, NSCLC patients with a high mutation burden have been shown to be particularly sensitive to immunotherapeutic agents that inhibit the PD-1 pathway [30,33,34], indicating that SCLC could potentially benefit from immunotherapy treatments, given its high mutation burden [35]. However, several mechanisms of immune resistance have been identified in SCLC, such as downregulation of major histocompatibility complex (MHC) antigens I and II, low PD-L1 expression, increase in regulatory T-cells, and poor tumor infiltration of effector T-cells, partially due to a lack of vasculature surrounding the tumor [36].These features likely diminish the response in SCLC compared to the observed immunotherapeutic response in NSCLC.

Immunotherapies targeted against programmed death ligand 1 (PD-L1) and its receptor (PD-1) have improved survival in a subset of NSCLC patients. PD-L1 protein expression has emerged as a biomarker that predicts which patients are more likely to respond to immunotherapy. Noticeably, PD-L1 expression has been demonstrated in SCLC, although at lower levels than NSCLC. In one retrospective study of 102 SCLC specimens, PD-L1 expression was observed in 72% of tumor cells and was associated with significantly longer overall survival (OS) [37]. In contrast, another study involving 94 clinical cases of small cell NE carcinoma found PD-L1 expression in 19% of cases, and only expressed in stromal cells [38]. Similarly, others have reported PD-L1 expression in tumor cells, ranging from 27% to 89% [39,40]. The inconsistency of these findings is most likely related to differences in PD-L1 detection methods, the utilization of different IHC platforms and antibodies, as well as the lack of standardized scoring methods [41].

Regulatory CD4+ T-cells play a major role suppressing other immune cells, lessening the immune response. A study demonstrated the ability of SCLC cell lines to induce a de novo differentiation from activated CD4+ T-cells to regulatory CD4+ T-cells (FOXP3+ T-cells), by secretion of IL-15 [42]. The same study, including data from a cohort of 65 SCLC patients, demonstrated the presence of IL-15 in SCLC biopsies, and a positive correlation between increase in tumor infiltration of FOXP3 T-cells and worse OS [42], opening new avenues for future immune therapies against SCLC.

MHC proteins are usually present at the cell surface of tumor cells, playing a central role in antigen presentation to activate cytotoxic T-cells and elicit antitumor immune response. Strikingly, it is another component of SCLC suppressed immunogenicity, as both MHC class I and II expression have been reported to be decreased in SCLC [43,44].

Overall, there is solid evidence that SCLC has the potential to be immunogenic. This could be achieved by counteracting the immunosuppressive nature of this tumor type, and for this purpose, further research is needed to understand the specific pathways that drive SCLC-mediated immunosuppression.

#### **7. SCLC Tumor Heterogeneity**

Evidence of intra-tumoral heterogeneity in SCLC was demonstrated many years ago by the diverse expression of antigens detectable with monoclonal antibodies [45]. Contemporary work from animal models such as genetically engineered mouse models (GEMMs) and patient-derived xenograft/cell-derived xenograft (PDX/CDX) models has shown new levels of cellular complexity in SCLC. The presence of several subpopulations of SCLC cells and their functional interactions may explain the outstanding plasticity of SCLC tumors and their striking metastatic potential [46,47]. Additionally, SCLC tumors that look similar at the histopathological level may represent distinct subtypes of tumors, and these differences have an impact on the response to specific therapeutic agents. A better understanding of genetic and cellular heterogeneity will guide the development of personalized approaches to help SCLC patients [46].

#### **8. Genetic Variation of SCLC**

SCLC is a molecularly complex disease comprising numerous genetic alterations, including variations in tumor suppressor genes, copy number variation, somatic mutations in transcription factors, chromatin modification, and receptor tyrosine kinases or their downstream signaling components [48] (Table 2). Notably, 95% of tumors reveal loss of *Retinoblastoma 1* (*RB1*) and more than 65% present *TP53* mutations [49–53]. In addition, frequent allelic loss involving chromosome arm 3p deletion has been reported in 91% of SLCC tumors [54]. Mutations in *v-myc avian myelocytomatosis viral oncogene homolog* (*MYC*) family genes, *BCL2*, *Phosphatase and tensin homolog* (*PTEN*), *Slit homolog 2* (*SLIT2*), *CREB binding protein* (*CREBBP*), *Ephrin type-A receptor 7* (*EPHA7*), and *Fibroblast Growth Factor Receptor 1* (*FGFR1*) mutations have been identified [35]. Other molecular abnormalities, such as increased expression of c-KIT, amplification of *MYC* family members (*MYC*, *MYC lung carcinoma-derived homolog 1* [*MYCL1*], *MYC neuroblastoma-derived homolog* [*MYCN*]), and loss of *PTEN* have also been described in subsets of SCLC [54–58].

Recently, comprehensive genomic research studies with cell lines, GEMMs, and PDX models have revealed biologically heterogenous SCLC subtypes (Table 3), based on mRNA expression profiles defined by the differential expression of four key transcription regulators: *achaete-scute homologue 1* (*ASCL1*; also known as *ASH1*), *neurogenic differentiation factor 1* (*NeuroD1*), *yes-associated protein 1* (*YAP1*), and *POU class 2 homeobox 3* (*POU2F3*). As a result of such studies, recent consensus suggested grouping SCLC into four subtypes defined by mRNA expression of *ASCL1*, *NEUROD1*, *POU2F3*, and *YAP1*. Consequently, a working nomenclature for SCLC subtypes defined by the relative expression of these four factors was proposed, classifying cases as: ASCL1 high (SCLC-A), NEUROD1 high (SCLC-N), POU2F3 high (SCLC-P), and YAP1 high (SCLC-Y) [12,59–61]. SCLC-Y is characterized by WT *RB1* enrichment [62] and low or absent expression of *ASCL1*, *NEUROD1*, and other

neuroendocrine markers, accounting for approximately 5% to 10% of SCLC tumors. In most instances of combined SCLC with NSCLC histologies, there is a strong evidence of clonality between SCLC and NSCLC components, likely indicating a common precursor cell in such cases [12,63]. When comparing the morphological features and considering the similar genetics and genomics characteristics between SCLC-Y (WT *RB1*) and LCNEC (WT *RB1*, WT *KEAP1*, WT *STK11*) subtypes, together with a low or absent expression of *ASCL1* and *NEUROD1*, it has been suggested that these two lung cancer subtypes belong to a single entity [62]; thus, implying potential impact in molecular classification and clinical implications. Recently, a first comprehensive IHC-based study in patient samples has confirmed highly distinct characteristics of SCLC tumors expressing *ASCL1* (SCLC-A) and/or *NEUROD1* (SCLC-N) (86%) compared to SCLC lacking these markers (14%) [59]. The former is associated with a high NE program (NE-markers high/TTF-1 high/DLL3 high) and a pure SCLC histology, whereas the latter exhibits an enrichment in a combined SCLC histology. The same study also confirmed a highly distinctive nature for *POU2F3* expressing tumors (SCLC-P), which account for 7% of SCLC [59]. Using tumor expression data and non-negative matrix factorization, Gay et al. identified four SCLC subtypes defined largely by differential expression of transcription factors ASCL1, NEUROD1, and POU2F3 or low expression of all three transcription factor signatures accompanied by an Inflamed gene signature (SCLC-A, N, P, and I, respectively). Stating that the SCLC-I subgroup may benefit from the addition of immunotherapy to chemotherapy, while the other subtypes each have distinct vulnerabilities, including to inhibitors of PARP, Aurora kinases, or BCL-2 [64].

**Table 3.** Molecular classification of Small cell lung carcinoma (SCLC) subsets.

Albeit uncommon, transformation of NSCLC into SCLC has been observed as a resistance mechanism upon treatment of *EGFR*-mutated NSCLC with *EGFR* tyrosine kinase inhibitors (TKI) (3% to 10% of *EGFR*-TKI resistant cases) [65,66]. Interestingly, NSCLC to SCLC transformation is dependent on EGFR mutation, as only rare instances of such transformation have been reported in EGFR-WT NSCLC, and it is often accompanied by mutations in *TP53*, *Rb1*, and *PIK3CA* [66–70].

#### **9. Conclusions**

SCLC represents one of the most lethal forms of cancer, with limited successful therapeutic options and consequently presenting striking low survival rates in late stages. It is reckoned as a high-grade neuroendocrine lung carcinoma and it is classified as either pure SCLC or combined SCLC, the latter when a portion of the tumor shows NSCLC features. Although the histological diagnosis is based mostly in H&E specimens, IHC can prove very helpful in the differential diagnosis setting. Intratumoral heterogeneity is thought to be related with its remarkable plasticity and striking metastatic potential. Histologically similar SCLC tumors may actually represent distinct subtypes of tumors, linked to disparate response outcomes to specific therapeutic agents. Recent molecular classifications approaches are paving the way to a deeper understanding of this entity and to related potential treatment approaches. Further knowledge of this entity, including both

its genetic features and cellular heterogeneity, as well as its tumor microenvironment, will guide the development of personalized therapeutic strategies for SCLC patients. In the authors opinion, an exciting new era of molecular driven treatment decisions is finally foreseeable in the near future for SCLC patients.

**Funding:** This work was funded by the Specialized Program of Research Excellence (SPORE) from the National Cancer Institute [Grant 1-P50-CA70907-01].

**Conflicts of Interest:** I.I.W. receives research funding from Genentech, Oncoplex, HTG Molecular, DepArray, Merck, Bristol-Myers Squibb, Medimmune, Adaptive, Adaptimmune, EMD Serono, Pfizer, Takeda, Amgen, Karus, Johnson & Johnson, Bayer, Iovance, 4D, Novartis, and Akoya. I.I.W. sits on the advisory board of the following companies: Genentech/Roche, Bayer, Bristol-Myers Squibb, Astra Zeneca/Medimmune, Pfizer, HTG Molecular, Asuragen, Merck, GlaxoSmithKline, Guardant Health, Oncocyte, and MSD. The funders had no role in the writing of the manuscript.

#### **References**

## *Review* **PARP Inhibitors in Small-Cell Lung Cancer: Rational Combinations to Improve Responses**

**Erik H. Knelson 1, Shetal A. Patel 2 and Jacob M. Sands 1,\***

**\*** Correspondence<EMAIL_ADDRESS>
**Simple Summary:** Small-cell lung cancer carries a dismal prognosis with few long-term treatment options. The enzyme poly-(ADP)-ribose polymerase (PARP), which functions to repair DNA breaks, has emerged as a promising therapeutic target, with modest response rates in early clinical trials prompting investigation of predictive biomarkers and therapeutic combinations. This review summarizes the development and testing of PARP inhibitors in small-cell lung cancer with an emphasis on developing treatment combinations. These combinations can be divided into three categories: (1) contributing to DNA damage; (2) inhibiting the DNA damage response; and (3) activating the immune system. An evolving classification of small-cell lung cancer subtypes and gene expression patterns will guide PARP inhibitor biomarker identification to improve treatments for this challenging cancer.

**Abstract:** Despite recent advances in first-line treatment for small-cell lung cancer (SCLC), durable responses remain rare. The DNA repair enzyme poly-(ADP)-ribose polymerase (PARP) was identified as a therapeutic target in SCLC using unbiased preclinical screens and confirmed in human and mouse models. Early trials of PARP inhibitors, either alone or in combination with chemotherapy, showed promising but limited responses, suggesting that selecting patient subsets and treatment combinations will prove critical to further clinical development. Expression of SLFN11 and other components of the DNA damage response (DDR) pathway appears to select for improved responses. Combining PARP inhibitors with agents that damage DNA and inhibit DDR appears particularly effective in preclinical and early trial data, as well as strategies that enhance antitumor immunity downstream of DNA damage. A robust understanding of the mechanisms of DDR in SCLC, which exhibits intrinsic replication stress, will improve selection of agents and predictive biomarkers. The most effective combinations will target multiple nodes in the DNA damage/DDR/immune activation cascade to minimize toxicity from synthetic lethality.

**Keywords:** SCLC; PARP; DDR; ICB; synthetic lethality; SLFN11; STING

#### **1. Introduction**

Small-cell lung cancer (SCLC) is a high-grade neuroendocrine malignancy with a poor prognosis that accounts for 13% of all lung cancer diagnoses [1,2]. First-line treatment for extensive-stage SCLC (ES-SCLC) is often effective, with a response rate of more than 60% to platinum-based chemotherapy, but prior to recent first-line advances, median overall survival was less than 11 months [2,3]. Immune checkpoint blockade (ICB) using inhibitors of the programmed cell death protein and its ligand (PD-1/PD-L1) initially showed promise in the third-line setting, and inclusion into first-line platinum-based therapy has demonstrated an overall survival benefit, becoming the new standard of care [4–7] with particular improvement in durable responses. Until recently, topotecan has been the only option approved by the United States Food and Drug Administration (FDA) in the second-line setting but has not been widely utilized due to concerns about toxicity

**Citation:** Knelson, E.H.; Patel, S.A.; Sands, J.M. PARP Inhibitors in Small-Cell Lung Cancer: Rational Combinations to Improve Responses. *Cancers* **2021**, *13*, 727. https:// doi.org/10.3390/cancers13040727

Academic Editors: Alessandro Morabito and Christian Rolfo

Received: 2 January 2021 Accepted: 8 February 2021 Published: 10 February 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

and only modest efficacy [8–10]. Despite this, multiple randomized studies with a topotecan control arm have been negative, highlighting the resistant disease state [11–13] after prior platinum-based therapy. One of the negative studies that failed to meet its primary overall survival endpoint was a recent combination of lurbinectedin and doxorubicin compared to a control arm of either topotecan or CAV (cyclophosphamide, doxorubicin, vincristine) [14], which followed prior accelerated FDA approval of single-agent lurbinectedin based upon impressive data in small-cell lung cancer from a basket trial [15]. National Comprehensive Cancer Network guidelines include multiple regimens that may be considered in the second-line setting and beyond, but clinical trial is one of the three preferred regimens, highlighting the need for more effective treatments [16].

SCLC is a transcriptionally active disease with common (up to 90%) loss-of-function genomic alterations in the tumor suppressor genes *TP53* and *RB1*, creating further genomic instability by preventing arrest of the cell cycle for important DNA repair [17–20]. This suggests the potential for synergy with treatments that disrupt replication enough to halt the process and lead to apoptosis. One such approach, poly-(ADP)-ribose polymerase (PARP) inhibitors, have been a compelling class of drugs in the ongoing efforts to improve outcomes in this cancer that has been so resistant to other treatment options. Overexpression of PARP1 in SCLC further suggests therapeutic potential for PARP inhibitors [21].

Recurrent, targetable genomic alterations have not been identified in SCLC, but epigenetic and gene expression studies have led to the description of four distinct molecular subtypes defined by transcriptional regulators [22]. Subtyping of SCLC may offer an opportunity for better identification of treatment options with a higher likelihood of generating durable responses and will likely be an important component of prospective studies, including those evaluating PARP inhibitors and combinations.

PARP inhibitors represent a therapeutic class that has become an important treatment option for multiple tumor types. Although there is evidence of response, PARP inhibitors are not currently part of the treatment armamentarium for SCLC, and single-agent efficacy is limited. There is substantial ongoing investigation incorporating PARP inhibitors into the treatment of SCLC, and the following sections outline the mechanisms and rationale for these promising therapeutic combinations.

#### **2. PARP Inhibitor Mechanism of Action**

Recognition and repair of DNA damage form an essential cellular function mediated by a number of interconnected pathways termed the DNA damage response (DDR; Figure 1). PARP enzymes are a family of proteins that function in recognition and repair of DNA breaks, chromatin remodeling, and transcriptional regulation [23]. PARP 1 and 2 enzymatic function is activated by binding single-strand DNA breaks (SSB) and involves poly-ADP ribosylation (PARylation) of various substrates and recruitment of proteins that mediate DNA repair (Figure 1). PAR groups are subsequently metabolized by Poly-(ADP) ribose glycohydrolase (PARG) and other enzymes as part of coordinated dePARylation critical to effective DNA repair [23]. In the absence of SSB repair by PARP1, the replication fork stalls and double strand breaks occur prompting repair via homologous recombination (HR) or non-homologous end joining (NHEJ). If DSBs are not correctly repaired, replication aberrancies such as mutations, deletions, chromosomal translocations, and amplifications can occur resulting in cell death, senescence or malignant transformation. PARP inhibitors were initially developed to sensitize tumor cells to standard treatments such as chemotherapy or radiation, which induce DNA damage [24]. However, the observation that tumor cells with defects in HR are highly sensitive to single-agent PARP inhibition accelerated their clinical development [25,26]. The activity of PARP inhibitors in patients with *BRCA1* or *BRCA2* mutant cancers was the first clinical demonstration of synthetic lethality for cancer therapy [27]. In this setting, by inhibiting PARP catalytic activity and trapping PARP on DNA, PARP inhibitors stall replication machinery leading to DNA double strand breaks (DSB). In the absence of BRCA1 or BRCA2, these breaks cannot be repaired by HR (Figure 1). Several PARP inhibitors are currently approved or in clinical trials. In

addition to differences in their selectivity for PARP 1/2, these agents differ in their PARP trapping function, with talazoparib being the most potent [28]. Further studies in tumors without HR deficiency suggest that PARP inhibitors could have a broader role in cancer therapy [29].

**Figure 1.** The Role of PARP in the DNA Damage Response. PARP = poly-(ADP)-ribose polymerase, OGG1 = 8-oxoguanine glycosylase, XRCC1 = X-ray repair cross-complementing protein 1, Pol β = DNA polymerase beta, PCNA = proliferating cell nuclear antigen, FEN1 = flap endonuclease 1, ATM = ataxia telangiectasia, mutated, MRN complex = Mre11 + RAD50 + NBS1/nibrin, RPA = replication protein A, BRCA2 = FANCD1 breast cancer susceptibility gene and DNA repair enzyme, Pol δ = DNA polymerase delta, Pol ε = DNA polymerase sigma, KU70/80 = lupus Ku autoantigen protein p70/p80, DNA PKcs = DNA-dependent protein kinase, catalytic subunit, XRCC4 = X-ray repair cross-complementing protein 4, XLF = XRCC4-like factor, and Pol μ = DNA polymerase mu. Created with BioRender.com; accessed on 21 January 2021.

PARP was initially identified as a potential therapeutic target in SCLC through seminal work by Byers et al., who performed unbiased proteomic analysis of cell lines using reverse-phase protein arrays (RPPA) to identify proteins that were differentially expressed in SCLC compared with non-small-cell lung carcinoma (NSCLC) [21]. PARP1 transcript and protein levels were significantly elevated in SCLC cell lines compared to NSCLC. Increased PARP1 protein expression was also confirmed by immunohistochemical (IHC) analysis of tissue microarrays. Notably, several other components of the DDR pathway were increased in SCLC, including the checkpoint kinases CHK1 and CHK2, the ataxia telangiectasia related protein ATR, and the DNA-dependent protein kinase catalytic subunit DNA PKcs, which may be important to maintain cell viability in light of high replication stress (Figure 1). Treatment of a series of lung cancer cell lines with AZD2281 (olaparib) demonstrated that SCLC lines were significantly more sensitive to PARP inhibition than other histologic subtypes of lung cancer. Combining PARP inhibition with chemotherapy further decreased tumor cell viability.

These observations led to the initial studies of PARP inhibitors in SCLC as single agents (Table 1). In a phase I trial of talazoparib, 23 patients with SCLC were treated at the recommended phase II dose of 1.0 mg daily [30]. Two patients had a partial response, for an objective response rate (ORR) of 9% with a duration of response of 12.0 and 15.3 weeks. Both patients with an objective response had a platinum-free interval of 6 months or less. An additional four patients had stable disease, for a clinical benefit rate of 26% at 16 weeks. The UK STOMP trial examined the role of olaparib in the maintenance setting, but failed to show an improvement in progression-free survival (PFS) [31].

**Table 1.** Studies including PARP inhibitors in SCLC with outcomes data.

PFS = progression-free survival, OS = overall survival, ES-SCLC = extensive-stage small-cell lung cancer, CE = cisplatin/etoposide, LDH = lactate dehydrogenase, TMZ = temozolamide, SLFN11 = schlafen family member 11, and PDX = patient-derived xenograft.

#### **3. Biomarkers of Response to PARP Inhibitors in SCLC**

Since only a subset of SCLC patients appears sensitive to PARP inhibition, identification of predictive biomarkers has been an important focus of translational research. Clinical trials have attempted to identify correlative markers of response. Based on their preclinical work Owonikoko et al. examined DNA-PKcs expression as a biomarker but did not observe a correlation with veliparib activity in their phase II trial [32]. Although elevated serum lactate dehydrogenase (LDH) levels and male gender are poor prognostic markers, in the veliparib arm, these correlated with improvement in PFS in multivariable analysis. Mutations in DNA damage response genes such as *BRCA1*, *BRCA2*, *ATM*, or *ATR* (Figure 1) are not frequently seen in SCLC. However, homologous recombination deficiency (HRD) assays have been used to identify *BRCA1*/*2* wildtype ovarian cancer patients with sensitivity to PARP inhibition. Using three different measures of HRD, Lok et al. analyzed a series of SCLC cell lines to determine if HRD predicted response to PARP inhibition [33]. While they did not observe any correlation between HRD scores and response to PARP inhibitors, gene expression analysis demonstrated that high levels of *schlafen family member 11* (*SLFN11*) transcript did correlate with PARP inhibitor sensitivity. SLFN11 has been identified as critical for SCLC cell line and patient-derived xenograft (PDX) response to chemotherapy [34,35], as well as a potential biomarker for PARP inhibitor response using unbiased screens in SCLC cell lines and PDXs [33,35]. SLFN11 is a protein that is recruited to sites of DNA damage, inhibits HR, and activates a replication-stress response. High levels of SLFN11 have been correlated with enhanced response to PARP inhibitors in many [33–37] but not all [38,39] SCLC trials and preclinical models. Furthermore, using clustered regularly interspaced short palindromic repeats (CRISPR) based gene editing, deletion of *SLFN11* was found to confer resistance to talazoparib [33]. Importantly for clinical translation, a SLFN11 IHC H-score predicted sensitivity of SCLC PDXs to PARP inhibition [34]. A bimodal expression pattern of *SLFN11* transcript levels was observed in SCLC from The Cancer Genome Atlas (TCGA) dataset [33].

Using the NCI-60 database to identify genomic correlates of sensitivity to talazoparib across multiple tumor types, Murai et al. also identified *SLFN11* among the top-ranking genes [37]. They observed that deletion of *SLFN11* conferred resistance to PARP inhibition but found that ATR inhibition could overcome this resistance. An integrated proteomic and transcriptomic analysis of SCLC PDX models also identified SLFN11 protein levels as predictive of response to PARP inhibition [40]. Additionally, low ATM and high E-cadherin expression correlated with sensitivity to PARP inhibition. Treatment with cisplatin or PARP inhibitors reduced SLFN11 expression in cell line models, raising the question of dynamic changes in this marker in response to prior therapy. Using gene expression derived from PDX models, Farago et al. identified an inflammatory gene signature *(CEACAM1*, TN-FSF10, OAS1, TGIF1) that selected for sensitivity to olaparib + temozolamide. Markers of epithelial-to-mesenchymal transition (EMT) and high *MYC* target gene expression correlated with resistance. Collectively, these studies demonstrate that high SLFN11 expression is a promising biomarker for sensitivity to PARP inhibitor activity in SCLC, but prospective validation is needed and integration of multiple markers may improve predictive ability. SLFN11 expression is being studied prospectively as a biomarker in a randomized phase II clinical trial of talazoparib as maintenance therapy with atezolizumab in patients with ES-SCLC (SWOG1929, NCT04334941). Recent preclinical work argues that SCLC subtype can also influence response to PARP inhibitors, with expression of the transcription factor *POU2F3* sensitizing to PARP inhibitors [41].

#### **4. PARP Inhibitors Combined with Chemotherapy**

Given limited single-agent activity, a number of preclinical and clinical studies have examined combinations of PARP inhibitors with chemotherapy, radiation, and targeted therapies to enhance therapeutic benefit (Figure 2, Table 1). Several groups have demonstrated that PARP inhibition can potentiate the activity of platinum-based chemotherapy in SCLC cell lines and xenografts [21,42,43]. Owonikoko et al. tested the combination of veliparib with cisplatin (75 mg/m2) and etoposide (100 mg/m2 on days 1–3) in a phase I/II randomized clinical trial (ECOG-ACRIN 2511) in patients with ES-SCLC [32,44]. The recommended phase II dose for veliparib in combination with cisplatin and etoposide was determined to be 100 mg twice daily on days 1–7. Patients treated with veliparib had a median PFS of 6.1 months (95% CI, 5.9 to 6.7) relative to 5.5 months for placebo (95% CI, 5.0 to 6.1). Overall survival was 8.9 months (95% CI, 8.3 to 11.3) in patients receiving placebo relative to 10.3 months (95% CI 8.9 to 12.0) with the addition of veliparib (stratified HR, 0.83; 80% CI 0.64 to 1.07; *p* = 0.17). Patients were stratified by sex and serum LDH levels. Male patients with high LDH levels derived benefit in PFS (HR 0.34; 80% CI 0.22 to 0.51), but no difference in OS by strata was observed. The combination was tolerable, with higher rates of lymphopenia and grade 3 or 4 neutropenia seen with the addition of veliparib. The combination of veliparib with carboplatin (AUC = 5) and etoposide (100 mg/m2 on days 1–3) has also been studied [45]. The recommended phase II dose for veliparib in this study was 240 mg twice daily for days 1–14, due to excess hematologic toxicity seen with continuous dosing. A randomized phase II study was performed with three arms: (A) carboplatin/etoposide + veliparib followed by veliparib, (B) carboplatin/etoposide + veliparib followed by placebo, and (C) carboplatin/etoposide + placebo followed by placebo. Median PFS in arm A was 5.8 months (80% CI 5.6 to 6.8), arm B 5.7 months (5.6 to 5.8) and 5.6 months (5.1 to 6.7) for arm C. Similarly, no significant differences in OS were observed.

PARP inhibitor and chemotherapy combinations have also been examined in patients with relapsed disease after platinum-based chemotherapy (Table 1). Temozolomide (TMZ) is an oral alkylating agent, previously demonstrated to have single-agent activity in SCLC [46]. TMZ methylates the O6 position of guanine, ultimately leading to DSBs. O6 methylguanine-DNA methyltransferase (MGMT) is involved in repair of these lesions; therefore, silencing of *MGMT* expression by promoter methylation has been correlated with improved clinical response to TMZ. Given that PARP proteins also have a role in repair of these lesions, it was hypothesized that the combination of PARP inhibitors and TMZ could have synergistic activity. Using talazoparib, Lok et al. evaluated the activity of TMZ and PARP inhibition in several SCLC models, demonstrating synergistic tumor growth inhibition, particularly in high *SLNF11*-expressing models [33]. *MGMT* expression did not correlate with sensitivity to TMZ + talazoparib. Murai et al. similarly observed synergistic

activity for talazoparib and TMZ in SCLC models with high *SLFN11* expression [37]. Two phase II studies in relapsed SCLC patients have evaluated the combination of TMZ and PARP inhibition. Pietanza et al. performed a randomized, double-blind, placebo-controlled study of veliparib (40 mg twice daily, days 1 to 7) or placebo and TMZ (150–200 mg/m2/day, days 1 to 5) on a 28-day cycle [36]. The primary endpoint of the study was 4-month PFS, with no significant differences observed between TMZ/veliparib (36%) and TMZ/placebo (27%, *p* = 0.19). Median PFS was 3.8 and 2.0 months (log-rank *p* = 0.39, HR 0.84; 95% CI 0.56 to 1.25) for the TMZ/veliparib and TMZ/placebo arms, respectively. OS was also similar between the 2 arms. ORR was higher for the combination of TMZ/veliparib (39%) versus TMZ/placebo (14%), in both platinum-sensitive and platinum-refractory patients. Biomarker analysis was performed for PARP-1 and SLFN11 expression by IHC. No association with PARP-1 expression and clinical outcomes was observed. SLFN11-positive tumors (H-score cutoff ≥ 1) treated with TMZ/veliparib had improved PFS (5.7 vs. 3.6 months, *p* = 0.009) and OS (12.2 vs. 7.5 months; *p* = 0.014). Notably, the authors highlighted that a low dose of veliparib was used in this study and veliparib has lower PARP trapping activity, both of which could have contributed to limited efficacy.

**Figure 2.** PARP Inhibitor Combinations: Enhancing Response in SCLC. PARP = poly-(ADP)-ribose polymerase, ADCs = antibody–drug conjugates, SSB = single-strand DNA break, DSB = double-strand DNA break, CDK = cyclindependent kinase, XRT = radiation therapy, HDAC = histone deacetylase, MSI = microsatellite instability, ssDNA = singlestrand DNA, ISGs = Interferon-stimulated genes, and ICB = immune checkpoint blockade. Created with BioRender.com; accessed 29 on January 2021.

Using the more potent PARP inhibitor olaparib in combination with TMZ, Farago et al. performed a phase I/II study in relapsed SCLC [39]. To facilitate biomarker analysis and mechanistic studies, a co-clinical trial with PDXs was performed. At the recommended phase II dose of olaparib (200 mg twice daily, day 1–7) and TMZ (75 mg/m2, day 1–7 of 21 days cycle), the ORR was 41%, with a median duration of response of 5.3 months. Across all dose levels, PFS was 4.2 months (95% CI, 2.8 to 5.7) with a median OS of 8.5 months (95% CI, 5.1 to 11.3). A phase II study of continuous talazoparib with intermittent low-dose TMZ (NCT03672773) in relapsed/refractory SCLC is ongoing. Additional studies are evaluating the combination of PARP inhibitors with agents that induce DNA damage such as pegylated SN-38, the active metabolite of irinotecan, an inhibitor of topoisomerase I activity (NCT04209595, Table 2).

**Table 2.** Ongoing studies in SCLC.

ES-SCLC = extensive-stage small-cell lung cancer, SLFN11 = schlafen family member 11, TMZ = temozolamide, and CE = cisplatin/etoposide.

> Antibody–drug conjugates (ADCs) are another class of therapeutic that acts by inducing DNA damage selectively in tumor cells after targeting to tumor-specific antigens. The first ADC to enter clinical development for SCLC targeted the Notch inhibitory protein delta-like ligand 3 (DLL3), identified as enriched on SCLC cells with impaired Notch signaling [47]. The DLL3 ADC, Rovalpituzumab tesirine (Rova-T), showed promise in

preclinical work and early trials [48], but ultimately failed to meet primary endpoints in phase II/III trials [12,49]. A phase I trial of an ADC targeting the tumor-associated calcium signal transducer Trop2 showed an acceptable safety profile and promising results with an ORR of 14% and clinical benefit in 34% of heavily pretreated patients with SCLC [50]. Further studies are ongoing, and ADCs may be of particular benefit in combination with other treatments including PARP inhibitors.

In addition to chemotherapy combinations, PARP inhibitors can also sensitize SCLC models to ionizing radiation [42,51]. Laird et al. noted that talazoparib is a more potent radiosensitizer than veliparib, suggesting that PARP trapping ability may play a role in sensitization to radiation. Talazoparib treatment led to increased DSBs compared to veliparib. Interestingly, radiosensitization was observed irrespective of SLFN11 expression. Several early phase trials are examining the combination of PARP inhibition and radiation in ES-SCLC (NCT03532880, NCT04170946, Table 2). Potential toxicity to normal tissues with these combinations is a concern and will be carefully evaluated in these studies.

#### **5. Synthetic Lethality Downstream of PARP Inhibitors**

In addition to synergy with chemotherapy and radiation, several preclinical studies have suggested that combinations with DDR inhibitors could enhance the therapeutic potential of PARP inhibitors in SCLC. These approaches aim to target multiple nodes of the DDR response to prevent resistance and promote synergistic antitumor activity. Since PARP inhibition prevents repair of single-strand DNA breaks, which subsequently progress to DSBs at stalled replication forks, PARP inhibitors are most effective when DSB repair is impaired. This strategy, referred to as synthetic lethality, was originally developed in the setting of *BRCA* germline mutations in ovarian cancer patients and holds particular appeal in SCLC, where defining mutations in *RB1* and *P53* combine with elevated tumor mutational burden from tobacco exposure to generate additional replication stress and dependence on DNA repair mechanisms [52,53]. SCLC is not associated with germline *BRCA* mutations, and global microsatellite instability in SCLC is rare. However, a "DNA-repair score" was shown to correlate with response to PARP inhibition in preclinical work using SCLC PDXs [54]. This prognostic score includes canonical DNA repair genes such as *PARP*, *BRCA*, *ATM*, *ATR*, *CHK*, *RAD50*, *53BP1*, *MSH2*, and *FANC* (Figure 1) [54], several of which can also be inhibited pharmacologically (Figure 2). Early phase clinical trials have opened for SCLC patients targeting the DNA damage response (Table 2), including for AZD1775 targeting WEE1 (NCT02593019, NCT02688907), VX-970 targeting ATR (NCT02157792, NCT02487095), and Prexasertib targeting CHK (NCT02735980) [52]. While these specific trials do not include PARP inhibitors, combination studies with PARP inhibition are also being developed.

CHK1 is a protein kinase that plays an important role in DNA damage-dependent cell cycle arrest, particularly in TP53-deficient tumors. CHK1 protein expression is increased in SCLC patient tumors [21,55]. Combination therapy with the CHK1 inhibitor LY2606368 and cisplatin or olaparib enhanced tumor regression and survival in mouse SCLC models [55].

WEE1 is a kinase involved in S phase and G2-M progression, by phosphorylating CDK1/2 and allowing for DNA repair prior to mitotic entry. Targeting WEE1 with inhibitors such as AZD1775 compromises DNA damage checkpoints, particularly in cancer cells that may be more dependent on the G2-M checkpoint. Lallo et al. studied the combination of olaparib and AZD1775 in SCLC PDXs and observed activity in both chemotherapy sensitive and resistant models [56]. This combination is being evaluated in a trial for patients with refractory solid tumors, including SCLC (NCT02511795).

Preclinical work using cell lines and patient specimens suggests that treatment with histone deacetylase (HDAC) and enhancer of zeste homology 2 (EZH2) inhibitors can restore epigenetically suppressed SLFN11 expression [57], suggesting potential synergy with PARP inhibition. In the majority of SCLC with low SLFN11 expression, resistance to PARP inhibition may be overcome by pharmacologic ATR inhibition [37], further supporting the role for DDR synthetic lethality in enhancing response to PARP inhibitors in SCLC.

#### **6. Combining PARP and Immune Checkpoint Inhibition**

ICB has been incorporated into the first-line treatment of SCLC [6,7]. Combining ICB and PARP inhibitors may offer synergy because of molecular signaling pathways linking cytosolic DNA to PD-L1 expression. DNA-sensing pathways, which evolved to protect against bacteria and viruses, also recognize self-DNA released from the nucleus when DDR is suppressed [58]. Double-stranded DNA is recognized by the enzyme cyclic GMP-AMP synthase (cGAS), which produces the cyclic dinucleotide second messenger 2'3' cGAMP, activating the Stimulator of Interferon Genes (STING) pathway, which upregulates interferon stimulated genes, including PD-L1. PARP inhibitors have been shown to activate STING and upregulate PD-L1 across cancer models regardless of *BRCA* mutation status, leading to synergy with PD-L1 inhibitors in preclinical mouse studies [59–63]. In SCLC, preclinical data suggest that synergy between PARP inhibitors and PD-L1 checkpoint inhibition may depend on intact tumor cell STING and innate immune activity downstream of cytosolic DNA released after PARP inhibition [64]. A phase II trial in relapsed SCLC combining durvalumab 1500 mg every 4 weeks with olaparib 300 mg twice a day showed an ORR of 10.5% (two patients out of nineteen) [65]. The treatment combination was well tolerated, with expected cytopenias from PARP inhibition but no evidence of overlapping toxicity. Of note, both responders exhibited an inflamed phenotype with CD8+ T cells contacting tumor cells in a pretreatment biopsy [65]. Co-mutation status and/or histology may influence response to combined PARP and immune checkpoint inhibition in SCLC, as one of the responders had a *BRCA* mutation that may have sensitized to PARP inhibition, and the other had *EGFR*-mutant transformed SCLC. Post-treatment biopsies confirmed increases in PD-L1 expression after PARP inhibition in 6/9 paired cases. However, these increases failed to correlate with T-cell infiltration. The disappointing response rates in this trial are similar to a previous phase II basket study including patients with relapsed SCLC that used the same doses of olaparib and durvalumab but with a 4-week olaparib run-in period [66]. These early results suggest that additional mechanisms suppress antitumor immunity in SCLC. The phase II trial of rucaparib + nivolumab in platinum-sensitive SCLC (NCT03958045) may identify a clinical context with residual disease where these agents are more effective [67].

Additional immune checkpoints, such as CTLA-4 (cytotoxic T-lymphocyte-associated protein 4), which binds to CD80/CD86, may suppress antitumor immunity downstream of PARP inhibition. A number of trials combining PARP and CTLA-4 inhibitors are currently underway, including a phase I trial of thoracic radiation combined with durvalumab +/− tremelimumab or olaparib in ES-SCLC after first-line chemotherapy (NCT03923270) [67]. Antibody dependent cellular cytotoxicity (ADCC) represents another promising approach to unleash antitumor immunity. Preclinical studies identified BMS-986012 as an antibody that can bind the tumor cell-specific ganglioside FucGM1, leading to ADCC [68]. This compound is currently being tested in phase I/II trials (NCT02247349, NCT02815592), either as part of first-line treatment for ES-SCLC alongside chemotherapy or in the relapsed setting alongside nivolumab. As the designs of these trials suggest, targeting multiple steps in DNA damage response concurrently (see Figure 2) may ultimately prove successful.

#### **7. Restoring Tumor Cell Inflammatory Signaling to Enhance PARP Inhibitor Response**

The majority of SCLC are "immune deserts" with minimal infiltration by CD8+ effector T cells [22]. However, a subset of non-neuroendocrine tumors demonstrates enhanced inflammatory infiltrates and markers of innate immunity including restored STING expression [69,70]. As suggested by the phase II data for durvalumab + olaparib [65], and confirmed in elegant preclinical work [41], the non-neuroendocrine inflamed subtype may represent a biomarker for response to this combination. To expand the patient population that can benefit from the combination of DDR inhibition and ICB, novel approaches to restore tumor cell inflammatory pathways are sorely needed. The neuroendocrine stress response inhibits inflammation, so strategies that target neuroendocrine lineage commitment could elicit antitumor immunity. Reversing EZH2 epigenetic programing to

de-repress antigen presentation and tumor cell STING expression represents one promising approach [70,71]. Indeed, EZH2 levels are higher in SCLC than any other tumor type in TCGA [72], and EZH2 inhibitors can restore *SLFN11* expression to potentially improve response to PARP inhibitors [34]. The combination of EZH2 and PARP inhibitors was effective in preclinical models of ovarian cancer [73], and is being developed in SCLC, where both approaches have shown promise as monotherapies [21,74]. Restoring tumor suppressive *NOTCH1* or inhibiting the Notch suppressive protein DLL3 to alter neuroendocrine differentiation could have similar effects, as recent evidence suggests that phenotype switching can uncover therapeutic vulnerabilities [41]. Targeting negative regulators of DNA-sensing including ectonucleotide pyrophosphatase/phosphodiesterase family member 1 (ENPP1), the enzyme that cleaves the STING second messenger 2 3 -cGAMP [75], may also potentiate the effects of DDR inhibition. ENPP1 can also metabolize PAR downstream of PARP in the DNA damage response [76]. While inhibiting PARylation and dePARylation simultaneously may seem counterproductive, both processes cooperate in DNA damage repair, and their concurrent inhibition shows promise in preclinical cancer models [77]. Combinations that disrupt coordinated DNA damage repair are more likely to stimulate innate antitumor immunity and response to immune checkpoint blockade.

#### **8. Orthogonal Approaches**