Most patients are diagnosed with SCLC at an advanced stage and rarely undergo surgical resection. As a result, small tissue biopsies or cytology samples are the only biological material available. Research into the tumorigenesis of SCLC has been largely hampered by the low number of samples available. However, new research techniques such as genetically engineered mouse models (GEMMs), development of SCLC cell lines, patient-derived in vivo models, as well as analysis of mechanisms of acquired therapeutic resistance through transcriptomic and proteomic approaches, have enabled a deeper understanding of SCLC.
3. Potential Clinical Utility of Emerging Subtypes
The detection of biological heterogeneity based on master transcriptional activators has been a huge advancement in the understanding of SCLC and has potential to translate into clinical practice in the future. Each of these subtypes have distinct biological activity and therapeutic vulnerabilities which will hopefully lead to the development of novel diagnostic, prognostic, and predictive biomarkers to aid in the pathologic assessment and guide clinical decisions for these aggressive tumors.
3.1. Subtypes as Emerging Diagnostic Biomarkers
Diagnosis of SCLC is made by histopathological examination. Because of the central location of the tumors, biopsies are most often obtained by bronchoscopy with or without endobronchial ultrasound. As a result, diagnosis is made on scant biopsy specimens or cytology. Due to the aggressive nature of SCLC, workup including diagnosis and staging should be performed as quickly as possible after presentation.
Morphologically, SCLCs are composed of small cells (<3 resting lymphocytes) with scant cytoplasm, finely granular nuclei, and inconspicuous nucleoli, indistinct cell boarders, high mitotic count, apoptosis, and frequent necrosis. Tumors are usually densely packed, forming a sheet-like pattern 
. Morphologically, SCLC are homogenous compared to NSCLC, although rare variations exist 
Historically, SCLC is a morphological diagnosis made on the haematoxylin and eosin (H&E) stain. However, IHC is widely used in pathology practice. Histologically, the differential diagnosis includes other neuroendocrine tumors, especially large cell neuroendocrine carcinoma (LCNEC), basaloid squamous cell carcinoma, undifferentiated SMARCA4 tumor, small round blue cell tumors, and lymphoma. Both clinical course and treatments vary widely across these malignancies, and therefore, accurate diagnosis is essential.
NE markers are positive in 90 to 95% of SCLC cases. However, 5–10% can be positive only with CD56, sometimes only weakly, or even negative, creating a major diagnostic challenge especially in those where morphology is not well preserved.
As a result, large IHC panels are needed to exclude other differentials, often delaying diagnosis. In such cases, or whenever the differential diagnosis with low-grade NE tumors, NSCLC or LCNEC, arises, the loss of RB IHC and a mutant staining for TP53 (either loss of TP53 or a strong diffuse staining) is helpful. This pattern is seen in SCLC representing its genetic hallmark 
. This approach is not yet validated but might be of great value in small or crushed biopsies. In difficult cases, the addition of p16 IHC might be of additional assistance; strong and diffuse p16 reflects loss of Rb through its negative regulatory loop 
POU2F3, the novel transcriptional factor expressed in 7% of SCLC, is showing great promise as a diagnostic biomarker in NElow/-
SCLCs. Baine et al. analyzed POU2F3 IHC expression in SCLC (n = 123) and other major lung cancer types (n = 433) 
. Expression was diffusely positive in 70% of SCLCs that were fully NE negative. Expression was negative in NE SCLCs and therefore, is best suited as a second step marker in suspected NElow/-
SCLC. Although not fully restricted to SCLC (positive staining was seen in basaloid SCC and LCNEC in 22% and 12% of cases, respectively), it is highly selective, and therefore is useful as a multipurpose IHC, similar to TTF-1. The addition of POU2F3 IHC, therefore, would aid in a more efficient diagnostic pathway for NElow/-
SCLCs, eliminating the use of larger IHC panels and conserving tissue for a further workup, such as detection of predictive biomarkers, in the future.
3.2. SCLC Subtypes as Prognostic Biomarkers
None of the four distinct subtypes give clear prognostic information. SCLC-A and SCLC-N are associated with immune cold tumors 
and low NE subtypes are associated with chemoresistance. However, few studies have evaluated the prognosis of these subtypes. SCLC-P has been identified as a poor prognostic factor in one study 
and conflicting results exist as to whether high YAP1 expression is associated with poor prognosis 
. For now, SCLC subtype behavior in a clinical context is still not clarified.
3.3. Predictive Biomarkers
In stark contrast to NSCLC, SCLC has been treated as a single disease, with a one-size-fits-all model, and with platinum-based chemotherapy remaining the cornerstone of treatment. Over 20 years, there has been no improvement in the survival or response rate to chemotherapy of SCLC patients and therefore, there is a desperate need for new approaches in this setting 
Currently, there are a number of promising biomarkers in the pipeline which may be used to guide clinical decisions for these patients in the future.
3.1. SCLC Subtypes as Predictive Biomarkers
The four described SCLC subtypes, SCLC-A, SCLC-N, SCLC-P, and SCLC-I, described by distinct translational activators, have been shown to have distinct therapeutic vulnerabilities. Using in vitro cytotoxic assays, Gay et al. identified that the SCLC-A subtype have increased sensitivity to BCL2 inhibitors, the SCLC-N subtype, to aurora kinase inhibitors, and the SCLC-P subtype, to PARPi and antimetabolites 
. The SCLC-P subtype had increased sensitivity to PARPi independent of SLFN11 (an emerging predictive biomarker for PARPi in SCLC, see below), suggesting there is an alternate pathway of efficacy which potentially widens the cohort of patients who will derive benefit from this promising new target in SCLC. Within SCLC-A, there is a bimodal expression of SLFN11, and if separated by high and low expression, there is a stark difference in cisplatin sensitivity and to PARPi with olaparib 
To identify therapeutic vulnerabilities, Gay et al. also identified unique protein expression patterns between groups which may serve as predictive biomarkers. SCLC-A and SCLC-N subtypes have a high expression of DLL-3, which is absent in SCLC-P and SCLC-I groups. A subset of SCLC-A also has high expression of SLFN11, which predicts a response to PARPi (see below). The SCLC-N group has a high expression of somatostatin receptor 2 (SSTR2), which can be targeted by somatostatin analogues such as octreotide.
The response of subtypes to these targeted therapies requires validation. However, the identification of distinct therapeutic vulnerabilities within these four subtypes highlights that a one-size-fits-all-model may not be appropriate for managing this disease. This will certainly pave the way for biomarker-driven clinical trials in the future introducing SCLC into the world of precision oncology.
3.2. SCLC-I Predicts Response to Immunotherapy
SCLCs have a high TMB and, therefore, are predicted to induce strong T-cell responses. NSCLC are also associated with a similarly high TMB, and as such, have shown responsiveness to immunotherapy. Immunotherapy by means of targeting PD1/PD-L1, either as a monotherapy or in combination with chemotherapy, has become the first-line treatment for patients with stage IV NSCLC lacking a driver mutation 
. PD-L1 expression on tumor cells (tumor proportion score) by IHC is a strong predictor of response, with cut offs of ≥1% and ≥50% used as selection criteria for combination therapy and monotherapy, respectively 
. Additionally, TMB status is an important predictive biomarker with high TMB predicting improved objective response to durable benefit, and progression-free survival independent of PD-L1 expression 
. High TMB in NSCLC is associated with immune cell infiltration and an inflammatory T-cell mediated response which may explain the increased sensitivity to immunotherapy 
Although NSCLC and SCLC have a similar TMB profile, they exhibit a differential response to immunotherapy. Clinical trials involving immunotherapy in SCLC have yielded disappointing results, with only a subset of patients deriving a benefit 
. PD-L1 IHC is rarely expressed in SCLC and does not predict a response to immunotherapy 
. The differential responsiveness between SCLC and NSCLC may lie within the interaction of SCLC and its surrounding environment, including immune surveillance 
. SCLC exhibits an extremely cold T-cell receptor (TCR) repertoire and lower immune infiltrate compared to NSCLCs despite having similar TMBs 
. SCLCs also have low infiltration by immune cells, specifically, cytotoxic T cells 
Currently, there are no biomarkers that predict a response to immunotherapy in SCLC.
The novel, low-NE subtype identified by Gay et al., SCLC-I, demonstrates an ‘inflamed’ phenotype and has a high expression of both CD8A and CD8B, suggesting greater cytotoxic T-cell infiltration 
The Impower133 trial was the first randomized trial demonstrating PFS and OS improvements with Immunotherapy in SCLC 
. Using data from this trial, Gay et al. retrospectively stratified survival data by SCLC subtype and the identified OS benefit was seen in the SCLC-I group compared to all others in the platinum-etoposide (EP) plus atezolizumab arm, but not in the EP plus placebo arm (HR, 0.566; 95% CI, 0.321–0.998) 
. This represents clinical data that the SCLC-I subtype is a candidate biomarker for predicting a response from the immune checkpoint blockade in SCLC.
Additionally, the SCLC-N subtype has been shown to have the most immunosuppressive TME and therefore, may derive no benefit from IO 
. These studies highlight the need to match tumor subtype to therapy to maximizes the response in patients. Future prospective trials are needed to elucidate the clinical relevance of the novel transcriptional subsets in SCLC.
3.3. SLFN11 as a Predictive Biomarker
Resistance to cell death and genomic instability are important hallmarks of cancer 
. Small molecule inhibitors that directly damage DNA response (DDR) have gained increasing interest over the years. Poly (ADP-ribose) polymerase (PARP) are a family of proteins who play a critical role in DNA repair. When induced by a DNA lesion, PARP catalyzes the addition of poly (ADP-ribose) chains to target proteins, which mediates the recruitment of additional DNA repair factors to the damaged DNA 
. PARP inhibitors (PARPi) have been studied most in cancers with deficient homologous recombination repair (HRR) pathways which depend on PARP-mediated base excision for repair.
Schlafen 11 (SLFN11) is a DNA/RNA helicase that induces an irreversible replication block and it has been identified as a predictive biomarker of DNA-damaging agents and PARPi in preclinical settings in SCLC 
, as well as many other solid tumors, including breast 
, ovarian 
, colorectal 
, and gastric cancer 
, and mesothelioma 
SLFN11 also predicts the response to other DNA-damaging agents, including platinum salts, topoisomerase I inhibitors, topoisomerase II inhibitors, alkylating agents, and antimetabolites. Low expression also predicts resistance to these agents 
Clinical detection of SLFN11 can be made using IHC. Multiple studies have demonstrated that detection of SLFN11 is made by the presence of any nuclear staining 
. Conversely, negative SLFN11 tumors have a complete absence of staining. Therefore, the distinction between SLFN11-positive and -negative tumors can be easily made in clinical practice by IHC.
Unlike other biomarkers, SLFN11 induces its cellular response to PARPi independently of HRR. Instead, ‘PARP trapping’ occurs, whereby PARPi traps PARP1/2 at the site of DNA damage preventing repair 
. SLFN11 induces cell cycle arrest in the S-phase, which leads to a stalling of replication forks resulting in fork breakage and replisome disassembly 
. SLFN11 and PARPi are believed to have synergistic effects through this ‘trapping’ mechanism 
. PARP trapping varies among different PARPi, with talazoparib being the strongest and veliparib being the weakest 
. The predictive strength of SLFN11, therefore, may be PARPi class dependent.
A phase II trial of relapsed SCLC treated with temolizomide plus veliparib (PARPi) versus temolizomide plus placebo found no significant difference in 4-month PFS or median OS 
. Retrospective analysis, identifying SLFN11-positive patients using IHC on archival tissue, identified that SLFN11-positive patients had a significantly longer PFS and OS compared to SLFN11-negative patients in the temozolomide plus veliparib group 
. This represented the first clinical data that SLFN11 is a candidate biomarker for PARPi in SCLC.
A phase II randomized clinical trial of atezolizumab in combination with talazoparib versus atezolizumab alone in SCLC is currently ongoing (NCT04334941) for patients with SLFN11-positive extensive-stage SCLC. As mentioned above, talazoparib has the strongest PARP-trapping ability and may confer additional benefit compared to other PARPi in SLFN11-positive patients. This is the first clinical trial selecting patients based on SLFN11 status.
Lurbinectedin, an alkylating agent, recently received FDA-accelerated approval as a second line treatment option for metastatic SCLC 
. SLFN11 has been shown as a candidate biomarker for lurbinectedin sensitivity. SLFN11-low SCLC cell lines are resistant to lurbinectedin 
. However, the addition of an ATR (Ataxia telangiectasia mutated and Rad3-related) inhibitor re-sensitizes these cells 
. This confirms SLFN11’s role as a master regulator of DNA damage response, independent of ATR, with the absence of SLFN11 leading to synthetic lethality when ATR is inhibited. Therefore, the addition of an ATR inhibitor to lurbinectedin in SLFN11-low tumors may overcome resistance.
SLFN11 has also been shown to be detectable in CTCs in SCLC and may represent a candidate biomarker to monitor patients via non-invasive liquid biopsy 
3.4. DLL3 as a Predictive Biomarker
Delta-like ligand 3 (DLL3) is a ligand that inhibits the Notch pathway and is upregulated and aberrantly expressed on the cell surface in up to 80% of SCLCs, specifically, NE subtypes (SCLC-A and SCLC-N) 
. In contrast, DLL3 is only expressed at very low levels in a few normal cells and is exclusively cytoplasmic 
. This selective expression has gained a lot of attention as an attractive biomarker in SCLC.
Rovalpituzumab tesirine (Rova-T) is an antibody–drug conjugate composed of a DLL3-targeting immunoglobulin G1 monoclonal antibody tethered to pyrrolobenzodiazepine (PBD), a toxic DNA crosslinking agent, by means of a protease-cleavable linker 
. Rova-T was designed to deliver cytotoxic treatment to DLL3, expressing SCLC cells while sparing healthy cells, reducing side effects. Phase II and III clinical trials using RovaT as a third line and beyond treatment for participants with relapsed or refractory DLL3 expressing SCLC exhibited an inferior OS compared to patients on current standard second-line chemotherapy, topotecan, and therefore, it was discontinued 
Despite this, DLL3 remains an attractive target due to its specificity and high expression in SCLC. Novel therapeutic targets, such as anti-DLL3 bispecific T-cell engager, are in development 
. Additionally, a recent study used a radiolabeled anti-DLL3 mAb SC16 with the therapeutic radioisotope Lutetium-177 in GEMMs. This radioisotope specifically attaches to SCLC cells to deliver targeted radiotherapy, minimizing radiation to healthy cells. Results showed antitumor efficacy and a low toxicity profile, making it a strong potential for clinical translation