In the absence of HPV, an increased risk of SPTs was found [32]. Consuming alcohol and smoking are the two most significant risk factors for HPV-negative LSCC and esophageal squamous cell carcinoma (ESCC). These carcinogens can directly damage and mutate DNA, increasing mutational burden, intercellular heterogeneity, and driving mutations [33]. TP53 tumor suppressor mutations have been shown to be the most common genetic variant and stable, well-distributed point mutation (exons 5–8) in LSCC, rendering this mutation a significant predictor of treatment outcome and valuable for clonality assessment [34]. Histopathological studies of ESPT in patients with HPV-negative LSCC have identified small TP53 mutated plaques (less than 200 cells in diameter) that precede the expansion of cancer [35]. But Chen et al. [36] conducted mutation analysis of the TP53 core binding domain and found no clonal relationship between LSCC and ESPT. The mutation pattern observed in the P53 gene exhibits considerable variability, suggesting a polyclonal nature of transformation. It is important to note that tumor development is a multifaceted process, necessitating additional genetic alterations and a large number of stromal cells (inflammatory cells, immune cells, etc.) for the transformation of plaques into extended fields. P53 gene mutations and sustained loss of cell differentiation, along with the entry of cells with damaged DNA into the S phase, lead to altered genetic characteristics and chromosomal aberrations, promoting tumor development [37]. Furthermore, P53 is essential for the repair of DNA damage and the initiation of apoptosis. Consequently, the mutation of the TP53 gene observed in patients with LSCC, attributed to smoking, hinders the timely repair of DNA damage, resulting in the continuous proliferation of cells carrying genetic abnormalities. This uncontrolled proliferation ultimately leads to the hypermutation of tumor cells [38, 39]. While the cancer stem cell hypothesis traditionally opposes the notion of polyclonal tumor origins, the interaction among numerous pluripotent stem cells presents a potential mechanism for polyclonal tumors origins [40].

Some studies have found that alcohol-induced LSCC and ESPT also have an independent origin, particularly in individuals with alcohol-induced facial flushing [41]. The alcoholic flush response is associated with a deficiency of acetaldehyde dehydrogenase (ALDH2), which results from excessive accumulation of acetaldehyde (AD). There is growing evidence that people who lack ALDH2 are at much higher risk of developing LSCC and ESCC from alcohol consumption than those with fully functional ALDH2, while ALDH2 × 1/2 heterozygotes are also at a heightened risk of developing LSCC and ESCC [42]. When significant quantities of AD accumulate in saliva, it results in direct contact between AD and the upper airway mucosa, potentially leading to genetic mutations [43]. AD has the ability to bind to DNA, forming stable DNA adducts that initiate DNA damage. Moreover, if these AD-induced DNA adducts manage to evade cellular repair mechanisms and persist, they can ultimately give rise to coding errors and genomic instability [44]. Genomic instability can have an impact on chromosome structure, chromosome number, and DNA sequence, and in certain cases, it can compromise genomic integrity across multiple levels simultaneously. In extreme cases, resulting in a huge number of mutations in tumor cells, the effects of genetic drift (random loss or immobilization of genotypes) can be amplified so that neutral or even harmful mutants can be retained during enlargement, leading to clonal diversity [45]. Therefore, there is a preference for independent clonal origin between HPV-negative LSCC and ESPT.

HPV-positive tumors behave biologically differently from HPV-negative tumors, thereby influencing the pathophysiology of the disease [46]. The early occurrence of HPV infection is observed in both LSCC and ESCC, with HPV16 being the predominant pathogenic subtype [47, 48]. HPV is a circular, double-stranded DNA virus with a genome size of approximately 8 kb, lacking an envelope, and belonging to the papillomavirus family. This epitheliophilic virus specifically targets basal cells in stratified epithelial tissues in mucosal or cutaneous regions. Infection is initiated when the virus reaches the basal cells and replicates viral DNA via the cellular DNA replication machinery, producing a small number of copies of the circulating virus. The viral genome is organized into three sections: the early genetic region (E), the late genetic region (L), and the long regulatory region (LCR) that connects the two. The late region encodes L1 and L2 proteins, which are responsible for encoding the primary and minor viral capsid proteins, respectively, the early region codes for E1-E5 genes primarily involved in viral genome replication and transcription [49]. It is essential for both E6 and E7 genes to be present in order to induce oncogenic transformation in host cells. Furthermore, the expression of E6 and E7 is typically elevated in advanced precancerous stages, leading to the transformation of infection [50]. The integration of viral DNA into the host cell genome is a crucial step in the progression of HPV to cancer [51].The whole cellular genome, including both gene-rich and gene-poor areas, is subject to viral integration. Initial investigations into viral integration causing cervical lesions indicate that this process is stochastic and may exhibit a preference for microhomologous regions, areas with strong transcriptional activity, common weak sites, or areas near microRNAs (miRNAs) [52]. Integration into or near genes can result in alterations in gene expression through various mechanisms, including the formation of viral-cellular fusion transcripts. However, the precise mechanism remains unclear [53]. Parfenov et al. [54] observed an elevated somatic DNA copy number in the integrated region and reported that HPV viral integration disrupts genetics through multiple crucial pathways, such as the loss of tumor suppressor function, increased expression of oncogenes, and rearrangement of gene expression. It is rare for the P53 gene to be altered in HPV-positive LSCC, which is usually eliminated by E6 [49]. The E6 protein, in a protein-dependent manner, binds to the core region of the P53 protein, resulting in the formation of the E6/E6AP complex. This complex facilitates the degradation of P53 through a ubiquitin-dependent pathway, thereby promoting tumor progression [55]. Additionally, the E7 protein interacts with various cell cycle regulatory proteins, influencing their levels and/or cellular activity. One such interaction involves the high affinity of E7 for pRb, which leads to feedback upregulation of p16INK4A [56]. This upregulation inhibits the interaction between pRb and the transcription factor E2F, which is responsible for controlling cell cycle G1/S changes. Consequently, this disruption of the cell cycle promotes oncogenic transformation and clonal amplification [57]. Moreover, tumor growth exhibits a clonal evolutionary trajectory characterized by sequential clonal amplification, genetic diversification, and clonal selection. The stochastic nature of viral integration sites adds to tumor cell genetic diversity, and integration sites are passed down through clonal amplification [58]. One study revealed the clonal origin of bilateral HPV16-positive tonsillar tumors by viral integration analysis, which finally supported the monoclonal hypothesis [59]. Therefore, based on DNA integration, a key step in HPV viral carcinogenesis, it was found that HPV-positive LSCC secondary to ESPT is likely to be the result of clonal amplification. However, one case study suggests that there may be no clonal relationship between the LSCC and ESPT, and the exact mechanism needs to be further elucidated [60].

Gene expression can be altered through epigenetic modification without altering nucleotide sequences. These changes can impede apoptosis, disrupt the cell cycle, facilitate the growth of precancerous cells, and result in the expansion of clonal cell populations that are susceptible to new carcinogens. Over time, these alterations can accumulate carcinogenic events and contribute to the development of secondary primary tumors [61]. Additionally, epigenetic modifications like histone alterations, DNA methylation, chromatin remodeling, and microRNA can serve as potential indicators of cancer growth and progression [62]. Altered DNA methylation patterns are commonly observed in LSCC. These patterns typically involve the hypermethylation of tumor suppressor oncogenes and the hypomethylation and transcriptional deletion of proto-oncogenes, followed by transcriptional reactivation [63]. In HPV-negative LSCC patients, genes such as CDKN2A, MGMT, MLH1, and DAPK are frequently methylated, resulting in the inhibition of gene transcription and gene silencing [64]. According to the principles of phylogenetic tree analysis, mutations in TP53 and copy number alterations at 3q (contains SOX2), 9p (contains CDKN2A), 11q (contains CCND1), and 2q (contains NFE2L2) are considered to be backbone variants. During the progression from intraepithelial neoplasia to malignancy, certain genes exhibit clonal dominance, resulting in the clonal diversity of tumor cells [65]. Additionally, tobacco smoke and alcohol can modify the cellular chromatin through histone modification and impact gene transcriptions [66]. DNA methylation status plays a crucial role in gene regulation and is closely associated with histone modifications. Active gene expression is linked to histone H3 lysine 9 (H3K9) acetylation and histone H3 lysine 4 (H3K4) biomethylation [67]. Furthermore, it has been observed that smoking and alcohol consumption have the potential to induce genetic damage in miRNA genes, particularly in regions characterized by single nucleotide polymorphisms. These regions are closely linked to the regulation of the P53 gene [68]. In the case of mutant TP53, there is an upregulation of programmed cell death ligand 1 (PD-L1) due to the modulation of miR-34 activity. Conversely, in wild-type TP53 tumor cells, DNA damage leads to an increase in miR-34 expression, which subsequently interacts with the 3’-untranslated region of PD-L1 and suppresses its protein expression. This TP53/miR-34/PD-L1 pathway highlights the significant disparity in PD-L1 production between TP53 mutant tumors and wild-type tumors [69]. It is hypothesized that the higher levels of PD-L1 exhibited by HPV-negative LSCC patients are likely due to the synchronous dual primary LSCC/ESPT with increased TP53 of the mutant type (Fig. 3A).

Key oncogenic mechanisms in HPV-negative and HPV-positive laryngeal cancers. A: Smoking and alcohol consumption cause mutations in the TP53 gene and affect DNA methyltransferase (DNMT)-dependent regulation of miRNA expression, leading to tumor transformation. B: By engaging the ubiquitin-protein ligase E3A (E6AP), E6 binds with p53 and promotes its degradation. Ubiquitinated TP53 also causes changes in the expression of miR-16, miR-15a, miR-143, miR-145, and miR-195. Cell cycle protein D1 expression begins when cell growth signaling occurs. Cell cycle protein E then activates CDK2, phosphorylates RB, releases E2F, and initiates cell cycle entry gene transcription

The degree of CpG island DNA methylation in HPV-positive LSCC was found to be significantly higher compared to HPV-negative LSCC. The most researched DNA methyltransferases are DNMT3a, DNMT3b, and DNMT1. It has been observed that the overexpression of DNMT1, DNMT3a, and DNMT3b is induced by the viral proteins E6 and E7, resulting in overall DNA hypermethylation [70]. This epigenetic alteration plays a role in the pathogenesis of respiratory papillomatosis recurrences, with some cases showing clonal changes in the progression of recurrences. Additionally, similar epigenetic events have been identified in two cases of HPV-infected laryngeal benign lesions that progressed to squamous cell carcinomas [71]. In the comparison between HPV-positive LSCC and cervical squamous cell carcinoma, there was an observed increase in the expression of miR-15a, miR-16, miR-195, miR-143, and miR-145. Significant overlap of these differentially expressed microRNAs was also found, suggesting that HPV-dependent microRNA expression disturbances are often present regardless of the anatomical location of the tumor [72]. Further investigations suggest that this miRNA expression may be directly influenced by viral E6 and E7 oncoproteins [73]. According to this research, viral integration events commonly cause host chromatin alterations, which further influence the carcinogenic process of HPV-positive malignancies [74]. The papillomavirus genome is connected with transcriptionally active host chromatin regions throughout the viral life cycle to enhance viral replication, transcription, DNA amplification, and persistence [75]. The primary drivers of these viral oncogenes are the oncoproteins E6 and E7, which not only induce infection but also contribute to epigenetic alterations associated with malignant transformation. These oncoproteins interact with cellular chaperones involved in the interdependent viral and cellular cycles within complexly differentiated epithelia [76]. Overall, HPV-positive LSCC exhibits susceptibility to DNA methylation, and its oncogenic mechanism primarily relies on the oncoproteins E6 and E7. Consequently, it is probable that LSCC and ESPT originate from a shared source and demonstrate shared genetic variations (Fig. 3B).

The process of cancer cell division, resulting in the emergence of tumor cells, induces notable molecular, cellular, and physical modifications in the surrounding tissues, thereby establishing a tumor microenvironment (TME). This interplay between cancer cells (seeds) and the microenvironment (soil) facilitates the progression of tumor growth [77]. TME is not totally homogeneous, as different regions of the tumor may exhibit diverse blood densities, lymphovascular networks, immune infiltrating cell populations, and extracellular matrix compositions. External oncogenic factors can not only modulate cell signaling to directly cause phenotypic diversity in tumor cells but can also act as selection pressures, leading to regional heterogeneity and supporting the cloning of cells that proliferate efficiently in the context of a given microenvironment [78]. For example, tumor cells with the same genotype within a clone can exhibit varied behavior in response to alterations in the microenvironment (such as hypoxia, immune monitoring, and additional extrinsic variables), leading to intratumor heterogeneity [79]. Likewise, microenvironmental factors, such as the proximity of cancer cells to cancer-associated fibroblasts or hypoxia, can affect the “quiescence” of cancer cells, causing the cells to exhibit more or less stem cell-like behavior. In addition, it is becoming clear that host-tumor reactivity, as mediated by immune cells in the tumor microenvironment, is important for tumor formation [80]. The immune cell-mediated host-tumor reactivity within the TME establishes a foundation for the development of clonal tumors.

For LSCC, although both HPV-negative and HPV-positive LSCC are among the cancer types with the highest immune filtration rates, the degree and composition of immune cell infiltration vary depending on the etiology [81, 82] (Fig. 4). HPV-negative LSCC is characterized by a “cold” immune response, while the presence of numerous random mutations or overexpression of cellular genes contributes to intra-tumoral immune heterogeneity [83]. Reduced numbers of dendritic cells, a subpopulation of specialized antigen-presenting cells that drive T-cell differentiation, were seen in the interstitial tumor region of smoking patients. Defects in dendritic cell maturation also affect regulatory T cells as immature dendritic cells transform into gene-tolerant dendritic cells and secrete higher levels of TGF-β1, activates naive T cells to become Treg cells [84]. TGF-β expression in the larynx is reported to be higher in malignancies than in dysplastic lesions, and it may be a valuable diagnostic for malignant transformation [85]. In addition, smoking leads to less infiltration of activated cytotoxic T lymphocytes (CTLs) in intraepithelial and mesenchymal areas, thereby suppressing the immune response to TME during smoking exposure [86]. To put it another way, smoking is highly likely to weaken the immunological response mediated by T cells. T cells are critical mediators of the adaptive immune response, and an imbalanced or incorrect T cell response may contribute to cancer progression and other immune disorders. Essentially, carcinogen exposure prevents the differentiation and maturation of precursors and progenitors. Under selection pressures, proliferating cells of any differentiation stage can be susceptible to introducing and accumulating mutations, and accumulate sufficient driver mutations to get the benefits of clonal proliferation [87]. Therefore, monitoring the functional status of T cells is particularly important in HPV-negative LSCC. Furthermore, we need to understand how damage changes the tissue microenvironment and why some mutations can be both harmful and advantageous to cells depending on the tissue microenvironment.

The outcome of clonal competition between HPV-positive and HPV-negative laryngeal cancers. In general, wild-type normal cells (green) are preferred over mutant cells (yellow). Nevertheless, alterations in the tumor immune microenvironment resulting from distinct etiologies (HPV-positive cell exhibiting “hot” immunity and HPV-negative cell exhibiting “cold” immunity) may grant a competitive edge to mutant cells, enabling them to surpass wild-type cells and establish dominance within the field. Consequently, mutant normal cells undergo further transformation into tumor cells (red) under the influence of the surrounding environment and subsequently remaining competitive

HPV-positive LSCC has been found to exhibit a higher abundance of Tc17 lymphocytes, naive CD4 + T cells, infiltrating CD8 + T cells, bone marrow dendritic cells, and TIL cells compared to HPV-negative LSCC [82]. It has been demonstrated that a high density of tumor-infiltrating CD8 + T cells has been shown to be indicative of favorable clinical outcomes in various cancer types, including laryngeal cancer [88]. There is controversy over CD4 + T cells’ role in anti-cancer immunity, although most studies suggest that tumor-infiltrating CD4 + T cells may serve as a prognostic marker for Treg, a crucial mediator of tumor immunosuppression [89]. During the early stages of HPV infection, the expression of E5 allows the virus to evade detection by anti-viral CD4 + and CD8 + T cells. This evasion mechanism leads to increased viral persistence, replication, and spread to neighboring cells, ultimately contributing to malignant transformation [90]. Moreover, cell cultures derived from HPV-positive LSCC patients exhibit significantly elevated levels of chemokines, including CXCL21, CXCL17, CXCL12, CCL10, and CCL9, as well as slightly higher levels of cytokines such as IL-23, IL-17, IL-2, and IFN-γ. These chemokines not only play a role in establishing a pro-tumor microenvironment and facilitating organ-directed metastasis, but also contribute to disease progression [91]. HPV-positive LSCC patients showed more mDCs and slightly more pDCs and monocytes/macrophages [82]. Abundant CD68 + macrophages are related with lymph node metastases, extraperitoneal dissemination, and advanced disease [92]. All of these were attributed to the persistent expression of E6 and E7 oncoproteins, finally leading to the immune escape of tumor cells and a more malignant phenotype [77]. Overall, HPV-positive LSCC demonstrates heightened activation and infiltration of immune cells, leading to stromal alterations that exert a direct influence on the adjacent tissues, thereby facilitating the phenomenon of field cancerization and ultimately fostering the proliferation of malignant clones. Consequently, the persistent expression of the early proteins E6 and E7, in LSCC presents a promising target for immunotherapeutic interventions.