Intratumoral genomic heterogeneity in primary head and neck cancer and corresponding metastases detected by dual-FISH

Intratumoral genomic heterogeneity, which can be defined as both intersample and intrasample heterogeneity, is still a poorly understood phenomenon in head and neck squamous cell carcinoma (HNSCC) with presumed implications on tumor behavior and even prognosis. We analyzed 89 tumor specimen from 37 HNSCC patients by fluorescence in situ hybridization (dual-FISH) using specific DNA probes binding to centromeric sites of 6 chromosomes to investigate intratumoral heterogeneity. A derivation from disomy in at least 1/6 chromosomes was detected in 88/89 (99%) specimen. In 33% of these samples, a change in ploidy could be suspected. Intrasample heterogeneity was detected in 68/89 (76%). Intrasample heterogeneity was more pronounced in primary tumors than in metastatic tumors. Analysis of the intersample heterogeneity revealed notable differences between the 6 chromosomes with the highest discordance detected for chromosome 3 (46%) and the lowest for chromosome 11 (27%). Following our results, it seems important to us to underline that intratumoral heterogeneity exists as intra- and sample heterogeneity in HNSCC. Altogether, trisomic cells were significantly more frequent in primary tumors than in metastases (p=0.01) while, in turn, monosomic cells were significantly more frequent in metastases (p=0.029). In individual cases the extent of discordance between corresponding samples made a common clonal precursor unlikely. In these cases, the synchronous development of a primary tumor and a carcinoma of unknown primary ('CUP syndrome'), otherwise undetected, should be considered.     

Clonal Heterogeneity in Human Esophageal Squamous Cell Carcinomas on DNA Analysis

Cancers are thought to arise through multistep accumulation of somatic mutations in the progeny of a single cell. Multiple mutations may induce molecular intratumor heterogeneity. Therefore, we examined molecular clonal heterogeneity in esophageal squamous cell carcinomas. Twenty-four esophageal squamous cell carcinomas and associated lymph node metastases were examined for microsatellite alterations, and abnormalities of the p53 and transforming growth factor-β type II receptor ( TGF-β RII ) genes. There were eight cases (33%) showing different patterns of loss of heterozygosity in primary tumors and metastatic lymph nodes with microsatellite markers. On the other hand, the abnormalities of p53 were identical in all these cases. No mutation was detected in the simple repeated sequences of the TGF-β RII gene. These results indicate that molecular clonal heterogeneity exists in esophageal squamous cell carcinomas. Therefore, care is necessary in preoperative genetic diagnosis using biopsy samples.     

Unknown primary head and neck squamous cell carcinoma: molecular identification of the site of origin

BACKGROUND: Unknown primary head and neck squamous cell carcinoma (HNSCC) presents as a cervical lymph node metastasis without identification of the primary tumor, despite thorough diagnostic work-up that includes physical examination, computed tomography, esophagoscopy, laryngoscopy, bronchoscopy, and multiple surveillance biopsies. We investigated whether the site of origin of the primary tumor could be localized in the upper aerodigestive tract mucosa by detection of genetic alterations identical to those found in metastatic lesions. METHODS: Microsatellite analysis was performed on metastatic tumors obtained from 18 patients with unknown primary HNSCC. Histologically benign surveillance biopsy specimens were also analyzed. Patients were followed up to 13 years with continuing surveillance for primary mucosal tumors. Most patients were treated with neck dissection followed by radiation therapy to the affected neck and ipsilateral Waldeyer's ring. RESULTS: In 10 (55%) of the 18 patients, at least one histopathologically benign mucosal biopsy specimen from defined anatomic sites (i.e., most likely sites for an occult primary tumor) demonstrated a pattern of genetic alterations identical to that present in cervical lymph node metastases. One patient harboring genetic alterations in the base of the tongue and two patients harboring genetic alterations in a tonsillar fossa subsequently developed HNSCC in the identical or adjacent mucosal region; all three of the primary head and neck mucosal tumors that eventually appeared between 1 and 13 years later in these patients had genetic changes identical to those in the benign mucosal biopsy specimens and in the metastatic lymph nodes. CONCLUSIONS: These data support the hypothesis that histopathologically benign mucosa of the upper aerodigestive tract may harbor foci of clonal, preneoplastic cells that are genetically related to metastatic HNSCC and that such mucosal sites are the sites of origin of unknown primary HNSCC. Microsatellite analysis may represent a clinically useful tool for determining such sites.     

The clonal evolution of metastases from primary serous epithelial ovarian cancers

Several models of evolution from primary cancers to metastases have been proposed; but the most widely accepted is the clonal evolution model proposed for colorectal cancer in which tumors develop by a process of linear clonal evolution driven by the accumulation of somatic genetic alterations. Various other models of cancer progression and metastasis have been proposed, including parallel evolution and the same gene model. The aim of this study was to investigate the evolution of metastases from primary cancer in 22 patients diagnosed with high‐grade serous epithelial ovarian cancer. We established somatic genetic profiles based on the pattern of loss of heterozygosity, in several different regions of tumor tissue within the primary tumor and metastatic deposits from each case. Maximum parsimony tree analysis was used to examine the evolutionary relationship between the primary and metastatic samples for each patient. In addition, we investigated the extent of genetic heterogeneity within and between metastatic tumors compared with primary ovarian tumors. Our data suggest that most, if not all, metastases are clonally related to the primary tumors. However, the data oppose a single model of linear‐clonal evolution whereby a late stage clone within the primary tumor acquires additional genetic changes that enable metastatic progression. Instead, the data support a model in which primary ovarian cancers have a common clonal origin, but become polyclonal, with different clones at both early and late stages of genetic divergence acquiring the ability to progress to metastasis. © 2009 Wiley‐Liss, Inc.     

A flow cytometric analysis of DNA content in primary and metastatic lesions of esophageal squamous cell carcinoma.

BACKGROUND. DNA content of malignant tumors has been considered a significant prognostic factor, but intratumor variations in DNA content and differences in DNA content between primary and metastatic lesions have been found in various tumors. METHODS. DNA indexes of multiple samples obtained from different sites in each tumor were determined by flow cytometry (FCM) in 27 esophageal squamous cell carcinomas. RESULTS. Intratumor variation in DNA indexes in primary lesions was found in 10 (37%) of the 27 specimens. Of the rest, all DNA indexes were diploid in 5 and all identically aneuploid in 12 samples. A DNA index differing from that of the metastatic lesion was found in four (50%) of eight primary lesions; however, a component of the DNA index identical to that of metastatic lesion was found in seven (88%) of eight primary lesions. CONCLUSIONS. Recurrence after a "curative" operation was frequent in patients with a tumor that had an aneuploid DNA index, with or without a variation in DNA indexes. There was no recurrence in the five patients with tumors that had only a diploid DNA index. These results suggest that differences in DNA content between primary and metastatic lesions reflect intratumor variation in DNA content in the primary lesions. The presence of a tumor cell population with an aneuploid DNA content, even when the DNA content varied in the primary lesion, may indicate an aggressive clinical course in patients with esophageal squamous cell carcinoma.     

Human papillomavirus genome detection by in situ hybridization in fine-needle aspirates of metastatic lesions from head and neck squamous cell carcinomas

BACKGROUND: Patients with head and neck squamous cell carcinoma (HNSCC) often present with metastatic disease. The diagnosis of metastatic lesions usually is determined by fine-needle aspiration. Human papillomavirus (HPV) is now being considered as a causative agent in a subset of HNSCC. The objectives of this study were, first; to search for the presence of HPV DNA by in situ hybridization (ISH) in metastatic lesions from HNSCC using alcohol-fixed, archival, cytopathologic material; second, to characterize the cytologic features of HPV-positive metastatic lesions of HNSCC; and, third, to determine whether there is a correlation between the presence of HPV DNA and the origin of metastatic lesions. METHODS: The authors performed chromogenic ISH analysis for HPV DNA on fine-needle aspiration materials from metastatic lesions from 26 patients with HNSCC. Along with the ISH analysis, a detailed cytologic review was performed, and cytopathologic features were recorded. The HPV DNA status in metastatic lesion was correlated with cytopathologic features and primary tumor location. RESULTS: The integration of HPV DNA was visualized microscopically on tumor cell nuclei in 15% of aspirates. The anatomic locations of the study samples were as follows: 16 lymph node aspirates (11 cervical lymph nodes and 5 lymph nodes at other sites other), 5 tracheostomy sites, and 5 miscellaneous sites located on the head and neck area. Cytologic review revealed 13 keratinized and 13 nonkeratinized metastatic tumors. HPV DNA was detected in four metastatic sites (three lymph nodes and one tracheostomy site). All HPV DNA-positive tumors were of the nonkeratinizing type (P < 0.05; Fisher exact test). The origins of HPV-positive tumors included two laryngeal sites, one nasopharyngeal site, and one oral cavity site. CONCLUSIONS: The current findings showed that archival cytology slides can be used for HPV DNA detection with ISH. The results also showed that HPV DNA-containing HNSCC has distinctively nonkeratinizing cytologic features. The authors concluded that HPV DNA not only is involved in the initiation of tumoral processes but also plays an important role in the development of metastatic disease.     

Spatial genomic diversity associated with APOBEC mutagenesis in squamous cell carcinoma arising from ovarian teratoma

Although the gross and microscopic features of squamous cell carcinoma arising from ovarian mature cystic teratoma (MCT-SCC) vary from case to case, the spatial spreading of genomic alterations within the tumor remains unclear. To clarify the spatial genomic diversity in MCT-SCCs, we performed whole-exome sequencing by collecting 16 samples from histologically different parts of two MCT-SCCs. Both cases showed histological diversity within the tumors (case 1: nonkeratinizing and keratinizing SCC and case 2: nonkeratinizing SCC and anaplastic carcinoma) and had different somatic mutation profiles by histological findings. Mutation signature analysis revealed a significantly enriched apolipoprotein B mRNA editing enzyme catalytic subunit (APOBEC) signature at all sites. Intriguingly, the spread of genomic alterations within the tumor and the clonal evolution patterns from nonmalignant epithelium to cancer sites differed between cases. TP53 mutation and copy number alterations were widespread at all sites, including the nonmalignant epithelium, in case 1. Keratinizing and nonkeratinizing SCCs were differentiated by the occurrence of unique somatic mutations from a common ancestral clone. In contrast, the nonmalignant epithelium showed almost no somatic mutations in case 2. TP53 mutation and the copy number alteration similarities were observed only in nonkeratinizing SCC samples. Nonkeratinizing SCC and anaplastic carcinoma shared almost no somatic mutations, suggesting that each locally and independently arose in the MCT. We demonstrated that two MCT-SCCs with different histologic findings were highly heterogeneous tumors with clearly different clones associated with APOBEC-mediated mutagenesis, suggesting the importance of evaluating intratumor histological and genetic heterogeneity among multiple sites of MCT-SCC.     

Circulating tumor DNA profiling reveals clonal evolution and real‐time disease progression in advanced hepatocellular carcinoma

Circulating tumor DNA (ctDNA) provides a potential non‐invasive biomarker for cancer diagnosis and prognosis, but whether it could reflect tumor heterogeneity and monitor therapeutic responses in hepatocellular carcinoma (HCC) is unclear. Focusing on 574 cancer genes known to harbor actionable mutations, we identified the mutation repertoire of HCC tissues, and monitored the corresponding ctDNA features in blood samples to evaluate its clinical significance. Analysis of 3 HCC patients' mutation profiles revealed that ctDNA could overcome tumor heterogeneity and provide information of tumor burden and prognosis. Further analysis was conducted on the 4th HCC case with multiple lesion samples and sequential plasma samples. We identified 160 subclonal SNVs in tumor tissues as well as matched peritumor tissues with PBMC as control. 96.9% of this patient's tissue mutations could be also detected in plasma samples. These subclonal SNVs were grouped into 9 clusters according to their trends of cellular prevalence shift in tumor tissues. Two clusters constituted of tumor stem somatic mutations showed circulating levels relating with cancer progression. Analysis of tumor somatic mutations revealed that circulating level of such tumor stem somatic mutations could reflect tumor burden and even predict prognosis earlier than traditional strategies. Furthermore, HCK (p.V174M), identified as a recurrent/metastatic related mutation site, could promote migration and invasion of HCC cells. Taken together, study of mutation profiles in biopsy and plasma samples in HCC patients showed that ctDNA could overcome tumor heterogeneity and real‐time track the therapeutic responses in the longitudinal monitoring.     

Detection of human papillomavirus-16 in fine-needle aspirates to determine tumor origin in patients with metastatic squamous cell carcinoma of the head and neck.

PURPOSE: Patients with head and neck squamous cell carcinoma (HNSCC) often clinically present with metastases to regional lymph nodes. Fine-needle aspiration of neck masses is routinely used to establish the presence of metastatic carcinoma and in turn to initiate a subsequent workup to determine the site of tumor origin. Human papillomavirus (HPV) 16 is an important etiologic agent for HNSCCs that arise from the oropharynx but less so for tumors from non-oropharyngeal sites. HPV16 detection thus provides a strategy for localizing an important subset of HNSCCs, but this approach has not been applied to fine-needle aspiration specimens. EXPERIMENTAL DESIGN: We did in situ hybridization for HPV16 on 77 consecutive aspirated neck masses diagnosed as metastatic squamous cell carcinoma. P16 immunohistochemistry was also done because p16 overexpression may serve as a surrogate marker of HPV-associated HNSCC. RESULTS: HPV16 was detected in 13 of the 77 (17%) aspirates. By site of origin, HPV16 was detected in 10 of 19 metastases from the oropharynx but in none of 46 metastases from other sites (53% versus 0%; P < 0.0001). HPV16 was not detected in 2 branchial cleft cysts misdiagnosed as metastatic squamous cell carcinoma, but it was detected in 3 of 10 metastases from occult primary tumors. P16 expression was associated with the presence of HPV16: 12 of 13 HPV16-positive metastases exhibited p16 expression, whereas only 4 of 62 HPV16-negative metastases were p16 positive (92% versus 6%; P < 0.0001). P16 expression also correlated with site of tumor origin: 13 of 19 oropharyngeal metastases were p16 positive, whereas only 1 of 46 non-oropharyngeal metastases was p16 positive (68% versus 2%; P < 0.0001). CONCLUSIONS: HPV16 status can be determined in tumor cells aspirated from the necks of patients with metastatic HNSCC. Its presence is a reliable indicator of origin from the oropharynx.     

Heterogeneity of cell surface antigen expression of human small cell lung cancer detected by monoclonal antibodies

Using immunohistochemistry, radiobinding, and indirect immunofluorescence assays, seven distinct cell surface antigens, detected by monoclonal antibodies, were analyzed for the degree of homogeneity or heterogeneity of antigen expression on a panel of human small cell lung cancers. The panel included 7 tumors taken directly from patients, 21 established cell lines (9 of which were derived from different metastatic sites of 3 patients), and 33 clonal derivatives of 3 lines. With all assays, considerable heterogeneity of antigen expression between tumors from different patients was observed. In both fresh tumors and in cell lines, as well as in cell lines established from different metastatic sites in an individual patient, we observed intratumor heterogeneity finding antigen positive and negative cells and variation in antigenic density, by immunohistochemistry and indirect immunofluorescence assays. Antigenic expression was not cell cycle dependent. In addition, when cell lines or patient samples expressing antigen positive and antigen negative tumor cells were cloned, heterogeneity of antigenic expression was still present in the clonal lines. This suggests that either the expression of the antigen was not heritable and/or the ability to regenerate antigenic heterogeneity is an intrinsic property of the tumor cells. The heterogeneity of antigen expression on lung cancer cells has significant implications for the use of these and other monoclonal antibodies in the study and therapy of lung cancer.     

Delineating an epigenetic continuum in head and neck cancer

A tissue field of somatic genetic alterations precedes the histopathological phenotypic changes of carcinoma. Genomic changes could be of potential use in the diagnosis and prognosis of pre-invasive squamous head and neck carcinoma (HNSCC) lesions and as markers for cancer risk assessment. Studies of sequential molecular alterations and genetic progression of pre-invasive HNSCC have not been clearly defined. Studies have shown recurring alterations at chromosome 9p21 (location of the CDKN2A) and TP53 mutations in the early stages of HNSCC. However, gene silencing via hypermethylation is still a relatively new idea in the development of HNSCC and little is known about the contribution of epigenetics to the development of neoplasia, its transformation, progression, and recurrence in HNSCC. This review examines the role of promoter hypermethylation of tumor suppressor genes in the progression continuum from benign papillomas to malignancy in HNSCC.     

Multi‐regional sequencing reveals intratumor heterogeneity and positive selection of somatic mtDNA mutations in hepatocellular carcinoma and colorectal cancer

Recent studies have revealed significant intratumor heterogeneity (ITH) of nuclear genome mutations and highlighted its function in tumor progression and treatment resistance. However, the ITH of somatic mitochondrial DNA (mtDNA) mutations detected in cancers remains unknown. In this study, we performed multiregional mtDNA sequencing of tumor and paratumor tissue samples from 12 hepatocellular carcinoma (HCC) and 13 colorectal cancer (CRC) patients. A substantial level of mtDNA mutations was found in paired non‐HCC inflammatory tissues, suggesting that these tissues might not be mtDNA‐genetically “normal.” Moreover, our data indicated that the ITH of somatic mtDNA mutations was a common feature in HCC and CRC patients. In addition, we found that shared mutations which were observed in at least 2 samples in each patient exhibited a significantly higher heteroplasmic level than mutations that were private to a specific tumor region from both HCC (p = 0.039) and CRC patients (p = 0.001). The heteroplasmic level of shared mutations was positively correlated with intratumoral recurrence of mtDNA mutations. We also found that shared mutations in tumor tissues with a higher degree of pathogenicity risk exhibited a higher heteroplasmic level and intratumoral recurrence in both HCC and CRC patients. These findings suggest that some mtDNA mutations may undergo positive selection during the clonal expansion. Taken together, our analyses identified various levels of ITH of somatic mtDNA mutations in HCC and CRC patients and provided evidence supporting the positive selection working on some somatic mtDNA mutations in tumor tissues.     

Synchronous cancers in patients with head and neck cancer

BACKGROUND:

Second primary malignancies (SPMs) are the leading cause of death in survivors of head and neck squamous cell carcinoma (HNSCC). Synchronous SPMs are of significant clinical interest because they potentially can be identified by screening procedures at the time of diagnosis of the index cancer. Recently, human papillomavirus (HPV) has emerged as a distinct risk factor for oropharyngeal head and neck squamous cell carcinoma (HNSCC), differing from classic tobacco/alcohol‐associated HNSCC, suggesting that there also may be distinct patterns of synchronous SPMs.

METHODS:

The authors performed a population‐based cohort study in 64,673 patients in the National Cancer Institute Surveillance, Epidemiology, and End Results registry (1979‐2008), defining risks of synchronous SPM in patients with HNSCC who were diagnosed before and after the emergence of prevalent HPV‐associated oropharyngeal HNSCC. Excess risk was calculated using standardized incidence ratios (SIR) and excess absolute risk per 100 patients.

RESULTS:

Among patients with HNSCC, the SIR of synchronous SPM was 5.0, corresponding to 2.62 excess cases per 100 patients. The site with the highest excess risk of a second cancer was the head and neck (SIR, 41.4), followed by the esophagus (SIR, 21.8), and lung (SIR, 7.4). The risk of synchronous SPM changed markedly over time for patients with oropharyngeal HNSCC. In the 1970s and 1980s, oropharyngeal cancers carried the highest risk of SPM. Risk began to dramatically decline in the 1990s; and currently, oropharyngeal cancers carry the lowest risk of synchronous SPM.

CONCLUSIONS:

The current data are consistent with the etiologic shift of oropharyngeal HNSCC, from a primarily tobacco‐associated malignancy associated with significant field cancerization of the upper aerodigestive mucosa, to a malignancy primarily caused by oncogenic human papillomavirus. Cancer 2013. © 2013 American Cancer Society.     

MicroRNA-138 suppresses invasion and promotes apoptosis in head and neck squamous cell carcinoma cell lines

Metastasis is a critical event in the progression of head and neck squamous cell carcinoma (HNSCC). To identify microRNAs associated with HNSCC metastasis, six paired HNSCC cell lines with different metastatic potential were examined. Using microarrays, a panel of differentially expressed microRNAs was identified, including reduction of miR-138 in highly metastatic cells. Ectopic transfection of miR-138 suppressed cell invasion and led to cell cycle arrest and apoptosis. Knockdown of miR-138 enhanced cell invasion and suppressed apoptosis. Thus, our results suggested miR-138 acts as a tumor suppresser and may serve as a therapeutic target for HNSCC patients at risk of metastasis.     

Multiregion whole-exome sequencing of matched primary and metastatic tumors revealed genomic heterogeneity and suggested polyclonal seeding in colorectal cancer metastasis

BackgroundDistant metastasis accounts for 90% of deaths from colorectal cancer (CRC). Genomic heterogeneity has been reported in various solid malignancies, but remains largely under-explored in metastatic CRC tumors, especially in primary to metastatic tumor evolution.Patients and methodsWe conducted high-depth whole-exome sequencing in multiple regions of matched primary and metastatic CRC tumors. Using a total of 28 tumor, normal, and lymph node tissues, we analyzed inter- and intra-individual heterogeneity, inferred the tumor subclonal architectures, and depicted the subclonal evolutionary routes from primary to metastatic tumors.ResultsCRC has significant inter-individual but relatively limited intra-individual heterogeneity. Genomic landscapes were more similar within primary, metastatic, or lymph node tumors than across these types. Metastatic tumors exhibited less intratumor heterogeneity than primary tumors, indicating that single-region sequencing may be adequate to identify important metastasis mutations to guide treatment. Remarkably, all metastatic tumors inherited multiple genetically distinct subclones from primary tumors, supporting a possible polyclonal seeding mechanism for metastasis. Analysis of one patient with the trio samples of primary, metastatic, and lymph node tumors supported a mechanism of synchronous parallel dissemination from the primary to metastatic tumors that was not mediated through lymph nodes.ConclusionsIn CRC, metastatic tumors have different but less heterogeneous genomic landscapes than primary tumors. It is possible that CRC metastasis is, at least partly, mediated through a polyclonal seeding mechanism. These findings demonstrated the rationale and feasibility for identifying and targeting primary tumor-derived metastasis-potent subclones for the prediction, prevention, and treatment of CRC metastasis.     

Receptor-type Protein Tyrosine Phosphatase �� Regulates Met Phosphorylation and Function in Head and Neck Squamous Cell Carcinoma

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer and has a high rate of mortality. Emerging evidence indicates that hepatocyte growth factor receptor (or Met) pathway plays a pivotal role in HNSCC metastasis and resistance to chemotherapy. Met function is dependent on tyrosine phosphorylation that is under direct control by receptor-type protein tyrosine phosphatase β (RPTP-β). We report here that RPTP-β expression is significantly downregulated in HNSCC cells derived from metastatic tumors compared to subject-matched cells from primary tumors. Knockdown of endogenous RPTP-β in HNSCC cells from primary tumor potentiated Met tyrosine phosphorylation, downstream mitogen-activated protein (MAP) kinase pathway activation, cell migration, and invasion. Conversely, restoration of RPTP-β expression in cells from matched metastatic tumor decreased Met tyrosine phosphorylation and downstream functions. Furthermore, we observed that six of eight HNSCC tumors had reduced levels of RPTP-β protein in comparison with normal oral tissues. Collectively, the results demonstrate the importance of RPTP-β in tumor biology of HNSCC through direct dephosphorylation of Met and regulation of downstream signal transduction pathways. Reduced RPTP-β levels, with or without Met overexpression, could promote Met activation in HNSCC tumors.     

Plasmacytoid dendritic cell subpopulations in head and neck squamous cell carcinoma

Human plasmacytoid dendritic cells (PDCs) are present in solid tumor tissue and metastatic cervical lymph nodes (CLN) in head and neck squamous cell carcinoma (HNSCC). We recently showed that classical PDC functions are heavily disturbed in the tumor microenvironment. In this study we present a new approach to the subject by introducing 3 PDC subsets in HNSCC, characterized by the surface markers CD25, CD56 and CD203c. The first subset, positive for CD25, is significantly induced by HNSCC in?vitro and present in metastatic lymph nodes in?vivo. This subset can be phenotypically subdivided into matured cells and into a group expressing early T cell markers. Functionally this subgroup is associated with the secretion of IL-8. The second subset, positive for CD56, constitutes 4-5% of all PDCs and is significantly down-regulated by HNSCC. Furthermore, this population sporadically expresses perforin/granzyme B and is absent in metastatic lymph nodes. The third subset, positive for the basophile marker CD203c, is inducible by crosslinking BDCA-2 in the presence of HNSCC and IL-4. Future studies will have to clarify the in vivo relevance of the different PDC subsets in HNSCC.