Targeting tumour-associated macrophages in hodgkin lymphoma using engineered extracellular matrix-mimicking cryogels.

Tumour-associated macrophages are linked with poor prognosis and resistance to therapy in Hodgkin lymphoma; however, there are no suitable preclinical models to identify macrophage-targeting therapeutics. We used primary human tumours to guide the development of a mimetic cryogel, wherein Hodgkin (but not Non-Hodgkin) lymphoma cells promoted primary human macrophage invasion. In an invasion inhibitor screen, we identified five drug hits that significantly reduced tumour-associated macrophage invasion: marimastat, batimastat, AS1517499, ruxolitinib, and PD-169316. Importantly, ruxolitinib has demonstrated recent success in Hodgkin lymphoma clinical trials. Both ruxolitinib and PD-169316 (a p38 mitogen-activated protein kinase (p38 MAPK) inhibitor) decreased the percent of M2-like macrophages; however, only PD-169316 enhanced the percentage of M1-like macrophages. We validated p38 MAPK as an anti-invasion drug target with five additional drugs using a high-content imaging platform. With our biomimetic cryogel, we modeled macrophage invasion in Hodgkin lymphoma and then used it for target discovery and drug screening, ultimately identifying potential future therapeutics.

Quantitative single-cell analysis of immunofluorescence protein multiplex images illustrates biomarker spatial heterogeneity within breast cancer subtypes.

BACKGROUND: The extent of cellular heterogeneity in breast cancer could have potential impact on diagnosis and long-term outcome. However, pathology evaluation is limited to biomarker immunohistochemical staining and morphology of the bulk cancer. Inter-cellular heterogeneity of biomarkers is not usually assessed. As an initial evaluation of the extent of breast cancer cellular heterogeneity, we conducted quantitative and spatial imaging of Estrogen Receptor (ER), Progesterone Receptor (PR), Epidermal Growth Factor Receptor-2 (HER2), Ki67, TP53, CDKN1A (P21/WAF1), CDKN2A (P16INK4A), CD8 and CD20 of a tissue microarray (TMA) representing subtypes defined by St. Gallen surrogate classification. METHODS: Quantitative, single cell-based imaging was conducted using an Immunofluorescence protein multiplexing platform (MxIF) to study protein co-expression signatures and their spatial localization patterns. The range of MxIF intensity values of each protein marker was compared to the respective IHC score for the TMA core. Extent of heterogeneity in spatial neighborhoods was analyzed using co-occurrence matrix and Diversity Index measures. RESULTS: On the 101 cores from 59 cases studied, diverse expression levels and distributions were observed in MxIF measures of ER and PR among the hormonal receptor-positive tumor cores. As expected, Luminal A-like cancers exhibit higher proportions of cell groups that co-express ER and PR, while Luminal B-like (HER2-negative) cancers were composed of ER+, PR- groups. Proliferating cells defined by Ki67 positivity were mainly found in groups with PR-negative cells. Triple-Negative Breast Cancer (TNBC) exhibited the highest proliferative fraction and incidence of abnormal P53 and P16 expression. Among the tumors exhibiting P53 overexpression by immunohistochemistry, a group of TNBC was found with much higher MxIF-measured P53 signal intensity compared to HER2+, Luminal B-like and other TNBC cases. Densities of CD8 and CD20 cells were highest in HER2+ cancers. Spatial analysis demonstrated variability in heterogeneity in cellular neighborhoods in the cancer and the tumor microenvironment. CONCLUSIONS: Protein marker multiplexing and quantitative image analysis demonstrated marked heterogeneity in protein co-expression signatures and cellular arrangement within each breast cancer subtype. These refined descriptors of biomarker expressions and spatial patterns could be valuable in the development of more informative tools to guide diagnosis and treatment.

Heterogeneity of Circulating Tumor Cell-Associated Genomic Gains in Breast Cancer and Its Association with the Host Immune Response.

Tumor cells that preferentially enter circulation include the precursors of metastatic cancer. Previously, we characterized circulating tumor cells (CTC) from patients with breast cancer and identified a signature of genomic regions with recurrent copy-number gains. Through FISH, we now show that these CTC-associated regions are detected within the matched untreated primary tumors of these patients (21% to 69%, median 55.5%, n = 19). Furthermore, they are more prevalent in the metastases of patients who died from breast cancer after multiple rounds of treatment (70% to 100%, median 93%, samples n = 41). Diversity indices revealed that higher spatial heterogeneity for these regions within primary tumors is associated with increased dissemination and metastasis. An identified subclone with multiple regions gained (MRG clone) was enriched in a posttreatment primary breast carcinoma as well as multiple metastatic tumors and local breast recurrences obtained at autopsy, indicative of a distinct early subclone with the capability to resist multiple lines of treatment and eventually cause death. In addition, multiplex immunofluorescence revealed that tumor heterogeneity is significantly associated with the degree of infiltration of B lymphocytes in triple-negative breast cancer, a subtype with a large immune component. Collectively, these data reveal the functional potential of genetic subclones that comprise heterogeneous primary breast carcinomas and are selected for in CTCs and posttreatment breast cancer metastases. In addition, they uncover a relationship between tumor heterogeneity and host immune response in the tumor microenvironment. SIGNIFICANCE: As breast cancers progress, they become more heterogeneous for multiple regions amplified in circulating tumor cells, and intratumoral spatial heterogeneity is associated with the immune landscape.

Whole-mount pathology of breast lumpectomy specimens improves detection of tumour margins and focality.

AIMS: Technical limitations in conventional pathological evaluation of breast lumpectomy specimens may reduce diagnostic accuracy in the assessment of margin and focality. A novel technique based on whole-mount serial sections enhances sampling while preserving specimen conformation and orientation. The aim of this study was to investigate assessment of focality and margin status by the use of whole-mount serial sections versus simulated conventional sections in lumpectomies. METHODS AND RESULTS: Two pathologists interpreted whole-mount serial sections and simulated conventional sections for 58 lumpectomy specimens by reporting the closest margin and focality. Measurements were compared by the use of McNemar's chi-squared test. Statistically significant differences were observed in the assignment of both margin positivity (P = 0.014) and multifocality (P = 0.021). A positive margin or multifocal disease was identified by the use of whole-mount serial sections but missed in the simulated conventional assessment in 10.3% and 17.2% of all cases, respectively. There was no case in which a positive margin was detected only in the simulated conventional assessment. CONCLUSIONS: The whole-mount technique is more sensitive than conventional assessment for identifying a positive margin or multifocal disease in breast lumpectomy specimens. Undersampling in conventional sections was implicated in almost all cases of discordance. The majority of positive margins or secondary foci identified only in whole-mount serial sections concerned in-situ disease.

Ki-67 Membranous Staining: Biologically Relevant or an Artifact of Multiplexed Immunofluorescent Staining.

In the process of developing a multiplex of 8 common breast cancer biomarkers (Her2/neu, estrogen receptor, progesterone receptor, Ki-67, aldehyde dehydrogenase-1, NaK-ATPase, cytokeratin 8/18, and myosin smooth muscle) on a single formalin-fixed paraffin-embedded slide using a sequential staining, imaging, and dye bleaching technology developed by General Electric Company, membranous Ki-67 staining was observed and colocalized with Her2/neu staining. Using immunohistochemistry as gold standards, we discovered that membranous Ki-67 was an artifact caused by the binding of cyanine 5-conjugated rabbit polyclonal Ki-67 antibody to a secondary cyanine 3-conjugated donkey anti-rabbit antibody which was previously applied and bound to rabbit Her2/neu antibody in our multiplexing experiment. After blocking with rabbit serum, a successful protocol for 8 biomarker multiplexing without cross-reactivity of antibodies from the same species was developed.

A novel, automated technology for multiplex biomarker imaging and application to breast cancer.

AIMS: Multiplexed immunofluorescence is a powerful tool for validating multigene assays and understanding the complex interplay of proteins implicated in breast cancer within a morphological context. We describe a novel technology for imaging an extended panel of biomarkers on a single, formalin-fixed paraffin-embedded breast sample and evaluating biomarker interaction at a single-cell level, and demonstrate proof-of-concept on a small set of breast tumours, including those which co-express hormone receptors with Her2/neu and Ki-67. METHODS AND RESULTS: Using a microfluidic flow cell, reagent exchange was automated and consisted of serial rounds of staining with dye-conjugated antibodies, imaging and chemical deactivation. A two-step antigen retrieval process was developed to satisfy all epitopes simultaneously, and key parameters were optimized. The imaging sequence was applied to seven breast tumours, and compared with conventional immunohistochemistry. Single-cell correlation analysis was performed with automated image processing. CONCLUSIONS: We have described a novel platform for evaluating biomarker co-localization. Expression in multiplexed images is consistent with conventional immunohistochemistry. Automation reduces inconsistencies in staining and positional shifts, while the fluorescent dye cycling approach dramatically expands the number of biomarkers which can be visualized and quantified on a single tissue section.

C4ST-1/CHST11-controlled chondroitin sulfation interferes with oncogenic HRAS signaling in Costello syndrome.

Costello syndrome is a pediatric genetic disorder linked to oncogenic germline mutations in the HRAS gene. The disease is characterized by multiple developmental abnormalities, as well as predisposition to malignancies. Our recent observation that heart tissue from patients with Costello syndrome showed a loss of the glycosaminoglycan chondroitin-4-sulfate (C4S) inspired our present study aimed to explore a functional involvement of the chondroitin sulfate (CS) biosynthesis gene Carbohydrate sulfotransferase 11/Chondroitin-4-sulfotransferase-1 (CHST11/C4ST-1), as well as an impaired chondroitin sulfation balance, as a downstream mediator of oncogenic HRAS in Costello syndrome. Here we demonstrate a loss of C4S, as well as a reduction in C4ST-1 mRNA and protein expression, in primary fibroblasts from Costello syndrome patients. We go on to show that expression of oncogenic HRAS in normal fibroblasts can repress C4ST-1 expression, whereas interference with oncogenic HRAS signaling in Costello syndrome fibroblasts elevated C4ST-1 expression, thus identifying C4ST-1 as a negatively regulated target gene of HRAS signaling. Importantly, we show that forced expression of C4ST-1 in Costello fibroblasts could rescue the proliferation and elastogenesis defects associated with oncogenic HRAS signaling in these cells. Our results indicate reduced C4ST-1 expression and chondroitin sulfation imbalance mediating the effects of oncogenic HRAS signaling in the pathogenesis of Costello syndrome. Thus, our work identifies C4ST-1-dependent chondroitin sulfation as a downstream vulnerability in oncogenic RAS signaling, which might be pharmacologically exploited in future treatments of not only Costello syndrome and other RASopathies, but also human cancers associated with activating RAS mutations.

Development and evaluation of a robust algorithm for computer-assisted detection of sentinel lymph node micrometastases.

AIMS: Increasing the sectioning rate for breast sentinel lymph nodes can increase the likelihood of detecting micrometastases. To make serial sectioning feasible, we have developed an algorithm for computer-assisted detection (CAD) with digitized lymph node sections. METHODS AND RESULTS: K-means clustering assigned image pixels to one of four areas in a colourspace (representing tumour, unstained background, counterstained background and microtomy artefacts). Four filters then removed 'false-positive' pixels from the tumour cluster. A set of 43 sections containing tumour (a total of 259 foci) and 59 sections negative for malignancy was defined by two pathologists, using light microscopy, and CAD was applied. For the clinically relevant task of identifying the largest focus in each section (micrometastasis in 22/43 sections), the sensitivity and specificity were 100%. Isolated tumour cells (ITCs) were identified in one slide initially considered to be negative. Identification of all 259 foci yielded sensitivities of 57.5% for ITCs (<0.200 mm), 89.5% for micrometastases, and 100% for larger metastases, with one false-positive. Reduced sensitivity was ascribed to variable staining. Nine additional metastases (<0.01-0.3 mm) that were not initially identified were detected by CAD. CONCLUSIONS: This algorithm is well suited to the task of sentinel lymph node evaluation and may enhance the detection of occult micrometastases.

Heightened aberrant deposition of hard-wearing elastin in conduit arteries of prehypertensive SHR is associated with increased stiffness and inward remodeling.

Elastin is a major component of conduit arteries and a key determinant of vascular viscoelastic properties. Aberrant organization of elastic lamellae has been reported in resistance vessels from spontaneously hypertensive rats (SHR) before the development of hypertension. Hence, we have characterized the content and organization of elastic lamellae in conduit vessels of neonatal SHR in detail, comparing the carotid arteries from 1-wk-old SHR with those from Wistar-Kyoto (WKY) and Sprague Dawley (SD) rats. The general structure and mechanics were studied by pressure myography, and the internal elastic lamina organization was determined by confocal microscopy. Cyanide bromide-insoluble elastin scaffolds were also prepared from 1-mo-old SHR and WKY aortas to assess their weight, amino acid composition, three-dimensional lamellar organization, and mechanical characteristics. Carotid arteries from 1-wk-old SHR exhibited narrower lumen and greater intrinsic stiffness than those from their WKY and SD counterparts. These aberrations were associated with heightened elastin content and with a striking reduction in the size of the fenestrae present in the elastic lamellae. The elastin scaffolds isolated from SHR aortas also exhibited increased relative weight and stiffness, as well as the presence of peculiar trabeculae inside the fenestra that reduced their size. We suggest that the excessive and aberrant elastin deposited in SHR vessels during perinatal development alters their mechanical properties. Such abnormalities are likely to compromise vessel expansion during a critical period of growth and, at later stages, they could compromise hemodynamic function and participate in the development of systemic hypertension.

Neuraminidase-1, a subunit of the cell surface elastin receptor, desialylates and functionally inactivates adjacent receptors interacting with the mitogenic growth factors PDGF-BB and IGF-2.

We recently established that the elastin-binding protein, which is identical to the spliced variant of beta-galactosidase, forms a cell surface-targeted complex with two proteins considered "classic lysosomal enzymes": protective protein/cathepsin A and neuraminidase-1 (Neu1). We also found that cell surface-residing Neu1 can desialylate neighboring microfibrillar glycoproteins and facilitate the deposition of insoluble elastin, which contributes to the maintenance of cellular quiescence. Here we provide evidence that cell surface-residing Neu1 contributes to a novel mechanism that limits cellular proliferation by desialylating cell membrane-residing sialoglycoproteins that directly propagate mitogenic signals. We demonstrated that treatment of cultured human aortic smooth muscle cells (SMCs) with either a sialidase inhibitor or an antibody that blocks Neu1 activity induced significant up-regulation in SMC proliferation in response to fetal bovine serum. Conversely, treatment with Clostridium perfringens neuraminidase (which is highly homologous to Neu1) decreased SMC proliferation, even in cultures that did not deposit elastin. Further, we found that pretreatment of aortic SMCs with exogenous neuraminidase abolished their mitogenic responses to recombinant platelet-derived growth factor (PDGF)-BB and insulin-like growth factor (IGF)-2 and that sialidosis fibroblasts (which are exclusively deficient in Neu1) were more responsive to PDGF-BB and IGF-2 compared with normal fibroblasts. Furthermore, we provide direct evidence that neuraminidase caused the desialylation of both PDGF and IGF-1 receptors and diminished the intracellular signals induced by the mitogenic ligands PDGF-BB and IGF-2.

Aldosterone induces elastin production in cardiac fibroblasts through activation of insulin-like growth factor-I receptors in a mineralocorticoid receptor-independent manner.

Aldosterone is known to regulate electrolyte homeostasis, but it may also contribute to other processes, including the maladaptive remodeling of postinfarct hearts. Because aldosterone has been implicated in the stimulation of collagen production in the heart, we investigated whether it would also affect elastin deposition in cultures of human cardiac fibroblasts. We first demonstrated that treatment with 1 to 50 nmol/L aldosterone leads to a significant increase in collagen type I mRNA levels and in subsequent collagen fiber deposition. Pretreatment of cells with the mineralocorticoid receptor antagonist spironolactone, but not with the glucocorticoid receptor antagonist RU 486, inhibited collagen synthesis in aldosterone-treated cultures. Most importantly, we demonstrated that aldosterone also increases elastin mRNA levels, tropoelastin synthesis, and elastic fiber deposition in a dose-dependent manner. Strikingly, neither spironolactone nor RU 486 eliminated aldosterone-induced increases in elastin production. We further discovered that the proelastogenic effect of aldosterone involves a rapid increase in tyrosine phosphorylation of the insulin-like growth factor-I receptor and that the insulin-like growth factor-I receptor kinase inhibitor AG1024 or an anti-insulin-like growth factor-I receptor-neutralizing antibody inhibits both insulin-like growth factor-I and aldosterone-induced elastogenesis. Thus, we have demonstrated for the first time that aldosterone, which stimulates collagen production through the mineralocorticoid receptor-dependent pathway, also increases elastogenesis via a parallel mineralocorticoid receptor-independent pathway involving I insulin-like growth factor-I receptor signaling.

Ellagic and tannic acids protect newly synthesized elastic fibers from premature enzymatic degradation in dermal fibroblast cultures.

Progressive proteolytic degradation of cutaneous elastic fibers, that cannot be adequately replaced or repaired by adult dermal fibroblasts, constitutes a major feature of aging skin. Our present investigations, employing monolayer cultures of human dermal fibroblasts and organ cultures of skin biopsies, were aimed at testing whether the hydrophilic tannic acid (TA) and lipophilic ellagic acid (EA) would protect dermal elastin from exogenous and endogenous enzymatic degradation. Results from both culture systems indicated that dermal fibroblasts, maintained with TA or EA, deposit significantly more elastic fibers than untreated control cultures despite the fact that neither polyphenol enhanced transcription of elastin mRNA or cellular proliferation. Results of a pulse and chase experiment showed that pretreatment with both polyphenols enhanced biostability of tropoelastin and newly deposited elastin. Results of in vitro assays indicated that both polyphenols bound to purified elastin and significantly decreased its proteolytic degradation by elastolytic enzymes belonging to the serine proteinase, cysteine proteinase, and metallo-proteinase families. Importantly, both polyphenols also synergistically enhanced elastogenesis induced by selected elastogenic compounds in cultures of dermal fibroblasts. We propose that EA and TA may be useful for preventing proteolytic degradation of existing dermal elastic fibers and for enhancing more efficient elastogenesis in aged skin.

Proteolytic digest derived from bovine Ligamentum Nuchae stimulates deposition of new elastin-enriched matrix in cultures and transplants of human dermal fibroblasts.

BACKGROUND: Diverse topical products and injectable fillers used for correcting facial wrinkles induce rather short-lived effects because they target replacement of dermal collagen and hyaluronan, matrix components of limited biologic durability. OBJECTIVE: Present studies were aimed at stimulation of fully differentiated human dermal fibroblasts to resume deposition of new extracellular matrix rich of elastin, the most durable and metabolically inert component of dermal ECM. METHODS: We have created a novel proteolytic digest of bovine ligamentum nuchae (ProK-60), and tested its potential biological effect on dermal fibroblasts derived from females of different ages. Northern blots, quantitative immunohistochemistry and metabolic assays were used to assess effects of ProK-60 on proliferation and matrix production in primary cultures of dermal fibroblasts, in cultures of skin explants and after implantation of stimulated fibroblasts into the skin of athymic nude mice. RESULTS: ProK-60 increased proliferation (25-30%) of cultured dermal fibroblasts and significantly enhanced their production of new elastic fibers (>250%) and collagen fibers (100%). These effects were mostly mediated by stimulation of cellular elastin receptor. In contrast, ProK-60 inhibited production of fibronectin (-30%) and chondroitin sulfate proteoglycans (-50%). ProK-60 also activated proliferation of dermal fibroblasts, mostly derived from the stratum basale and induced deposition of elastic fibers in cultures of skin explants. Moreover, human fibroblasts pre-treated with ProK-60 produced abundant elastic fibers after their injection into the skin of athymic nude mice. CONCLUSION: The described biological effects of ProK-60, including its unique elastogenic property, encourage use of this compound in cosmetic formulations stimulating rejuvenation of aged skin.

Retrovirally mediated overexpression of versican v3 reverses impaired elastogenesis and heightened proliferation exhibited by fibroblasts from Costello syndrome and Hurler disease patients.

The phenotypic resemblance of patients with Costello syndrome and Hurler disease has been linked to impaired formation of elastic fibers that coincides with elevated cellular proliferation. Impaired elastogenesis in these diseases associates with respective abnormal accumulation of chondroitin sulfate and dermatan sulfate proteoglycans that induce cell surface shedding of elastin-binding protein (EBP) normally required for intracellular chaperoning of tropoelastin and its assembly into elastic fibers. A variant of the chondroitin sulfate proteoglycan versican, V3, which lacks chondroitin sulfate, has recently been shown to stimulate elastic fiber assembly and decrease proliferation when expressed by retroviral transduction in arterial smooth muscle cells. However, the mechanism(s) by which V3 influences this phenotype is not known. We now demonstrate that transduction of skin fibroblasts from Costello syndrome and Hurler disease patients with cDNA to versican V3 completely reverses impaired elastogenesis and restores normal proliferation of these cells. This phenotypic reversal is accompanied by loss of chondroitin sulfate from the cell surface and increased levels of EBP. Versican V3 transduction of skin fibroblasts from GM(1)-gangliosidosis patients, which lack EBP, failed to restore impaired elastogenesis. These results suggest that induction of elastic fiber production by gene transfer of versican V3 in skin fibroblasts is mediated by rescue of the tropoelastin chaperone, EBP.

Concomitant loss of mitochondria and the DNA repair protein hOGG1 in clear cell carcinoma of the kidney.

The kidney is subjected to DNA oxidative damage from reactive oxygen species generated by free radicals and toxic metabolites, leading to formation of DNA base lesions. One such DNA lesion is 8-oxoguanine, which, if not sufficiently removed, is potentially mutagenic because it can cause G:C to T:A transversion in subsequent DNA replication. The human 8-oxoguanine DNA glycosylase 1 (hOGG1) gene on chromosome 3, a region (3p25-26) that shows frequent loss of heterozygosity in clear cell renal cell carcinoma (CC-RCC), encodes for a DNA repair enzyme capable of excision repair of 8-oxoguanine. Of the known isoforms of the hOGG1 enzyme (types Ia, Ib, Ic, Id, and II), only 1, Ia, is found in the nucleus, whereas the rest show a mitochondrial distribution. We investigated, by an immunohistochemical staining method, the expression of hOGG1 protein in 40 cases of CC-RCC, using archival formalin-fixed tissue. To localize the hOGG1 enzyme in normal and tumor tissue, immuno-staining against cytochrome c, a specific mitochondrial enzyme, was also performed. The results showed marked reduction in hOGG1 expression in the majority of tumors, with complete loss of staining seen in 26 (65%) and moderate and weak positive staining present in 9 (22.5%) and 5 (12.5%) of the cases, respectively. Strong hOGG1 protein expression was present in normal tubular epithelium, located in the mitochondria. The results correlated with the expression patterns of cytochrome c. The findings indicate that loss of hOGG1 expression may have a role in development or progression of CC-RCC.

Induced expression of syndecan-1 in the stroma of head and neck squamous cell carcinoma.

Syndecan-1 (CD138), a cell-surface heparan sulfate proteoglycan, is involved in cell-cell, cell-matrix interaction and growth factor binding. Loss of expression of syndecan-1 in tumor cells leads to decreased intercellular cohesion, increased potential for tumor invasiveness, and metastatic spread. Furthermore, induction of syndecan-1 expression in the tumor stroma has been postulated to promote tumor angiogenesis via its binding to growth factors such as basic fibroblast growth factor. Although syndecan-1 expression within tumor cells has been investigated in head and neck squamous cell carcinoma, stromal expression has not been studied in detail. We analyzed 38 cases of head and neck squamous cell carcinoma by immunohistochemical staining for syndecan-1 expression within the stroma. The expression of syndecan-1 within tumor cells of various histologic grades of differentiation, squamous cell carcinoma in situ cells, and benign squamous epithelium was also determined. Variable levels of diminished syndecan-1 expression were noted within the dysplastic cells of 9 of 16 (60%) squamous cell carcinoma in situ lesions and in all 38 (100%) invasive squamous cell carcinoma. In general, higher levels of syndecan-1 expression were observed in the well-differentiated tumors, in contrast to significant reduction of expression seen in poorly differentiated tumors. Syndecan-1 expression was observed within the stroma (in fibroblasts) surrounding infiltrating carcinoma cells in 28 of 38 (74%) cases. The intensity of syndecan-1 staining within the stroma showed generally an inverse correlation with the degree of tumor cell differentiation. Syndecan-1 expression was not detected in the stroma beneath normal squamous epithelium or adjacent to areas of squamous cell carcinoma in situ. We conclude that induced expression of syndecan-1 in the stroma surrounding tumor cells of invasive head and neck squamous cell carcinoma is a frequent event. The increased stromal syndecan-1 expression, coupled with its loss from the surface of carcinoma cells, may contribute to tumor cell invasion and the development of metastases.

Promoter hypermethylation and inactivation of hMLH1, a DNA mismatch repair gene, in head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is a multistage process during which adverse genetic alterations accumulate resulting in loss of cell cycle control, selective cell overgrowth, and ultimately formation of malignancy. Among various genetic alterations in HNSCC is increased microsatellite instability (MSI). hMLH1 is one of the major mismatch DNA repair genes, the inactivation of which caused increased MSI in a variety of human cancers including HNSCC. While somatic mutation is a major mechanism of the hMLH1 gene inactivation in hereditary form of human cancer, promoter hypermethylation appears to be primarily involved in the inactivation of the hMLH1 gene in sporadic form of human cancers. In the current study, we analyzed 78 cases of HNSCC for hMLH1 protein expression and promoter hypermethylation by IHC and methylation-specific PCR (MSP). Twenty-four of 78 cases (31%) of HNSCC contained markedly reduced levels of the hMLH1 protein. Based on the IHC results, 8 cases without and 8 with hMLH1 protein expression (total of 16) were further analyzed by MSP. Seven of 8 cases (88%) that were negative for the hMLH1 protein displayed promoter hypermethylation, whereas 7 of 7 cases (100%) strongly positive for the protein were free of promoter methylation. This study confirms our previous conclusion that promoter hypermethylation represents a major mechanism of the hMLH1 gene inactivation in HNSCC.

Promoter hypermethylation: an important epigenetic mechanism for hMLH1 gene inactivation in head and neck squamous cell carcinoma.

OBJECTIVE: The hMLH1 gene is one of the mismatch DNA repair genes. Inactivation of the hMLH1 gene has been implicated in the tumorigenesis of many types of human cancers. In most sporadic forms of human cancers, promoter hypermethylation is responsible for hMLH1 gene inactivation. Lack of hMLH1 protein expression has been found in a subset of head and neck squamous cell carcinomas (HNSCCs). The purpose of this study was to investigate whether promoter hypermethylation causes hMLH1 gene inactivation in HNSCCs. STUDY DESIGN: hMLH1 protein expression was determined by immunohistochemical staining in 62 cases, whereas hMLH1 gene promoter methylation was analyzed by methylation-sensitive restriction enzyme digestion, followed by polymerase chain reaction, in 35 cases of HNSCCs. RESULTS: Sixteen (26%) of 62 cases of HNSCCs showed near-complete loss of hMLH1 protein expression on immunohistochemical staining. Twelve (92%) of 13 cases that were negative for the hMLH1 protein displayed promoter hypermethylation, whereas 17 (77%) of 22 cases positive for the protein were free of promoter methylation. CONCLUSIONS: Promoter hypermethylation may be an important mechanism for hMLH1 gene inactivation in a subset of HNSCCs.