Incidence of significant prostate cancer after negative MRI and systematic biopsy in the FUTURE trial.

OBJECTIVES: To assess the proportion of clinically significant (cs) prostate cancer (PCa) found during follow-up in patients with negative systematic biopsy (SB) followed by non-suspicious multiparametric magnetic resonance imaging (mpMRI) and persistent clinical suspicion of PCa compared to the general population. PATIENTS AND METHODS: A prospective study in a subgroup of patients from a multicentre randomized controlled trial was conducted between 2014 and 2017, including 665 men with prior negative SB with a persistent elevated prostate-specific antigen and/or suspicious digital rectal examination undergoing mpMRI. All patients with negative SB and Prostate Imaging-Reporting and Data System (PI-RADS) ≤2 on mpMRI entered biochemical follow-up. Follow-up data until December 2021 were collected by reviewing institutional hospital records and the Dutch Pathology Registry (PALGA). The primary outcome was the observed number of csPCa (Gleason ≥3 + 4/International Society of Urological Pathology grade group ≥2) cases during follow-up compared to the expected number in the general population (standardized incidence ratio [SIR]). RESULTS: In total, 431 patients had non-suspicious mpMRI and entered biochemical follow-up. After a median (interquartile range) follow-up of 41 (23-57) months, 38 patients were diagnosed with PCa, of whom 13 (3.0%) had csPCa. The SIR for csPCa was 4.3 (95% confidence interval 2.3-7.4; total excess of eight cases). A higher risk of a positive biopsy for (cs)PCa based on the European Randomized Study of Screening for Prostate Cancer risk calculator and a suspicious repeat MRI (PI-RADS ≥3) were significant predictive factors for csPCa. CONCLUSION: After negative prior biopsy and non-suspicious mpMRI the risk of csPCa is low. However, compared to the general population, the risk of csPCa is increased despite the high negative predictive value of mpMRI. More research focusing on biochemical and image-guided risk-adapted diagnostic surveillance strategies is warranted.

The estimation of tumor cell percentage for molecular testing by pathologists is not accurate.

Molecular pathology is becoming more and more important in present day pathology. A major challenge for any molecular test is its ability to reliably detect mutations in samples consisting of mixtures of tumor cells and normal cells, especially when the tumor content is low. The minimum percentage of tumor cells required to detect genetic abnormalities is a major variable. Information on tumor cell percentage is essential for a correct interpretation of the result. In daily practice, the percentage of tumor cells is estimated by pathologists on hematoxylin and eosin (H&E)-stained slides, the reliability of which has been questioned. This study aimed to determine the reliability of estimated tumor cell percentages in tissue samples by pathologists. On 47 H&E-stained slides of lung tumors a tumor area was marked. The percentage of tumor cells within this area was estimated independently by nine pathologists, using categories of 0-5%, 6-10%, 11-20%, 21-30%, and so on, until 91-100%. As gold standard, the percentage of tumor cells was counted manually. On average, the range between the lowest and the highest estimate per sample was 6.3 categories. In 33% of estimates, the deviation from the gold standard was at least three categories. The mean absolute deviation was 2.0 categories (range between observers 1.5-3.1 categories). There was a significant difference between the observers (P<0.001). If 20% of tumor cells were considered the lower limit to detect a mutation, samples with an insufficient tumor cell percentage (<20%) would have been estimated to contain enough tumor cells in 27/72 (38%) observations, possibly causing false negative results. In conclusion, estimates of tumor cell percentages on H&E-stained slides are not accurate, which could result in misinterpretation of test results. Reliability could possibly be improved by using a training set with feedback.

Orphan granzymes find a home.

Cytotoxic lymphocytes are armed with granules that are released in the granule-exocytosis pathway to kill tumor cells and virus-infected cells. Cytotoxic granules contain the pore-forming protein perforin and a family of structurally homologues serine proteases called granzymes. While perforin facilitates the entry of granzymes into a target cell, the latter initiate distinct apoptotic routes. Granzymes are also implicated in extracellular functions such as extracellular matrix degradation, immune regulation, and inflammation. The family of human granzymes consists of five members, of which granzyme A and B have been studied most extensively. Recently, elucidation of the specific characteristics of the other three human granzymes H, K, and M, also referred to as orphan granzymes, have started. In this review, we summarize and discuss what is currently known about the biology of the human orphan granzymes.

Notch1 functions as a tumor suppressor in mouse skin.

Notch proteins are important in binary cell-fate decisions and inhibiting differentiation in many developmental systems, and aberrant Notch signaling is associated with tumorigenesis. The role of Notch signaling in mammalian skin is less well characterized and is mainly based on in vitro studies, which suggest that Notch signaling induces differentiation in mammalian skin. Conventional gene targeting is not applicable to establishing the role of Notch receptors or ligands in the skin because Notch1-/- embryos die during gestation. Therefore, we used a tissue-specific inducible gene-targeting approach to study the physiological role of the Notch1 receptor in the mouse epidermis and the corneal epithelium of adult mice. Unexpectedly, ablation of Notch1 results in epidermal and corneal hyperplasia followed by the development of skin tumors and facilitated chemical-induced skin carcinogenesis. Notch1 deficiency in skin and in primary keratinocytes results in increased and sustained expression of Gli2, causing the development of basal-cell carcinoma-like tumors. Furthermore, Notch1 inactivation in the epidermis results in derepressed beta-catenin signaling in cells that should normally undergo differentiation. Enhanced beta-catenin signaling can be reversed by re-introduction of a dominant active form of the Notch1 receptor. This leads to a reduction in the signaling-competent pool of beta-catenin, indicating that Notch1 can inhibit beta-catenin-mediated signaling. Our results indicate that Notch1 functions as a tumor-suppressor gene in mammalian skin.

Noncytotoxic inhibition of cytomegalovirus replication through NK cell protease granzyme M-mediated cleavage of viral phosphoprotein 71.

Granzyme M (GrM) is highly expressed in cytotoxic granules of NK cells, which provide the first line of defense against viral pathogens. GrM knockout mice show increased susceptibility toward murine CMV infection. Although GrM is a potent inducer of cell death, the mechanism by which GrM eliminates viruses remains elusive. In this paper, we show that purified human GrM in combination with the perforin-analog streptolysin O (SLO) strongly inhibited human CMV (HCMV) replication in fibroblasts in the absence of host cell death. In a proteomic approach, GrM was highly specific toward the HCMV proteome and most efficiently cleaved phosphoprotein 71 (pp71), an HCMV tegument protein that is critical for viral replication. Cleavage of pp71 occurred when viral lysates were incubated with purified GrM, when intact cells expressing recombinant pp71 were challenged with living cytotoxic effector cells, and when HCMV-infected fibroblasts were incubated with SLO and purified GrM. GrM directly cleaved pp71 after Leu(439), which coincided with aberrant cellular localization of both pp71 cleavage fragments as determined by confocal immunofluorescence. In a luciferase reporter assay, cleavage of pp71 after Leu(439) by GrM completely abolished the ability of pp71 to transactivate the HCMV major immediate-early promoter, which is indispensable for effective HCMV replication. Finally, GrM decreased immediate-early 1 protein expression in HCMV-infected fibroblasts. These results indicate that the NK cell protease GrM mediates cell death-independent antiviral activity by direct cleavage of a viral substrate.

In vivo bladder cancer diagnosis by high-volume Raman spectroscopy.

We studied the feasibility of Raman spectroscopy for the diagnosis of bladder cancer in vivo. Since the invasion stage is crucial for the treatment choice, a high-volume based Raman probe was used to investigate the potential of determining the invasiveness of bladder cancer. High quality spectra were obtained from suspicious and nonsuspicious bladder locations during the procedure of transurethral resection of bladder tumors (TURBT) with collection times of 1-5 s. Multivariate analysis was used to generate the classification models. The algorithm was able to distinguish bladder cancer from normal bladder locations with a sensitivity of 85% and a specificity of 79%. The Raman spectra of bladder cancer stages showed a gradual increase in the intensity of specific amino acid peaks and, most likely, an increase in the intensity of DNA peaks.

Biology of granzyme M: a serine protease with unique features.

The granule-exocytosis pathway is the major mechanism for cytotoxic lymphocytes to kill tumor cells and virus-infected cells. Cytotoxic granules contain the pore-forming protein perforin and a set of structurally homologues serine proteases called granzymes. Perforin facilitates the entry of granzymes into a target cell, allowing these proteases to initiate distinct cell death routes by cleaving specific intracellular substrates. The family of granzymes consists of multiple members, of which granzyme A and granzyme B have been studied most extensively. Since the cloning of the granzyme M cDNA in the early 1990s, it has remained an "orphan" granzyme for many years and only during the past few years the interest in this protease has increased. Granzyme M appears to be a potent inducer of tumor cell death with morphological hallmarks that are unique among all granzymes. In this review, we summarize the characteristics of granzyme M that are currently known, including its cellular expression, substrate specificity, physiological functions, and inhibitors.

Is There Still a Need for Repeated Systematic Biopsies in Patients with Previous Negative Biopsies in the Era of Magnetic Resonance Imaging-targeted Biopsies of the Prostate?

BACKGROUND: The role of targeted prostate biopsies (TBs) in patients with cancer suspicious lesions on multiparametric magnetic resonance imaging (mpMRI) following negative systematic biopsies (SBs) is undebated. However, whether they should be combined with repeated SBs remains unclear. OBJECTIVE: To evaluate the value of repeated SBs in addition to TBs in patients with a prior negative SB and a persistent suspicion of prostate cancer (PCa). DESIGN, SETTING, AND PARTICIPANTS: A prospective study as part of a multicenter randomized controlled trial conducted between 2014 and 2017, including 665 men with a prior negative SB and a persistent suspicion of PCa (suspicious digital rectal examination and/or prostate-specific antigen >4.0ng/ml). INTERVENTION: All patients underwent 3T mpMRI according to Prostate Imaging Reporting and Data System (PI-RADS) v2. Patients with PI-RADS ≥3 were randomized 1:1:1 for three TB techniques: MRI-TRUS fusion TB (FUS-TB), cognitive registration fusion TB (COG-TB), or in-bore MRI TB. FUS-TB and COG-TB were combined with repeated SBs. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Clinically significant prostate cancer (csPCa) was defined as Gleason ≥3+4. Differences in detection rates of csPCa, clinically insignificant PCa (cisPCa), and overall PCa between TBs (FUS-TB and COG-TB) and repeated SBs were compared using McNemar's test. RESULTS AND LIMITATIONS: In the 152 patients who underwent both TB and SB, PCa was detected by TB in 47% and by SB in 32% (p<0.001, 95% confidence interval [CI]: 6.0-22%). TB detected significantly more csPCa than SB (32% vs 16%; p<0.001, 95% CI: 11-25%). Clinically significant PCa was missed by TB in 1.3% (2/152). Combining SB and TB resulted in detection rate differences of 6.0% for PCa, 5.0% for cisPCa, and 1.0% for csPCa compared with TB alone. CONCLUSIONS: In case of a persistent suspicion of PCa following a negative SB, TB detected significantly more csPCa cases than SB. The additional value of SB was limited, and only 1.3% of csPCa would have been missed when SB had been omitted. PATIENT SUMMARY: We evaluated the role of systematic biopsies and magnetic resonance imaging (MRI)-targeted biopsies for the diagnosis of prostate cancer in patients with prior negative systematic biopsies. MRI-targeted biopsies perform better in detecting prostate cancer in these patients. The value of repeated systematic biopsies is limited.

Downregulation of SERPINB13 expression in head and neck squamous cell carcinomas associates with poor clinical outcome.

Tumorigenesis of head and neck squamous cell carcinomas (HNSCC) is associated with various genetic changes such as loss of heterozygosity (LOH) on human chromosome 18q21. This chromosomal region maps a gene cluster coding for a family of intracellular serine protease inhibitors (serpins), including SERPINB13. As SERPINB13 expression in HNSCC has recently been shown to be downregulated both at the mRNA and protein levels, here we investigated if such a low SERPINB13 expression is associated with histopathological and clinical parameters of HNSCC tumors and patient survival. By generating specific antibodies followed by immunohistochemistry on a well-defined cohort of 99 HNSCC of the oral cavity and oropharynx, SERPINB13 expression was found to be partially or totally downregulated in 75% of the HNSCC as compared with endogenous expression in non-neoplastic epithelial cells. Downregulation of SERPINB13 protein expression in HNSCC was significantly associated with the presence of LOH at the SERPINB13 gene in the tumors (p = 0.006), a poor differentiation grade of the tumors (p = 0.001), the presence of a lymph node metastasis (p = 0.012), and a decreased disease-free (p = 0.033) as well as overall (p = 0.018) survival of the patients. This is the first report demonstrating that downregulation of SERPINB13 protein expression in HNSCC is positively associated with poor clinical outcome. Therefore, SERPINB13 seems to act as an important protease inhibitor involved in the progression of HNSCC.

Expression of cellular FLICE inhibitory protein, caspase-8, and protease inhibitor-9 in Ewing sarcoma and implications for susceptibility to cytotoxic pathways.

PURPOSE: Ewing sarcoma is a common pediatric bone tumor with an unfavorable prognosis for metastatic or recurrent disease. Cellular immunotherapy may provide new treatment options and depends on the cytolytic death receptor and perforin/granzyme pathways. Expression of death receptor pathway inhibitor cellular FLICE inhibitory protein (cFLIP), initiator caspase-8, and granzyme B inhibitor protease inhibitor-9 (PI-9) have been reported to determine susceptibility to cell- and chemotherapy-mediated killing in several tumor types. Here, we have studied their in vitro and in vivo expression in Ewing sarcoma and the implications for susceptibility to cytotoxicity. EXPERIMENTAL DESIGN: Ewing sarcoma cell lines (n = 8) were tested for cFLIP, PI-9, and caspase-8 expression. Functional significance was tested by anti-Fas antibody (death receptor pathway) or natural killer cell (perforin/granzyme pathway) treatment. Immunohistochemistry was done on 28 sections from 18 patients. In half of the cases, sequential material, including metastases, was available. RESULTS: Although all tested Ewing sarcoma cell lines expressed cFLIP, resistance to CD95/Fas-mediated apoptosis was only observed in two cell lines lacking caspase-8 expression. PI-9 was expressed at low levels in four of eight Ewing sarcoma cell lines, but positive cell lines remained susceptible to perforin/granzyme-mediated killing. In primary Ewing sarcoma, including metastases, cFLIP was abundantly expressed in 18 of 18 patients. Caspase-8 was expressed in all patients but showed more intertumoral and intratumoral variation in both intensity and heterogeneity of staining. PI-9, in contrast, was undetectable. CONCLUSIONS: The expression patterns of cFLIP, caspase-8, and the absence of PI-9 provide a rationale to preferentially exploit the perforin/granzyme pathway in cytotoxic therapies against Ewing sarcoma.

Late (> 5 years) regional lymph node metastasis of oral squamous cell carcinoma (SCC), proven by p53 mutation analysis.

BACKGROUND: A late (> 5 years) neck nodal metastasis of oral cancer, poses a problem to the clinician: is it a late metastasis or a metastasis of a (unknown) second primary tumour? METHODS: A 50-year-old male was seen with a contralateral lymph node metastasis, 5 1/2 years after treatment of a pT2N1M0 carcinoma in the floor of the mouth. Both the late metastasis and the original tumour specimen were analysed for p53 mutations. RESULTS: Both specimens showed an identical p53 mutation, thereby confirming the lymph node to be a late metastasis. CONCLUSIONS: A lymph node metastasis can occur more than 5 years after treatment of an oral squamous cell carcinoma. p53 mutation analysis is of help to discriminate it from a second primary tumour.

Human mast cells produce and release the cytotoxic lymphocyte associated protease granzyme B upon activation.

Mast cells are widely distributed throughout the body and express effector functions in allergic reactions, inflammatory diseases, and host defense. Activation of mast cells results in exocytosis of preformed chemical mediators and leads to novel synthesis and secretion of lipid mediators and cytokines. Here, we show that human mast cells also express and release the cytotoxic lymphocyte-associated protease, granzyme B. Granzyme B was active and localized in cytoplasmic granules, morphologically resembling those present in cytotoxic lymphocytes. Expression and release of granzyme B by mast cell-lines HMC-1 and LAD 2 and by cord blood- and mature skin-derived human mast cells depended on the mode of activation of these cells. In mast cell lines and cord blood-derived mast cells, granzyme B expression was mainly induced by non-physiological stimuli (A23187/PMA, Compound 48/80) and substance P. In contrast, mature skin-derived mast cells only produced granzyme B upon IgE-dependent stimulation. We conclude that granzyme B is expressed and released by human mast cells upon physiologic stimulation. This suggests a role for granzyme B as a novel mediator in mast cell biology.

Maintenance of head and neck tumor gene expression profiles upon lymph node metastasis.

Spread of cancer and development of solid metastases at distant sites is the main cause of cancer-related deaths. To understand and treat metastases, it is important to determine at which stages the most pivotal steps for development of metastases occur. In head and neck squamous cell carcinoma (HNSCC), metastasis nearly always occurs first in local lymph nodes before development of distant metastasis. Here, we have investigated gene expression patterns in HNSCC lymph node metastases using DNA microarrays. Several types of analyses show that the gene expression patterns in lymph node metastases are most similar to the corresponding primary tumors from which they arose, as long as samples contain sufficient proportions of tumor cells. Strikingly, gene expression patterns of metastatic primary HNSCC are largely maintained upon spread to the lymph node. Only a single gene, metastasis-associated gene 1 (MTA1), was found to show consistently changed expression between a large number of matched primary tumor-lymph node metastasis pairs. The maintained expression pattern includes the predictive signature for HNSCC lymph node metastasis. These results underscore the importance of the primary tumor gene expression profile for development and treatment of metastasis. The findings also agree with the concept that disseminated cancer cells alter the surrounding tissue into a metastatic environment that resembles the primary tumor microenvironment.

Added Value of 50-Gene Panel Sequencing to Distinguish Multiple Primary Lung Cancers from Pulmonary Metastases: A Systematic Investigation.

Differentiation between multiple primary lung cancers and pulmonary metastases (PM) has important implications in staging, prognosis, and treatment strategies. Clinical and immunohistopathologic criteria have been standardized; however, a substantial number of cases remain difficult to classify. Using next-generation sequencing, it is now possible to improve the classification of multiple lung cancer lesions. This study systematically investigated the value of routine morphologic and IHC characteristics, p53 protein expression, TP53 mutation analysis, and 50-gene panel sequencing (GPS) in 111 lesions from 50 patients with multiple lung lesions. Based on immunohistopathologic criteria, 32 paired lesions were classified as multiple primary lung cancer (MPLC) and 21 as PM. TP53 mutation analysis indicated MPLC in 23 and PM in 6 pairs, but in the majority of cases (n = 28, 49%) no mutation was observed and no conclusion could be drawn. In contrast, only 2 pairs were not conclusive using GPS. In a significant number of matching tumor samples (n = 19, 39%), sequencing results were contradictory to the initial immunohistopathology diagnosis. No separation in overall survival for classifications based on immunohistopathology was observed, while a clear but nonsignificant trend was observed concerning survival in MPLC patients (hazard ratio = 3.98) using 50-gene GPS. In about one-third of the patients, GPS provided additional information to improve the differentiation between MPLC and PM.

Inflammasome components NALP 1 and 3 show distinct but separate expression profiles in human tissues suggesting a site-specific role in the inflammatory response.

Several autoinflammatory disorders such as Muckle-Wells syndrome are characterized by mutations in the NALP3/cryopyrin gene. NALP3 and NALP1 proteins can assemble to inflammasomes that activate caspase-1, resulting in the processing of pro-inflammatory cytokines IL-1beta and IL-18. The present study was designed to determine which cells and tissues express NALP1 and NALP3. Monoclonal antibodies were developed and their use revealed distinct distribution profiles of NALP1 and NALP3. Granulocytes, monocytes (very weakly), dendritic cells, and B and T cells all express NALP1 and NALP3. Highest levels of NALP1 are found in T cells and Langerhans cells. Furthermore, NALP1 is present in glandular epithelial structures such as stomach, gut, lung, and, surprisingly, in neurons and testis. In contrast to NALP1, NALP3 shows a more restricted tissue distribution with expression mainly in non-keratinizing epithelia in the oropharynx, esophagus, and ectocervix. Moreover, NALP3 expression is found in the urothelial layer in the bladder. Likewise, a difference in subcellular distribution between NALP1 and NALP3 is observed because NALP1 is localized mainly in the nucleus, whereas NALP3 is predominantly cytoplasmic. We propose that the presence of NALP3 in epithelial cells lining the oral and genital tracts allows the rapid sensing of invading pathogens, thereby triggering an innate immune response.

Performance of immuno-positron emission tomography with zirconium-89-labeled chimeric monoclonal antibody U36 in the detection of lymph node metastases in head and neck cancer patients.

PURPOSE: Immuno-positron emission tomography (PET), the combination of PET with monoclonal antibodies (mAb), is an attractive option to improve tumor detection and to guide mAb-based therapy. The long-lived positron emitter zirconium-89 ((89)Zr) has ideal physical characteristics for immuno-PET with intact mAbs but has never been used in a clinical setting. In the present feasibility study, we aimed to evaluate the diagnostic imaging performance of immuno-PET with (89)Zr-labeled-chimeric mAb (cmAb) U36 in patients with squamous cell carcinoma of the head and neck (HNSCC), who were at high risk of having neck lymph node metastases. EXPERIMENTAL DESIGN: Twenty HNSCC patients, scheduled to undergo neck dissection with or without resection of the primary tumor, received 75 MBq (89)Zr coupled to the anti-CD44v6 cmAb U36 (10 mg). All patients were examined by computed tomography (CT) and/or magnetic resonance imaging (MRI) and immuno-PET before surgery. Six patients also underwent PET with (18)F-fluoro-2-deoxy-d-glucose. Immuno-PET scans were acquired up to 144 hours after injection. Diagnostic findings were recorded per neck side (left or right) as well as per lymph node level (six levels per side), and compared with histopathologic outcome. For this purpose, the CT/MRI scores were combined and the best of both scores was used for analysis. RESULTS: Immuno-PET detected all primary tumors (n = 17) as well as lymph node metastases in 18 of 25 positive levels (sensitivity 72%) and in 11 of 15 positive sides (sensitivity 73%). Interpretation of immuno-PET was correct in 112 of 121 operated levels (accuracy 93%) and in 19 of 25 operated sides (accuracy 76%). For CT/MRI, sensitivities of 60% and 73% and accuracies of 90% and 80% were found per level and side, respectively. In the six patients with seven tumor-involved neck levels and sides, immuno-PET and (18)F-fluoro-2-deoxy-d-glucose PET gave comparable diagnostic results. CONCLUSION: In this study, immuno-PET with (89)Zr-cmAb U36 performed at least as good as CT/MRI for detection of HNSCC lymph node metastases.

A genetic explanation of Slaughter's concept of field cancerization: evidence and clinical implications.

The concept of "field cancerization" was first introduced by Slaughter et al. [D. P, Slaughter et al., Cancer (Phila.), 6: 963-968, 1953] in 1953 when studying the presence of histologically abnormal tissue surrounding oral squamous cell carcinoma. It was proposed to explain the development of multiple primary tumors and locally recurrent cancer. Organ systems in which field cancerization has been described since then are: head and neck (oral cavity, oropharynx, and larynx), lung, vulva, esophagus, cervix, breast, skin, colon, and bladder. Recent molecular findings support the carcinogenesis model in which the development of a field with genetically altered cells plays a central role. In the initial phase, a stem cell acquires genetic alterations and forms a "patch," a clonal unit of altered daughter cells. These patches can be recognized on the basis of mutations in TP53, and have been reported for head and neck, lung, skin, and breast cancer. The conversion of a patch into an expanding field is the next logical and critical step in epithelial carcinogenesis. Additional genetic alterations are required for this step, and by virtue of its growth advantage, a proliferating field gradually displaces the normal mucosa. In the mucosa of the head and neck, as well as the esophagus, such fields have been detected with dimensions of >7 cm in diameter, whereas they are usually not detected by routine diagnostic techniques. Ultimately, clonal divergence leads to the development of one or more tumors within a contiguous field of preneoplastic cells. An important clinical implication is that fields often remain after surgery of the primary tumor and may lead to new cancers, designated presently by clinicians as "a second primary tumor" or "local recurrence," depending on the exact site and time interval. In conclusion, the development of an expanding preneoplastic field appears to be a critical step in epithelial carcinogenesis with important clinical consequences. Diagnosis and treatment of epithelial cancers should not only be focused on the tumor but also on the field from which it developed.

Molecular clonality assessment shows high performance to predict malignant B-cell non-Hodgkin's lymphoma using cytological smears.

AIMS: Molecular PCR-based clonality analysis is helpful for identification of monoclonal B-cell or T-cell populations and to distinguish malignant lymphoma from reactive lymphoid hyperplasia. Typically, clonality assessment on fine-needle aspiration cytology (FNAC) requires freshly obtained aspirates, but the collection and processing of these samples are often challenging in daily practice. In this study, we assessed the routine diagnostic value of the EuroClonality/BIOMED-2 assay for B-cell clonality on air-dried archived Giemsa-stained smears. METHODS: This study comprised a retrospective analysis of a consecutive diagnostic cohort of 192 FNAC samples from 184 patients with at least 2-year follow-up. The results from the clonality assay were integrated with cytomorphological assessment and evaluated for their accuracy in detecting malignant disease. EuroClonality expert re-evaluation was performed for all cases with ambiguous results and for cases in which the diagnosis did not match the follow-up data. RESULTS: The clonality assay showed a high accuracy of 93% for detection of malignancy, with a sensitivity of 93% and a specificity of 92%. All 64 cases with monoclonal Ig heavy chain (IGH)/Ig kappa chain (IGK) rearrangements were confirmed malignant by histology or clinical follow-up. Expert re-evaluation changed the definite diagnosis for five cases (3%), mainly because of low signals or no proper duplicate results. We discuss and elucidate all cases for which the clonality results did not match the disease follow-up. CONCLUSIONS: This study showed that EuroClonality/BIOMED-2 assay can successfully be performed on cytological Giemsa-stained smears and inclusion in daily practice can assist in better identification of malignant lymphoma.

Genetically altered fields as origin of locally recurrent head and neck cancer: a retrospective study.

PURPOSE: Surgeons treating patients with head and neck squamous cell carcinoma (HNSCC) rely heavily on histology to decide whether the resection margins are tumor free and subsequent adjuvant treatments can be omitted. However, despite the presence of tumor-free margins, 10-30% of HNSCC patients still develop a locally recurrent tumor. Evidence is available that recurrent cancer develops from either (a). outgrowth of a relatively small number of tumor cells that have not been detected by the pathologist or (b). a precursor lesion in which additional genetic alterations have led again to invasive cancer. EXPERIMENTAL DESIGN: In a retrospective study on 13 HNSCC cases, we analyzed the primary tumor, its surrounding histologically tumor-free resection margins, and local recurrences for loss of heterozygosity (22 microsatellite markers on 6 chromosomes) and TP53 mutations to determine the origin of the recurrent cancer. RESULTS: A precursor lesion was absent in 5 of 13 (39%) cases, and the genetic similarity of the primary and recurrent cancer was high, providing evidence that residual cancer cells were the origin of recurrence. For the remaining eight cases (61%) a genetically related precursor lesion (field) was detected, and for five of these cases, evidence was found that both the primary and recurrent carcinoma originated from this field. The remaining three cases were less conclusive. CONCLUSIONS: This study explains the pathobiology of locally recurrent HNSCC in patients with histologically tumor-free resection margins and indicates that the development of novel therapies to decrease the local recurrence rates in HNSCC should not only be focused on eradicating residual cancer cells but also on the precursor lesions that are left behind.

Molecular diagnosis of surgical margins and local recurrence in head and neck cancer patients: a prospective study.

PURPOSE: Approximately 10-30% of surgically treated head and neck cancer patients develop local recurrences while the resection margins are histologically tumor free. These recurrences may arise from cancer cells left behind but not detected by the pathologist, or they may develop from precursor lesions adjacent to the tumor that were not completely resected. We have investigated whether TP53-mutated DNA in the surgical margins is suitable to identify patients with head and neck squamous cell carcinoma at risk for local and locoregional recurrence. EXPERIMENTAL DESIGN: In a prospective cohort study of 76 patients with histologically tumor-free margins, the presence of TP53-mutated DNA was determined in the surgical margins using the phage plaque assay and correlated to clinical outcome. Immunostaining of the molecular-positive margins for mutated p53 protein was used to identify whether unresected precursor lesions or residual tumor cells were left behind. RESULTS: The absence of TP53-mutated DNA in surgical margins was significantly associated with remaining free of local and locoregional recurrence (P = 0.027 and P = 0.028, respectively). Moreover, the presence of TP53-mutated DNA in the surgical margins was an independent prognosticator for locoregional recurrence (relative risk = 7.1; P = 0.021; 95% confidence interval, 0.9-56). In 20% of the cases, the presence of TP53-mutated DNA in the surgical margins was found to be related to the presence of tumor-related precursor lesions. CONCLUSIONS: This study shows the value of TP53-mutated DNA as a molecular marker to predict locally recurrent head and neck squamous cell carcinoma. The observation that all patients who were negative for TP53-mutated DNA in the surgical margins remained free of local recurrence raises hope that molecular analysis of histologically tumor-free surgical margins can be exploited to decide on postoperative radiotherapy. Furthermore, our data provide evidence that local recurrences originate mainly from tumor cells left behind but also originate, in part, from unresected precursor lesions.