Elevated Mitotic Rate Research Articles

Abstract LB237: Functional characterisation of TRACERx reveals mechanisms of NSCLC evolution

Abstract Chromosomal instability (CIN), the increase in the rate of whole/partial chromosome gains and losses, is a driving feature of cancer identified in ~90% of solid tumours. CIN generates intratumor heterogeneity (ITH), drives evolutionary adaptation, and is associated with highly aggressive, drug-resistant tumours and poor prognosis. Aberrations in numerous pathways have been suggested to increase CIN. However, how CIN is initiated and maintained in non-small cell lung cancer (NSCLC) is largely unknown. Here we have analysed multi-region WES sequencing data from patients of the TRACERx study, and identified novel genes involved in the DNA damage response (DDR) that may contribute to chromosomal instability. Using orthogonal methods, we have identified that genetic alteration in 6 genes, particularly FAT1, culminates in deficiencies in homologous recombination repair (HRR). This is manifested by a reduction of RAD51 and BRCA1 foci formation, reduced end-resection rate and increased 53BP1 bodies. We find that these deficiencies in the HRR pathway can lead to an increase in structural CIN and deviation from the modal cjromosome number. Furthermore, we observed an early selection of FAT1 mutations in the TRACERx421 LUSC cohort, which resulted in more genome-doubling. We reprot mirrored sub-clonal allelic imbalance (MSAI) events at the FAT1 gene locus, indicating parallel evolution. We have proceeded to validate these observations using several cell lines, showing that FAT1 loss promotes whole genome doubling (WGD) through the generation of actively replicating binucleated cells through an elevated mitotic error rate. Importantly, the elevated CIN induced by FAT1 loss could be partially ameliorated by co-depletion of the oncogene YAP1. We hypothesize that elevated CIN could synergize with WGD to generate increased intratumor heterogeneity. To investigate this, we modelled the relationship between FAT1 loss and genome doubling in an isogenic cell culture system using PC9 cells which are sensitive to the EGFR inhibitor, Osimertinib. Indeed, we observed an elevated rate of acquired resistance in FAT1 KO genome-doubled PC9 cells, which we attribute to the high CIN level generated by FAT1 loss. In line with our hypothesis, we also observed a significant increase in genome content in FAT1 KO cells, suggesting FAT1 loss induces a second WGD event to escape targeted therapy. In conclusion, FAT1 is one of the most frequently mutated genes both in the TRACERx421 cohort and in somatic tissues (Martincorena et al, Science 2015). We postulate that FAT1 loss attenuates DDR and exacerbates CIN to enhance tumour heterogeneity, early in lung cancer evolution. Our observation that FAT1 loss leads to elevated EGFRi resistance also provides a unique opportunity to understand how EGFRi resistance arises through elevated CIN. Citation Format: Wei-Ting Lu, Lykourgos-Panagiotis Zalmas, Chris Bailey, Oriol Pich, Carlos M. Ruiz, James Black, Georgia Stavrou, Dhruva Biswas, Francisco Gimeno-Valiente, Kevin Litchfield, Jiri Bartek, Nicholas McGranahan, Nnennaya Kanu, Charles Swanton. Functional characterisation of TRACERx reveals mechanisms of NSCLC evolution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB237.

Predictive accuracy of elevated mitotic rate on lymph node positivity and recurrence in thin melanomas.

Mitotic rate (MR) is considered an important prognostic factor for melanoma but is not currently used for staging because its nuanced effect is not yet well-delineated. We sought to determine if T category-specific MR is predictive of sentinel lymph node (SLN) positivity, recurrence, and melanoma-specific mortality (MSM). A retrospective review of patients with primary cutaneous melanoma from 1994 to 2020 at a single academic center was performed. Patient demographics and tumor characteristics were recorded. MR was considered elevated for each AJCC8-defined T category if it was ≥2 mitoses/mm2 for T1, ≥4 mitoses/mm2 for T2, ≥6 mitoses/mm2 for T3, or ≥7 mitoses/mm2 for T4. Statistical analysis was performed to assess the predictive accuracy of MR on selected outcomes while controlling for ulceration. Data from 2,984 patients with complete records were analyzed. Along with Breslow thickness and ulceration, elevated MR was associated with higher risk of MSM (HR 1.816, P=0.0001). There was no difference among patients with ulcerated T1 or T2 tumors regardless of MR, but those with non-ulcerated T1 or T2 tumors and elevated MR were more likely to have positive SLNs (P<0.0001 and P=0.0043, respectively) and recurrence (P=0.0007 and P=0.0004, respectively) compared to counterparts with low MR. There were no notable differences for T3 or T4 tumors based on MR. Elevated MR is associated with SLN positivity and recurrence in thin melanomas, independent of ulceration. SLN biopsy should therefore be strongly considered for patients with non-ulcerated lesions <0.8mm thick if the MR is ≥2 mitoses/mm2.

Microsecretory adenocarcinoma of the skin harboring recurrent SS18 fusions: A cutaneous analog to a newly described salivary gland tumor.

Microsecretory adenocarcinoma (MSA) is a newly described salivary gland neoplasm characterized by MEF2C::SS18 fusions. MSA was previously thought to occur exclusively in salivary glands. Here, we expand the spectrum of known primary sites of this tumor by describing a series of cutaneous tumors with analogous findings. We identified four cutaneous primary tumors with histopathologic features identical to MSA of the salivary glands. These cases were evaluated by immunohistochemistry, fluorescence in situ hybridization (FISH) for SS18 rearrangement and targeted RNA-sequencing. We also queried a pan-tumor database of advanced carcinomas for MEF2C::SS18. The cases occurred in men ranging from 61 to 74 years (mean, 68). They arose from the skin of the nose, chin, scalp, and external auditory canal. All included cords/microcysts of eosinophilic cells with bland oval nuclei and bluish mucin within fibromyxoid stroma. The scalp tumor also exhibited high-grade transformation (marked atypia, elevated mitotic rate, and necrosis), a feature unreported in salivary MSA. By immunohistochemistry, all cases were positive for S100. Two showed a myoepithelial component positive for p40 and smooth muscle actin or calponin. Three cases harbored MEF2C::SS18 by RNA sequencing, while one with limited tissue had SS18 rearrangement via FISH. Two patients had no evidence of recurrence or metastasis in limited follow-up (3 and 6 months). The pan-tumor database query also did not identify MEF2C::SS18 in any advanced cutaneous carcinomas. This report expands the sites that can be involved by MSA. Similar to salivary cases, MEF2C::SS18 represents a recurrent fusion in MSA of the skin. Unusual features in cutaneous cases not seen in salivary MSA include one case with high-grade transformation and two cases with a myoepithelial cell component. Identification of this fusion expands the spectrum of salivary-analog cutaneous tumors and aids in precise tumor classification.

MERLIN_001: A prospective registry study of a primary melanoma gene-signature to predict sentinel node (SN) status and determine its prognostic value for more accurate staging of patients with SN-negative melanoma.

TPS9606 Background: For patients with cutaneous melanoma, sentinel lymph node biopsy (SLNB) provides important staging and prognostic information that guides surveillance and adjuvant systemic therapy decisions. At most centers, SLNB is indicated for patients with cutaneous melanoma with at least a 5% risk of having nodal metastases, typically melanomas ≥ 0.8 mm in thickness or thinner lesions with high-risk features such as elevated mitotic rate and/or ulceration. SLNB, generally involving a separate incision, does carry a small but measurable risk of complications including seroma, infection and rarely lymphedema, and most patients have negative sentinel lymph nodes. Currently, there is an unmet clinical need to identify patients who may safely forgo SLNB due to a low (&lt;5%) risk of nodal metastasis, who otherwise meet established criteria for SLNB. Previously, a model consisting of gene expression profile (GEP) of the primary tumor combined with clinicopathological features (CP) has been developed to identify melanoma patients with a low risk of having a positive SLNB. The model has also been validated in multiple retrospective studies. The aim of the MERLIN_001 registry study is to prospectively validate the CP-GEP model in an independent multicenter cohort of primary cutaneous melanoma patients who undergo SLNB for standard indications. Methods: In the next two years, a total of 10 centers across the US will enroll 2,340 patients with clinically node-negative cutaneous melanoma undergoing SLNB using current guideline indications and will follow these patients for 5 years (ClinicalTrials.gov identifier: NCT04759781). Enrollment of patients started in September 2021 and 242 patients have been enrolled as of February 1, 2022. FFPE material from the initial melanoma biopsy will be used to assess the GEP of the primary melanoma. The CP-GEP probability scores will be expressed as a binary classification (Low Risk or High Risk for nodal metastasis) and will be compared to SLN pathology. Performance metrics for CP-GEP will be evaluated and will include: Negative Predictive Value, Positive Predictive Value, Sensitivity and Specificity, and the corresponding 95% confidence intervals. Risk for nodal metastasis will be calculated for Low Risk and High Risk CP-GEP patients. Finally, the performance of CP-GEP to stratify patients according to risk of recurrence (local, regional, distant, death) will also be studied, since data will be collected for up to 5 yrs. Clinical trial information: NCT04759781.

Abstract 421: Characteristics of melanoma diagnosed in patients with underlying HIV infection

Abstract Background: Melanoma is diagnosed more frequently in patients living with HIV (PLWH) compared to the general US population and HIV-infected melanoma patients also experience higher cancer-specific mortality. The mechanisms underlying these outcome disparities are poorly understood, and data describing tumor pathology and non-mortality outcomes for melanoma patients with HIV are lacking. Objective: To describe differences in tumor presentation and disease course among melanoma patients presenting with or without underlying HIV infection in the era of effective HIV therapy. Methods: We conducted an initial retrospective chart review of 29 HIV+ and 29 HIV- patients, matched as possible to HIV+ patients on age and disease stage, who presented to Moffitt Cancer Center between 2004 and 2019. Data abstracted included patient demographics, tumor pathology, treatment details, and clinical outcomes. Results: Certain melanoma pathology features differed by HIV status, with a higher prevalence of elevated mitotic rate (78.9% vs 41.2%, p=0.02) and vertical growth phase (90% vs 55%, p=0.02) in melanoma patients with HIV. Nearly all patients underwent lesion excision, but post-treatment complications were greater in PLWH (44.8% vs 24%, p=0.09). Among 16 patients who received systemic therapy after excision (HIV+ 6; HIV- 10), progressive disease was more frequent in PLWH (100% vs 30%, p=0.01), with further suggestion of higher severe side effect rates, defined as events interrupting systemic therapy (66.7% vs 30%; p=0.18). Despite these differences in progression, overall survival rate were similar in PLWH (79.3%) and uninfected patients (82.8%). Conclusions: These preliminary results suggest that PLWH and melanoma experience a unique disease course. Higher prevalence of post-excision complications and disease progression following systemic therapy in particular warrant further attention as age-related cancers, such as melanoma, are expected to grow in the US HIV population in coming years. Citation Format: Grace Wei, Sweta Sinha, Jane Messina, Anna E. Coghill. Characteristics of melanoma diagnosed in patients with underlying HIV infection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 421.

Clinical and Histopathologic Characteristics of Metastatic and Locally Advanced Cutaneous Basal Cell Carcinomas.

Locally advanced or metastatic basal cell carcinomas (laBCCs or mBCCs) are rare, with few case series providing information on their epidemiology. We aimed to describe the clinical and histologic features of locally advanced and metastatic basal cell carcinomas. Forty cases of laBCC or mBCC were identified by searching Vanderbilt's database from 1984 to January 2019. A retrospective chart review was performed. Pathology slides were available for 23 cases (13 mBCCs and 10 laBCCs). Twenty-one of 23 cases were Clark level IV or V, with a mean depth of invasion of >7 mm for both types. The mean mitotic rate was 4.4 mitoses/mm2 for laBCCs and 3.3 mitoses/mm2 for mBCCs. Ulceration was identified in 7 laBCC and 8 mBCC cases. Perineural invasion was present in 2 laBCC and 6 mBCC cases, with 3 mBCCs invading nerves >0.1 mm. Of 13 mBCC cases, histologic subtypes included infiltrative (n = 9), nodular (n = 7), morpheaform (n = 4), and superficial (n = 2), with multiple patterns present in some specimens. 10 of 13 patients with mBCC had local recurrence before metastasis. In summary, we identified several potential markers of high-risk BCC, including perineural invasion, deep invasion, elevated mitotic rate, and local recurrence of the primary tumor.

Risk factors and patterns of recurrence after sentinel lymph node biopsy for thin melanoma.

While having a thin melanoma (defined as AJCC 8 T1 stage tumor ≤ 1.0mm) with negative sentinel lymph node biopsy (SLNB) provides an excellent prognosis, some patients still develop recurrence and die. To determine risk factors for any recurrence (local/in-transit, nodal, distant) in thin melanoma patients with negative SLNB and assess survival outcomes. Retrospective review of thin melanomas with negative SLNB from 1999 to 2018 was performed. Two hundred and nine patients were identified. Clinicopathologic characteristics of the primary melanoma were collected. Patterns of recurrence for local/in-transit, nodal or distant recurrence and survival outcomes were analyzed. Eighteen patients (8.6%) developed recurrence: 3 (1.9%) local/in-transit, 4 (2.9%) regional/nodal, and 11 (5.3%) distant recurrence during a median follow-up time of 62months. A multivariate Cox regression model showed that head and neck site (HR 3.52), ulceration (HR 10.8), and mitotic rate (HR 1.39) were significant risk factors for recurrence. Median time to first recurrence was 49months. Patients with recurrence had a significantly worse 5year overall survival than those without recurrence (82.2 vs 99.2%). A retrospective single center study and limited sample size. Did not factor in possible false negative SLNBs when calculating hazard ratios. For thin melanoma patients with negative SLNB, heightened surveillance is warranted for those with ulceration, primary tumor location on the head or neck, and elevated mitotic rate.

Reappraisal of the prognostic significance of mitotic rate supports its reincorporation into the melanoma staging system.

Mitotic rate is a strong, independent prognostic factor in patients with melanoma. However, incorporating it into the melanoma staging system has proved challenging. The prognostic impact of mitotic rate was assessed in a melanoma cohort comprising 5050 patients from 2 geographically distinct populations. Computer-generated cut points for mitotic rate were constructed to determine its impact on melanoma-associated survival using Kaplan-Meier and multivariate regression analyses. The impact of mitotic rate also was assessed in randomly split training and validation sets. Mitotic rate had a nonlinear impact on survival, as evidenced by unequally spaced cut points. An index incorporating these cut points that was constructed from one population produced significantly more accurate predictions of survival in the other population than using the entire scale of mitotic rate. An index constructed from the combined cohort was found to be independently predictive of survival, with an impact comparable to that of ulceration. Optimal high-versus-low cut points for mitotic rate were generated separately for each T category (<2 mitoses/mm2 vs ≥2 mitoses/mm2 for T1 melanoma, <4 mitoses/mm2 vs ≥4 mitoses/mm2 for T2 melanoma, <6 mitoses/mm2 vs ≥6/mitoses/mm2 for T3 melanoma, and <7 mitoses/mm2 vs ≥7 mitoses/mm2 for T4 melanoma). Using Kaplan-Meier analysis, elevated mitotic rate was found to have an impact on survival comparable to that of ulceration within each T category. Application of the index for mitotic rate that was constructed from the training data set demonstrated an independent impact in the validation data set, with a significance similar to that of ulceration. The results of the current study demonstrated the comparable prognostic impact of mitotic rate and ulceration, providing support for its reincorporation into the T category.

SMARCB1 (INI-1)-Deficient Adenocarcinoma of the Sinonasal Tract: A Potentially Under-Recognized form of Sinonasal Adenocarcinoma with Occasional Yolk Sac Tumor-Like Features

The classification of sinonasal adenocarcinoma (SNAC) is complex. The high-grade, non-intestinal SNAC group is particularly heterogeneous, with tumors showing widely variable morphology. SMARCB1 (INI-1)-deficient sinonasal carcinoma is a newly described, aggressive tumor that usually resembles sinonasal undifferentiated carcinoma (SNUC) or non-keratinizing squamous cell carcinoma; however, glandular differentiation has been rarely reported and this feature may be under-recognized. We present a dedicated series of 12 SMARCB1-deficient SNACs. All tumors had an oncocytoid/plasmacytoid cytomorphology with variable degrees of glandular differentiation consisting of tubules and cribriform structures with foci of intracellular or intraluminal mucin. Three of 12 tumors exhibited foci of yolk sac tumor-like histologic features. The tumors were uniformly high-grade, with nuclear pleomorphism, elevated mitotic rates and frequent necrosis. By immunohistochemistry, all tumors were entirely SMARCB1-deficient, and 10 of 12 were CK7-positive. Occasional expression of CDX2 (4 of 12), CK20 (3 of 12), and p40 (3 of 10) was seen. Expression of yolk sac markers was variably present in tumors that harbored yolk sac-like areas but also tumors that did not: glypican-3 (10 of 11), SALL4 (6 of 11), HepPar-1 (4 of 11), PLAP (1 of 10), and AFP (1 of 11). SMARCB1-deficient sinonasal carcinoma, particularly the oncocytoid/plasmacytoid form, can demonstrate variable degrees of glandular differentiation. This unexpected morphology combined with variable immunohistochemical results may lead to misdiagnoses of high-grade intestinal or non-intestinal SNAC, myoepithelial carcinoma, or even yolk sac tumor or metastatic hepatocellular carcinoma.

Early Melanoma Nodal Positivity and Biopsy Rates Before and After Implementation of the 7th Edition of the AJCC Cancer Staging Manual

There has been a continued increase in the incidence of newly diagnosed melanomas, most of which are T1 melanomas. The associations between changes in tumor staging, implemented with the 7th edition of the AJCC Cancer Staging Manual (AJCC 7), and sentinel lymph node biopsy rates and nodal positivity rates remain to be seen. To evaluate the change that the implementation of the AJCC 7 had on staging criteria and the distribution of thin melanomas requiring nodal surgery and nodal positivity rates. Retrospective cross-sectional study from 2004 through 2013 of all adults (≥18 years) diagnosed with a T1 (Breslow depth ≤1.0 mm) melanoma using The National Cancer Database that captures 70% of all newly diagnosed cancers from accredited Commission on Cancer organizations, including both academic and community settings. Data were analyzed in May 2017. Patients were grouped together based on year of diagnosis, before and after 2009. To determine the sentinel lymph node biopsy rate before and after the implementation of the AJCC 7. A total of 141 280 patients met inclusion criteria. Of 86 846 patients diagnosed from 2004 through 2009, 53.7% (49 644) were male and had a mean (SD) age of 57.7 (16.4) years. Of 54 434 patients diagnosed from 2010 through 2013, 54.3% (31 086) were male and had a mean (SD) age of 59.5 (15.9) years. After 2010, there was a 3.8% decrease in the number of nodal surgeries performed (32 485 of 86 846 patients [37.6%] vs 18 379 of 54 434 patients [33.8%]; P < .001). The nodal positivity rate decreased 1.0% from (9.8% [3166 of 86 846] to 8.8% [1618 of 54 434]) (P < .001). An increase in the proportion of T1b melanomas being evaluated, from 48.8% to 62.2%, was seen (P < .001). Of T1b melanomas that underwent nodal evaluation from 2004 through 2009, 74.0% had Clark level IV (invasion of the reticular dermis) or Clark level V (invasion of the deep, subcutaneous tissue) and 9.5% were ulcerated. From 2010 through 2013, of the T1b melanomas undergoing nodal evaluation, 82.6% had an elevated mitotic rate only, 3.7% were ulcerated, and 13.7% had both ulceration and an elevated mitotic rate. It appears that after the institution of AJCC 7, there was an overall decrease in the number of T1 melanomas undergoing nodal biopsy without a clinically relevant change in sentinel lymph node positivity, with an increase in the number of T1b melanomas undergoing nodal evaluation.

Distinct Dynamics of Mitotic Transition in B-Cell Lymphoma and Reactive B-Cell Lymphoproliferations Determined by H3S10 Phosphohistone Immunolabeling

Objectives: Clonal selection in the follicular germinal centers in lymphatic tissues is accompanied by an intense proliferation of polyclonal B cells in a precisely regulated fashion. In contrast, B-cell neoplasias proliferate autonomously due to endogenous stimuli. The cell kinetic activity is obvious at many levels including progressive chromatin modification and elevated mitotic rates. We asked if there are differences in the kinetics of histone H3S10 phosphorylation required for mitotic entry between highly proliferating B cells of reactive germinal centers and in B-cell lymphomas with different proliferative capacity. Material and Methods: Phospho-H3 histone (pH3S10)-specific immunohistochemistry was applied to cultivated cell, reactive and selected indolent and aggressive lymphoma samples (diffuse large B-cell lymphoma, Burkitt lymphoma, lymphoblastic lymphoma, follicular lymphoma and small lymphocytic lymphoma). Microscopic quantification of the “dot-type” (representing late G₂ phase) and “mitotic” immunolabeling patterns per field of view was performed and compared with classical cell proliferation markers. Results: In addition to the dense homogeneous chromatin labeling highlighting mitotic figures, we stated a selective dot-type nuclear labeling representing ongoing chromatin condensation in premitotic G₂ phase cells. While cell proliferation and mitotic counts correlated in general with histology, statistical analysis indicated an accumulation of G₂ phase pH3S10 pattern in the reactive germinal centers in contrast to lymphomas. The dot-type G₂ staining pattern was surprisingly overrepresented (1,321.7 ± 356.5/10 HPF) in the reactive germinal centers compared to aggressive lymphomas (101.3 ± 33.1) (p < 0.005). The relative G₂/M value was significantly higher (4.6 ± 0.6) in reactive germinal center B cells than in any lymphoma entity evaluated (0.7 ± 0.2 in Burkitt lymphoma, 0.9 ± 0.4 in grade 3b follicular lymphoma, 1.3 ± 1.1 in diffuse large B-cell lymphoma, 1.5 ± 0.6 in lymphoblastic lymphoma, and 0.9 ± 0.2 in small lymphocytic lymphoma). Conclusions: pH3S10 immunohistochemistry enabled the presentation of significant differences in the cell cycle kinetics between reactive and neoplastic B-cell lymphoproliferations. Accumulation of G₂ phase B cells in reactive folliculi directs to physiological G₂/M checkpoint blockade. In contrast, accelerated G₂/M transition in lymphomas is potentially associated with impaired genomic repair and cell death mechanisms.

Detailed pathologic evaluation of low dose CT (LDCT) detected stage I/0 lung adenocarcinomas (ADCA).

8543 Background: Although LDCT lung screening improves survival, some worry that indolent BAC-like ADCA may be over-detected/treated. Comprehensive histologic subtyping has recently been adopted to improve grading and thus risk stratification in lung ADCA. We report a detailed pathologic comparison of stage I/0 lung ADCAs detected by LDCT screening vs. incidentally discovered ADCAs stratified by NCCN risk criteria. Methods: We performed comprehensive histologic subtyping on 35 consecutive stage I/0 LDCT screen detected ADCAs in patients meeting NCCN group 1/2 high risk (HR) criteria and compared to incidentally detected stage I/0 ADCAs meeting HR (n = 41) or low-risk (LR, n = 28) criteria. Results: Screen detected and incidentally detected ADCAs from HR groups show an equally low-rate of indolent, non-invasive/minimally invasive ADCAs (9%) compared to LR patients (36%). Aggressive solid predominant ADCAs were equally more frequent in HR patients from screen and incidentally detected patients (19%) compared to LR patients (6%). The rate of angiolymphatic invasion (59%) and air space invasion (32-34%) were similarly elevated in HR screen and incidental patients compared to LR patients (39% &amp; 17% respectively). A collection of lepidic predominant (BAC-like) ADCAs associated with aggressive non-predominant cribriform and/or solid patterns, elevated mitotic rates, and high proportion of lymphatic invasion were uniquely observed in the screen-detected group (12%). Subgroup analysis of screen detected NCCN group 1 vs. 2 shows group 2 tumors exhibit histologic features which are at least as aggressive as group 1 tumors, albeit at smaller invasive sizes (1.2 cm vs. 1.5 cm) and lower stage (IA 75% vs. 61%). Conclusions: This is the first detailed pathologic comparison of LDCT screen detected vs incidentally discovered stage I/0 ADCA. Tumors from HR patients are pathologically more aggressive than LR tumors. Not all BAC-like tumors in LDCT screen detected patients should be presumed indolent given that approximately half of these bear histologic features of aggressive ADCAs while still in a lepidic predominant phase. NCCN group 2 tumors are similar to group 1 and should be further studied.

Histological Predictors of Outcome in Ependymoma are Dependent on Anatomic Site Within the Central Nervous System

Ependymomas originate in posterior fossa (PF), supratentorial (ST) or spinal cord (SC) compartments. At present, grading schemes are applied independent of anatomic site. We performed detailed histological examination on 238 World Health Organization grade II and III ependymomas. Among PF ependymomas, the presence of hypercellular areas, necrosis, microvascular proliferation and elevated mitotic rate (all P < 0.01) were significantly associated with worse progression-free survival (PFS), while extensive ependymal canal formation was not (P = 0.89). Similar to the PF tumors, microvascular proliferation (P = 0.01) and elevated mitotic rate (P = 0.03) were significantly associated with worse PFS in the ST tumors. However, in contrast to PF tumors, extensive ependymal canals (P = 0.03) were associated with worse clinical outcome in ST ependymomas, but hypercellularity (P = 0.57) and necrosis (P = 0.47) were not. On multivariate Cox regression, after adjusting for relevant clinical variables, individual histological factors and a composite histological score remained significant among ST and PF ependymoma. In contrast to both PF and ST ependymoma, histological features were not found to be associated with PFS in SC tumors. Taken together, the clinical relevance of specific histological features in ependymoma appears to be related to the anatomic site of origin and suggests that site-specific grading criteria be considered in future classification systems.

32. Sentinel lymph node biopsy in patients with thin melanoma

Background Most new cases of melanoma are thin (≤1 mm) tumours. A small but significant proportion of patients with thin melanoma suffer disease progression and melanoma-related death. We sought to determine clinico-pathological factors predictive poor clinical outcomes in patients with thin melanoma who underwent sentinel lymph node (SLN) biopsy. Methods Patients diagnosed with a single primary thin cutaneous melanoma who underwent SLN biopsy between 1992 and 2009 were identified from the database of the Melanoma Institute Australia. Thin melanomas were defined as having a Breslow thickness ≤1.00 mm. Clinico-pathological factors associated with SLN positivity, regional lymph node recurrence and survival were analysed. Results There were 432 patients (226 male, 206 female, median age 49.5 years). SLNs were positive in 29 (6.7%) patients. No SLN positivity was detected in 37 patients with tumour thickness ≤0.50 mm. Breslow thickness (OR = 52.0, 95%CI 1.4-1948.7) and presence of lymphovascular invasion (OR = 8.5, 95%CI 1.8-41.4) were the only independent predictors of SLN positivity. Anatomic location in head/neck region (OR = 8.5, 95%CI 1.8-40.2; compared to location in the limbs) was the only independent predictor of regional lymph node recurrence. Mitotic rate was the only independent predictor of melanoma-specific survival (HR=1.2, 95% CI 1.1-1.3). SLN positivity in Clark level III tumours (HR=8.0, 95%CI 1.8-36.0) and anatomic location in head/neck region in Clark level IV tumours (HR = 23.7, 95% CI 2.0-277, compared to limbs) were the only independent predictors of poorer disease-free survival. Conclusions Patients with thicker tumours (>0.5 mm) and other adverse prognostic factors (e.g., ulceration, elevated mitotic rate, lymphovascular invasion) have a greater risk of occult nodal metastasis and are more likely to benefit from SLN biopsy.

Carcinoid of the Rectum Risk Stratification (CaRRs): A Strategy for Preoperative Outcome Assessment

Predicting rectal carcinoid behavior based exclusively on tumor size is imprecise. We sought to identify factors associated with outcome and incorporate them into a pre-operative risk stratification scheme. Seventy rectal carcinoid patients evaluated at our institution were identified. Demographic, clinical, and histopathologic data were collected and correlated with recurrence and survival. The mean age of our cohort was 53.6 years. Fifty-seven percent of patients were female. The mean tumor size was 1.3 cm (range: 0.1-5 cm). Twenty-five percent of patients had deeply invasive tumors (into the muscularis propria or deeper); an equal percentage had tumors with lymphovascular invasion (LVI) or an elevated mitotic rate (> or =2/50 HPF). Eleven patients (17%) had distant metastases at presentation. Sixty-one patients were followed for a median of 22 months (2-308 months), during which seven patients developed recurrence and seven died of disease (2/7 who developed recurrence). Poor outcome was associated with large tumor size, deep invasion, presence of LVI, and elevated mitotic rate. These factors were incorporated into a carcinoid of the rectum risk stratification (CaRRS) score. CaRRS predicted recurrence-free and disease-specific survival better than any single factor alone. Poor prognostic features of rectal carcinoids include: large size, deep invasion, LVI, and elevated mitotic rate. The CaRRS score incorporates these features and accurately predicts outcome. Because the CaRRS score is based upon values available on pre-operative biopsy, it can identify patients with very favorable prognosis as well as those with poor prognosis that may benefit from additional staging or surveillance.

Cellular Fibromas of the Ovary: A Study of 75 Cases Including 40 Mitotically Active Tumors Emphasizing Their Distinction From Fibrosarcoma

Cellular fibroblastic tumors of the ovary are currently classified as either cellular fibroma (CF) or fibrosarcoma. The former are characterized by bland nuclei, 3 or fewer mitotic figures per 10 high-power fields (MFs/10 HPFs), and a low malignant potential, whereas fibrosarcomas usually have severe nuclear atypia, > or = 4 MFs/10 HPFs, and an aggressive clinical course. The prognosis of cellular fibromatous tumors with > or = 4 MFs/10 HPFs and low-grade cytology is not established and it is the purpose of this study to investigate that aspect. It has been our anecdotal experience that otherwise typical CFs with > or = 4 MFs/10 HPFs usually have a benign clinical course, suggesting that such tumors should be regarded as "mitotically active cellular fibroma" (MACF) rather than fibrosarcoma. Seventy-five cellular fibromatous neoplasms were analyzed to determine their clinicopathologic features and the appropriateness of "MACF" as a designation for otherwise typical CFs with > or = 4 MFs/10 HPFs. The mean age of patients with CF (n = 35, 0 to 3 MFs/10 HPFs) and MACF (n = 40, > or = 4 MFs/10 HPFs) was 51 and 41 years, respectively. Patients most commonly presented with symptoms related to a pelvic mass. All tumors were unilateral. The mean tumor size of CFs was 8.0 cm and 9.4 cm for MACFs. The majority of the tumors were solid; approximately one-third of them had a cystic component. Ovarian surface adhesions, involvement of the ovarian surface, or both, was present in 6% of CFs and 10% of MACFs. Eleven percent of CFs and 13% of MACFs were associated with extraovarian involvement. All tumors consisted of cellular, intersecting bundles of spindle cells with bland nuclear features. The mean highest mitotic count for MACFs was 6.7 MFs/10 HPFs (range 4 to 19 MFs/10 HPFs). Follow-up of 3 months to 12 years (mean 4.75 y) was available in 18 of the 40 patients with MACFs and was uneventful in all cases. We conclude that cellular fibromatous neoplasms with bland cytology and elevated mitotic counts are associated with favorable patient outcome and should be diagnosed as MACF rather than fibrosarcoma, which usually have moderate to severe atypia and elevated mitotic rates. As prior observations have shown that even typical CFs can occasionally recur locally, particularly if they are associated with rupture or adherence, long-term follow-up for patients with CFs and MACFs is appropriate.

Prognostic criteria in nonfunctioning pancreatic endocrine tumours.

To identify prognostic subgroups among non-functioning (nonsyndromic) pancreatic endocrine tumours, a series of 61 tumours were analysed systematically for macroscopic, histopathological and immunohistochemical variables potentially predictive of malignancy. High-grade nuclear atypia, elevated mitotic rate and multifocal necrosis allowed us to separate 5 poorly differentiated carcinomas from 56 well differentiated tumours. Among the latter, 29 well-differentiated carcinomas showing gross local invasion or metastases were identified. Vascular or perineural microinvasion, Ki67 proliferative index > 2%, mitotic rate > or = 2, size > or = 4 cm, capsular penetration, nuclear atypia, lack of progesterone receptors and presence of calcitonin were among the variables correlated with malignancy. The first two were the most sensitive and specific. Their presence or absence was used in the 27 tumours lacking evidence of malignancy at the time of surgery to separate 11 cases with increased risk of malignancy (in 2 of which metastases developed during follow-up) from 16 cases with limited risk. The resulting four prognostic groups of non-functioning pancreatic endocrine tumours (limited- and increased-risk tumours, well-differentiated carcinomas and poorly differentiated carcinomas) showed distinct survival curves, which were significantly affected by vascular microinvasion, Ki67 proliferative index and metastases.

The role of recombination and RAD52 in mutation of chromosomal DNA transformed into yeast.

While transformation is a prominent tool for genetic analysis and genome manipulation in many organisms, transforming DNA has often been found to be unstable relative to established molecules. We determined the potential for transformation-associated mutations in a 360 kb yeast chromosome III composed primarily of unique DNA. Wild-type and rad52 Saccharomyces cerevisiae strains were transformed with either a homologous chromosome III or a diverged chromosome III from S. carlsbergensis. The host strain chromosome III had a conditional centromere allowing it to be lost on galactose medium so that recessive mutations in the transformed chromosome could be identified. Following transformation of a RAD+ strain with the homologous chromosome, there were frequent changes in the incoming chromosome, including large deletions and mutations that do not lead to detectable changes in chromosome size. Based on results with the diverged chromosome, interchromosomal recombinational interactions were the source of many of the changes. Even though rad52 exhibits elevated mitotic mutation rates, the percentage of transformed diverged chromosomes incapable of substituting for the resident chromosome was not increased in rad52 compared to the wild-type strain, indicating that the mutator phenotype does not extend to transforming chromosomal DNA. Based on these results and our previous observation that the incidence of large mutations is reduced during the cloning of mammalian DNA into a rad52 as compared to a RAD+ strain, a rad52 host is well-suited for cloning DNA segments in which gene function must be maintained.

Patterns of Hemocyte Production and Release Throughout the Molt Cycle in the Penaeid Shrimp Sicyonia ingentis.

The production and release of hemocytes was evaluated throughout the molt cycle in the shrimp Sicyonia ingentis. Hematopoiesis occurs in paired epigastric hematopoietic nodules (HPN) which consist of an extensive network of vessels. Hemocytes are produced within the walls of these tubules and released into the vessel lumens. During molt stage C (intermolt), few cells were present in the tubule wall; most of these were hematopoietic stem cells. Elevated mitotic rates during stages C to D1-2 (2-4%) led to the production and rapid release of individual hemocytes, primarily granulocytes. Although the mitotic rate progressively declined from stage D3-4 until after ecdysis (stage A1), the maturing hemocytes accumulated within the tubule walls. Around ecdysis, production of hyaline hemocytes exceeded that of granulocytes. Large groups of these hemocytes were channeled into the vessel lumens immediately after molting. Mitotic rates increased again during stages A2 and B with the number of hemocytes in the tubules reaching seven times that of stage C. Morphological stages in the transition of hematopoietic stem cells into hyaline hemocytes and granulocytes are described, and a model of decapod hemocyte maturation is presented.

Iron as a synergist for hepatocellular carcinoma induced by polychlorinated biphenyls in Ah-responsive C57BL/10ScSn mice

Male Ah-responsive C57BL/10ScSn mice received a single dose of iron-dextran (600 mg Fe/kg) and were fed a diet containing 0.01% of the PCBs mixture Aroclor 1254 for up to 12 months. Iron caused a marked synergistic increase in liver size from 2 months with greatly elevated mitotic rates, numbers of basophilic foci and incidences of bile duct and oval cell proliferation. Although at 4 months much of the liver enlargement was due to iron-depleted hyperplastic regions and lipid accumulation, by 8 months seven out of nine mice had nodules (hepatocellular adenomas) whereas none were observed in the Aroclor alone group. After 12 months, 16 out of 18 mice had multiple nodules and/or hepatocellular carcinomas whereas only one of 16 mice was positive in a group not given iron. Basophilic nodules were more common than clear cell nodules in those mice with carcinomas than in those animals without. Preloading with iron also greatly enhanced the development of cholangiofibrosis at 8 and 12 months. Preliminary experiments with the polybrominated biphenyl mixture Firemaster BP-6 indicated a similar synergistic interaction with iron. No effects of iron and Aroclor 1254 on the liver were observed in the Ah-nonresponsive strain DBA/2. Iron potentiated the development of uroporphyria after exposure to those chemicals in C57BL/10ScSn mice but not in the DBA/2 mice. Therefore in C57BL/10ScSn mice the carcinogenicity of PCBs and possibly PBBs, is modulated by iron status and probably not at a late stage where these chemicals may act in a promotional manner.