Einar Gudlaugsson

Phosphohistone H3 expression has much stronger prognostic value than classical prognosticators in invasive lymph node-negative breast cancer patients less than 55 years of age

The proliferation factor mitotic activity index is the strongest prognostic factor in early breast cancer, but it may lack reproducibility. We analyzed the prognostic value of phosphohistone H3, a marker of cells in late G2 and M phase, measuring highly standardized immunohistochemical nuclear phosphohistone H3 expression by subjective counts and digital image analysis. Expression was compared with classical clinico-pathologic prognostic variables and the mitotic activity index in 119 node-negative invasive breast cancers in patients less than 55 years old treated with adjuvant systemic chemotherapy with long-term follow-up (median 168 months). Nineteen patients (16%) developed distant metastases and 16 (13%) died. Strong phosphohistone H3 expression occurred preferentially in the peripheral growing front; counts were highly reproducible between observers (R=0.92) and highly consistent with digital image analysis (R=0.96). Phosphohistone H3 correlated (P<0.05) with tumor diameter, estrogen receptor, carcinoma grade, and mitotic activity index. Phosphohistone H3 values were systematically (80%) higher than the mitotic activity index. Receiver-operating curve analysis objectively showed that phosphohistone H3 <13 (n=53; 45% of all cases) vs phosphohistone H3≥13 (n=66; 55% of all cases) was the strongest prognostic threshold, with 20-year recurrence-free survival of distant metastases of 96 and 58%, respectively (P=0.0002, HR=9.6). Mitotic activity index was the second strongest prognostic variable (P=0.003, HR=3.9). In multivariate analysis, phosphohistone H3 <13 vs≥13 exceeded the prognostic value of the mitotic activity index. None of the other classical prognostic factors examined offered prognostic value additional to phosphohistone H3. Phosphohistone H3 is by far the strongest prognostic variable in early invasive node-negative breast cancer patients less than 55 years old with long-term follow-up.

Comparison of the effect of different techniques for measurement of Ki67 proliferation on reproducibility and prognosis prediction accuracy in breast cancer

Gudlaugsson E, Skaland I, Janssen E A M, Smaaland R, Shao Z, Malpica A, Voorhorst F & Baak J P A 
(2012) Histopathology
Comparison of the effect of different techniques for measurement of Ki67 proliferation on reproducibility and prognosis prediction accuracy in breast cancer

Biologic profiling of lymph node negative breast cancers by means of microRNA expression.

Breast cancer is a heterogeneous disease. Different subgroups can be recognized on the basis of the steroid receptors, HER-2, cytokeratin expression and proliferation patterns. As a result of mRNA-profiling studies, five major groups can be recognized, of which the triple-negative and basal-like tumors have the worst prognosis. Many of these tumors have a high proliferation that has the strongest prognostic value in node negative breast cancer. In the current study we analyzed the microRNA pattern in 103 lymph node negative breast cancers and compared these profiles with different biological characteristics and clinicopathological features. Unsupervised hierarchical cluster analysis divides the patients into four main groups, of which the basal-like/triple-negative group is the most prominent (11% of all cases), the luminal A cancers containing the Her2 negative and estrogen receptor/progesterone receptor-positive tumors is the largest group (57%), and the group of luminal B (32%) is more heterogeneous and contains the Her2 positive/estrogen receptor-negative patients as well. The highest overall classification values by analysis of variance followed by cross validation (leave one sample out and reselect genes) were found for cytokeratin 5 and 6, triple-negative and estrogen receptor, with 97, 90 and 90% accuracy, respectively. MiR-106b gene is prominent in all of these signatures and correlates strongest with high proliferation. Other interesting observations are the presence of several microRNAs (miR532-5p, miR-500, miR362-5p, and miR502-3p) located at Xp11.23 in cancers with a triple-negative signature, and the upregulation of several miR-17 cluster members in estrogen receptor-negative tumors. The current study shows that estrogen receptor negativity and cytokeratin 5 and 6 expression are important, and specific biological processes in lymph node negative breast cancer, as microRNA signatures are strongest in these subgroups.

Local immune response in the microenvironment of CIN2-3 with and without spontaneous regression

Fifteen to thirty percent of cases with histologically confirmed CIN2–3 in cervical biopsies regress spontaneously (ie, show CIN1 or less in the follow-up cervical cone). The balance between immune-reactive cells from the host and high-risk human papillomavirus (hrHPV) genotypes may provide a biological explanation for this phenomenon. We retrospectively studied 55 cases of CIN2–3 in a cervical biopsy with subsequent cervical cone to assess whether hrHPV genotypes (by AMPLICOR and Linear Array tests) CD4, CD8, CD25, CD138 and Foxp3 cells (by quantitative immunohistochemistry) in the cervical biopsies can predict regression (defined as CIN1 or less in the follow-up cone biopsy). Eighteen percent of the CIN2–3 cases regressed (median biopsy–cervical cone time interval: 12.0 weeks, range: 5.0–34.1 weeks). HPV-16 correlated with low CD8+ and high CD25+. None of the regressing CIN2–3 lesions contained HPV-16. The regressing CIN2–3 lesions had lower numbers of stromal CD138+ and higher numbers of stromal CD8+cells; higher stromal and intra-epithelial ratios of CD4+/CD25+ cells; higher ratios of CD8+/CD25+ cells and lower ratios of CD8+/CD4+, CD138+/Foxp3+ and CD25+/Foxp3+cells in the stroma. With multivariate survival analysis, stromal CD8+ cell numbers, CD4+/CD25+ cell ratios and CD138+ cell numbers are found to be independent regression predictors. In conclusion, in non-HPV-16 CIN2–3 lesions, assessing stromal immune cells can be a useful prognostic indicator of regression or persistence.

Comparison of different commercial methods for HPV detection in follow-up cytology after ASCUS/LSIL, prediction of CIN2-3 in follow up biopsies and spontaneous regression of CIN2-3

OBJECTIVE Different Human Papilloma Virus (HPV) tests are currently used. An integrated comparison of the Amplicor, Cobas4800, PreTect HPV-Proofer and APTIMA HPV tests has not been done. METHODS We compared the high-risk HPV detection power of these HPV tests in 528 consecutive population-based follow-up Liquid-Based Cytology samples (LBC) after ASCUS/LSIL index cytology. Their sensitivity and specificity to detect HPV in LBC, their predictive values of histopathologic CIN2-3 in follow-up punch biopsies and CIN2-3 regression in the subsequent cones was assessed. The HPV subtypes detected by the Linear Array genotyping-test (LA), PreTect HPV-Proofer and Cobas4800 were also compared. The follow-up histopathology was consensus expert-reviewed and Ki67/p16-supported. The predictive values of the HPV results in LBC by the different tests for presence of CIN2-3 in follow-up biopsies, and regression in subsequent cones, was assessed. RESULTS Amplicor, Cobas4800 and APTIMA show good agreement for HPV-positivity/negativity. PreTect HPV-Proofer has many discrepancies versus any of the other methods. The sensitivities for Amplicor, Cobas4800 and APTIMA to detect CIN2-3 were very high (96-100%), but rather low for PreTect HPV-Proofer (53%). Specificity in case of CIN1 or less in follow-up biopsies of Amplicor and Cobas4800 is lower than APTIMA and highest for PreTect HPV-Proofer. HPV subtyping by LA agreed in 90% with Cobas4800 but 70% with PreTect HPV-Proofer. CONCLUSIONS The Amplicor, Cobas4800 and APTIMA give comparable results but PreTect HPV-Proofer differs from the other tests, with low sensitivity but higher specificity. None of the methods predicted regression of CIN2-3.

Benign Peritoneal Cystic Mesothelioma

BackgroundBenign peritoneal cystic mesothelioma (BPCM) is a rare tumor of unknown origin, most frequently encountered in women of reproductive age. Etiology is unknown; definitions and terminology are confusing, and preoperative diagnosis is difficult. Several differential diagnoses must be considered.MethodsBased on our own clinical experience and a review of the relevant literature, we address clinical challenges and controversies of importance.ResultsCurrent literature on BPCM is mostly based on small case reports. Complete surgical resection is recommended if possible. Nevertheless, recurrent disease is not uncommon. Clinical positive effects of various adjuvant medical treatments remain to be shown.ConclusionsLack of consistent definitions, various treatment approaches, and mostly short follow-up times make it difficult to draw any firm conclusions from published reports. The natural history of this rare disease is less than well clarified. When possible, in an individual patient, surgical resection with curative intent seems to be the treatment of choice.

Validation of Expression Patterns for Nine miRNAs in 204 Lymph-Node Negative Breast Cancers

Introduction Although lymph node negative (LN-) breast cancer patients have a good 10-years survival (∼85%), most of them still receive adjuvant therapy, while only some benefit from this. More accurate prognostication of LN- breast cancer patient may reduce over- and under-treatment. Until now proliferation is the strongest prognostic factor for LN- breast cancer patients. The small molecule microRNA (miRNA) has opened a new window for prognostic markers, therapeutic targets and/or therapeutic components. Previously it has been shown that miR-18a/b, miR-25, miR-29c and miR-106b correlate to high proliferation. Methods The current study validates nine miRNAs (miR-18a/b miR-25, miR-29c, miR-106b, miR375, miR-424, miR-505 and let-7b) significantly correlated with established prognostic breast cancer biomarkers. Total RNA was isolated from 204 formaldehyde-fixed paraffin embedded (FFPE) LN- breast cancers and analyzed with quantitative real-time Polymerase Chain Reaction (qPCR). Independent T-test was used to detect significant correlation between miRNA expression level and the different clinicopathological features for breast cancer. Results Strong and significant associations were observed for high expression of miR-18a/b, miR-106b, miR-25 and miR-505 to high proliferation, oestrogen receptor negativity and cytokeratin 5/6 positivity. High expression of let-7b, miR-29c and miR-375 was detected in more differentiated tumours. Kaplan-Meier survival analysis showed that patients with high miR-106b expression had an 81% survival rate vs. 95% (P = 0.004) for patients with low expression. Conclusion High expression of miR-18a/b are strongly associated with basal-like breast cancer features, while miR-106b can identify a group with higher risk for developing distant metastases in the subgroup of Her2 negatives. Furthermore miR-106b can identify a group of patients with 100% survival within the otherwise considered high risk group of patients with high proliferation. Using miR-106b as a biomarker in conjunction to mitotic activity index could thereby possibly save 18% of the patients with high proliferation from overtreatment.

In Patients Younger Than Age 55 Years With Lymph Node–Negative Breast Cancer, Proliferation by Mitotic Activity Index Is Prognostically Superior to Adjuvant!

PURPOSE In breast cancer, different tools are used for prognostication and adjuvant systemic therapy selection. We compared the accuracy of the online program Adjuvant!, the Norwegian Breast Cancer Group (NBCG) guidelines, and the proliferation factor mitotic activity index (MAI) in patients with lymph node (LN) -negative disease (pN0). PATIENTS AND METHODS Adjuvant! and MAI thresholds were set to 90% to 95% breast cancer-specific survival (BCSS) rates. These thresholds were 95% for Adjuvant!, 3 for MAI, and as follows for NBCG: pT1 grade 1 + pT1a-b grade 2 to 3; all pN0M0 and estrogen receptor/progesterone receptor positive versus all others. In 516 patients younger than age 55 years (T1-3N0M0) without adjuvant systemic therapy, univariable and multivariable 10-year BCSS rates were estimated. RESULTS Median follow-up time was 118 months. The concordance between MAI and Adjuvant! or NBCG was fair (κ = 0.35 and κ = 0.29, respectively). Adjuvant!, NBCG, and MAI were all prognostically significant (P ≤ .001). In the univariable analysis, the 10-year BCSS of MAI less than 3 versus ≥ 3 was 95% v 71%, respectively, with a hazard ratio of 7.0. In multivariable analysis, MAI was superior to Adjuvant! and NBCG. The 10-year survival of Adjuvant! ≥ 95% versus less than 95% was 91% v 74%, respectively, but stratification by MAI identified subgroups with different prognosis. Similar results occurred for NBCG and MAI. Adjuvant! and NBCG were not prognostic to each other. CONCLUSION MAI is superior to Adjuvant! and NBCG in prognostication of patients with LN-negative breast cancer younger than age 55 years.

Chromogranin A, a Marker of the Therapeutic Success of Resection of Neuroendocrine Liver Metastases: Preliminary Report

Recent publications indicate that life may be prolonged by surgical debulking of neuroendocrine tumors. A minimum 90% reduction of liver metastases has been suggested to alleviate symptoms of the carcinoid. We have used the tumor marker chromogranin A (CgA) to assess hepatic resection in patients with neuroendocrine metastatic tumor disease. Since 1998, seven patients (3 men) of median age 73 years (range 64–84 years) with carcinoid primary tumors in the ileum who had solitary (n = 2) or multiple (n = 5) liver metastases underwent hepatic resections. Two patients had synchronous small intestinal and liver resections; the rest had deferred hepatic resections after intestinal resection. Hormonal manifestations in the form of loose stools or diarrhea or flushing were observed in five patients, and five had abdominal symptoms from partial obstruction of the small bowel. The resection was deemed radical in five patients. Two patients with non-radical resection needed postoperative octreotide treatment, and symptoms were alleviated or improved in the others. All seven patients are alive with an observation period from 6 to 64 months (median 36 months). Median CgA (normal < 30 ng/ml) was 292 ng/ml (range 79-14,000 ng/ml) before liver surgery. Postoperatively, CgA became normal in three of the radically resected patients, whereas in two others, it decreased to a lowest median level of 79 ng/ml (range 52–105 ng/ml). In two palliatively resected patients, one had a near normalization to 65 ng/ml, and the last patient had a reduction from 14,000 to 2400 ng/ml following debulking surgery. A similar postoperative reduction was noted for 24 hr urinary 5-HIAA excretion. Postoperative octreotide scintigraphy suggested residual hepatic or extrahepatic tumors in three of the patients thought radically resected, whereas two had no clear sign of disease corresponding to a normal CgA value. The CgA values, however, reflected the extent of positive scintigraphy findings. Serum CgA levels monitored the extent and short-term course of the disease and corresponded well with scintigraphy findings and 5-HIAA excretion, but prolonged follow-up in more patients may be necessary before decisive conclusions are allowed to be drawn.

The impact of epithelial biomarkers, local immune response and human papillomavirus genotype in the regression of cervical intraepithelial neoplasia grades 2-3.

Background and aims 15–30% of cases of cervical intraepithelial neoplasia grades 2–3 (CIN2–3) detected in punch biopsies regress spontaneously (ie, show CIN1 or less in the follow-up cone). Epithelial retinoblastoma protein (pRb), tumour suppressor protein (p53), HPV genotype and immunoreactive cells have been reported to be helpful in predicting regression but their interaction in regression prediction is unknown. Material and methods 55 cases of CIN2–3 in cervical biopsies with subsequent cervical cones were studied retrospectively to assess how epithelial biomarkers, immunoreactive cells (with immunohistochemistry) and high-risk (hr) HPV genotypes (by the AMPLICOR and linear array tests) prognostically interact with epithelial pRb and p53. Results 18% of CIN2–3s regressed (median biopsy–cone interval of 12.0 weeks, range 5.0–34.1 weeks). CIN2–3s that regressed had higher epithelial pRb and p53, lower stromal CD25+ and CD138+, and higher CD8 cells than persistent lesions. They also had higher ratios of CD4+/CD25+ and CD8+/CD25 in stroma and epithelium. HPV16 correlated with low pRb and low CD8+. With multivariate analysis a combined high ratio of CD8+/CD25+ in the stroma, high epithelial pRb and p53 expression had independent value to predict the regression. Conclusions CIN2–3 lesions with a non-hrHPV16 infection, high ratios of stromal CD8+/CD25+ and high epithelial expression of pRb or p53 are associated with spontaneous regression.