Precision needle-punch tumor enrichment from paraffin blocks improves the detection of clinically actionable genomic alterations and biomarkers.

Background: While many molecular assays can detect mutations at low tumor purity and variant allele frequencies, complex biomarkers such as tumor mutational burden (TMB), microsatellite instability (MSI), and genomic loss of heterozygosity (gLOH) require higher tumor purity for accurate measurement. Scalable, quality-controlled, tissue-conserving methods to increase tumor nuclei percentage (TN%) from tumor specimens are needed for complex biomarkers and hence necessary to maximize patient matching to approved therapies or clinical trial enrollment. We evaluated the clinical utility and performance of precision needle-punch enrichment (NPE) compared with traditional razor blade macroenrichment of tumor specimens on molecular testing success. Methods: Pathologist-directed NPE was performed manually on formalin-fixed, paraffin embedded (FFPE) blocks. Quality control of target capture region and quantity of residual tumor in each tissue block was determined via a post-enrichment histologic slide recut. Resultant tumor purity and biomarker status were determined by the computational analysis pipeline component of the FDA-approved next-generation sequencing (NGS) assay, FoundationOne®CDx. Following NPE implementation for real-world clinical samples, assay performance and biomarker (MSI, TMB, gLOH) detection were analyzed. Results: In real-world clinical samples, enrichment rate via NPE was increased to ~50% over a 2.5-year period, exceeding the prior use of razor blade macro-enrichment (<30% of cases) prior to NPE implementation due to proven efficacy in generating high quality molecular results from marginal samples and the ease of use for both pathologist and histotechnologists. NPE was associated with lower test failures, higher computational tumor purity, and higher rates of successful TMB, MSI and gLOH determination when stratified by pre-enriched (incipient) tumor nuclei percentage. In addition, challenging cases in which tumor content was initially insufficient for testing were salvaged for analysis of biomarker status, gene amplification/deletion, and confident mutant or wild-type gene status determination. Conclusions: Pathologist-directed precision enrichment from tissue blocks (aka NPE) increases tumor purity, and consequently, yields a greater number of successful tests and complex biomarker determinations. Moreover, this process is rapid, safe, inexpensive, scalable, and conserves patient surgical pathology material. NPE may constitute best practice with respect to enriching tumor cells from low-purity specimens for biomarker detection in molecular laboratories.

Cell-free tumor DNA, CA125 and HE4 for the objective assessment of tumor burden in patients with advanced high-grade serous ovarian cancer.

BACKGROUND: The present prospective study aimed at determining the impact of cell-free tumor DNA (ct-DNA), CA125 and HE4 from blood and ascites for quantification of tumor burden in patients with advanced high-grade serous epithelial ovarian cancer (EOC). METHODS: Genomic DNA was extracted from tumor FFPE and ct-DNA from plasma before surgery and on subsequent post-surgical days. Extracted DNA was subjected to hybrid-capture based next generation sequencing. Blood and ascites were sampled before surgery and on subsequent post-surgical days. 20 patients (10 undergoing complete resection (TR0), 10 undergoing incomplete resection (TR>0)) were included. RESULTS: The minor allele frequency (MAF) of TP53 mutations in ct-DNA of all patients with TR0 decreased significantly, compared to only one patient with TR>0. It was not possible to distinguish between patients with TR0 and patients with TR>0, using CA125 and HE4 from blood and ascites. CONCLUSIONS: Based upon the present findings, ct-DNA assessment in patients with high-grade serous EOC might help to better determine disease burden compared to standard tumor markers. Further studies should prospectively evaluate whether this enhancement of accuracy can help to optimize management of patients with EOC.

ALK alterations in salivary gland carcinomas.

Salivary gland carcinomas represent a heterogeneous group of poorly characterized head and neck tumors. The purpose of this study was to evaluate ALK gene and protein aberrations in a large, well-characterized cohort of these tumors. A total of 182 salivary gland carcinomas were tested for anaplastic lymphoma kinase (ALK) positivity by immunohistochemistry (IHC) using the cut-off of 10% positive cells. ALK positive tumors were subjected to FISH analysis and followed by hybrid capture-based next generation sequencing (NGS). Of the 182 tumors, 8 were ALK positive by IHC. Further analysis using hybrid capture NGS analysis revealed a novel MYO18A (Exon1-40)-ALK (exon 20-29) gene fusion in one case of intraductal carcinoma. Additional genomic analyses resulted in the detection of inactivating mutations in BRAF and TP53, as well as amplifications of ERBB2 and ALK. ALK rearrangements are a rare entity in salivary gland carcinomas. We identified a potentially targetable novel ALK fusion in an intraductal carcinoma of minor salivary glands.

Multisite Tumor Sampling Reveals Extensive Heterogeneity of Tumor and Host Immune Response in Ovarian Cancer.

BACKGROUND/AIM: Ovarian cancer (OVCA) is characterized by genomic/molecular intra-patient heterogeneity (IPH). Tissue histology and morphological features are surrogates of the underlying genomic/molecular contexture. We assessed the morphological IPH of OVCA tumor compartments and of lymphocytic infiltrates in multiple matched samples per patient. MATERIALS AND METHODS: We examined 294 hematoxylin & eosin (H&E) OVCA tumor whole sections from 70 treatment-naïve patients who had undergone cytoreductive surgery. We assessed morphological subtypes as immunoreactive (IR), solid - proliferative (SD), papilloglandular (PG), and mesenchymal transition (MT); subtype load per patient; stromal tumor-infiltrating lymphocyte (sTIL) density as average per sample; and, as maximal sTIL values (max-TILs) among all samples per patient, ovaries and implants. RESULTS: Among all 294 tumor sections, the most frequent primary morphological subtype was PG (n=150, 51.0%), followed by MT (71, 24.1%), SD (48, 16.3%) and IR (15, 5.1%). Subtype combinations were observed in 67/294 sections (22.8%) and IPH in 48/70 patients (68.6%). PG prevailed in ovaries (p<0.001), SD and MT in implants (p=0.023 and p<0.001, respectively). sTILs were higher in SD compared to non-SD (p=0.019) and lower in PG, respectively (p<0.001). sTIL density was higher in implants than in ovaries (p<0.001). Higher max-TILs were associated with stage IV disease (p=0.043), upper abdominal dissemination (p=0.024), endometrioid histology (p=0.013), and grade 3 tumors (p=0.021). Favorable prognosticators were higher max-TILs per patient (PFS, OS) and higher SD-load (PFS). CONCLUSION: Clinically relevant morphological and host immune-response IPH appear to be the norm in OVCA. This may complicate efforts to decipher sensitivity of the tumor to certain treatment modalities from a single pre-operative biopsy.

Pathogenic BRCA1 mutations may be necessary but not sufficient for tissue genomic heterogeneity: Deep sequencing data from ovarian cancer patients.

BACKGROUND: Tissue genomic heterogeneity (t-HET) in patients with epithelial ovarian cancer (OVCA) is related to tissue plasticity, i.e., flexibility to adapt to adverse molecular environments. Here, we interrogated the presence and clinical relevance of OVCA t-HET. METHODS: We applied high-depth (>2000×) sequencing on 297 paraffin tissue samples (fallopian tubes, ovaries, intra-abdominal metastases) from 71 treatment-naïve patients who subsequently received first-line platinum-based chemotherapy. Based on tissue mutation patterns, we distinguished tissue genotypes into: no mutation (33/297 samples; 11.1%), stable (173; 58.2%) and unstable (91; 30.7%). We profiled genotypes per patient and assessed t-HET in 69 patients. Predicted pathogenic mutations refer to germline and/or tissues. RESULTS: Among all 71 patients, 46 (64.8%) had pathogenic BRCA1 mutations and 15 (21.7%) had BRCA1/2 disruption (i.e., pathogenic mutations with position-LOH). We classified 29 patients with t-HET (42%), all with pathogenic BRCA1; t-HET was observed in 64% with such mutations (p < 0.001). As opposed to non-t-HET, matched tissues in t-HET shared pathogenic BRCA1 (p < 0.001) but not BRCA2 and TP53. Germline BRCA1 mutations in tissues exhibited position-LOH; heterozygous status; or, partial loss of the inherited allele accompanied by additional clonal mutations. Patients with t-HET had worse outcome (log-rank p = 0.048 [progression-free]; p = 0.037 [overall survival]), including 12/15 patients with disrupted BRCA1/2 and 3 BRCA1 carriers with partial germline loss in tissues. CONCLUSIONS: Pathogenic BRCA1 mutations appear necessary but may not be sufficient for the establishment of t-HET. t-HET may be associated with worse outcome, including in patients with disrupted BRCA1/2, which is usually considered as a favourable marker. OVCA t-HET may need to be addressed for treatment decisions.

Mismatch repair deficiency and aberrations in the Notch and Hedgehog pathways are of prognostic value in patients with endometrial cancer.

The aim of this study was to investigate the prognostic value of the Hedgehog (Gli, Patched-1, Shh, Smo) and Notch (Jag1, Notch2, Notch3) pathway members, in comparison to a panel of proteins (ER, PgR, HER2/neu, Ki67, p53, p16, PTEN and MMR) previously suggested to be involved in the pathogenesis of endometrial cancer, in association with clinical outcome and standard clinicopathological characteristics. A total of 204 patients with histological diagnosis of endometrial cancer treated from 2004 to 2013 were included. The evaluation of protein expression was assessed by immunohistochemistry. Univariate analysis showed that higher Ki67 labeling, expression of PTEN, p16, Notch2 and Notch3 proteins, as well as MMR proficiency were associated with increased relapse and mortality rate. Additionally, Patched-1 protein expression was associated with worse DFS, while p53 overexpression was associated with worse OS. In multivariate analyses, patients with MMR proficient tumors had more than double risk for death than patients with MMR deficient (MMRd) tumors (adjusted HR = 2.19, 95% CI 1.05-4.58, p = 0.036). Jag1 positivity conferred reduced mortality risk (HR = 0.48, 95% CI 0.23-0.97, p = 0.042). However, as shown by hierarchical clustering, patients fared better when their tumors expressed high Jag1 protein in the absence of Notch2 and Notch3, while they fared worse when all three proteins were highly expressed. Patched-1 positivity conferred higher risk for relapse (HR = 2.04, 95% CI 1.05-3.96, p = 0.036). Aberrant expression of key components of the Notch and Hedgehog signaling pathways, as well as MMRd may serve as independent prognostic factors for recurrence and survival in patients with endometrial cancer.

Phase II study of panitumumab combined with capecitabine and oxaliplatin as first-line treatment in metastatic colorectal cancer patients: clinical results including extended tumor genotyping.

This clinical trial assessed the efficacy and toxicity of panitumumab combined with oxaliplatin and capecitabine as first-line treatment in KRAS exon 2 wild-type metastatic colorectal cancer (mCRC) patients. Patients with exon 2 KRAS wild-type mCRC received panitumumab 9 mg/Kg, oxaliplatin 130 mg/m2, and capecitabine 2000 mg/m2 repeated every 3 weeks. The primary endpoint was objective response rate (ORR, minimum 42 responses). We retrospectively assessed mutations in genes implicated in CRC with massively parallel sequencing; ERBB2 and EGFR amplification with fluorescence in situ hybridization, and tumor-infiltrating lymphocyte density. Among 78 patients enrolled, 45 (57.7%) completed 6 cycles. Most common grade 3-4 toxicities were skin rash (19.2%), diarrhea (18%), and neuropathy (6.4%). Among 5 (6.4%) potentially treatment-related deaths, 2 (2.6%) were characterized toxic. Objective response occurred in 43 (55.1%) of the patients (complete 6.4% and partial response 48.7%; stable 17.9% and progressive disease 7.7%), while 3.8% were non-evaluable and 15% discontinued their treatment early. Additional mutations in KRAS/NRAS/BRAF were found in 11/62 assessable (18%) tumors. After 51 months median follow-up, median progression-free (PFS) was 8.1 and overall survival 20.2 months, independently of KRAS/NRAS/BRAF or PI3K-pathway mutation status. Patients with TP53 mutations (n = 34; 55%), as well as those with left colon primary tumors (n = 66; 85%), had significantly better PFS, also confirmed in multivariate analysis. Although the clinical trial met its primary endpoint, according to the current standards, the efficacy and tolerability of the drug combination are considered insufficient. Extended genotyping yielded interesting results regarding the significance of TP53 mutations.ClinicalTrials.gov identifier: NCT01215539, Registration date: Sep 29, 2010.

Genomic and Functional Fidelity of Small Cell Lung Cancer Patient-Derived Xenografts.

Small cell lung cancer (SCLC) patient-derived xenografts (PDX) can be generated from biopsies or circulating tumor cells (CTC), though scarcity of tissue and low efficiency of tumor growth have previously limited these approaches. Applying an established clinical-translational pipeline for tissue collection and an automated microfluidic platform for CTC enrichment, we generated 17 biopsy-derived PDXs and 17 CTC-derived PDXs in a 2-year timeframe, at 89% and 38% efficiency, respectively. Whole-exome sequencing showed that somatic alterations are stably maintained between patient tumors and PDXs. Early-passage PDXs maintain the genomic and transcriptional profiles of the founder PDX. In vivo treatment with etoposide and platinum (EP) in 30 PDX models demonstrated greater sensitivity in PDXs from EP-naïve patients, and resistance to EP corresponded to increased expression of a MYC gene signature. Finally, serial CTC-derived PDXs generated from an individual patient at multiple time points accurately recapitulated the evolving drug sensitivities of that patient's disease. Collectively, this work highlights the translational potential of this strategy.Significance: Effective translational research utilizing SCLC PDX models requires both efficient generation of models from patients and fidelity of those models in representing patient tumor characteristics. We present approaches for efficient generation of PDXs from both biopsies and CTCs, and demonstrate that these models capture the mutational landscape and functional features of the donor tumors. Cancer Discov; 8(5); 600-15. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 517.

Genotyping and mRNA profiling reveal actionable molecular targets in biliary tract cancers.

Biliary tract cancer (BTC) represents a heterogeneous disease with dismal outcome. Herein, we examined genotype and angiogenesis features in BTC. We applied genotyping (Sanger, qPCR, 101-gene panel NGS), mRNA relative quantification methods, and ß-catenin immunohistochemistry in 84 FFPE BTC (55 gallbladder [GBC], 14 intrahepatic [ICC], 15 extrahepatic [ECC] carcinomas). We identified 541 mutations in 68 (81%) tumors. Top mutated genes were CTNNB1 (36%); PTEN (33%); TP53 (31%); PIK3R1 (29%); PIK3CA (13%); BRCA2 and KRAS (12%); BRCA1 (11%). Six GBCs were hypermutated [hm] displaying a distinct mutational pattern. Mutations in TP53 and PI3K, Wnt and RAS components were prevalent among non-hypermutated tumors. All hmGBCs carried mutations in BRCA2 and other homologous recombination repair (HRR) genes, in PD1, but not in CTNNB1 and KRAS. None of the pathogenic BRCA2 p.D2723G and BRCA1 p.Q563* and c.5266dupC was present at frequencies expected for germline mutations. We observed copy gains (>6 copies) in EGFR (9% of informative tumors), PRKAR1A (7%), PIK3CA (6%), ERBB2 (5%) and MET (4%). TP53 mutations were prevalent in GBC (P<0.001) and PRKAR1A copy gains in ICC (P=0.007). PTEN was frequently co-mutated with CTNNB1 (P<0.001). Unrelated to CTNNB1 mutations, nuclear ß-catenin was detected in 45% of tumors, among them in 5/6 hmGBC. We observed strong mRNA expression correlation of the two neuropilins (NRP1 and NRP2) with each other (Spearman's rho 0.59) and with the endothelin receptor (NRP2 rho 0.66; NRP2 rho 0.51), and between VEGFA and its receptors (FLT1 rho 0.49; KDR rho 0.45). All PIK3CA mutated tumors expressed endothelin 1 mRNA (P=0.010). Most tumors expressing nuclear ß-catenin were negative for VEGFC (P=0.009) and FLT4 (P=0.002) mRNA expression. In conclusion, we confirmed the presence of known genomic aberrations in BTC and different genotypes between BTC subsets. Novel findings are the coexistence of PI3K and WNT pathway gene alterations in BTC, their association with angiogenesis, and the hypermutated GBCs with HRR gene mutations, all of which may be considered for new treatment options in this difficult to treat disease.

Concordance between Comprehensive Cancer Genome Profiling in Plasma and Tumor Specimens.

INTRODUCTION: Detection of somatic genomic alterations in the plasma of patients with cancer ("liquid biopsy") are increasingly being used in the clinic. However, the concordance of alterations identified in liquid biopsies with those detected in cancer specimens is not routinely being determined. METHODS: We sought to systematically compare alterations found by a massively parallel sequencing liquid biopsy assay covering 39 genes (NEOliquid [NEO New Oncology GmbH, Köln, Germany]) with those identified through routine diagnostic testing in a certified central pathology laboratory in a cohort of patients with nonsquamous NSCLC. NEOliquid is based on enrichment of the genomic territory of interest by hybrid capture and is thus capable of detecting point mutations, small insertions and deletions, copy number alterations, and gene rearrangements/fusions in a single assay. RESULTS: In a cohort of 82 patients with matched blood/tissue samples, the concordance between NEOliquid and tissue-based routine testing was 98%, the sensitivity of NEOliquid was higher than 70%, and the specificity was 100%. Discordant cases included those with insufficient amounts of circulaating tumor DNA in plasma and cases in which known driver mutations (e.g., isocitrate dehydrogenase (NADP(+)), 1 systolic gene [IDH1] R132H, kinesin family member 5B gene [KIF5b-ret proto-oncogene [RET], or MNNG HOS Transforming gene [MET] exon 14) were found in the plasma but were not interrogated by routine tissue analyses. CONCLUSIONS: In summary, NEOliquid offers accurate and reliable detection of clinically relevant driver alterations in plasma of patients with cancer.

Comparison of immunohistochemistry with PCR for assessment of ER, PR, and Ki-67 and prediction of pathological complete response in breast cancer.

BACKGROUND: Proliferation may predict response to neoadjuvant therapy of breast cancer and is commonly assessed by manual scoring of slides stained by immunohistochemistry (IHC) for Ki-67 similar to ER and PgR. This method carries significant intra- and inter-observer variability. Automatic scoring of Ki-67 with digital image analysis (qIHC) or assessment of MKI67 gene expression with RT-qPCR may improve diagnostic accuracy. METHODS: Ki-67 IHC visual assessment was compared to the IHC nuclear tool (AperioTM) on core biopsies from a randomized neoadjuvant clinical trial. Expression of ESR1, PGR and MKI67 by RT-qPCR was performed on RNA extracted from the same formalin-fixed paraffin-embedded tissue. Concordance between the three methods (vIHC, qIHC and RT-qPCR) was assessed for all 3 markers. The potential of Ki-67 IHC and RT-qPCR to predict pathological complete response (pCR) was evaluated using ROC analysis and non-parametric Mann-Whitney Test. RESULTS: Correlation between methods (qIHC versus RT-qPCR) was high for ER and PgR (spearman´s r = 0.82, p < 0.0001 and r = 0.86, p < 0.0001, respectively) resulting in high levels of concordance using predefined cut-offs. When comparing qIHC of ER and PgR with RT-qPCR of ESR1 and PGR the overall agreement was 96.6 and 91.4%, respectively, while overall agreement of visual IHC with RT-qPCR was slightly lower for ER/ESR1 and PR/PGR (91.2 and 92.9%, respectively). In contrast, only a moderate correlation was observed between qIHC and RT-qPCR continuous data for Ki-67/MKI67 (Spearman's r = 0.50, p = 0.0001). Up to now no predictive cut-off for Ki-67 assessment by IHC has been established to predict response to neoadjuvant chemotherapy. Setting the desired sensitivity at 100%, specificity for the prediction of pCR (ypT0ypN0) was significantly higher for mRNA than for protein (68.9% vs. 22.2%). Moreover, the proliferation levels in patients achieving a pCR versus not differed significantly using MKI67 RNA expression (Mann-Whitney p = 0.002), but not with qIHC of Ki-67 (Mann-Whitney p = 0.097) or vIHC of Ki-67 (p = 0.131). CONCLUSION: Digital image analysis can successfully be implemented for assessing ER, PR and Ki-67. IHC for ER and PR reveals high concordance with RT-qPCR. However, RT-qPCR displays a broader dynamic range and higher sensitivity than IHC. Moreover, correlation between Ki-67 qIHC and RT-qPCR is only moderate and RT-qPCR with MammaTyper® outperforms qIHC in predicting pCR. Both methods yield improvements to error-prone manual scoring of Ki-67. However, RT-qPCR was significantly more specific.

Interaction Between Beta-Catenin and EGFR Expression by Immunohistochemistry Identifies Prognostic Subgroups in Early High-risk Triple-negative Breast Cancer.

Wnt and epidermal growth factor receptor (EGFR) pathway abnormalities and de-stabilization of cell adhesion are all important aspects of the pathogenesis of triple-negative breast cancer (TNBC). Herein we investigated how the expression of related protein markers may affect the outcome of patients bearing TNBC treated in the adjuvant setting. Immunohistochemistry for beta-catenin, Myc (Wnt pathway), E-cadherin, P-cadherin (cell-adhesion), EGFR and cytokeratin 5 (CK5) (identification of basal-like tumors) was carried out in 364 centrally confirmed TNBCs. Survival analysis was performed with Cox-regression models according to dichotomized continuous protein expression data and marker interactions. In 352 evaluable tumors, 81.5% were basal-like TNBC. E-cadherin and P-cadherin were positively associated, with co-expression being present in 68% of tumors. Individual markers did not affect patient outcome. However, a statistically significant interaction was shown such that low expression of beta-catenin in the cell membrane, defined as expression below the median of the H-score distribution, was associated with unfavourable disease-free survival among tumors that expressed EGFR, but not in the absence of EGFR expression (interaction p=0.0085). The interaction persisted after correcting for clinicopathological variables. A considerable number of TNBC co-expresses E-cadherin and P-cadherin, while membranous localization of beta-catenin may predict patient outcome in an EGFR-dependent manner. This novel interaction seems worthy for validating with regards to its biological and clinical relevance.

Immunohistochemical study of immunological markers: HLAG, CD16, CD25, CD56 and CD68 in placenta tissues in recurrent pregnancy loss.

INTRODUCTION: Recurrent pregnancy loss (RPL) of unknown etiology is correlated with immunological alterations during pregnancy. Normally, changes in leukocyte subpopulations and HLA expression take place in pregnant uterus in order to tolerate the semi-allogenic embryo. OBJECTIVE: Our research tries to enlighten the immunological changes that take place in the uterus of women with recurrent abortions of unknown etiology during first trimester of pregnancy. MATERIALS AND METHODS: The miscarriage group was obtained from 25 women who miscarried between the ages of 35 to 42 years and controls consisted of 25 healthy women between the ages of 27 to 39 years, who had electively terminated their pregnancies during the first trimester of pregnancy. The abortion was processed and specimens taken were studied using immunohistochemical methods. Specimens were taken from decidua basalis and decidua parietalis. Monoclonal antibodies were used against HLAG (Human Leukocyte Antigen G), CD68( Cluster of Differentiation 68), CD56, CD16 and CD25. The results were statistically analysed with Mann-Whitney test. RESULTS: HLA-G expression in decidua basalis from miscarriage group was found to be decreased. CD25+ cell expression was found to be invariable in deciduas from both groups. CD16+ cell and CD68 + cell expression was found to be increased in deciduas from the miscarriage group. CD56+ cell expression was found to be increased in decidua parietalis from miscarriage group. Conclusion : Several differences in the immunological profile of deciduas from RPL group were observed. Changes in feto-protective HLA-G expression and a possible implication of macrophages and NK cells were found.

Low expression of progesterone receptor A in intermediate trophoblast of miscarriages.

OBJECTIVE: To examine the potential differences in the expression of Progesterone Receptor A and Estrogen Receptor A in intermediate trophoblastic cells at the implantation site in elective abortions and miscarriages by immunohistochemistry. STUDY DESIGN: Twenty two (22) samples of miscarriages and eighteen (18) samples of elective abortions were obtained during gestational weeks 6 to 12. Monoclonal antibodies against Cytokeratin 7 and prolactin were used to help discriminate between trophoblastic and decidual cells at the feto-maternal interface on formalin-fixed paraffin-embedded sections. Samples were then stained with ERA and PRA antibodies. Nuclear expression was considered positive. Staining intensity was measured according to a 4 grade scale. Statistical analysis of the results was performed using the Mann-Whitney test and the Wilcoxon signed rank test. RESULTS: PRA expression in intermediate trophoblastic cells was significantly higher in elective abortions (control group) compared to miscarriages. ERA expression was uniformly negative in both groups. CONCLUSION: PRA expression is significantly lower in intermediate trophoblastic cells of miscarriages compared to elective abortion pregnancies. Although this could be solely a result of a secondary event, it is still an important finding in the effort to unravel the complex molecular pathobiology of spontaneous abortions.