A Targeted Mass Spectrometry Strategy for Developing Proteomic Biomarkers: A Case Study of Epithelial Ovarian Cancer.

Protein biomarkers for epithelial ovarian cancer are critical for the early detection of the cancer to improve patient prognosis and for the clinical management of the disease to monitor treatment response and to detect recurrences. Unfortunately, the discovery of protein biomarkers is hampered by the limited availability of reliable and sensitive assays needed for the reproducible quantification of proteins in complex biological matrices such as blood plasma. In recent years, targeted mass spectrometry, exemplified by selected reaction monitoring (SRM) has emerged as a method, capable of overcoming this limitation. Here, we present a comprehensive SRM-based strategy for developing plasma-based protein biomarkers for epithelial ovarian cancer and illustrate how the SRM platform, when combined with rigorous experimental design and statistical analysis, can result in detection of predictive analytes.Our biomarker development strategy first involved a discovery-driven proteomic effort to derive potential N-glycoprotein biomarker candidates for plasma-based detection of human ovarian cancer from a genetically engineered mouse model of endometrioid ovarian cancer, which accurately recapitulates the human disease. Next, 65 candidate markers selected from proteins of different abundance in the discovery dataset were reproducibly quantified with SRM assays across a large cohort of over 200 plasma samples from ovarian cancer patients and healthy controls. Finally, these measurements were used to derive a 5-protein signature for distinguishing individuals with epithelial ovarian cancer from healthy controls. The sensitivity of the candidate biomarker signature in combination with CA125 ELISA-based measurements currently used in clinic, exceeded that of CA125 ELISA-based measurements alone. The SRM-based strategy in this study is broadly applicable. It can be used in any study that requires accurate and reproducible quantification of selected proteins in a high-throughput and multiplexed fashion.

Identification of Liver Epithelial Cell-derived Ig Expression in µ chain-deficient mice.

Growing evidence indicates that B cells are not the only source of immunoglobulin (Ig). To investigate this discovery further, we used µMT mice, which have a disruption of the first transmembrane exon of the µ heavy chain and do not express the membrane form of IgM. These mice lack mature B cells and thus serve as a good model to explore Ig expression by liver epithelial cells. We found that Ig heavy chains (µ, δ, γ and α) and light chains (κ and λ) were expressed in sorted liver epithelial cells of µMT mice. Surprisingly, each heavy chain class showed its respective variable region sequence characteristics in their variable region, instead of sharing the same VDJ usage, which suggests that class switching does not occur in liver epithelial cells. Moreover, the γ and α chains, but not the µ and δ chains, showed mutations in the variable region, thus indicating that different classes of Ig have different activities. Our findings support the concept that non-B cells, liver epithelial cells here, can produce different classes of Ig.

Serum immune-related proteins are differentially expressed during hibernation in the American black bear.

Hibernation is an adaptation to conserve energy in the face of extreme environmental conditions and low food availability that has risen in several animal phyla. This phenomenon is characterized by reduced metabolic rate (∼25% of the active basal metabolic rate in hibernating bears) and energy demand, while other physiological adjustments are far from clear. The profiling of the serum proteome of the American black bear (Ursus americanus) may reveal specific proteins that are differentially modulated by hibernation, and provide insight into the remarkable physiological adaptations that characterize ursid hibernation. In this study, we used differential gel electrophoresis (DIGE) analysis, liquid chromatography coupled to tandem mass spectrometry, and subsequent MASCOT analysis of the mass spectra to identify candidate proteins that are differentially expressed during hibernation in captive black bears. Seventy serum proteins were identified as changing by ±1.5 fold or more, out of which 34 proteins increased expression during hibernation. The majority of identified proteins are involved in immune system processes. These included α2-macroglobulin, complement components C1s and C4, immunoglobulin µ and J chains, clusterin, haptoglobin, C4b binding protein, kininogen 1, α2-HS-glycoprotein, and apoplipoproteins A-I and A-IV. Differential expression of a subset of these proteins identified by proteomic analysis was also confirmed by immunodetection. We propose that the observed serum protein changes contribute to the maintenance of the hibernation phenotype and health, including increased capacities for bone maintenance and wound healing during hibernation in bears.

[Primary amyloidosis associated with IgD-lambda M-proteinemia].

We describe here a case of primary AL amyloidosis associated with IgD monoclonal gammopathy of undetermined significance. A 73-year-old man was referred to our hospital with suspected multiple myeloma due to renal failure and urinary Bence Jones protein. Although serum electrophoresis revealed IgDlambda monoclonal protein, the bone marrow did not showed plasma cell proliferation. Systemic bone survey disclosed no lytic bone lesions. Because the patient had macroglossia and multiple ecchymosis in the face and neck, primary amyloidosis was suspected. Skin biopsy revealed extensive deposition of amyloid which was positively stained by Congo red dye. A diagnosis of primary AL amyloidosis associated with IgD monoclonal gammopathy was made. The patient was also complicated renal failure that eventually needed hemodialysis. To our knowledge, this is the first report of primary AL amyloidosis associated with IgD monoclonal gammopathy with undetermined significance.

Mu-heavy chain disease associated with systemic amyloidosis.

mu-heavy chain disease (HCD) is very rare, with only 30 cases reported in the literature. We report a patient with mu-HCD associated with systemic amyloidosis. The diagnosis of mu-HCD was based on findings of mu-heavy chain fragments in the serum, Bence Jones proteinuria and vacuolated plasma cells in the bone marrow. To our knowledge, this is the third case in which systemic amyloidosis led to the patient's death.

[A case of Sjögren's syndrome with marked lacrimal gland enlargement, atypical onset and IgA-M-proteinemia].

Since July, 1999, a 66 year-old man had been complaining of dry cough. He noticed submandibular swelling, lacrimal gland enlargement and dry eye. Keratoconjuctivitis sicca was detected by an ophthalmologist. Sjögren's syndrome was diagnosed based on microscopic findings of a labial salivary gland biopsy although anti-SS-A and anti-SS-B antibodies were negative. Hypergammaglobulinemia (IgG 3916 mg/dl) and IgA-M-proteinemia were pointed out. Swelling of mediastinal and abdominal lymph nodes was detected together with enlargement of salivary and lacrimal glands. We suspected the existence of malignant lymphoma, but a biopsy of lacrimal glands showed only lymphocytic and plasma cell infiltration and immunohistochemical analysis denied monoclonality of lymphoid line. An administration of corticosteroids caused rapid diminution in size of lacrimal and submandibular glands and lymph nodes. Clinical symptoms were also improved, but IgA-M proteinemia remains. The characteristics of our case were enlargement of lacrimal glands, the negativity of anti SS-A and SS-B antibodies, atypical onset and M-proteinemia. We discussed about these characteristics of Sjögren's syndrome in our case.

Ontogeny of the murine Ig joining chain gene and protein.

We used Northern blot analysis in order to investigate the ontogeny of the murine joining (J)-chain gene. No J-chain expression was detected in embryonic tissues, including liver, spleen and intestine, but an expression of mu heavy chain was detected in foetal liver at day 17. J-chain expression was detected in the spleen at day 9 and in the intestine at day 15 after birth. Western blot analysis was carried out in order to compare the protein levels of J and mu heavy chains in serum from day 8 to day 24 after birth, using antihuman J chain and antimouse mu chain antibodies. Although mu chain protein could be detected in serum from day 8, J-chain protein was detectable only at day 24. These results suggest that the expression of J chain is a later event than the mu chain in the mouse, which thus differs in embryogenesis from humans.

Cast nephropathy in mu heavy chain disease.

The occurrence of kidney diseases was very rarely reported in heavy chain diseases (HCD). At variance with gamma and alpha HCD in which there is no free light chain secretion, about two-thirds of mu HCD patients have urinary Bence Jones (BJ) proteins. We report on a 66 year-old man affected with typical mu HCD who developed renal failure after a 3-year follow-up. He had presented with chronic lymphocytic leukemia with bone marrow vacuolated plasma cells, serum mu HCD protein and serum and urine BJ protein. After an apparent hematological remission following fludarabine therapy, anemia and blood hyperlymphocytosis recurred together with microscopic hematuria, proteinuria and increased creatininemia. Kidney biopsy showed numerous tubular eosinophilic casts which stained for kappa chain determinants by immunofluorescence and an interstitial infiltration by lymphocytes and plasma cells. The hematological and renal condition improved after reinitiation of chemotherapy. This appears to be the first documented report of a light chain-dependent visceral complication in HCD.

An analysis of clinical and laboratory features of acute lymphocytic leukemias with emphasis on 35 children with pre-B leukemia.

In 35 of 191 patients with acute lymphocytic leukemia (ALL) malignant cells were similar in phenotype to B-lymphocyte precursors. Both these patients' lymphoblasts and normal pre-B-cells contain cytoplasmic immunoglobulin (Ig) mu heavy chains, but have no surface Ig. In patients with pre-B leukemias, lymphoblasts containing cytoplasmic mu chains alone were often accompanied by cells of identical morphology that expressed no Ig and less frequently by lymphoblasts bearing scant amounts of surface mu. This spectrum of cellular Ig expression suggests that "null," pre-B, and intermediate pre-B/B ALLs represent closely related malignancies with complete or partial arrests at different stages of maturation. When pre-B, B, T, and "null" cell categories of ALL were compared for 22 different clinical and laboratory features, including remission rate and short-term remission duration, no statistical differences were observed between the pre-B and "null" groups. These early results suggest that pre-B-cell leukemias represents a relatively good prognostic subclass of ALL, do not require more intensive treatment than that proven to be effective for "null" cell ALL, and should be distinguished from the less common, but more clinically aggressive, B-cell subclass of ALL. Longer follow-up will be required to confirm these preliminary conclusions.

Acute lymphoblastic leukemia with pre-B-cell characteristics.

Blast cells from 6 of 50 patients with acute lymphoblastic leukemia (ALL) displayed intracytoplasmic mu chains in the absence of detectable light chains and surface immunoglobulins. These cells also expressed lalike and common ALL antigens. Terminal deoxynucleotidyltransferase was detectable in 2 of 5 cases tested. These blast cells are probably related to early B-cell precursors (pre-B cells). In 4 of 6 cases the disease had a tumoral presentation; the prognostic significance of this new subgroup, which accounts for 20% of patients with non-T non-B ALL, remains to be established.