BRCA Data Share Database

Breast Cancer: is also known as mammary cancer results from several factors such as ionizing radiation, diety, familiar history or exposure to genetic mutagens. Carcinomas of the breast are the eight most prevalent form of cancer and fourth among the most common causes of cancer deaths in the US. It is the most common femal malignancy in most industrialized countries, estimated to affect about 10% of the female population during their lifespan. Breast cancer is the primary killer of women. One in eight American women will develop reast cancer in her lifetime. An estimated 3 million women in the US are living with breast cancer.

Genetic Risks: 

BRCA1 gene:

About 1 out of 800 in the general femaile population has a mutation in the BRCA1 gene sequence that places her at high genetic risk of developing breast cancer and ovarian cancer. In any one year, a variation of unkown clinical significiance is identified in about 15 per% of women having tests for unkonw variations in the BRCA1 gene. 

The discovery of BRCA1 and BRCA2 has expanded our knowledge of genetic factors which can contribute to familial breast cancer. Germ-line mutations within these two loci are associated with a 50-80% lifetime risk of breast and/or ovarian cancer. However, it is likely that other, non-genetic factors also have a significant effect on the etiology of the disease.

Detection: 

The principal manner of identifying breast cancer is through detection of the presence of dense tumorous tissue. This may be accomplished to varying degrees of effectiveness by direct examination of the outside of the breast, or through mammography or other X-ray imagin methods. Mammograms are not recommended for women under 50 since this group is not as likely to develop breast cancers as are older women and mammogram have a small risk of inducing breast tumors.

Diagnostic genetic testing for the existence of BRCA mutations is also an important consideration in the provision of clinical care for breast or ovarian cancer. This testing provides a patient with information on her risk for hereditary breast and ovarian cancers, and thus aids in the difficult decision regarding whether to undertake preventive options, including prophylactic surgery. Myriad Genetics is a leader in this type of testing.

Classification: 

Gene expression profiling information dervied using DNA microarrays to analyse expression from multiple thousands of genes has led to the subdivision of breast cancer into at least 5 different subtypes. One subtype “basal-like” is the most aggressive and is thought to arise from basal epithelial cells (WO 2010/127399). The most studied and widely used method of breast tumour-grading is the Elston-Ellis modified  Scarff, Bloom, Richardson grading system, also known as the Nottingham grading system (NGS). The NGS is based on a phenotypic scoring procedure that involves the microscopic evaluation of morphologic and cytologic features of tumour cells including degree of tubule formation, nuclear pleomorphism and mitotic count. The sume of these scores stratifies breast tumours into Grade I (G1) (well-differentiated, slow growing), Grade II (G2) (moderately differentiated), and Grade III (G3) (poorly-differentiated, hihgly proliferative) malignancies. Untreated patients with G disease have about a 95% five year survival rate whereas those with G2 and G3 have survival rates of about 75 and 50%, respecitively (WO 2008/048193).

Treatment: 

One or more of a variety of treatments such as surgery, radiotherapy, chemotherpay and hormone therapy are used. The treatment course for a certain type of breast cancer is usually selected based on various prognostic parameters, for example, an analysis of specific tumor markers.  For example, one factor considered in prognosis and treatment decisions for breast cancer is the presence or absence of the estrogen receptor (ER). ER-positive breast cancers typically respond better to hormonal therapies such as tamoxifen, which acts as an anti-estrogen breast tissue, than ER-negative cancers. A correlation between the expression of TLE3 (transducin-like enhancer of split 3) and a cancer’s response to chemotherapy has also been found. Binding of TLE3 antibodies in samples from treated ovarian cancer patients correlates with improved prognosis (US 2011/0015259). 

Antibodies against the HER-2 protoongogene: The HER2 protooncogene encodes a 185-kDa transmembrane protein (p185HER2) with extensive homology to the epidermal growth factor (EGF) receptor. Clinical and experimental evidence supports a role for overexpression of the HER2 protooncogene in the progression of human breast, ovarian, and non-small cell lung carcinoma. The anti-p185HER2 murine monoclonal antibody (muMAb) 4D5 is one of over 100 monoclonals that was derived following immunization of mice with cells overexpressing p185HER2. The monoclonal antibody (see Herceptin below) is directed at the extracellular (ligand binding) domain of this receptor tyrosine kinase and presumably has its effect as a result of modulating receptor function. (Shepard J Clin Immunol. 1991 May;11(3):117-27.)

–HERCEPTIN® (Genetech Inc) is an antibody that has been used successfully to treat breast cancer. HERCEPTIN is a recombinant DNA derived humanized monoclonal antibody that selectively binds to the extracellular domain of the human epidermal growth factor recetpor 2 (HER2) proto-oncogene. HER2 overexpression is observed in 25-30% of primary breast cancers. Perjeta (pertuzumab) (also from Genentech) is a monoclonal antibody/HER-dimerization inibitor that targets a diffferent epitope on the HER2 receptor than Herceptin and can be used in combination with Herceptin (trastuzumab) and Taxotere (docetaxel) for metastatic HER2/neu-overexpressing breast cancer. In a phase 3 Cleopatra trial, women who received the Perjeta-Herceptin-Taxotere combination achieved a 6.1 month improvement in pregression-free survival over those in the control group receiving Herceptin and Taxotere plus placebo. 

A recombinant humanized version of the murine HER2 antibody 4D5 (huMAb4D5-8, rhuMab HER2, Trastuzumab or HERCEPTIN (see US5,821,337) is clinically active in patients with HER2 overexpressing metastatic breast cancers that have recieved extensive prior anti-cancer therapy (Baselga, J. Clin Oncol. 14 (1996) 737-744). Trastuzumab received marketing form the FDA in 1998. 

Relapse: 

Despite earlier diagnosis of breast cancer, about 1-5% with newly diagnosed breast cancer have a distant metastasis at the time of the diagnosis. In addition, about 50% of the patients with local disease who are primarily diagnosed eventually relapse with metastasis. 85% of these recurrences take place within the first 5 years after the primary manifestation of the disease. The most common site for distant metastasis is the bone followed by lung and liver. The median survival from the manifestation of distant metastases is about 3 years.

See also antibodies as specific cancer treatment 

Hodgkin’s Lymphoma: 

Hodgkin’s lymphoma usually arises in the lymph nodes of young adults. It can be divided into classical subtype and a less common nodular lymphocytic predominant subtype. The most common features at presentation are painless enlargement of lymph nodes, usually in the neck, but occasionally in the inguinal region. 

The difference between non-hodgkin’s and Hodgkin’s lymphoma is that Hodgkin’s requires the presence of a specific type of abnormal cell called a Reed-Sternber cell. RS express CD15, CD25, CD30 and transferrin receptor. In most cases, the RS cells do not express CD45, a feature that aids in distinguishing this disease form NHL. Epstein Barr virus has been shown to be present in RS in about 1/3 of hodgkin’s lymphoma but its role is unclear. 

Non-Hodgkin’s Lymphoma: 

Non-Hodgkin’s lymphoma (NHL) are a large group of cancesr which starts in lymphocytes. RITUXAN (Genentech) have been used successfully to treat non-Hodgkin’s lympoha. HERCEPTIN is a recombinant DNA dervied humanized monoclonal antibody that selectively binds to the extracellular domain of the human epidermal growth factor receptor 2 (HER2) proto-oncogene. HER2 protein overexpression is observed in 25-30% of primary breast cancers. RITUXAN is a genetically engineered chimeric murine/human monoclona antibody directed against the CD20 antigen found on the surface of normal and malignant B lymphocytes.  NHL is much more common than Hodgkin’s and in the US is the sixth most common cancer amoung males and the fifth most common cancer amoung females. 

Acute (B Cell) lymphocytic leukemia (ALL): is a bone marrow based neoplasm largely affecting children between 1-5. Most common symptoms include fatigue, lethargy, fever and bone and joint pain. Precise diagnosis relies on a bone aspirate and biopsy. The demonstration of lymphoblasts in the bone marrow is diagnostic of ALL. 

B cell prolymphocytic leukemia (PLL): once a variant of CLL is a distinct disease of elderly men and characterized by a very high white blood count (greater than 200.times.10.sup.9/L) and splenomegaly. Additional symptoms include anemia and thrombocytopenia.

Burkitt’s Lymphoma: is an aggressive B cell lymphoma typically observed in children and young adults and is usually assocaited with builky disease of the jaw and/or abdomen. About 20% of patients have bone marrow involvement. An endmic form of Burkitt’s lympohma involves Epstein-Barr virus (EBV) infection of malignant cells. 

Chronic lymphocytic leukemia (CLL)/Small B cell lymphocytic lymphoma (SLL): is the most common type of leukemia. When the disease involves the periopheral blood and bone marrow it is referred to as Cll. However, when the lymph nodes and other tissues are infiltrated by cells that are identical to those in CLL, but where leukemic characteristis of the disease are absent, it is referred to as SLL. This dsease largely afflicts the elderly with a greater incidence in men. Painless lymphadenopathy is the most common finding at presentation. Asymptomatic patients are frequently diagnosed during routine blood coutns (lymphocyte count of over 5000.times.10.sup.9/L). 

Cutaneous T-cell Lymphoma: is a type of non-Hodgin lymphoma. Mycosis Fungoides and Sezary syndrome are two of the most common cutaenous T cell lymphomas. A sign of mycosis fungoides is a red rash on the skin. Mycosis fungoides may go through a premycotic phase which includes a scaly, red rash in areas of the body not usually exposed to sun. This rash does not cause any symptoms a may last for years. a patch phase where there is a reddened eczema like rash. a plaque phase shown by small raised bumps or hardened lesions on the skin which may be redenned. a tumor phase. In Sezary syndrome cancerous T cells are found in the blood. 

Diffuse Large B cell Lymphoma (DLBCL): is the most common NHL and can arise form small B cell lymphoma, folicular lymphoma or marginla zone lymphoma. Typcially, pateints present with lymphadenopathy; however, a large percent of patients present in extranodal sites as well, with gastrointestinal involvement being the most common. 

Extranodal marginal zone lymphoma: is an extranodal lymphoma that occurs in organs normally lacking organized lymphoid tissues (e.g., stomach, salivary glands, lung and thyroid glands). It largely affects older adults over 60. Often, chronic inflammation or autoimmune processes precede development of the lymphoma. Gastric mucosal associated lympohic tissue (MALT) lymphoma, the most common type of marginal-zone lymphoma, is associated with Helicobacter pylori infection. Studies have shown a resuolution of symptoms with eradication of the H. plori infection following an antibiotic regimen. Symptoms for gasric MALT lymphoma include epigastric pain, nausea, gastrointestinal bleeding and anemia. 

Follicular lymphoma (FL): is a slow-growing, chronic, and usually incurable type of non-Hodgkin lymphoma (NHL) that affects white blood cells called B-lymphocytes. It’s the second most common type of NHL, accounting for 20–30% of all cases. FL occurs when white blood cells clump together to form lumps in the lymph glands or organs.

Follicular lymphoma can progress very slowly – so slowly that at first you may not need treatment. This is especially true if you have fewer or smaller tumors (“low tumor burden”) and you don’t have any symptoms. Studies suggest to start treatment if your lymph nodes get larger, you experience fever and night sweats or you have low blood counts. 

Follicular lymphoma is hard to cure. Treatment keeps symptoms under control and can keep the cancer at bay for years at a time. But many people eventually relapse, and it can happen more than once. If your disease progresses after one type of treatment, your doctor will recommend that you move onto another.

Hairy Cell Leukemia (HCL): is a rare, indolent chronic leukemia affecting more men than women and largely those of middle age. Symptoms include massive splenomegaly and pancytopenia. The periopheral blood and bone marrow contain the typical “hairy cells” which are B lymphoctyes with cytoplasmic projections. 

Nodal marginal zone lymphoma: has a heterogeneous cytology and morphology. Due to its relatively high proportion of large cells this lymphoma, unlike the other marginal lymphomas (splenic and extranodal) cannot be classified as true low grade B cell lymphoma.

Splenic Marginal Zone Lymphoma: is an indolent micro-nodular B cell lymphoma with a prominnet splenomegaly and infiltration of the peripheral blood and the bond marrow.

Although there are more than 200 different types of cancer, four of them –breast, lung, colorectal and prostate — acount for over half of all new cases. 

Acute Myeloid Leukemia:

Treatments: 

–Antibody/drug conjugates:

MYLTARG (cemtuzumab ozogamicin) is a commercially avaialbe antibody/drug conjguate which is approved for teh treatment of acute myeloid leukemia in elderly patients. ((Boghaert, WO 2006/031653). 

Breast Cancer  see outline

Cervix Cancer: See outline

Chronic myeloid (myelogenous, myelocytic, granulocytic) leukemia (“CML”): 

CML is a cancer of the blood and bone marrow characterized by overproduction of white blood cells. CML accounts for 7-20% of all luekemias adn affects an estimated 1-2/100,000 persons in the general population. CML is caused by a specific cytogenetic abnormality, the Philadelphia chromosome, which results in a clonal myeloproliferative disorder of pluripotent hematopietic stem cells. Current treatment options in the chronic phage of CML include buslfan, hydroxyurea, interfron based regimens, specific kinase inhibitor for bcr/abl or bone marrow/stem cell transplatnation (US 2006/0079458). 

Colorectal cancer (CRC) (“colon cancer”): See outline

Digestive System Cancers: 

Digestive system cancers including pancreatic cancer and stomach cancer, often are silent, and are not detected until they have reached a relatively advanced stage. As cuh, digestive tract cancers are associated with substantial morbidity and mortality. When detected at an early enough state, they can be treated by surgery, radiation therapy, chemotherapy or a combined modality therapy such as surgery to debulk the tumor, followed by chemotherapy to kill remaining tumor cells, including any metastatic disease.

Esophageal cancer (ECA): is the ninth most common malignancy in the world. Even when localized the 5 year survival rate is less than 20%. The most common approach to pateints with localized carcinoma of the esophagus is preoperative chemotradiotherapy.

Gastric cancer: is the second leading cause of cancer-related deaths worldwide and early detection is the key in its management. Endoscopy is widely used for screening, but the method is invasive. One patent describes a process of detecting gastric cancer by determing an expression lvel of inter-alpha trypsin inhibitor heavy chain H3 (ITIH3) (US 2012/0028269).

Pancreatic cancer: is the most fequent adenocarcinoma and has the wrost progrnosis of all cancers, with a five year survival rate. Pancreatic cancer occurs with afrequency of about 9 patients per 100k individuals making it the 11th most common cancer in the US. Currently the only curative treatment is surgery, but only 10-20% of patients are candidates for surgery at the time of presentation and of this goup, only 20% of patients who undergo a curative oepration are alive after five years. Lubman (WO2007/112082). 

Glioblastoma: 

Glioblastoma is the most common form of brain cancer. There are about 10,000 new cases in the U.S. each year. A very aggressive cancer gene is responsible for about a third of all glioblastomas. However, a new vaccine (EGFRvIII) has been shown to be able to educate the immune system to produce antibodies against the tumor. 

Leukemia: see outline
Lung Cancer:  see outline

Multiple Myeloma:

Multiple Myeloma is a malignancy of plasma cells. Neoplastic cells are located in the bone marrow, and osteolytic bone lesions are characteristic. Bone pain related to multiple lytic lesions is the mos common clinical presentation. 

Multile myeloma is treated with chemotrherapeutic agents that are often initially effective, only for the blood malignance to retun months or years later. Accordingly, many patients go through 4-or more rounds of potent drug treatment to reduce the mounting myeloma cells. 

An antigen, called MB281 or Protein M, is known to non-specifically bind to immunoglobulins with high affinity. MG281 protein has the properties of a B-cell super antigens in that immunoglobulin reactivity with this protein is non-specific. The protein is about 556 amino acids long and contains a 16-36 amino acid transmembrane domain. 

treatment: 

Anti-CD38:  DARZOLEX (Johnson & Johnson) is an anti-CD38 antibody that kills multiple myeloma cells by attaching to CD38 which is present at greater amounts on myeloma cells. 

Myelodysplastic Syndromes (MDS):

MDS are a hterogeneous group of clonal hematologic disorders characterized by ineffective ematopoiesis and dysplasia. In MDS, genomic abnormalities accumulate in a hematopoietic stem cell leading to peripheral cytopenias of varying degrees of severity, as a consequence of multilineage differentiation impairment, and in the early phases, bone marrow (BM) apoptosis. Morbidity and mortality in the disease results form cytopenias or transformation to acute myeloid leukemia, which may both lead to serious infectious diseaes, anemia or hemorrhage caused by dysfunciton and reduction of blood cells. Cytopenia (low blood cell count) resutls form a high rate of apoptosis within the bone marrow environment and consequent lack of release of cells into the periopheral blood circulation. (Albitar, US Patent No: 10,604,801).

Diagnosis of MDS:

Proepr abnd early diagnosis is very important for treating and managing progression of MDS becasue a chance for remission is much higher if MDS is detected prior to the stage where it has progressed to leukemia. Diagnosing MDS can be cahllening, especially during early stages when a patient’s symptoms include cytopenia without an increase in blasts. Tehre are numerous reactive processes that cause cytopenia including drug reaction, nutritional or hormonal deficiencies, and autoimmune diases or chronic infection. The majro criteria for diagnosing MDS are the presence of periopheral cytopenia and dysplasia. However, evaluating dysplasia is subjective and can be difficult without bone marrow biopsy. It was discovered that periopheral blood plasma is enriched with tumor specific DNA in patietns with MDS. DNA is beleived to be due to the relatively high apoptosis of neoplastic cells in MDS. These neoplastic cells die in bone marrow and rleease their DNA in circulation. Most likely this DNA is in the form of apoptotic bodeis, microvessels or protein-DNA complex. (Albitar, US 10,227,657)

–Next Generation Sequencing (NGS) on cell-free DNA:

Albitar, (US 10,227,657) disclsoses a method of treating a patient for myelody plastic syndrome (MDS) that includes a step of identifying the patient as an MDS patient by performing a mutation analysis comprising next geenration sequencing (NGS) on cell-free DNA from the patient’s periopheral blood plasma or serum. In some embodiments, the mutation analysis is performed on MDS assocaited genes such as ASXL1, ETV6, EZH2, IDH1, IDH2, NRAS, CBI, RUNX1, SF3B1, SRSF2, TET2, TP53, U2AF1 and ZRSR2. NGS analysis includes any DNA sequencing method which is performed by adhering a DNA sample to a solid substrate and without separating DNA molecules according to their lenght by electrophoretic gel. In some embodimetns, the NGS can be performed with ILLUMINA (SOLEXA) SEQUENCING kit, ROCHE 454 SEQUENCING, ION TORRENT: PROTON?PGM SEQUENCING (Thermofisher) and SOLID SEQUENCING (Thermofisher). (Albitar, US 10,227,657)

Treatment:

Known treatments that can be adminsitered to a patient diagnosed by the NGS mutation anlysis include transfusion therpay, erythropoiesis-stimulating agents, antibiotics, drug therapy, chemotherpay with or without stem cell therapy, entry into clinical trials or a combination thereof. (Albitar, US 10,227,657)

Myeloproliferative Neoplasms (MPNs):

MPNs are chronic myeloid cancers that are characterized by the overproduction of mature blood cells, and that may evolve into acute myeloid leukemia. In addition to chronic myeloid leukemia with the BCR-ABL fusion gene, the three most common myeloproliferative neoplasms are essential thrombocythemia, polycythemia vera, and myelofibrosis. (Albitar, US Patent No: 10,550,435)

Diagnosis of MPNs:

Chronic myeloproliferative neoplasms (MPNs) are diagnosed and confirmed by morphologic and characteristic molecular abnormalities. Almost all cases with polycythemia vera (P-Vera) are characterized by a mutation in MAK2. Primary myelofibrosis (PMF) and essential thrombocythemia (ET) show JAK2 mutation in 40% of cases and MPL gene mutation in 2% of cases. Tests for these JAK2 mutations have greatly simplified the diagnosis of MPNs and are part of the standard screenign mechanisms. However, distinguishing essential thrombocythemia with nonmutated JAK2 form teh more common reactive thrombocytosis remains a diagnostic challenge. Albitar, US Patent No: 10,550,435)

Somatic mutations in the calreticulin gene CALR are detected in periopheral blood in 65-85% of essentail thrombocytheia (EG) and primary myelofibrosis (PMF) thatients that are JAK2 and MPL mutation negative. Moelcular anlysis of these three genes now allows these markers to be identified in >90% of MPN patients, helping to classify the disease and differentiate it form a reactive process. CALR mutation testing also has prognostic value as CALR mutations are assocaited with longer survival and fewer thrombotic evens compared to JAK2 mutations. Calreticulum is a calcium-bhding protein invovled in signaling and protein expression taht is believed to be responsbile for clearing misfolded prtoeins and involved in expression regulation. CALR mutations reported in myeloproliferative neoplasms create translation frameshifts in exon 9 which truncate the C terminal calcium binding domain and create a novel C-terminal peptide. Albitar, US Patent No: 10,550,435)

–Fragment Lenght analysis (FLA) of circulating tumor DNA (ctDNA): 

A non-invasive method for disease monitoring is through analyzing tumor derived circulating DNA found in blood plasma. Circulating DNA in blood palsma can be found either as cell free circualting tumor DNA (ctDNA) or in circulating tumor cells (CTCs). The current udnerstanding is that tumors udnergoing necrosis or apoptosis may depsit cell free fragmetns of DNA into the bloodstream, which may correlate with prognosis and tumor staging. Fragment lenght analysis (FLA) is a reliable technique for detecting mutations such as JAK2 V617F mutant allele and JAK2 V617F mutations. Albitar, US Patent No: 10,550,435)

Albitar, (US Patent No: 10,550,435) discloses a method for screening a patient for a myeloproliferative neoplasms which incldues extracting cell free DNA from periopheral blood plasma or serum, performing fragment lenght analysis of calreticulin human gene (CALR) in the cell free DNA and determining tumor laod and biallelic mutation in calreticulin human gene (CALR). At least in some embodimetns, the fragment lenght analysis includes a step of labeling CALR fragments with one or mroe fluorescent dyes, amplifying the labeled fragmetns using PCR, separating the labeled fragments by size using capillary electrophoresis and analyzing the data using software to determine the size of the amplified labeled fragments and genotype. 

Non-Hodgkin’s and Hodgkin’s lymphoma (NHL): See B-cell lymphomas on right hand panel. 

Neuroblastoma: 

Treatment:

Anti-Ganglioside GD2: 

Neuroblastoma is one of the most common solid tumors in children. Over 95% of the pateints present with non-resectable primary tumors and disseminated metastasis to distant organ sites at diagnosis. The overall survival rate of such patients has not been significanlty imporved during the last 20 year despite high dose chemotherapy follwed by allogeneic or autologous hematopoietic stem cell transplantation and differentiation therapy with 13-cis retinoic acid. One concept in the treatment is targeting of tumor cells with mAbs directed against the glycolipid antien ganglioside GD2, which is pexressed at high desnity in most neuroblastoma cases. In contrast, limited expression bhas been detected in normal tissues. A variety of murine and human mouse chimeric anti-GD2 antibodies have been generated. such as CH14.18. (Zeng, Molecular Immunology 42 (2005) 1311-1319). 

Ganglioside GD2 is a glycolipid that is strongly expressed on the surface of nueroblastoma cells. There is little intra or intertumor heterogeneity of the GD2 expression. The ch14.18 antibody is a chimeric construct using murine variable region H and L chain genes and human constant region genes for H IgG and L chain kappa. This antibody binds GD2 and activates complement. It mediates ADCC jby neutorphiles and NKs and lymphokine activated killer (LAK) cells. (J Clin Oncol 27:85-91, 2008). 

Ovarian:

Prostate 

Mesothelioma is a rare and aggressive cancer.

Non Hodgkin’s Lymphoma (NHL): 

NHL is a broad category encompassing 20 different malignancies of B-cell or T-cell origin, which vary considerably in proliferation rate, histology, immunophenotype, cytogenetics, and ultimately in response to therapy. Since the early 1980s, following the first report of Burkitt’s like lymphoma in homosexual men, there has been a steady increase in the reported incidence of NHL developing in patients with AIDS. Cyclophosphamide/doxorubicin/vincristine/prednisone (CHOP) has been the standard of therapy for aggressive NHLs, curidng greater than 30% of patients with diffuse large-cell NHL. A cHOP/rituximab comnbination has also become a popular regimen based on results from separate phase II/III trials evaluating the combination using different schedules in elderly patients with aggressive B-cell NHLs. Ritximab is a chimeric antibody that binds to the CD20 antigen, which is expressed on a majority of B-cell lymphomas.

Skin cancers  See outline

Renal cell carcinoma (RCC) accounts for about 3% of all adult malignancies in the US and is a clinicopathologically heterogeneous disease that includes several histologically distinct cellular subtypes. Evidence suggests that proximal renal tubules are the sites from which malignant RCC cells originiate, although such cells may also originate from distal tubules. A number of genetic syndromes predispose to the development of RCC (US 2009/0258002).

Thyroid Cancer: is the fastest growing cancer in the US with an estimated 46k plus new cases expected in 2012 according to the American Cancer Society. Diagnosis is by thyroid nodule fine needle aspirations (FNAs), a minimally invasive procedure to extract suspicious cells for examination under a microscope. However, FNA produces ambiguous results in up to 30% of cases. Because thyroid cancer is highly treatable, current guidelines recommend that most of these patients undergo thyroid resection for a definitive diagnosis. Post-surgical results, however, show that only 20-30% of these patients have the cancer. A company called Veracyte is using expert cytopathology of thyroid nodule FNA samples together with the company’s Afirma Gene Expression Classifier to resolved indeterminate results and thus avoid unnecessary surgery. Genzyme is also developing and marketing thyrotropin alfa for infection for patients with well-differentiated thyroid cancer under the trademark Thyrogen®. Thyrogen® is approved in the US as an adjunctive treatment for radioiodine ablation of thyroid tissue remants in patients who have unergone a near total thyroidectomy for well differeniated thyroid cancer and who do not have evidence of metastic thyroid cancer.

Vaginal cancers are rare entitiels, which are frequently associated with cervical or vulvar cancers. The upper vagina seems to be involved in most instances. If the cervix is involved with an invasive cancer with concomitant vaginal involvement, it is diagnosed as a primary cervical caner with extension into the vagina. The incidence of the disease is between 35 and 90 years, with most seen in the 60-79 year age range. Depending upon where the carcinoma is located, extension with regard to lymph node metastasis mimics the adjacent organ. If disease is in the upper vagina, it follws a spead pattern of cervical cancer, with metastasis to the obturator, iliac, and hypogastric lymph nodes.

Treatment is tailored to the extent of the disease. Large cancers are treated initially with external irradiation, which hopefully will shrink the tumor so local therapy will be more effective. External irradiation in a dose of 4000-5000 cGy is given initially for bulky stage I and II cancers. Some type of local therapy is then used, which may consist of vaginal ovids or a similar applicaitonthat will cover the whole vagina, delivering a surface dose of 6000 cGy, or higher doses is two applicaitons are used. One study concluded that a tChimeric antigen receptor (CAR) T cell therapy:otal radiation dose over 75 Gy including brachytherapy does great than 30 Gy provide a significant increase in residual-free survival. 

Depending upon the location of the primary tumor, the draining lymph nodes are also treated with external irradiation.

Pancreatic Cancer:

Pancreatic Ductal Adenocarcinoma: is resistant to current immuotherapies and remains one of the most letal cancer in humans. The resistance of pancreatic cancer to immunotherpay could be due in part to a paucity of neoantigen-reactive tumor-infltrating lymphocytes resulting form the low mutational buden of the diase; such lymphocytes may be critical mediators of immunotherpay responses in solid cancers. (Leidner, “Neoantigen T-cell Receptor gene therapy in pancreatic caner” NEJM, 382; 22, 2022.)

–-Cimeric antigen receptor (CAR) T cell therapy: targetting T-cell therapy targeting CD19 and B cell maturation antigen has revolutionized the treatment of hematologic cancers. However, the transfer of T cells that have been genetically enigneered to express CAR agaisnt overexpressed pancreatic-cancer antigens such as mesothelin, CD133 and epidermal growth factor receptor has been alrgely inefective in patients with pancreatic ancer. Leidner, “Neoantigen T-cell Receptor gene therapy in pancreatic caner” NEJM, 382; 22, 2022.

Leidner, “Neoantigen T-cell Receptor gene therapy in pancreatic caner” NEJM, 382; 22, 2022) disclsoes that a patient with progressive metasatic pancratic cancer was treated with a single infusion of 16.2×109 autologus T cells that had been genetically enigneered to clonally express two allogenic HLA-C*08.02 restricted T cell receptors (TCRs) tagetting mutation KRAS G12D expressed by the tumors. The pateint had regression of visceral metastases. The engineered T cells constituted more than 2% of all the cirulating periopheral blood T cells 6 months after the cell transfer. Note that HLA-C*08:02 is expressed by about 8% of white persons and 11% of black persons in teh US and by lower percetnages of persons in most other racial and ethnic groups, a situation that limits this therapy to a relatively low percetnage of potential patients. However, additional TCRs targeting KRAS G12D and other hot-spot KRAS mutants restricted by different HLA molecules that have been ienntified could extend TCR gene therapy agaisnt mutant KRAS to a larger number of patients. 

Synovial Sarcoma:

Synovial sarcoma is a rare form of cancer in which malignant cells develop and form a tumor in soft tissues of the body. This type of cancer can occur in many parts of the body, most commonly developing in the extremities. The cancerous cells may also spread to other parts of the body. Each year, synovial sarcoma impacts about 1,000 people in the U.S. and most often occurs in adult males in their 30s or younger. 

Treatments:

Treatment typically involves surgery to remove the tumor and may also include radiotherapy and/or chemotherapy if the tumor is larger, returns after being removed or has spread beyond its original location.

–TCR Targetting MAGE-A4:

TTecelra is also the first FDA-approved T cell receptor (TCR) gene therapy. The product is an autologous T cell immunotherapy composed of a patient’s own T cells. T cells in Tecelra are modified to express a TCR that targets MAGE-A4, an antigen (substance that normally triggers your immune system) expressed by cancer cells in synovial sarcoma. The product is administered as a single intravenous dose. Among the 44 patients in the trial who received Tecelra, the overall response rate was 43.2% and the median duration of response was six months.

One of the most useful diagnostic tests for cancer is the use of imagining systems such as follows:

–Radiographs/XRays: are very useful in describing the density of bone. Where cancer has infiltrated bone, the image will not appear as white as normal bone. A disadvantage of xrays is that not much can be determined about soft tissues.

–Sonography: use sound waves to determine whether a mass is solid (which will appear whiter).

–Computer Tomography (CT): or CAT scans use serial X-rays to reconstruct a 3D image of tissue.

–Magnetic Resonance Imaging (MRI): (see right hand panel)

See also “diagnostic assays” under “diagnostic techniques”

Galleri multi-cancer early detection test  (screens for more than 50 different cancers by searching a blood sample for tiny scrps of DNA cast off by cancer cells)

Diagnosis of cancer is of the greatest importance since if cancers can be detected at early , they can often be surgical removed and the patient cured.

Imaging: see outline

Histopathology: 

PAP (Papanicolao) smear: have been extremely effective at eradicating cervical cancer. In a PAP test, a collection of cells from the cervix of the uterine is taken and examined under the microscope to look for abnormalities.

Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) has been used in resolving proteins in biological specimens and used to differentiate ovarian, breat, prostate and liver cancers from healthy controls (Zhang, Gynecol Oncol, 102:61-66, 2006).

Look for aberrant levels of specific proteins in Blood/Cell.

For example, the protein CA125 is abnormally expressed with ovarian cancer and PSA is abnormally expressed with prostate cancer.

Density gradient centrifugation:Park (US 13/302678) discloses a method of separating cancer cells in a biological sample such as blood using a ligand which is specific to a surface marker on a target cell. The ligand is attached/bound to a particle and then allowed to specifically bind to a surface marker on a target cancer cell to form a particle target cell complex which will thus have a density difference with other cells and can thereby be separated using the density difference. 

Look for specific biomarkers/antigens on Exosomes:

Exosomes are small vesicles that are released into the extracellular environment from a variety of different cells, including tumor cells. An exosome is typically created when a segment of the cell membrane invaginates the exosome which is exocytosed. Exosomes may also be referred to as a microvescile, nanovesicle, vesicle, dexosome, bleb, blebby, prostasome, microparticle, intralumental vesicle, endosomal like vesicles or exocytosed vehicles.

–Isolation of Vesicles with antibodies specific to vesicle biomarker:

Spetzler (US2013/0178383) discloses isolation of a vesicle from a biological sample using a capture agent such as an antibody that binds to a biomarker (examples include CD63) on a vesicle. The binding agent can be bound to particles such as beads and the antibody bound particle is used to isolate the vesicle. In one embodiment, a vesicle having a cancer specific antigen on its surface can be captured using an atnibody specific for the antigen and then the vesicle is detected using that antibody which has been labeled with a fluorescent dye. In an alternative embodiment, once the vesicle is captured with the antibody, the caputred vesicles are then detected using detector antibodies against the same or different vesicle antigens of interest.

Taylor (Molecular Biology, 728, pp. 235-246) disclose protocls for size exlcusion chromatography, magentic beads with anti-EpCAM antibodies and ultracentrifugation for isolation of exosomal components from women diangosed with stage III serous adenocarcinoma of the ovary.

Look for circulating Tumor DNA (ctDNA):

Blood contains cellular componlated DNA within the bloostream originating from various cell types. The portion of the cfDNA in the blood of cancer patients released from tumor cells via apoptosis, necrosis, or active release is commonly refered to as circulating tumor DNA (ctDNA) The ctDNA has gained increasing attention becasue of the potentail to detect early cancer metastases. (Park, “Clinical circulating Tumor DNA testing for Prescision oncology” Cancer Res Treat. 2023; 55(2), 351-366). 

Commercial Products:

RaDaR Assay (Neogenomics – Inivata): 

Signatera (Natera): is a test which involves tumor tissue sequencing followed by the design of patient-specific panels for sequencing cell-free NA in blood. (see US Patent No: 11,530,454), which covers perfomring whole-exome sequencing or whole-genome sequencing on a tumor sample of the subject to identify a plurality of tumor-specific SNV mutations, targeted multiplex amplification of 10-500 loci from cell-free NA isolated form a plasma sample, and sequencing the resulting amplicons to detect one or more of the tumor specific mutations.  

Imagining Techniques:

Electron microscopy (EM) has demonstrated the presence of microvesicles and exosomes isolated from biological fluids of cancer patients. The composition of these vescicles can then be determeined by Western immunoblotting. Dynamic light scattering (DLS) can be used to measure these vesicles in suspension and determine size by light scattered from all vesicles under Brownian motion. The number and size of circulating vesicles has also been investigated using nanoparticle tracking analysis (NTA) where quantum dots are conjugated to anti-tumor antigens (anti-CD63 or anti-Ep-CAM antibodies using a Qdot 585 Antibody Conjugation kit. These conjugates were then added with the vesicles and analyzied by NTA. (Taylor Analytical Biochemistry 428 (2012) 44-53).

Detection of Autoantibodies to Cancer Specific Proteins/Peptides/Antigens

This type of detection method is much like when HIV peptides are detected by looking for whether people have developed antibodies (auto-antibodies) to such peptides. 

Taylor (WO 2011/063232) discloses methods for detecting or staging cancer by contacting a sample having IgG type (particularly IgG2 subclass) antibodies with a tumor associated antigen and then detecting the formation of the antibody-antigen complex where the presence of the autoantibodies incidates the presence or stage of cancer. In a separate paper by Taylor (Gynecologic Oncology, 115 (2009) 112-120) a diagnostic array utilized specific exosome dervied antigens to detect reactive IgG in patient’s sera.

Taylor (WO2008/092164) also disclsoes providing a biological sample suspected of comprising autoantibodies from a cancer subject and then contacting that sample with an cancer antigen which is a peptide that has been determined to be immunoreactive with an autoantibody isolated from an exosome from a cancer patient and then detecting the imuno-conjugate as with ELISA or Western blotting.

Antibody or Protein G which binds to Heavy Chain of IgG bound extracellular vesicle

Taylor (US13/795708, published as 2014/0186293; 15/152.901, published as US 2017/0097352) discloses methods of diagnosing cancer by isolating an IgG bound extracellular vesicle. The patent is based on the discovery that IgG binds to tumor antigens on extracellular vesicle in incaner patients. By using an agent such as protein G which binds to the heavy chain contstant domain of IgG, the IgG bound extracellular vesical can be isolated. 

Carcinogenes is is the process by which cancer develops. By the time the cancer has been detected, it contains billions of cells, often including many normal cells like fibroblasts in the supporting connective tissue associated with the carcinoma. These normal connective cells of a tumor (neoplastic mass) are sometimes referred to as the stroma whereas the neoplastic cells of the tumor are called the parenchyma. However, There is evidence that cancers originate from a single abnormal cell. While most malignant tumors are monoclonal in origin, this does not mean that a singe mutation is by itself enough to . In fact, evidence indicates the the genesis of a cancer typically requires several independent rare accidents that occur in the lineage of one cell.

Cancers seem to arise by a process in which an initial population of slightly abnormal cells which are descendants of a single mutant ancestor, evolve from bad to worse through successive cycles of mutation. Such evidence comes from epidemiological studies of the incidence of cancer as a function of age. If a single mutation were responsible, occurring with a fixed probability per year, the chance of developing cancer in any given year should be independent of age. In fact, for most types of cancer the incidence rises steeply with age as would be expected if cancer is caused by a slow accumulation of numerous random mutations in a single line of cells.

Animal models also confirm that a single genetic alteration is insufficient to cause cancer but rather that multiple mutations are required in many genes (perhaps ten or more). For those cancers that have a discernible external cause, cancer also does not usually become apparent until long after exposure to the causal agent. The incidence of lung cancer, for example, does not begin to rise steeply until 1-2- after heavy smoking. The incidence of luekemias from radiation similarly do not show a marked rise until about 5 years after exposure.

Other factors which are important for the development of cancer cells and how they evade host immune response are covered in a separate disease mechanisms section (see right hand panel). 

Cancer Mutations

All cancers are believed to be due to mutations. (Albitar, US 10,227,657)

Myeloid differentiation primary response gene 88 (MYD88): has significant therapeutic and diagnostic value in a range of cancer types, including Waldenstrom’s Macroglobulinemia (WM), diffuse alrge B-cell lymphoma (DLBCL), monoclonal gammopathy of unknown significance (MGUS), and spenic marginal zone lymphoma (SMZL). MYD88 is an adaptor molecule in a TLR and interleukin-1 receptor signaling pathway. Mutation in MYD88 results in over-activation of Toll-interleukin-receptor pathways, subsequent phosphorylation of IRAK1/4, and release of nuclear factor factor kappa B (NF-kB) drive cell survival and proliferation. It has been demonstrated in DLBCL and WM that inbhibition of MYD88 signaling resutls in decreased NF-kB activity and reduced cell survival. (Albitar, US 10,227,657)5P positive cells. However, inherent limtiations in this methodology prohibit AS-PCR from detecting variants other than those previously described or specifically designed for this purpose. Wild-type blocking PCR (WTB-PCR) using locked nucleic acid (LNA) has demonstrated high sensitivity and versatility in the detection of low percentage mutant populations. By adding an LNA oligo (10-12NT), complementary to the region of the hotspot, amplifcaiton of the WT allele is inhibited, leading to experimentally driven positive selection for mutant alleltes. This is accomplished by designing the LnA oligo so that it anneals to the template strand during the primer annealing step of PCR and melts form mutant template DNA, but not WT DNA, during extension. Becasue a single nucleotide mistmatch in the LnA-DNA hybrid greatly decreases its melting temeprature, only mutant template DNA is free to complete its extension. Albitar, US 10,227,657)

Most mutations in MYD88 occur at condom L265, converting leucine to proline; however, mutations at M232, P258, L103 and Q143 have also beenr eported. Allele-specific polymerase chain reaction (PCR) based assay have been developed for MYD88 L265P and dmonstrated the ability to detect minute fractions of L26

Cancer genomics and proteomics Types of Cancer How Cancer StartsTreatment and Strategies
Links of interest: Cancer Immunome Database Cancer Research Institute Memorial Sloan-Kettering Cancer Center  (see also Memorial Sloan free physician referral service at 800-525-2225 to gain access to world-renowened physicians for treamtent or a second opinion).  Cancer Genome Atlas (TCGA)   International Cancer Genome Consortium  

American Cancer Society   American Joint Committe on Cancer (AJCC) World Health Organization   Clinical Trials  Center for Cancer Research Clinical Trials   Cancer.net (list links for finding clinical trials)

US Oncology Network

Advocacy Groups of Cancer:  New Cures for Cancer 

Cancer is the second leading cause of death in the United States, after heart disease. More than one million Americans are diagnosed with cancer each year, with this number likely to increase as the population ages. Cancer kills one out of six people in the developed world. Among men, the three most commonly diagnosed cancers are prostate, lung and colorectal cancer in developed countries.

Solid tumors are composed to two prinicpal structures; (1) the malignant cell itself, and supporting cells, including connective tissue and vessels that comprise the stroma, which plays a fundamental role in nutritional support and the removal of waste products for tumor cells. One type of tumor associated stromal cell is the myofibroblast which develop in the neoplastic environment as tumor cells grow and progress and play an essential role in tumor cell metastasis by stimulating angiogenesis via secretion fo stromal derived factor (SDF_-1 and by controlling the formation of the extracellular matrix via secretion of matrix metalloproteases. Cho (Gynecologic Oncology, 123 (2011) 379-386

Cancer is a class of diseases in which a group of cells display uncontrolled growth, invasion and sometimes metastasis. Cancer begins as a single cell that progresses through mutations to a malignancy. “Cancer” or “tumor” refers to any neoplastic growth in a subject. The cancer can be of the liquid or solid tumor type. Liquid tumors include tumors of hematological origin (e.g., mylomas, luekemias, lymphomas). Solid tumors can originate in organs (e.g., lungs, breasts, prostate, colon, cervix, liver). While there are over 100 distinct types of cancer, transformed malignant cells in all of the cancers can exhibit the following common properties:

• loss of anchorage dependence: whereas normal cells need to attach to surfaces in order to survive, cancerous cells can be found floating in suspension cultures
• loss of contact inhibition: whereas normal cells will arrest growth after they have filled up a defined area, malignant cells have no respect for this contact inhibition
• focus formation; in tissue cultures a single cell is transformed leading to a single cancerous colony
• genetic abnormalities such as deletions, translocations and chromosomal ploidy
• evasion of programmed cell death or apoptosis
• larger nuclear to cytoplasmic ratios
• sustained angiogeniesis through the secretion of growth factors
• Tissue invasion & metastasis of cancerous cells to other tissues
• Limitless replicative potential
• Insensitivity to anti-growth signals
• Self-sufficiency in growth signals

  A  neoplasm is an abnormal mass, the growth of which is purposeless, autonomous and with a tendency to be an atypical and aggressive, new growth.  Oncology is the study of such neoplasms.

  Challenges in Cancer Research: 

  The success rate for new drugs in all areas of cevelopment is dismal. Out of 5-10k chemicals that enter the drug development pipeline only one will enter the market. The failure rate for oncology drugs is even higher. See forlifeonearth.org  for a list of references in the literature dealing with failure of animal models. 

  Development of cancer and response to anticancer drug therapy not only depends on discrete genetic alterations in the maligant clone but also on specific interactions between tumor cells and surrounding tissue components. Most established in vitro models fail to reflect the complex tissue archtiecture of an individual tumor. With respect to cancer research such as preclinical breast cancer resarch, the majority of such research is based on established cells lines. However, these cell lines frequently have undergone multiple changes influencing their biological behanior and thus no longer reflect the primary tumor of origin. Also, it is difficult to adapt the cells of many tumors to in vitro conditions when establishing a primary epithelial culture. In addition, it is most likely that separated tumor cells will behave differently in vitro, as both cell-cell and cell-matrix interactions are highly different compared to the in vivo situation (Heiko van der Kuip, BMC Cacer 2006, 6: 86, p. 2).

  To investigate tumor cell behavior ex vivo it is necessary to maintain or reconstitute an environment closely resembling the tumor tissue. To simulate such conditions either three dimensional tissue cultures using several biomatrices or co-culture experiments with tumor fibroblasts have been performed. However, these sytemems can not mimic the complex tissue architecture and the high degree of variability seen in individual tumors. One possibility to maintain the tissue architecture ex vivo is the direct cultivation of fresh and intact tumor material. A problem here too is restricted diffusion of oxygen and nutrients leading to cell death in the center of the tissue cubes. This problem can be overcome by the introduction of tissue microtome establishing the prepartion of thin tissue slices. The use of confocal laser scan miroscopy based technique has also enabled one to identify cell type and test cell viability in tissue slices maintained for at least 4 days in culture (Heiko van der Kuip, BMC Cacer 2006, 6: 86, p. 2).

Hemolytic anemia due to immune function is one of the major causes of acquired hemolytic anemia. (Rosse, “Immune-Mediated hemolytic anemia Hematology 2004, pp. 48-62). 

CHAPLE Disease:

Causes: CHAPLE disease “CD55 deficiency with hyperactivaiton of complement, angiopathic thrombosis, and protein-losing enteropathy” is an autosomal recessive disorder casued by loss of function mutations in CD55 (also known as decay accelerating factor, DAF). CD55 is a glycophosphatidylinositol (GPI)-anchored membrane proteins that inhibits the enzymatic activity of C3b and C4b, thus preventing the formation of C3 and C5 convertases that lead ultimately to the assembly of the membrane-attack complex (C5b-C9). Thus, the absence of CD55 causes overaction of the complement system. (Zhang, US Patent Application No: 17/078,309, published as US 2021/0139573). 

CD55 enodes the decay-accelerating factor (DAF), a widely expressed glycosylphosphatidylinositol-linked membrane protein. DAF regulates complement activaiton by inhibiting C3 and C5 cleavage and activation, thus protecting cells against complement induced self-injury. (“loss of CD55 in eculizumab-responsive Protein-losing enteropahy” NEJM, 2017). 

Symptoms: Signs and symptoms of CHAPLE can include hypoproteinemia (low serum levels of albumin and immunoglobulins), hyoproteinermia leads to facial and extremeity edema and recurrent infections, malabsorption syndrome (chronic diarrhea, failure ot thrive, anemail and micronutrient deficiencies), complement overaction, intestinal lymphangiectasis and bowel inflammation; and/or increased susceptibility to visceral thrombosis. 

Ozen “CD deficiency, early-onset protein-losing enteropathy, and thrombosis” NEJM, 2017) discribe 11 patients with abdominal pain and diarrhea casued by early onset protein losing enteropathy with primary intestinal lympahngietasis, edema due to hypoproteinenia, malabsorption and less fr3quently, bowel inflammation, recurrent infections and angiopathic thromboembolic diase; the disorder followed an autosomal recessive pattern of inheritance. Whole exome sequence enditified loss of function mtuations in the gene encoding CD55 (decay-accelerating factor). Patietns’ T lymphocytes hsowed increased complement activaiton causing surface deposition of complement and the generaiton of soluble C5a. CD55 deficiency with hyperactivaiton of complement, angiopathic thromosis, and protein losing enteropathy (the CHAPLE syndrome) is casued by absnormal complement activaiton due to biollelic loss of function mutations in CD55. 

Treatment: 

–anti-C5 antibodies:

CD55 deficient protein losing enteropathy (CD55 deficient PLE) is a rare disease also referred to as complement hyperactivation, angiopathic thrombosis, protein-losing enteropathy (CHAPLE disease) that can be treated by 5 blockage. (David, (Regeneron Pharmaceuticals), US Patent Application No: 17/078,309, published US 20210139573). 

—-Eculizumab (Soliris):

Lenardo (US2019/0256915) discloses methods of treating CD55 deficieincy, hyperactivation of complement, angiopathic thrombosis and protein losing enteropathy (CHAPLE) by administering a composition that includes at least one complement inhibitor such as the therapeutic antibody eculizumab. 

—-Pozelimab (REGN3918): 

David, (Regeneron Pharmaceuticals), US Patent Application No: 17/078,309, published US 20210139573) discloses adminstering an anti-C5 antibody such as REGN3918 to a subject sufffering form a C5 assocaited disease (e.g., PNH, aHUS, MG or CHAPLE).  In one embodiment, the regimen is one or more dsoes of about 30 mg/Kg intravenously and then one of more subcutaneous dsoes given weekly starting about 7 days after the first dose based on body weight of the pateint.  In one embodiment, the dosing regimen is one or more doses of about 30 mg/kg intravenously and then one or mroe doses of about 800 mg subcutaneously. 

Cold agglutinin disease (CAD)

Chronic cold agglutinin disease (CAD) is a subgroup of autoimmune hemolytic anemia (AIHA). (Berentsen, Hematology, October 2007, 12(5): 361-370). CAD is an autoimmune disorder characterized by autoantibodies that bind to the I antigen on red blood cells at colder temperatures. Once bound, the antibodies facilitate hemagglutination and complement mediated hemolysis of the cells. Tehre are two forms of CAD -primary and secondary. Primary is a form of CAD in which there is no underlying disease and secondary is associated with a pre-existing condition such as an infection or a neoplasms. For example, secondary CAD can be associated with HIV infection, Mycoplasma pneumonia infection, non-Hodgkin’s lymphoma or Waldenstrom’s macroglobulinemia. Symptoms of CAD can include, e.g., pain and reduction of circulation at the extremitiees, as well as symptoms of hemolytic anemia such as fatigue, jaundice and blood in the urine.

CAD is characterized by immunoglobulin M (IgM) mediated hemagglutination and robust complement activation leading to intravascular hemolysis and hemolysis related symptoms including anemia, fatiue, dyspnea, hemoglobinuria dn acrocyanosis. Allthough CAD can have a mild course, it may also be life threatining and its chronic nature leads to transfusion dependence in about 50% of cases. Roth “long-term efficacy of the ocmplement inhibitor eculizumab in cold agglutinin disease” Blood, 113, 2009, 3885-3886). 

Treatment:  

C5 antibodies: Eculizumab dosed at 600 mg intravenously every 7 days for 4 weeks, at 900 mg 7 days later adn then chronically at 900 mg every 14 days significantly reduced hemolysis as shown by reduction in levels of serum LDH from a mean of 850 UL in the year before treatment to 456 the year after treatment, improviement of anemia from a mean hemoglobin level of 9.8 g/dL to 11.7 g/dL in the year after treatment, improvement in fatirue and quality of life. Roth “long-term efficacy of the ocmplement inhibitor eculizumab in cold agglutinin disease” Blood, 113, 2009, 3885-3886).

Paroxysmal Cold Hemoglobinuria (PCH)

PCH, also known as Donath-Landsteiner syndrome, is characterized by the sudden presence of hemoglobin in the urine (called hemoglobinuria), typically after exposure to cold temperatures. In PCH, a polyclonal IgG anti-P autoantibody binds to red blood cell surface antigens in the cold. PCH, unliked cold reacting IgM, does not cause red cell agglutination but similar to cold reacting IgM it is able to fix complement. The mechanisms of hemolysis in PCH is similar to that in CAD. As the blood circulates to the periphery, it cools and the antibody and the first two components of complement are fixed to the RBC surface. PCH can follow a variety of infections 9e.g., syphilis) or neoplasms such as non-Hodgkin’s lymphoma. PCH can be an acute even or can be recurrent. Symptoms include pain, fever, pallor, icterus, urticarial dermal eruption, hemoglobinuria, hemoglobinemia and anemia. In some cases, symptoms can progress to renal disease or death resulting from anemia and massive hemolysis associated multi-organ failure.

Paroxysmal Nocturnal Hemoglobinuria (PNH): 

Causes:

PNH is an uncommon blood disorder where red blood cells are compromised and thus destroyed more rapidly than normal red blood cells. It is an acquired hematologic disease that results from clonal expansion of hematopoietic stem cells with somatic mutations in the X-linked gene called PIG-A. Mutations in PIG-1 lead to an early block in the synthesis of glycosylphosphatidyllinositol (GPI)-anchors, which are required to tether many proteins to the cells surface. Intravascular hemolysis is a prominent feature of PNH and a direct result of the absence of the GPI-linked proteins that protect cells from complement mediated attack. Two such proteins, CD55 and CD59, are absent from PNH type III erythrocytes, platelets, and other blood cells. CD55 regulates early complement activation by inhibiting C3 convertases whereas complement regulatory protein CD59 inhibits the assembly of the membrane-attack complex C5b-C9 by interacting with C8 and C9.

PNH is defined by the presence of a large populaiton of blood cells that, as a result of an inactivating somatic mutation in the X-linked gene PIGA, are deficient in all proteins that are normally tethered to the cell membrane through a glycosyl-phosphatidyl-inositol (GPI) molecule. Becasue these include the complement regulatory surface proteins CD59 and CD55, GPI deficient red cells are exquisitely sensitive to lysis by activated complement. (Risitano “Advances in understanding the pathogenesis of acquired aplastic anaemia” 2018)

Signs & Symptoms:

Excessive or persistent intravascular hemolysis in PNH patients results in hemolytic anemia (a decreased number of red blood cells) but also hemoglobinuria (the presence of hemoglobin in the bloodstream) and clinical sequelae related to the release of the erythrocyte contents into the circulation including fatigue, thrombosis, abdominal pain, dysphagia, erectile dysfunction and pulmonary hypertension.

PNH is characterized by chronic uncontrolled terminal complement activaiton and hemolysis. Uncontrolled complement activaiton leads to red clood cells (RBC) hemolysis, platelet activation and subsequently thromboembolism (TE), renal and other organ impairment, pain, severe fatigue, poor quality of life and early mortality. 

Clinically, PNH is typified by the triad of severe haemolysis, thrombosis and cytopenias with high reticulocytosis, very high serum lactate dehydrogenase and erythroid hyperplasia in the bone marrow. (Risitano “Advances in understanding the pathogenesis of acquired aplastic anaemia” 2018). 

Diagosis: The laboratory evaluation of hemolysis normally include hematologic, serologic and urine test. Hematologic tests include an examination of the blood smear for morphologic abnormalities of RBCs, and the meaurement of the reticulocyte count in whole blood (to determine bone marrow compensation for RBC loss).

Serologic tests include lactate dehydrogenase (LDH) as a direct measure of hemolysis. LDH levels, in the absence of tissue damage in other organs, can be useful in the diagnosis and mointoring of patients with hemolysis. An indicator of cell ysis is the apeparance of abnormally high levels of lactate dehydrogenase (LDH) in the serum. An LDH serum level of greater than 1.5 or 2 is a marekr of uncontrolled complement activaiton that has been used in multinational PNH clinical trils. Normal serm levels of LDH can vary depending on the laboratory and methods used for measurements; however, in children, the normal level is about 60-170 U/L and in adults it is about 100-190 U/L. 

The gold standard diagnostic test for PNH is the analysis of GPI linked molecules on the surface of hemtopoietic cells by flow cytometry. The minimum requirement to diagnosise PNH is the demonsration that a propotion of red cells are deficient in at least two different GPI linked molecules. (Rosse, “Immune-Mediated hemolytic anemia Hematology 2004, pp. 48-62).

Treatment: Many PNH patients depend on blood transfusions to maintain adequate erythrocyte hemoglobin levels. 

–Anti-C5 antibodies:

Payton (US Patent Applicaiton No: 16/757,512, published as US 20200254092) discloses a method for treating PNH or aHUS that includes adminstiering an anti-C5 antibody at 2400-3000 mg for a patient weighing 40-60kg, 2700-3300 mg for a pateint weighting greater than 60 and less than 100 kg or 3000-36000 mg for a patient weighing more than 100 kg. Exemplary antibodies that can be adminstiered include eculizumab (also known as Soliris), rayulizumab (also known as Ultomiris, ALXN1210 and BNJ441), 7086 antibody (described in US Patent Nos: 8,241,628 and 8,883,158), 305LO5 antibody (described in US 2016/0176954), the SKY59 antibody (described in Fukuzawa T., et al. Rep. 2017, Apr 24; 7(1): 1080) and REGN3918 antibody (alsko known as H4H12166PP and described in US 20170355757). The anti-C5 antibodies can also include substitutions that enhance the binding affinity of the antibody Fc constant region for FcRn such as the M252Y/S254T/T256E triple substitution (described by Dall’Acqua et al., 2006 J Biol Chem 281: 23514-23524), the M428L or T250Q/M428L substituions (described in Hinton (2004) J Biol Chem 279: 6213-6216 and Hinton (2006) J Immunol 176: 346-356) and the N434A or T307/E380A/N434A substitutions (described in Petkova (2006) Int Immunl 18(12): 1759-69). 

—-Eculizumab (Soliris®) which is a humanized monoclonal antibody directed against ther terminal complement protein C5 has been shown to reduce intravascular hemolysis and transfusion requirements. For example, Hillmen (N Engl J Med 2004; 350, 552-9) disclose a method for treating PNH by administering an effective amount of eculizumab which binds to the terminal complement protein C5, inhibiting its cleavage into C5a and C5b. In one embodiment, Hillmen teaches a dose at 600 mg every for four weeks followed one week later by a 900 mg dose and then by 900 mg every other week through week 12. Hill (Haematologica 2005) also teaches treating patients afflicted with PNH by administering eculizumab that specifically target C5. 

The variable regions of eulizumab are described in PCT/US1995/005688 and US Patent No: 6,355,245. The full heavy and light chains of eculizumab are described in PCT/US2007/006606.

—-Ravulizumab (trade name: ULTOMIRIS; also known as BNJ441 and ALXN1210): is a long-acting C5 complement inhibitor administered every eight weeks for the treatment of adult patients with PNH. It is advantageously administered every eight weeks and was shown to be equally efficacioues as SOLIRIS above wich is adminsitered bi-weekly. The drug was devleoped by Alexion Pharmaceuticals and approved by the FDA in 2018. 

Ravulizumab is described in PCTUS2015/019225 and US Patent No: 9,079,949.  Ravulizumab-cwvz is a humanized mAb produced in CHO cells. it consists of 2 dientical 448 amino acid H chains and 2 identical 214 amino acid L chains and has a MW of aobut 148 kDa. The cosntant regions of ravulizumab-cwvz include the human kappa light chain constant region and the protein engineered IgG2/4 H chain constant region. The H cahin CH1 domain, hinge region and the first 5 amin o acids of the CH2 domain matach the human IgG2 amioo acid sequence, residues 6-36 in the CH2 region (common to both human IgG2 and IgG4 amino acid sequence), while the remainder of the CH2 domain and the CH3 domain match the human IgG4 amino acid sequence. The H and L chain variable regions that form the human C5 binding site consits of human framework regions grafted to murine complementaryity-deterining region. (“Ultomeris (ravltizumab-cwvs) inection, for intravenous use, initial U.S. Approval: 2018). 

Andrien (US 2017/0298123) discloses that BNJ441 (“Ravulizumab”) relative to exulizumab contains four amino acid substitutions in the H chain, Tyr-27-His, Ser-57-His, Met-429-Leu and Asn-435 Ser (note that positions 429 and 435 of BNJ441 correspond to positions 428 and 434 uner the EU numbering system). These mutations were engineered to enable an extended dosing interval verus exulizumab by increasing the circulating half-life by reducing antibody clearnce and increasing the eficiency o f the FcRn-medaited antiboy recylcing. Adrian teaches taht the antibodies are useful for treating a variety of complement associated disorders such as aHUS, PNH and myasthenia gravis. 

The recommended dosing regimen for adult pateints (greater than 18 years of age) with PNH consists of a loading dose followed by maintenance dosing, adminsitered by intravenous infusion. Doses are administered based on the patient’s body weight (bod weight range (kg) of 40-60, 60-100 greater or equal to 100 are 2,400, 2700 and 3000 mg with maintenance dose of 3,000, 3,300 and 3,600 mg respetively. (see “Ultomiris (ravlulizumab-wvz)infection, for intravenous use, initial U.S. Approal: revised 12/2018) (also see “Ultomiris (ravalulizumab-cwvz) -new orphan drug approval, December 21, 2018).

—-REGN3918 (poxelimab): 

David, (Regeneron Pharmaceuticals), US Patent Application No: 17/078,309, published US 20210139573) discloses a REGN3918 (pozelimab) dosing regiment, including a subcutaneous dosing component for treating C5 related disorders such as PNH. REGN3918 adminsitered 30 mg/kg IV followed by 800 mg once weekly SC demonstrated robust inhibition of intravascular hemolysis, with normalizaiton of LDH in human patients with PNH. 

Zhang, (US Patent Application No: 17/078,309, published as US 2021/0139573) discloses treament of PNH by administering REGN3918 (poxelimab) over 26 weeks of treatmnet with a single loading dose of 30 mg/kg intravenously on day 1, then a dose not greater than 800 mg subcutaneously once weekly to week 26 or one or more doeses of about 30 mg/Kg of the anti-C5 antigen binding protein intravenously, then one or more subcutaneous doses (given weekly starting about 7 days after the first dose) based on body weight as follows: for <10 kg, 125 mg, for BW >10kg and <20 kg, 200 mg, for BW>20 and <40 kg, 350 mg, for BW >40 kg and < 60 kg, 500 mg and for BW>60 kg, 800 mg. Sose switching from eculizumab to REGN3918 resulted in normalization of serum C5 concentraitons and mainteained suppression of hemolytic activity. 

–Compstatin Analogs:

—-Pegcetacoplan (Empaveli) (compstatin anallog that binds to C3):

Deschatelets (US 20230076527) discloses methods of treatment of PNH using compstatin analogs, Compstatin is a cyclic peptide that binds to C3 and inhibits complement activation. US Patent No: 6,319,897 describes a petpide haivng the sequence Ile-[Cys-Val-Val-Gon-Asp-Trp-Gly-His-His-Arg-Cys]-Thr with the disulfide bond between the two cysteines denoted by brackets. The name compstatin was not used in this patent but was subsequently adopted in the scientific patent literature. It is beleived that extravascular hemolysis, one of the parameters contributing to the ongoing need for RBC transfusion despite eculizumab therpay, is mediated by C3b opsonization frather than C5 dependent MAC mediated intravascular hemolysis. While eclizumab is effective in addressing CD59 deficiency by preventing C5 dependent MAC meidated hemolysis, PNH cells are also deficient in CD55, which normally accelerates the dissociation of C3-convertase enzymes, inhibiting the production of C3 fragments and subsequent opoization. As a result, in the setting of eculizumab, surviging PNH RBCs become opsonized with C3b, targeting them for clearance through extravascular hemolysis by macrophages bearing complement receptors in the liver nad spleen. 

In May 2021, systemic pegcetacoplan was approved by the FDA with the brand name EMPAVELI for the treatment of adult patients with PNH. In December 2021, the European Commission approved systemic pegcetacoplan with the brand name Aspaveli for the treatment of adults with PNH who are anemic after treatment with a C5 inhibitor for at least three months. In January 2022, pegcetacoplan was also approved as EMPAVELI in Saudi Arabia and Australia. (See 10k for Apellis Pharmaceuticals For the fiscal year ended December 31, 2022).

See also Immune-mediated hemolytic anemia (outline)

Definitions

Hemolysis is the breakage of the red blood cells’ (RBC’s) membrane, causing the release of the hemologin and other internal components into the surrounding fluid. It is visually detected by showing a pink to red tinge in serum or plasma. There are a number of pathological conditions which can cause hemolysis.

Hemoglobin: is a tetrameric potein molecule composed of two identical alpha globin subunits (alpha 1 and 2) and two identical beta globin subunits (beta 1 and 2. In a hemaglobin tetramer, each alpah globin is associated with the beta globin to form two stable alpha/beta dimers, which in turn associated to form the tetramer. The subunits are noncovalently associated through Van der Waals forces, hydrogen bonds and salt bridges. A heme moleucle is incorporated into each of the alpha and beta globins. Heme is a large organic molecules coordinated around an iron atom. Heme that does not contain iron is called protoporphyrin IX (PIX) and is non-funcitonal (cannot bind a ligand). In the deoxygenated state, the four subunits form a tetrahedron. During ligand binding, the alpha1beta1 and alpha2beta2 interfaces remain relatively fixed while the alpa1beta2 and alpha2beta1 interfaces exhibit considerable movement. When the hemoglobin molecule is oxygenated, the intersubunit distances are increased relative to the deoxygenated distances and the moelcule assumes a relaxed state confirguation which is the predominant form of the molecule when a ligand is bound to the heme.

Hemophilia

Hemophila is due in over 80% of cases to a dificiency in factor VIII activity (hemophilia A), while in most of the remainder it is due to a deficiency in factor IX (hemophilia B). Global incidence of hemophilia is estimaited at between 200 to 400 thousand, with hemophilia A having an average incidence of about 2 per 10k people. 

Treatment:

Disease severity is linked by the percentage of residual active factor produced by the patient, and the idsease is treated via intravenous administration of the missing clotting factor. Before the introduction of clotting factor concentrates in the 1960s, the life expectancy was about 15-25 years. In the early 1980s, before the advent of screening tests of HIV in donated blood, large numbers of hemophiliacs contracted AIDS from clotting factors purified from human plasma. The introduction of recombinant clotting factors beginning in the ealry 1990s drastically reduced dependence on plasma derived products, and the 2017 global market value for such recombinant products stood at 8.5 billion. Treatment costs typically vary from 30k to several huundred thousand dollars annually. Clotting factor pdoucts are usually adminsitered therapeutically to control active bleeding or prophylactically to reduce frequency of future bleeding events. Administration for therapeutic purposes is tailed to individual circumstances while prophylactic adminsitration of unmodified (first generation) factors typically occurs three times weekly. Engineering to increase serum half life has relied on PEGylation, Fc fusion. (Gary Wash “Biopharmaceutical benchmarks 2018” Nature Biotechnology, 36(12), 2018)

Hemoglobinopathies/Hemoglobin Mutants:

Hemoglobinopathies, inherited disorders of hemoglobin structure and funciton, consist of structural hemogloin variatns and the thalassemias, inherited defects that reduce production in the synthesis of the alpha and beta globins of the human adult hemogloin. (Hedrick, “Population genetics of malaria resistance in human” Heredity 2011, 107, 283-304).

Two alpah and two beta subunits compose the tetrameric protein backbone of adult hemoglobin, and the alpha and beta subunits are more up of 141 and 146 amino acids, respectively, each with three exons. The genes that conde for alpha dn beta globins, HBA (there are actually two genes, HBA1 and HBA2, with identical amino acid sequnces) adn HBB, are unlinked and are locatge don chromsomes 11 and 16, respectively. (Hedrick, “Population genetics of malaria resistance in human” Heredity 2011, 107, 283-304).

Hudnreds of structural variatns of adult hemoglobin have been documented, but only three, all at the HBB locus, S, C and E, have reached substantial frequenceis in any popualtions. Two of these three, S and C, are different mutatns (with different phenotypes) at the same amino acid position 6 and although E is a structural variant at amino acid position 26, it also affects regulation in a manner similar to the thalassemias. 

Sickle Cell Anemia:

The sickle-cell allele (symbolized as S) was one of the frist human genetic variants that was associated with a specific moleuclar caused disease. The sickle-cell allele is widely known as a varaint that causes red blood cells to be deformed into a sickle shape when deoxygenated in AS heterozygotes, in which A incides the non-mutant form of the beta-globin gene, and also provides resistance to malaria in AS heterozygotes. In SS homozygotes, S casues the severe dsiease sicke-cell anemia. It is generally assumed that individuals with genotype SS had very low or zero fitness when, or if, there was no modern medical care. The greatest documented impact of S is that it protets agaisnt death or severe malarial disease in heterozygotes, and it apepars that S has less effect on the rate of infection. The S allele differs from the normal A allele at one nucleotide at codon position 6 in the beta globin molecule, which translate into a change form the amino acid glutamic acid to valine. The frequency of allele S is up to 0.2 in some parts of sub-Saharan Africa, Greece and India. There apepars to be five indpendent origins, or mutaitons, of the S allele form the A allele, based on theri existence in nearly non-overlapping geographical regions. (Hedrick, “Population genetics of malaria resistance in human” Heredity 2011, 107, 283-304).

Sickle cell anemia is a blood condition seen most commonly in people of African ancestry. It is inherited form both parents. If you inherit the sickle cell gene form only one of your parents, you will have a sickle cell trait but not the symptoms of sickle cell anemia. It is caused by an abnormal type of hemoglobin called hemologin S. Hemologlobin is a protein inside red blood cells that carries oxygen. The Hemoglobin S changes the shape of red blood cells so that they become shaped like crescents or sickles. These fragile, sicke-shaped cells deliver less oxygen to the body’s tissue and can also get stuck more easily in small blood vessels, as well as break into pieces that can interrupt healthy blood flow. The sickle gene has a genetic advantage in that it protects heterozygous carries from succumbing to Plasmodium falciparum malaria infection. Symptoms include painful episodes which cause pain in the bones of the back, the long bones and the chest. Treatment of SCD typically begins with early intervention involving newborn screen and prophylactic penicillin. One of the key components in the management of patients with SCD is transfusion, usually packed red blood cell therpy. As is noted by Staurt, however, sickle cell disease “is a systemic disease of monumental complexity” (Lancet, 364, 2004, p. 1357, last ¶).

The high motality and widespread impact of matlaria have resulted in this disease being the strongest evolutionary selective force in recent human history, and genes that confer resistance to malaria provide some of the best known case studies of strong positive selection in modern humans. (Hedrick, “Population genetics of malaria resistance in human” Heredity 2011, 107, 283-304). See also evolution

Sickle cell disease (SCD), a group of inherited blood disorders characterized by abnormal hemoglobin, reduces life expectancy by >20 years. SCD primarily affects persons whose ancestors came from Africa, where malaria is endemic, because the carrier state (sickle cell trait, inheritance of a sickle cell gene from only one parent) confers a selective advantage by protecting against the harmful effects of malaria. Thus, >90% of the estimated 100,000 persons in the United States with SCD are non-Hispanic Black or African American (Black), and an estimated 3%–9% are Hispanic or Latino (Hispanic). In persons with SCD, red blood cells become rigid and deform into a crescent or sickle shape. Sickled cells die early and often become lodged in small blood vessels, compromising blood flow, which can lead to serious health problems. SCD-associated complications include anemia; acute and chronic pain; infections; pneumonia and acute chest syndrome; stroke; and kidney, liver, and heart disease. Despite their extensive health care needs, many persons with SCD have difficulty accessing appropriate care and report feeling stigmatized and having their symptoms dismissed when they do seek care. SCD comprises four main genotypes; among these, the hemoglobin SS and hemoglobin Sβ0-thalassemia genotypes are the more severe forms and are collectively referred to as sickle cell anemia (SCA). SCA accounts for an estimated 75% of SCD cases in the United States. See CDC 

Given that SCA is a common cause of childhood stroke, children and adolescents aged 2–16 years with SCA be screened annually with transcranial Doppler (TCD) ultrasound to identify high cerebral blood velocity, an indicator of elevated stroke risk. Chronic blood transfusion therapy, the recommended intervention, substantially reduces stroke occurrence in children and adolescents identified as being at risk. Children and adolescents aged ≥9 months with SCA (including asymptomatic children) should also be offered treatment with hydroxyurea, a medication shown to be efficacious in preventing or reducing severe pain episodes, acute chest syndrome, and other SCA-associated complications and increasing patient survival. See CDC

Cause/etiology/symptoms

The cause of IPF is unknown; it may arise either from an autoimmune disorder or as a result of infection. Cigarette smoking has been identified as a potential risk factor and one case control study has suggested an assocaition between antidepressants and the risk of development of pulmonary fibrosis, alhtough this inding in the pathogenesis of IPF is unknown. (Am J Respir Grit Care Med 161: 646-664)

IPF is defined as a specific form of chronic fibrosing interstitial pneumonia limited to the lung and associated with the histologic appearance of usual interstitial pneumonia (UIP) on surgical (thoracoscopic or open) lung biopsy. (Am J Respir Grit Care Med 161: 646-664).

Symptoms include dyspenea (shortness of breach) and dry cough. Death can result from hypoxemia, right heart failure, heart attack, lung embolism, stroke or lung infection. 

Idiopathic pulmonary fibrosis (IPF): is characterized by inflammation and eventually fibrosis of lung tissue.

Treatment

Treatment includes corticosteroids (e.g., prednisone), penicillamine, and various anti-neoplastics. Other treatments include oxygen administration and in extreme cases, lung transplantation.

A small population of bone marrow stem cells which have the CXCR4 receptor are believed to move to the lung in the pathology of IPF in response to the chemokine stromal-dervied factor-1 (SDF-1) which is secreted by neutrophils and macrophages. These stem cells then differentiate into fibrocytes and elaborate collagen, contributing to fibrotic damage.